Mechanisms of the prostaglandin F2-induced rise in [Ca2+]i in rat intrapulmonary arteries
1 Department of Asthma, Allergy and Respiratory Science, King's College London School of Medicine, King's College London, London SE1 1UL, UK
Abstract
The mechanisms by which prostaglandin F2 (PGF2) increases intracellular Ca2+ concentration [Ca2+]i in vascular smooth muscle remain unclear. We examined the role of store-, receptor- and voltage-operated Ca2+ influx pathways in rat intrapulmonary arteries (IPA) loaded with Fura PE-3. Low concentrations (0.01–1 μM) of PGF2 caused a transient followed by a plateau rise in [Ca2+]i. Both responses became maximal at 0.1 μM PGF2. At higher concentrations of PGF2, a further slower rise in [Ca2+]i was superimposed on the plateau. The [Ca2+]i response to 0.1 μM PGF2 was mimicked by the FP receptor agonist fluprostenol, whilst the effect of 10 μM PGF2 was mimicked by the TP receptor agonist U-46619. The plateau rise in [Ca2+]i in response to 0.1 μM PGF2 was insensitive to diltiazem, and was abolished in Ca2+-free physiological salt solution, and by pretreatment with La3+, 2-APB, thapsigargin or U-73122. The rises in [Ca2+]i in response to 10 μM PGF2 and 0.01 μM U-46619 were partially inhibited by diltiazem. The diltiazem-resistant components of both of these responses were inhibited by 2-APB and La3+ to an extent which was significantly less than that seen for the response to 0.1 μM PGF2, and were also much less sensitive to U-73122. The U-46619 response was also relatively insensitive to thapsigargin. When Ca2+ was replaced with Sr2+, the sustained increase in the Fura PE-3 signal to 0.1 μM PGF2 was abolished, whereas 10 μM PGF2 and 0.05 μM U-46619 still caused substantial increases. These results suggest that low concentrations of PGF2 act via FP receptors to cause IP3-dependent Ca2+ release and store operated Ca2+ entry (SOCE). U-46619 and 10–100 μM PGF2 cause a TP receptor-mediated Ca2+ influx involving both L-type Ca2+ channels and a receptor operated pathway, which differs from SOCE in its susceptibility to La3+, 2-APB and thapsigargin, does not require phospholipase C activation, and is Sr2+ permeable.
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Introduction
The eicosanoids PGF2 and thromboxane A2 (TXA2) are powerful vasoconstrictors which have been implicated in a number of pathological states, including acute lung injury (Zamora et al. 1993; Goff et al. 1997) and cerebral vasospasm (Takeuchi et al. 1999). Both drugs bind to prostanoid receptors, which have been classified into five main types, designated as DP, EP, FP, IP and TP (Breyer et al. 2001). Both PGF2 and TXA2 cause vasoconstriction primarily through the TP prostanoid receptor (Dorn et al. 1992; Boersma et al. 1999; Sametz et al. 2000; Walch et al. 2001; Daray et al. 2003), although FP receptors have also been reported to mediate constriction to PGF2 in human umbilical vein (Daray et al. 2003).
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Vasoconstrictions caused by both PGF2 and TXA2 (or, more commonly, its stable analogue U-46619) have been shown to involve Ca2+ sensitization (Bradley & Morgan, 1987; Himpens et al. 1990; Hori et al. 1992; Janssen et al. 2001; Nobe & Paul, 2001; Ito et al. 2003; Ding & Murray, 2005), as well as increases in [Ca2+]i (Himpens et al. 1990; Hisayama et al. 1990; Balwierczak, 1991; Dorn et al. 1992; Hori et al. 1992; Tosun et al. 1998; Martinez et al. 2000; Nobe & Paul, 2001; Ding & Murray, 2005). In several types of vascular smooth muscle, PGF2 and U-46619 cause the release of intracellular Ca2+ stores (Himpens et al. 1990; Dorn et al. 1992; Hori et al. 1992; Kurata et al. 1993; Martinez et al. 2000), and both TP and FP receptors are known to couple to Gq protein to stimulate the DAG/IP3 second messenger system (Breyer et al. 2001). L-type Ca2+ channels are also involved in the responses evoked by these agonists, since selective inhibitors of these channels generally attenuate the contractions or [Ca2+]i increases they evoke (Hori et al. 1992; Tosun et al. 1998; Ding & Murray, 2005). For example, Cogolludo et al. (2003) presented evidence that increases in [Ca2+]i caused by U-46619 in rat intrapulmonary arteries were associated with a PKC-mediated inhibition of KV channels, causing depolarization and a contraction which was strongly suppressed by nifedipine. Store-operated and/or receptor-operated Ca2+ influx pathways also probably contribute to the TP-receptor stimulated contraction, as Tosun et al. (1998) reported that the verapamil-resistant rise in [Ca2+]i to U-46619 was eliminated by either Ni2+ or SKF-96365.
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Although these studies have revealed that a number of the mechanisms play a role in elevating [Ca2+]i during the contractions caused by these agonists, a detailed understanding of the relative contributions of these different pathways is lacking. Because much less work has been done with PGF2 than with U-46619, it is also unclear whether the fact that the former activates both FP and TP receptors, whereas the latter is much more selective for TP receptors (Narumiya et al. 1999; Breyer et al. 2001), has any bearing on their respective effects on [Ca2+]i in vascular smooth muscle. In light of observations that PGF2 seems to be more efficacious in causing contraction of small pulmonary arteries than are other agonists at G-protein coupled receptors (Leach et al. 1992; Aaronson et al. 2006), we evaluated in more detail the pathways mediating increases in [Ca2+]i in rat IPA during contractions to PGF2, and also to U-46619. We report here that both agonists stimulate TP receptors to activate a Ca2+ influx pathway which has properties consistent with those of a receptor operated channel (ROC). In addition, PGF2 at low concentrations also acts through FP receptors to cause the release of intracellular Ca2+ stores and store operated Ca2+ entry (SOCE).
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Methods
Male Wistar rats (200–300 g) were killed by cervical dislocation as approved by the UK Home Office Inspector. The lungs were excised and placed in cold physiological salt solution (PSS) containing (mM): 118 NaCl, 24 NaHCO3, 1 MgSO4, 0.435 NaH2PO4, 5.56 glucose, 1.8 CaCl2, and 4 KCl. Small (inner diameter 250–500 μm, median 400 μm) intrapulmonary arteries (IPA) were dissected free of adventitia, mounted in a temperature controlled myograph (Danish Myo Technology A/S, Aahrus, Denmark) at 37°C and gassed continuously with 95% air–5% CO2 (pH 7.35). The arteries were then normalized to an equivalent pulmonary transmural pressure of 30 mmHg and equilibrated with three 2 min exposures to PSS containing 80 mM KCl (KPSS, isotonic replacement of NaCl by KCl) (Ward & Snetkov, 2004).
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To prevent precipitation in contraction experiments involving La3+, air-bubbled Hepes-buffered PSS was used containing (mM): 135 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 11 glucose, 10 Hepes, pH 7.4 with NaOH. It should be noted that maximal agonist responses in this solution were smaller that in phosphate–bicarbonate buffered PSS.
Simultaneous tension and [Ca2+]i recording
Isolated IPA were mounted on a modified Cambustion AM-10 myograph (Cambustion Ltd, Cambridge, UK). After normalization and equilibration as described above, IPA were incubated for 1.5 h at room temperature in PSS containing 4 μM Fura PE-3/AM. Then temperature was increased to 37°C for 30 min to accomplish intracellular cleavage of the ester, and IPA were washed with fresh PSS. The myograph module was mounted on an inverted microscope (Nikon Diaphot, Nikon UK Ltd, Kingston-upon-Thames, UK) with a x 10 Fluor objective combined with a double-excitation microfluorimeter (Cairn Research Ltd, Faversham, UK). Tension was recorded simultaneously with light emitted by the whole artery at > 510 nm at excitation wavelengths 340 and 380 nm. The ratio of the emission intensities R340/380 in ratio units (RU) was taken as a measure of [Ca2+]i. For some experiments, an imaging setup (Molecular Devices Corporation, Sunnyvale, CA, USA) built around a Zeiss Axiovert 200 microscope was used in conjunction with confocal wire myograph (Danish Myo Technology).
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In most experiments, the rise in [Ca2+] caused by PGF2 or U-46619 was recorded in the absence and then presence of some type of an inhibitor. The effect of the inhibitor was then quantified as the ratio of the 2nd and 1st responses. Where we wanted to obtain an indication of the amplitude of the control response to an agonist, the change in R340/380 caused by this agonist was expressed as a fraction of the response to KPSS, which was recorded in each artery.
Membrane permeabilization
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-Toxin permeabilized arteries were studied using a method previously described (Kitazawa et al. 1989; Evans et al. 1999; Thomas et al. 2006). Briefly, arteries were mounted on a myograph as described above, but incubated at 26°C rather than 37°C. After obtaining a contractile response to 80 mM K+ the arteries were equilibrated in Ca2+-free relaxing solution containing 1 mM EGTA, and then permeabilized by incubation at pCa 6.5 with 60 μg ml–1-toxin until the resulting contraction reached a plateau. The vessels were then re-equilibrated with solution containing 10 mM EGTA, and a submaximal contraction was brought about by raising pCa to 7.0–6.8. Agonists were added to the bathing solution once contraction reached a steady state. Details of the solutions used have been published previously (Horiuti, 1986). pCa was regulated by adjusting the ratio of K2EGTA and CaEGTA. Agonist-induced contractions did not require inclusion of GTP in the solution, as this was presumably retained in sufficient concentration in cells to allow G-protein activation (Thomas et al. 2006).
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Statistics
Results are expressed as the mean ±S.E.M., and means are compared using paired or unpaired Student's t test as appropriate (SigmaStat, SPSS Inc., Chicago, IL, USA). A difference was deemed significant if P < 0.05.
Materials
Prostaglandin F2 tromethamine and U-46619 (Biomol Intl, Exeter, UK) were used as 10 mM and 1 mM stock solutions in distilled water and DMSO, respectively. Fura PE-3/AM, diltiazem, carbachol, thapsigargin, fluprostenol, AL-8810, U-73122 and -toxin were from Sigma-Aldrich Co. Ltd, Poole, UK; SQ-29584 was from Alexis Corp., Nottingham, UK; 2-aminoethoxydiphenylborane (2-APB) was from Tocris Bioscience, Bristol, UK.
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Results
PGF2 caused a concentration-dependent contraction of rat small IPA (Fig. 1A and B). The threshold for contraction was approximately 1 μM PGF2, the EC50 was 5.8 ± 2.4 μM and the maximum response was 118 ± 33% of that recorded in KPSS (n= 11). The contraction was highly dependent on extracellular Ca2+; in a nominally Ca2+-free solution it fell by 59 and 58% at 10 and 30 μM PGF2, respectively. Inclusion of EGTA in the solution further reduced the response; for example the response to 20 μM PGF2 was reduced by 86 ± 2% (n= 12) in the presence of 1 mM EGTA.
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A, a typical record showing [Ca2+]i (top traces) and tension (bottom traces) responses to ascending concentrations (0.001–100 μM) of PGF2. In this and other figures, ‘RU’ refers to ratio units as defined in Methods. For example the bar in this trace represents a change in the RU of 0.02. The arrow next to the response to 100 μM PGF2 shows the amplitude of the initial Ca2+ transient. B, concentration dependence of transient () and sustained () [Ca2+]i responses and force () induced by PGF2 in IPA. Results shown are the mean ±S.E.M. for 7 ([Ca2+]i) and 11 (force) experiments. C, raising the PGF2 concentration from 0.1 to 10 μM (left) caused a rise in [Ca2+]i which resembled that evoked by 0.05 μM U-46619 (right), in that the initial transient component was greatly attenuated.
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Based on these results, we hypothesized that PGF2 was exerting two separate effects on [Ca2+]i which developed over different concentration ranges. At concentrations of 1 μM and below, PGF2 was causing a transient rise in [Ca2+]i which was followed by the establishment of a lower plateau level of [Ca2+]i which remained constant or fell gradually. This response appeared to become maximal at 0.1 μM PGF2, and was not associated with contraction. At higher concentrations, PGF2 was in addition causing a somewhat slower increase in [Ca2+]i which tended to abate with time, and which was associated with contraction. Based on the reported difference between the affinities of PGF2 for FP and TP receptors (0.003 versus9 μM, respectively, Breyer et al. 2001), we further hypothesized that the transient and plateau rises in [Ca2+]i were due to the stimulation of FP receptors, whereas the slower increase in [Ca2+]i was due to TP receptors.
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In order to characterize these two putative responses, which we will hereafter term the ‘FP’ and ‘TP’ responses, we examined in more detail the effects of 0.1 μM PGF2, which would be expected to produce only the FP response, and 10 μM PGF2, which should produce a mixed FP/TP response. We also studied the effects of U-46619, which should cause a relatively selective stimulation of TP receptors at concentrations 0.1 μM (Narumiya et al. 1999; Breyer et al. 2001). The validity of this strategy is supported by the observation that the rise in [Ca2+]i caused by 10 μM PGF2 added in the presence of 0.1 μM PGF2 closely resembled that evoked by 0.1 μM U-46619 (Fig. 1C), consistent with the prediction that following a maximal stimulation of the FP response by 0.l μM PGF2 (Fig. 1B), a ‘pure’ TP response would be all that could be elicited when a higher PGF2 concentration was applied. Note that both 10 μM PGF2 and 0.1 μM U-46619 caused a contraction, while 0.1 μM PGF2 did not.
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Endothelial denudation was confirmed by the absence of vasodilatation to carbachol (1 μM). Similar results were obtained in 4 other arteries.
Characterization of the rise in [Ca2+]i caused by 0.1 μM PGF2: the FP response
The temporal profile of the Ca2+ signal elicited by 0.1 μM PGF2 suggested that this response might involve Ca2+ release from intracellular stores, followed by a sustained Ca2+ influx. In agreement with this concept, the sustained rise in [Ca2+]i in response to application of 0.1 μM PGF2 was abolished (n= 5) in Ca2+-free solution containing 30–100 μM EGTA (Fig. 3A) or in the presence of 10 μM La3+ (n= 9) (Fig. 3B). In contrast, the initial [Ca2+]i transient remained largely intact both in Ca2+-free and La3+-containing solutions (70 ± 3%, n= 5 and 67 ± 8%, n= 10, of control, respectively).
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Removal of extracellular Ca2+ (A), or application of 10 μM La3+ (B) abolished the sustained rise in [Ca2+]i to 0.1 μM PGF2, while leaving the initial transient [Ca2+]i increase largely intact.
Based on these results, we reasoned that the sustained increase in [Ca2+]i caused by 0.1 μM PGF2 might be due to store operated Ca2+ entry (SOCE), which can be elicited by the SERCA antagonist thapsigargin and is then blocked by low micromolar concentrations of La3+ in these arteries (Snetkov et al. 2003). In this case, prior depletion of SR/ER Ca2+ with thapsigargin should prevent this response. In agreement with this prediction, Fig. 4A illustrates that pretreatment with thapsigargin (1 μM), which caused a large sustained rise in [Ca2+]i, virtually abolished the response to 0.1 μM PGF2 (95 ± 4% inhibition, n= 5); pretreatment with 0.1 μM thapsigargin had a similar effect (93 ± 3% inhibition, n= 3, not shown). Moreover, as depicted in Fig. 4B, the sustained response to 0.1 μM PGF2 was completely eliminated in the presence of 2-APB (75 μM), a putative blocker of IP3 receptors and SOCE (n= 7). 2-APB reduced the initial Ca2+ transient to a similar extent as La3+ or Ca2+-free solution (to 71 ± 11% of control, n= 7), suggesting that it had little effect on Ca2+ release at this concentration. On the other hand, Fig. 4C illustrates that the L-type voltage-gated Ca2+ channel blocker diltiazem (10 μM) had no measurable effect on the sustained response to 0.1 μM PGF2 (n= 6). The [Ca2+]i rise evoked by 0.1 μM PGF2 was also unaffected by the TP receptor antagonist SQ-29548 at a concentration (1 μM) which should completely block this receptor (Narumiya et al. 1999) (Fig. 4D, n= 4).
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A, both transient and sustained increases in [Ca2+]i caused by 0.1 μM PGF2 were abolished following application of the SERCA inhibitor thapsigargin (1 μM), which itself caused a sustained rise in [Ca2+]i. B, pretreatment with 2-APB (75 μM) eliminated the sustained but not transient rises in [Ca2+]i evoked by 0.1 μM PGF2. C, the L-type Ca2+ channel blocker diltiazem (10 μM) had no measurable effect on the sustained rise in [Ca2+]i caused by 0.1 μM PGF2. D, the TP receptor antagonist SQ-29548 (1 μM) also had no apparent effect on the sustained rise in [Ca2+]i caused by 0.1 μM PGF2.
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The FP receptor agonist fluprostenol (0.1 μM) caused a rise in [Ca2+]i which was indistinguishable from that of 0.1 μM PGF2 and was also insensitive to 1 μM SQ-29548 (Fig. 5A) (n= 4) and to 10 μM diltiazem (Fig. 5B, n= 5), but was almost eliminated (93 ± 3% reduction, n= 3) by pretreatment with 0.1 μM thapsigargin (Fig. 5C).
A, the selective FP receptor agonist fluprostenol (0.1 μM) caused a rise in [Ca2+]i which closely resembled that of 0.1 μM PGF2 and was similarly unaffected by the TP receptor antagonist SQ-29548 (1 μM). The response to fluprostenol was also insensitive to diltiazem (10 μM, B), but was abolished by pretreatment with thapsigargin (1 μM, C).
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On average, the initial transient rise in [Ca2+]i to 0.1 μM PGF2 amounted to 85 ± 15% (n= 68) of the rise in [Ca2+]i caused by KPSS The subsequent plateau rise [Ca2+]i, measured after 10 min, was 28 ± 4% of the KPSS response. Experiments examining the relationship between [Ca2+]i and contraction in the presence of a range of elevated concentrations of K+ (not shown) suggested that increases in [Ca2+]i of these magnitudes would be expected to cause contractions of approximately 75 and 20%, respectively, of that caused by KPSS, indicating that the lack of contraction during the FP response was not simply due to an insufficient rise in [Ca2+]i.
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In summary, the results shown in Figs 1–5 suggested that 0.1 μM PGF2 was acting on FP receptors to cause release of Ca2+ from the sarcoplasmic/endoplasmic reticulum, leading to activation of SOCE. The resulting rise in [Ca2+]i, though substantial, was not coupled to contraction. We went on to assess whether 10 μM PGF2 also caused a rise in [Ca2+]i mainly through SOCE.
Effects of 10 μM PGF2 and U-46619 on [Ca2+]i and contraction: the TP response
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In agreement with previous findings (e.g. Dorn et al. 1992), the contraction induced by 10 μM PGF2 was abolished by SQ-29548 (n= 10), suggesting that at this concentration PGF2 was activating TP receptors, and that this was responsible for tension development. In contrast to its lack of effect on the response to 0.1 μM PGF2, diltiazem (10 μM) caused a significant inhibition of the rise in [Ca2+]i evoked by 10 μM PGF2, as illustrated in Fig. 6A. On average, the increase in [Ca2+]i was inhibited by 37 ± 11% (n= 6) in arteries preincubated with diltiazem. Diltiazem caused a similar degree of inhibition of contraction over a range of the PGF2 concentrations (e.g. 30 ± 9% at 10 μM PGF2; n= 15; Fig. 6B).
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A, diltiazem (10 μM) depressed the rise in [Ca2+]i caused by 10 μM PGF2 when added either before (upper trace) or during (lower trace) the application of agonist. B, diltiazem also partially suppressed contraction to a range of concentrations of PGF2 (n= 15). In each experiment, responses to ascending concentrations of PGF2 applied cumulatively were recorded before and after application of diltiazem (10 μM). Asterisks indicate a significant inhibition of contraction (P < 0.05).
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In order to determine whether the diltiazem-insensitive rise in [Ca2+]i evoked by 10 μM PGF2 might be due to SOCE, we assessed the effect of 2-APB on this response in the presence of diltiazem. As depicted in Fig. 7A, 2-APB (75 μM) strongly reduced the sustained rise in [Ca2+]i caused by 10 μM PGF2 applied in the presence of diltiazem, with inhibition amounting to 78 ± 3% in four arteries. Similarly, 2-APB markedly inhibited the contraction to PGF2 (Fig. 7B). On the other hand, pretreatment with 10 μM La3+, which had virtually abolished the sustained [Ca2+]i rise caused by 0.1 μM PGF2, caused a smaller attenuation of the [Ca2+]i increase caused by 10 μM PGF2 in both the presence (47 ± 17% inhibition, n= 5) or absence (32 ± 21% inhibition, n= 6) of diltiazem (P < 0.01 compared to La3+-mediated inhibition of the [Ca2+]i increase to 0.1 μM PGF2 in both cases) (Fig. 7C).
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A, an example trace illustrating that 75 μM 2-APB strongly suppressed but did not abolish the 10 μM PGF2 -induced increase in [Ca2+]i observed in the presence of diltiazem. B, both 30 and 75 μM 2-APB (n= 6 and 7, respectively) strongly suppressed the contractions caused by a range of concentrations of PGF2, when applied in the presence of diltiazem to prevent Ca2+ influx through L-type channels. C, pretreatment with La3+ (10 μM) abolished the [Ca2+]i rise to 0.1 μM PGF2, but only attenuated the [Ca2+]i response to 10 μM PGF2.
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The results shown in Figs 6 and 7 indicated that the [Ca2+]i rise in response to 10 μM PGF2 could be distinguished from that caused by 0.1 μM PGF2 in that it demonstrated diltiazem-sensitive and -insensitive components, the latter of which was relatively La3+ resistant compared to the SOCE elicited by 0.1 μM PGF2. Moreover, the temporal profile of the increase in [Ca2+]i caused by 10 μM PGF2 was different from that of the FP response (Fig. 1A). Taking into account the reported high affinity of PGF2 for the FP receptor (Narumiya et al. 1999) and our evidence that the rise in [Ca2+]i to 0.1 μM PGF2 represented the effect of maximal simulation of the FP receptor (Fig. 1B), it seemed evident that it must be activation of TP receptors which was responsible for activation of these additional components of Ca2+ entry by 10 μM PGF2. In order to focus specifically on the components of the response coupled to the TP receptor in the absence of the complicating factor introduced by the coactivation of FP receptors and therefore SOCE, we next examined the response to the TXA2 analogue and selective TP agonist U-46619.
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As expected, force development elicited by 0.1 μM U-46619 was abolished by the TP receptor antagonist SQ-29548 (1 μM) in all five arteries tested. Moreover, the rise in [Ca2+]i in response to 50 and 100 nM U-46619 was abolished by 1 μM SQ-29548 in 2 of 2 and 4 of 5 arteries examined, respectively, and fell by 40% in the remaining artery tested at 0.1 μM U-46619.
U-46619 caused a concentration-dependent contraction which was increasingly attenuated by 10 μM diltiazem at progressively higher concentrations of U-46619 (Fig. 8A). As shown in Fig. 8B, diltiazem also partially inhibited the rise in [Ca2+]i evoked by U-46619 (0.05 μM); in six arteries this was reduced by 44 ± 11%. These results echo those observed with 10 μM PGF2, suggesting that while a fraction of the U-46619 contraction was dependent upon the opening of L-type voltage-gated Ca2+ channels, another Ca2+ influx pathway was making an important contribution to this response. In support of this possibility, both 30 and 75 μM 2-APB, when applied in the presence of 10 μM diltiazem, strongly inhibited contraction over a range of U-46619 concentrations (Fig. 8C). 2-APB at 75 μM also reduced the rise in [Ca2+]i caused by 0.05 μM U-46619 by 76 ± 5% (n= 4) in the presence of diltiazem, a value similar to that found for its effect on the [Ca2+]i response to 10 μM PGF2.
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A, the effect of diltiazem on the contraction caused by a range of concentrations of U-46619 (n= 13). In each experiment, responses to ascending concentrations of U-46619 applied cumulatively were recorded before and after application of diltiazem (10 μM). B, an example of the effect of 10 μM diltiazem on the rise in [Ca2+]i to 0.05 μM U-46619. C, both 30 and 75 μM 2-APB (n= 7 and 8, respectively) strongly suppressed the contractions caused by a range of concentrations of U-46619 when applied in the presence of diltiazem.
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As described above, 10 μM La3+ abolished the sustained rise in [Ca2+]i in response to 0.1 μM PGF2 but reduced the diltiazem-insensitive increase in [Ca2+]i in response to 10 μM PGF2 by only 50%. Moreover, 75 μM 2-APB caused a greater suppression of the increase in [Ca2+]i caused by 0.1 μM PGF2 than it did of the diltiazem-insensitive rises in [Ca2+]i caused by 10 μM PGF2 and 50 nM U-46619. This difference, although not as pronounced as that for La3+, was highly significant (P < 0.01). These results suggested that TP receptors could activate a non-voltage-gated Ca2+ influx pathway different from the SOCE induced by FP receptor stimulation.
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More direct evidence for this possibility was obtained in experiments in which Ca2+ was replaced by Sr2+ (Fig. 9). Under these conditions, whereas 0.1 μM PGF2 still caused an initial transient increase in the Fura PE-3 signal, the sustained increase in signal present in Ca2+ was absent (Fig. 9A). This result is in accordance with previous reports that Sr2+ does not permeate SOCE very well (Broad et al. 1999; Ma et al. 2000). On the other hand, application of either 10 μM PGF2 (Fig. 9B) or 50 nM U-46619 (Fig. 9C) led to a sustained increase in the Fura PE-3 signal which was not greatly different from that observed in Ca2+-containing solution. The results of these experiments, which are summarized in Fig. 9D, show that TP receptor stimulation was activating a Sr2+ permeable pathway different from SOCE. This pathway was not due to L-type Ca2+ channels, since it was not significantly affected by 10 μM diltiazem (n= 5) or, in one experiment, by 10 μM verapamil (not shown).
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A, substitution of 4 mM Sr2+ for 1.8 mM Ca2+ abolished the sustained increase in the Fura PE-3 signal caused by 0.1 μM PGF2, but had a much smaller effect on the increase in this ratio to either 10 μM PGF2 (B) or 0.05 μM U-46619 (C). D, mean ±S.E.M. results from 5 experiments with each agonist, shown as the sustained (i.e. after 10 min) increase in the Fura PE-3 signal in the presence of Sr2+ expressed as a percentage of that observed in Ca2+. In panels A–C, bars beneath the traces show application of agonist. Sr2+ was used at a concentration of 4 mM because the high K+ contraction under these conditions was similar to that observed in normal PSS.
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Additional evidence that the response to the TP receptor was occurring via a non-SOCE pathway was obtained in experiments utilizing the phospholipase C inhibitor U-73122. As shown in Fig. 10A, both the transient and sustained rises in [Ca2+]i induced by 0.1 μM PGF2 were very markedly suppressed by pretreatment with 3 μM U-73122; in eight experiments these responses were inhibited by 87 ± 3% and 92 ± 4%, respectively. On the other hand, the additional rise in [Ca2+]i caused by 10 μM PGF2 (i.e. when 10 μM PGF2 was applied directly after 0.1 μM PGF2 as shown in Fig. 10A) was suppressed to a significantly smaller extent by U-73122 (29 ± 5% inhibition; n= 5), and the accompanying contraction was not significantly affected (5 ± 11% increase, n= 7, not shown). U-73122 had similar effects on the U-46619-induced rise in [Ca2+]i, which was reduced by 27 ± 12% in five experiments (Fig. 10B) and on the U-46619-induced contraction (7 ± 14% increase; n= 6, n.s., not shown). These results were consistent with the concept that the lower concentration of PGF2 was activating SOCE by causing IP3-mediated Ca2+ release, whilst both U-46619 and the higher concentration of PGF2 were increasing [Ca2+]i mainly by stimulating a separate pathway which was largely independent of both IP3 and SOCE. In accordance with this idea, pretreatment of arteries with thapsigargin (1 μM), which had virtually eliminated the response to 0.1 μM PGF2 and fluprostenol (Figs 4A and 5C), caused only a partial (38 ± 8%, n= 9) inhibition of the response to 0.1 μM U-46619. Figure 10B illustrates a typical experiment of this type.
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A, U-73122 (3 μM) suppressed the rise in [Ca2+]i to 0.1 μM PGF2 to a much greater extent than it did the increase in [Ca2+]i to 10 μM PGF2 or 0.01 μM U-46619. B, thapsigargin (1 μM) caused a partial inhibition of the rise in [Ca2+]i elicited by 0.01 μM U-46619.
Contractions in -toxin permeabilized IPA
Both PGF2 and U-46619 caused contractions in -toxin permeabilized IPA, and in both cases these contractions were reversed by SQ-29548. Figure 11A and B illustrates an example of the effect of 1 μM SQ-29548 on the contraction to 100 μM PGF2 and 1 μM U-46619, respectively, recorded in the presence of 10 μM cyclopiazonic acid (to block any effects on Ca2+ stores) at pCa 7.0. Conversely, in four arteries tested, fluprostenol (1 and 10 μM) did not cause a contraction in permeabilized IPA either in absence or presence of 1 μM GTP (Fig. 11C).
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PGF2 (30 μM) was then added to confirm that the artery was responsive to TP receptor stimulation. The TP receptor antagonist SQ-29548 (1 μM) reversed the responses to PGF2 and U-46619.
Discussion
As far as we are aware, these results represent the first characterization in intact arteries of distinct store- and receptor-activated [Ca2+]i influx pathways activated by a single vasoconstricting agonist. The data are consistent with previous reports in isolated smooth muscle cells and smooth muscle-derived cell lines that vasoconstrictors stimulate the opening of separate SOCs and ROCs (Byron & Taylor, 1995; Broad et al. 1999; Iwamuro et al. 1999; Furutani et al. 2002; Large, 2002; Moneer & Taylor, 2002; Albert & Large, 2003), and the recent demonstration by Jernigan et al. (2006) that cyclopiazonic acid and UTP activate a SOC and ROC, respectively, in rat IPA.
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The FP response
At a concentration of 0.1 μM, PGF2 caused a marked transient and sustained rise in [Ca2+]i which was mimicked by the FP receptor agonist fluprostenol. The rises in [Ca2+]i caused by both agonists at this concentration were also similar in that neither was associated with a contraction, and neither was affected by the TP receptor antagonist SQ-29548 or the L-type Ca2+ channel antagonist diltiazem. On the other hand, both transient and sustained rises in [Ca2+]i to 0.1 μM PGF2 were strongly suppressed by the phospholipase C antagonist U-73122 and virtually abolished by pretreatment with thapsigargin. The initial [Ca2+]i transient persisted in Ca2+-free solution or in the presence of 10 μM La3+, but the sustained rise in [Ca2+]i was abolished by both. These results indicate that at a concentration which was subthreshold for contraction, PGF2 nonetheless caused a rise in [Ca2+]ivia the activation of FP receptors. This rise in [Ca2+]i was likely initiated by an IP3-mediated Ca2+ release from the sarcoplasmic reticulum, and the effects of La3+ and 2-APB suggest that it was sustained by SOCE, consistent with the known coupling of the FP receptor to Gq-protein and phospholipase C (Narumiya et al. 1999). Interestingly, 75 μM 2-APB had little effect on the initial Ca2+ transient (Fig. 4B), suggesting that at this concentration it was not strongly antagonizing the IP3 receptor.
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We have previously found that SOCE stimulated by thapsigargin caused some tension development in IPA (Snetkov et al. 2003), although thapsigargin-induced SOCE causes large tension-independent rises in [Ca2+]i in a number of other resistance arteries and arterioles (Flemming et al. 2002; Snetkov et al. 2003). Experiments in which [Ca2+]i was elevated to varying degrees using progressively higher concentrations of [K+] suggested that the amplitude of the rise in [Ca2+]i caused by 0.1 μM PGF2 would have been expected to cause some contraction in IPA. There are a number of possible explanations for the lack of contraction associated with the FP response, one being that the rise in [Ca2+]i due to SOCE is somehow localized primarily to regions of the cell which lack elements of the contractile apparatus (Flemming et al. 2002). It is interesting in this regard that Gordienko et al. (2001) and Pucovsky & Bolton (2006) have shown that the primary Ca2+ discharge region of the sarcoplasmic reticulum both basally and during stimulation by agonists is located in close apposition to the nucleus, and that Abrenica et al. (2003) have reported that thapsigargin causes a rise in [Ca2+]i which is mainly restricted to the nucleus and the perinuclear cytoplasm. SOCE is enhanced in proliferating as compared to quiescent pulmonary artery myocytes, and its inhibition suppresses proliferation (Golovina et al. 2001). In addition, Dorn et al. (1992) reported that PGF2 caused SQ-29548-resistant (i.e. presumably FP receptor-mediated) increases in [Ca2+]i and phosphatidylinositol turnover in cultured aorta smooth muscle cells with EC50 values for both responses of < 0.05 μM, while in intact aorta the threshold and EC50 for PGF2-induced contraction were > 0.1 and 5 μM, respectively. These results are consistent with the possibility that nanomolar concentrations of PGF2 may act via the FP receptor and SOCE to cause a rise in cytoplasmic [Ca2+] which is specifically funnelled into the nucleus, allowing activation of transcription factors and promoting proliferation. It is also possible that the inability of SOCE stimulated by FP receptor activation to cause contraction in part reflects an absence of Ca2+ sensitization (Fig. 11), especially since contraction in IPA seems to be particularly dependent on the rhoA pathway (Hyvelin et al. 2004). This would also concur with our observation that rises in [Ca2+]i to U-46619, which caused Ca2+ sensitization, were often no larger than those to 0.1 μM PGF2, yet were always associated with contraction. A resolution of these issues will require further study using methods capable of assessing compartmentalization of [Ca2+]i during stimulation with FP- and TP-receptor selective agonists.
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The TP response
At a higher concentration (10 μM) PGF2 caused a contraction which was abolished by SQ-29548, indicating that it resulted from TP receptor stimulation. Although our experiments in -toxin skinned IPA and previous studies in other arteries (Bradley & Morgan, 1987; Himpens et al. 1990; Ito et al. 2003; Ding & Murray, 2005) showed that this contraction is partially dependent on Ca2+ sensitization, it also clearly requires the presence of Ca2+ for its full development, and is associated with a rise in [Ca2+]i which involves multiple mechanisms.
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Although 10 μM PGF2 would be expected to stimulate SOCE, the properties of the rise in [Ca2+]i caused by this concentration of agonist showed that it involved two additional pathways of Ca2+ influx. For example, when 10 μM PGF2 was applied directly after 0.1 μM PGF2 the temporal profile of the increment in [Ca2+]i caused by the higher concentration of agonist differed from that caused by the lower concentration. This increment in [Ca2+]i typically lacked a, or had a much smaller, initial transient component, and developed gradually over several minutes, unlike SOCE, which began immediately and remained essentially constant (Figs 1 and 2). This increment in [Ca2+]i was relatively insensitive to U-73122, suggesting that it did not require IP3-mediated Ca2+ release. Similar properties were demonstrated for the response to U-46619, again indicating that TP receptors were involved. Although diltiazem partially inhibited the [Ca2+]i increases elicited by 10 μM PGF2 and U-46619, the greater part of both responses was diltiazem insensitive. Moreover, this diltiazem-resistant rise in [Ca2+]i was mainly independent of SOCE, since it was comparatively insensitive to 10 μM La3+, which abolished SOCE (Fig. 3B, Robertson et al. 2000).
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More direct evidence that TP receptor stimulation by either U-46619 or 10 μM PGF2 was activating a Ca2+ entry mechanism which was not mediated by either SOCE or L-type Ca2+ channels came from experiments in which Ca2+ was replaced by Sr2+, which has been shown to permeate receptor- but not store-operated channels in A7r5 and other types of cell lines (Broad et al. 1999; Ma et al. 2000). SOCE caused by 0.l μM PGF2 did not cause a sustained rise in the Fura PE-3 signal when Ca2+ was replaced with Sr2+. In contrast, both U-46619 and 10 μM PGF2 induced large diltiazem-insensitive responses with Sr2+ in the solution. Also, the rise in [Ca2+]i to U-46619 was only partially prevented by pretreatment with thapsigargin, whereas those caused by 0.1 μM PGF2 or fluprostenol were abolished.
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The results therefore show that 10 μM PGF2, in addition to stimulating SOCE via FP receptors, also acted on TP receptors to activate Ca2+ influx through both L-type Ca2+ channels and another Ca2+ pathway which was Sr2+ permeable, relatively La3+ resistant, and which seemed to be mostly independent of IP3-mediated Ca2+ release, since it was little affected by the phospholipase C antagonist U-73122, which has previously been shown to inhibit Ca2+ release (Hansen et al. 1995).
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Evidence that vasoconstrictors acting on G-protein coupled receptors may activate receptor operated non-selective cation channels (ROCs) different from those opened by store depletion has emerged from electrophysiological and Ca2+ imaging studies in isolated or cultured smooth muscle cells (Byron & Taylor, 1995; Broad et al. 1999; Iwamuro et al. 1999; Large, 2002), and from pharmacological studies in intact arteries (Furutani et al. 2002; Jernigan et al. 2006). Vascular smooth muscle demonstrates a number of ROCs with diverse properties (Large, 2002) which may coexist in single types of cells (Iwamuro et al. 1999). The best-characterized ROC in vascular smooth muscle is the noradrenaline-activated ICAT (Large, 2002) of which TRPC6 is thought to be a crucial component (Inoue et al. 2001). One apparent difference between ICAT and the ROC in IPA is that activation of ICAT is potently inhibited by U-37122 (Helliwell & Large, 1997), which, however, had little effect on the TP-receptor-stimulated ROC at a concentration (3 μM) which almost abolished Ca2+ release and SOCE, indicating almost complete suppression of phospholipase C activity (Fig. 10A). Whether this implies that the channels involved are different, or alternatively that the 1 and TP receptors are coupled to the same channel through separate pathways, remains to be determined.
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Several non-SOCE pathways, as defined by their insensitivity to both L-type Ca2+ channel antagonists and prior treatment with Ca2+ store releasing agents, have recently been found to be activated in small pulmonary arteries by other vasoconstrictors. Ca2+ influx stimulated by 5-HT was observed to be insensitive to nitrendipine, to the lanthanide Gd3+, and to prior Ca2+ store release with cyclopiazonic acid (Guibert et al. 2004). This Ca2+ influx was also blocked by the DAG lipase inhibitor RHC-80267, suggesting that arachidonic acid was involved as a second messenger (Guibert et al. 2004), much as it is thought to be important in vasopressin-induced activation of a ROC in A7r5 cells (Broad et al. 1999). We did not explore whether arachidonic acid plays a similar role during TP receptor-associated ROC activation, although the lack of effect of U-73122 suggests that if this fatty acid is involved, it is not coming from DAG produced by phospholipase C. We also found that a rise in [Ca2+]i induced by sphingosylphosphorylcholine in IPA was insensitive to diltiazem, U-73122, and to prior Ca2+ store release with thapsigargin (Thomas et al. 2006), and was also Sr2+ permeable (Thomas et al., unpublished observation). UTP-stimulated Ca2+ entry in these arteries persisted in the presence of diltiazem, cyclopiazonic and 10 mM Ni2+, but was abolished by 50 μM SKF-96365. Conversely, SOCE activated by cyclopiazonic acid was completely blocked by Ni2+ but was unaffected by SKF-96365 (Jernigan et al. 2006). Whether these various receptor-operated Ca2+ influx pathways can be ascribed to the same ROC remains unresolved. It is noteworthy that a reciprocal relationship between SOC- and ROC-mediated Ca2+ entry has previously been described in a number of types of cells, including A7r5 (Moneer & Taylor, 2002). Intriguingly, the initial Ca2+ transient caused by PGF2 fell significantly at the highest concentration of this agonist (Fig. 1), suggesting that a reciprocal relationship could also exist between ROC-mediated Ca2+ entry and Ca2+ release, although confirmation of this possibility will require additional study.
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In summary, our results show that PGF2 raises [Ca2+]ivia multiple pathways. At a low concentration, this agonist activates FP receptors to cause an IP3-mediated release of intracellular Ca2+ stores. This in turn leads to SOCE. However, neither Ca2+ release nor SOCE results in contraction. At higher concentrations, which cause contraction, PGF2 also acts on TP receptors to activate a separate Ca2+ entry pathway which can be distinguished from SOCE in that it is relatively insensitive to U-73122, La3+ and thapsigargin, and is permeable to Sr2+. This pathway, which would appear to represent a ROC, seems to closely resemble those shown to be activated by 5-HT, and by sphingosylphosphorylcholine in these arteries (Guibert et al. 2004; Thomas et al. 2006). At the same time, TP receptor stimulation causes Ca2+ influx through L-type Ca2+ channels, which are presumably opened by depolarization arising as a result of Na+ influx through the ROC and/or K+ channel inhibition. The effect of the resulting rise in [Ca2+]i on contraction is amplified by TP receptor-dependent Ca2+ sensitization.
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References
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Abstract
The mechanisms by which prostaglandin F2 (PGF2) increases intracellular Ca2+ concentration [Ca2+]i in vascular smooth muscle remain unclear. We examined the role of store-, receptor- and voltage-operated Ca2+ influx pathways in rat intrapulmonary arteries (IPA) loaded with Fura PE-3. Low concentrations (0.01–1 μM) of PGF2 caused a transient followed by a plateau rise in [Ca2+]i. Both responses became maximal at 0.1 μM PGF2. At higher concentrations of PGF2, a further slower rise in [Ca2+]i was superimposed on the plateau. The [Ca2+]i response to 0.1 μM PGF2 was mimicked by the FP receptor agonist fluprostenol, whilst the effect of 10 μM PGF2 was mimicked by the TP receptor agonist U-46619. The plateau rise in [Ca2+]i in response to 0.1 μM PGF2 was insensitive to diltiazem, and was abolished in Ca2+-free physiological salt solution, and by pretreatment with La3+, 2-APB, thapsigargin or U-73122. The rises in [Ca2+]i in response to 10 μM PGF2 and 0.01 μM U-46619 were partially inhibited by diltiazem. The diltiazem-resistant components of both of these responses were inhibited by 2-APB and La3+ to an extent which was significantly less than that seen for the response to 0.1 μM PGF2, and were also much less sensitive to U-73122. The U-46619 response was also relatively insensitive to thapsigargin. When Ca2+ was replaced with Sr2+, the sustained increase in the Fura PE-3 signal to 0.1 μM PGF2 was abolished, whereas 10 μM PGF2 and 0.05 μM U-46619 still caused substantial increases. These results suggest that low concentrations of PGF2 act via FP receptors to cause IP3-dependent Ca2+ release and store operated Ca2+ entry (SOCE). U-46619 and 10–100 μM PGF2 cause a TP receptor-mediated Ca2+ influx involving both L-type Ca2+ channels and a receptor operated pathway, which differs from SOCE in its susceptibility to La3+, 2-APB and thapsigargin, does not require phospholipase C activation, and is Sr2+ permeable.
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Introduction
The eicosanoids PGF2 and thromboxane A2 (TXA2) are powerful vasoconstrictors which have been implicated in a number of pathological states, including acute lung injury (Zamora et al. 1993; Goff et al. 1997) and cerebral vasospasm (Takeuchi et al. 1999). Both drugs bind to prostanoid receptors, which have been classified into five main types, designated as DP, EP, FP, IP and TP (Breyer et al. 2001). Both PGF2 and TXA2 cause vasoconstriction primarily through the TP prostanoid receptor (Dorn et al. 1992; Boersma et al. 1999; Sametz et al. 2000; Walch et al. 2001; Daray et al. 2003), although FP receptors have also been reported to mediate constriction to PGF2 in human umbilical vein (Daray et al. 2003).
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Vasoconstrictions caused by both PGF2 and TXA2 (or, more commonly, its stable analogue U-46619) have been shown to involve Ca2+ sensitization (Bradley & Morgan, 1987; Himpens et al. 1990; Hori et al. 1992; Janssen et al. 2001; Nobe & Paul, 2001; Ito et al. 2003; Ding & Murray, 2005), as well as increases in [Ca2+]i (Himpens et al. 1990; Hisayama et al. 1990; Balwierczak, 1991; Dorn et al. 1992; Hori et al. 1992; Tosun et al. 1998; Martinez et al. 2000; Nobe & Paul, 2001; Ding & Murray, 2005). In several types of vascular smooth muscle, PGF2 and U-46619 cause the release of intracellular Ca2+ stores (Himpens et al. 1990; Dorn et al. 1992; Hori et al. 1992; Kurata et al. 1993; Martinez et al. 2000), and both TP and FP receptors are known to couple to Gq protein to stimulate the DAG/IP3 second messenger system (Breyer et al. 2001). L-type Ca2+ channels are also involved in the responses evoked by these agonists, since selective inhibitors of these channels generally attenuate the contractions or [Ca2+]i increases they evoke (Hori et al. 1992; Tosun et al. 1998; Ding & Murray, 2005). For example, Cogolludo et al. (2003) presented evidence that increases in [Ca2+]i caused by U-46619 in rat intrapulmonary arteries were associated with a PKC-mediated inhibition of KV channels, causing depolarization and a contraction which was strongly suppressed by nifedipine. Store-operated and/or receptor-operated Ca2+ influx pathways also probably contribute to the TP-receptor stimulated contraction, as Tosun et al. (1998) reported that the verapamil-resistant rise in [Ca2+]i to U-46619 was eliminated by either Ni2+ or SKF-96365.
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Although these studies have revealed that a number of the mechanisms play a role in elevating [Ca2+]i during the contractions caused by these agonists, a detailed understanding of the relative contributions of these different pathways is lacking. Because much less work has been done with PGF2 than with U-46619, it is also unclear whether the fact that the former activates both FP and TP receptors, whereas the latter is much more selective for TP receptors (Narumiya et al. 1999; Breyer et al. 2001), has any bearing on their respective effects on [Ca2+]i in vascular smooth muscle. In light of observations that PGF2 seems to be more efficacious in causing contraction of small pulmonary arteries than are other agonists at G-protein coupled receptors (Leach et al. 1992; Aaronson et al. 2006), we evaluated in more detail the pathways mediating increases in [Ca2+]i in rat IPA during contractions to PGF2, and also to U-46619. We report here that both agonists stimulate TP receptors to activate a Ca2+ influx pathway which has properties consistent with those of a receptor operated channel (ROC). In addition, PGF2 at low concentrations also acts through FP receptors to cause the release of intracellular Ca2+ stores and store operated Ca2+ entry (SOCE).
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Methods
Male Wistar rats (200–300 g) were killed by cervical dislocation as approved by the UK Home Office Inspector. The lungs were excised and placed in cold physiological salt solution (PSS) containing (mM): 118 NaCl, 24 NaHCO3, 1 MgSO4, 0.435 NaH2PO4, 5.56 glucose, 1.8 CaCl2, and 4 KCl. Small (inner diameter 250–500 μm, median 400 μm) intrapulmonary arteries (IPA) were dissected free of adventitia, mounted in a temperature controlled myograph (Danish Myo Technology A/S, Aahrus, Denmark) at 37°C and gassed continuously with 95% air–5% CO2 (pH 7.35). The arteries were then normalized to an equivalent pulmonary transmural pressure of 30 mmHg and equilibrated with three 2 min exposures to PSS containing 80 mM KCl (KPSS, isotonic replacement of NaCl by KCl) (Ward & Snetkov, 2004).
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To prevent precipitation in contraction experiments involving La3+, air-bubbled Hepes-buffered PSS was used containing (mM): 135 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 11 glucose, 10 Hepes, pH 7.4 with NaOH. It should be noted that maximal agonist responses in this solution were smaller that in phosphate–bicarbonate buffered PSS.
Simultaneous tension and [Ca2+]i recording
Isolated IPA were mounted on a modified Cambustion AM-10 myograph (Cambustion Ltd, Cambridge, UK). After normalization and equilibration as described above, IPA were incubated for 1.5 h at room temperature in PSS containing 4 μM Fura PE-3/AM. Then temperature was increased to 37°C for 30 min to accomplish intracellular cleavage of the ester, and IPA were washed with fresh PSS. The myograph module was mounted on an inverted microscope (Nikon Diaphot, Nikon UK Ltd, Kingston-upon-Thames, UK) with a x 10 Fluor objective combined with a double-excitation microfluorimeter (Cairn Research Ltd, Faversham, UK). Tension was recorded simultaneously with light emitted by the whole artery at > 510 nm at excitation wavelengths 340 and 380 nm. The ratio of the emission intensities R340/380 in ratio units (RU) was taken as a measure of [Ca2+]i. For some experiments, an imaging setup (Molecular Devices Corporation, Sunnyvale, CA, USA) built around a Zeiss Axiovert 200 microscope was used in conjunction with confocal wire myograph (Danish Myo Technology).
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In most experiments, the rise in [Ca2+] caused by PGF2 or U-46619 was recorded in the absence and then presence of some type of an inhibitor. The effect of the inhibitor was then quantified as the ratio of the 2nd and 1st responses. Where we wanted to obtain an indication of the amplitude of the control response to an agonist, the change in R340/380 caused by this agonist was expressed as a fraction of the response to KPSS, which was recorded in each artery.
Membrane permeabilization
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-Toxin permeabilized arteries were studied using a method previously described (Kitazawa et al. 1989; Evans et al. 1999; Thomas et al. 2006). Briefly, arteries were mounted on a myograph as described above, but incubated at 26°C rather than 37°C. After obtaining a contractile response to 80 mM K+ the arteries were equilibrated in Ca2+-free relaxing solution containing 1 mM EGTA, and then permeabilized by incubation at pCa 6.5 with 60 μg ml–1-toxin until the resulting contraction reached a plateau. The vessels were then re-equilibrated with solution containing 10 mM EGTA, and a submaximal contraction was brought about by raising pCa to 7.0–6.8. Agonists were added to the bathing solution once contraction reached a steady state. Details of the solutions used have been published previously (Horiuti, 1986). pCa was regulated by adjusting the ratio of K2EGTA and CaEGTA. Agonist-induced contractions did not require inclusion of GTP in the solution, as this was presumably retained in sufficient concentration in cells to allow G-protein activation (Thomas et al. 2006).
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Statistics
Results are expressed as the mean ±S.E.M., and means are compared using paired or unpaired Student's t test as appropriate (SigmaStat, SPSS Inc., Chicago, IL, USA). A difference was deemed significant if P < 0.05.
Materials
Prostaglandin F2 tromethamine and U-46619 (Biomol Intl, Exeter, UK) were used as 10 mM and 1 mM stock solutions in distilled water and DMSO, respectively. Fura PE-3/AM, diltiazem, carbachol, thapsigargin, fluprostenol, AL-8810, U-73122 and -toxin were from Sigma-Aldrich Co. Ltd, Poole, UK; SQ-29584 was from Alexis Corp., Nottingham, UK; 2-aminoethoxydiphenylborane (2-APB) was from Tocris Bioscience, Bristol, UK.
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Results
PGF2 caused a concentration-dependent contraction of rat small IPA (Fig. 1A and B). The threshold for contraction was approximately 1 μM PGF2, the EC50 was 5.8 ± 2.4 μM and the maximum response was 118 ± 33% of that recorded in KPSS (n= 11). The contraction was highly dependent on extracellular Ca2+; in a nominally Ca2+-free solution it fell by 59 and 58% at 10 and 30 μM PGF2, respectively. Inclusion of EGTA in the solution further reduced the response; for example the response to 20 μM PGF2 was reduced by 86 ± 2% (n= 12) in the presence of 1 mM EGTA.
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A, a typical record showing [Ca2+]i (top traces) and tension (bottom traces) responses to ascending concentrations (0.001–100 μM) of PGF2. In this and other figures, ‘RU’ refers to ratio units as defined in Methods. For example the bar in this trace represents a change in the RU of 0.02. The arrow next to the response to 100 μM PGF2 shows the amplitude of the initial Ca2+ transient. B, concentration dependence of transient () and sustained () [Ca2+]i responses and force () induced by PGF2 in IPA. Results shown are the mean ±S.E.M. for 7 ([Ca2+]i) and 11 (force) experiments. C, raising the PGF2 concentration from 0.1 to 10 μM (left) caused a rise in [Ca2+]i which resembled that evoked by 0.05 μM U-46619 (right), in that the initial transient component was greatly attenuated.
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Based on these results, we hypothesized that PGF2 was exerting two separate effects on [Ca2+]i which developed over different concentration ranges. At concentrations of 1 μM and below, PGF2 was causing a transient rise in [Ca2+]i which was followed by the establishment of a lower plateau level of [Ca2+]i which remained constant or fell gradually. This response appeared to become maximal at 0.1 μM PGF2, and was not associated with contraction. At higher concentrations, PGF2 was in addition causing a somewhat slower increase in [Ca2+]i which tended to abate with time, and which was associated with contraction. Based on the reported difference between the affinities of PGF2 for FP and TP receptors (0.003 versus9 μM, respectively, Breyer et al. 2001), we further hypothesized that the transient and plateau rises in [Ca2+]i were due to the stimulation of FP receptors, whereas the slower increase in [Ca2+]i was due to TP receptors.
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In order to characterize these two putative responses, which we will hereafter term the ‘FP’ and ‘TP’ responses, we examined in more detail the effects of 0.1 μM PGF2, which would be expected to produce only the FP response, and 10 μM PGF2, which should produce a mixed FP/TP response. We also studied the effects of U-46619, which should cause a relatively selective stimulation of TP receptors at concentrations 0.1 μM (Narumiya et al. 1999; Breyer et al. 2001). The validity of this strategy is supported by the observation that the rise in [Ca2+]i caused by 10 μM PGF2 added in the presence of 0.1 μM PGF2 closely resembled that evoked by 0.1 μM U-46619 (Fig. 1C), consistent with the prediction that following a maximal stimulation of the FP response by 0.l μM PGF2 (Fig. 1B), a ‘pure’ TP response would be all that could be elicited when a higher PGF2 concentration was applied. Note that both 10 μM PGF2 and 0.1 μM U-46619 caused a contraction, while 0.1 μM PGF2 did not.
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Endothelial denudation was confirmed by the absence of vasodilatation to carbachol (1 μM). Similar results were obtained in 4 other arteries.
Characterization of the rise in [Ca2+]i caused by 0.1 μM PGF2: the FP response
The temporal profile of the Ca2+ signal elicited by 0.1 μM PGF2 suggested that this response might involve Ca2+ release from intracellular stores, followed by a sustained Ca2+ influx. In agreement with this concept, the sustained rise in [Ca2+]i in response to application of 0.1 μM PGF2 was abolished (n= 5) in Ca2+-free solution containing 30–100 μM EGTA (Fig. 3A) or in the presence of 10 μM La3+ (n= 9) (Fig. 3B). In contrast, the initial [Ca2+]i transient remained largely intact both in Ca2+-free and La3+-containing solutions (70 ± 3%, n= 5 and 67 ± 8%, n= 10, of control, respectively).
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Removal of extracellular Ca2+ (A), or application of 10 μM La3+ (B) abolished the sustained rise in [Ca2+]i to 0.1 μM PGF2, while leaving the initial transient [Ca2+]i increase largely intact.
Based on these results, we reasoned that the sustained increase in [Ca2+]i caused by 0.1 μM PGF2 might be due to store operated Ca2+ entry (SOCE), which can be elicited by the SERCA antagonist thapsigargin and is then blocked by low micromolar concentrations of La3+ in these arteries (Snetkov et al. 2003). In this case, prior depletion of SR/ER Ca2+ with thapsigargin should prevent this response. In agreement with this prediction, Fig. 4A illustrates that pretreatment with thapsigargin (1 μM), which caused a large sustained rise in [Ca2+]i, virtually abolished the response to 0.1 μM PGF2 (95 ± 4% inhibition, n= 5); pretreatment with 0.1 μM thapsigargin had a similar effect (93 ± 3% inhibition, n= 3, not shown). Moreover, as depicted in Fig. 4B, the sustained response to 0.1 μM PGF2 was completely eliminated in the presence of 2-APB (75 μM), a putative blocker of IP3 receptors and SOCE (n= 7). 2-APB reduced the initial Ca2+ transient to a similar extent as La3+ or Ca2+-free solution (to 71 ± 11% of control, n= 7), suggesting that it had little effect on Ca2+ release at this concentration. On the other hand, Fig. 4C illustrates that the L-type voltage-gated Ca2+ channel blocker diltiazem (10 μM) had no measurable effect on the sustained response to 0.1 μM PGF2 (n= 6). The [Ca2+]i rise evoked by 0.1 μM PGF2 was also unaffected by the TP receptor antagonist SQ-29548 at a concentration (1 μM) which should completely block this receptor (Narumiya et al. 1999) (Fig. 4D, n= 4).
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A, both transient and sustained increases in [Ca2+]i caused by 0.1 μM PGF2 were abolished following application of the SERCA inhibitor thapsigargin (1 μM), which itself caused a sustained rise in [Ca2+]i. B, pretreatment with 2-APB (75 μM) eliminated the sustained but not transient rises in [Ca2+]i evoked by 0.1 μM PGF2. C, the L-type Ca2+ channel blocker diltiazem (10 μM) had no measurable effect on the sustained rise in [Ca2+]i caused by 0.1 μM PGF2. D, the TP receptor antagonist SQ-29548 (1 μM) also had no apparent effect on the sustained rise in [Ca2+]i caused by 0.1 μM PGF2.
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The FP receptor agonist fluprostenol (0.1 μM) caused a rise in [Ca2+]i which was indistinguishable from that of 0.1 μM PGF2 and was also insensitive to 1 μM SQ-29548 (Fig. 5A) (n= 4) and to 10 μM diltiazem (Fig. 5B, n= 5), but was almost eliminated (93 ± 3% reduction, n= 3) by pretreatment with 0.1 μM thapsigargin (Fig. 5C).
A, the selective FP receptor agonist fluprostenol (0.1 μM) caused a rise in [Ca2+]i which closely resembled that of 0.1 μM PGF2 and was similarly unaffected by the TP receptor antagonist SQ-29548 (1 μM). The response to fluprostenol was also insensitive to diltiazem (10 μM, B), but was abolished by pretreatment with thapsigargin (1 μM, C).
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On average, the initial transient rise in [Ca2+]i to 0.1 μM PGF2 amounted to 85 ± 15% (n= 68) of the rise in [Ca2+]i caused by KPSS The subsequent plateau rise [Ca2+]i, measured after 10 min, was 28 ± 4% of the KPSS response. Experiments examining the relationship between [Ca2+]i and contraction in the presence of a range of elevated concentrations of K+ (not shown) suggested that increases in [Ca2+]i of these magnitudes would be expected to cause contractions of approximately 75 and 20%, respectively, of that caused by KPSS, indicating that the lack of contraction during the FP response was not simply due to an insufficient rise in [Ca2+]i.
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In summary, the results shown in Figs 1–5 suggested that 0.1 μM PGF2 was acting on FP receptors to cause release of Ca2+ from the sarcoplasmic/endoplasmic reticulum, leading to activation of SOCE. The resulting rise in [Ca2+]i, though substantial, was not coupled to contraction. We went on to assess whether 10 μM PGF2 also caused a rise in [Ca2+]i mainly through SOCE.
Effects of 10 μM PGF2 and U-46619 on [Ca2+]i and contraction: the TP response
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In agreement with previous findings (e.g. Dorn et al. 1992), the contraction induced by 10 μM PGF2 was abolished by SQ-29548 (n= 10), suggesting that at this concentration PGF2 was activating TP receptors, and that this was responsible for tension development. In contrast to its lack of effect on the response to 0.1 μM PGF2, diltiazem (10 μM) caused a significant inhibition of the rise in [Ca2+]i evoked by 10 μM PGF2, as illustrated in Fig. 6A. On average, the increase in [Ca2+]i was inhibited by 37 ± 11% (n= 6) in arteries preincubated with diltiazem. Diltiazem caused a similar degree of inhibition of contraction over a range of the PGF2 concentrations (e.g. 30 ± 9% at 10 μM PGF2; n= 15; Fig. 6B).
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A, diltiazem (10 μM) depressed the rise in [Ca2+]i caused by 10 μM PGF2 when added either before (upper trace) or during (lower trace) the application of agonist. B, diltiazem also partially suppressed contraction to a range of concentrations of PGF2 (n= 15). In each experiment, responses to ascending concentrations of PGF2 applied cumulatively were recorded before and after application of diltiazem (10 μM). Asterisks indicate a significant inhibition of contraction (P < 0.05).
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In order to determine whether the diltiazem-insensitive rise in [Ca2+]i evoked by 10 μM PGF2 might be due to SOCE, we assessed the effect of 2-APB on this response in the presence of diltiazem. As depicted in Fig. 7A, 2-APB (75 μM) strongly reduced the sustained rise in [Ca2+]i caused by 10 μM PGF2 applied in the presence of diltiazem, with inhibition amounting to 78 ± 3% in four arteries. Similarly, 2-APB markedly inhibited the contraction to PGF2 (Fig. 7B). On the other hand, pretreatment with 10 μM La3+, which had virtually abolished the sustained [Ca2+]i rise caused by 0.1 μM PGF2, caused a smaller attenuation of the [Ca2+]i increase caused by 10 μM PGF2 in both the presence (47 ± 17% inhibition, n= 5) or absence (32 ± 21% inhibition, n= 6) of diltiazem (P < 0.01 compared to La3+-mediated inhibition of the [Ca2+]i increase to 0.1 μM PGF2 in both cases) (Fig. 7C).
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A, an example trace illustrating that 75 μM 2-APB strongly suppressed but did not abolish the 10 μM PGF2 -induced increase in [Ca2+]i observed in the presence of diltiazem. B, both 30 and 75 μM 2-APB (n= 6 and 7, respectively) strongly suppressed the contractions caused by a range of concentrations of PGF2, when applied in the presence of diltiazem to prevent Ca2+ influx through L-type channels. C, pretreatment with La3+ (10 μM) abolished the [Ca2+]i rise to 0.1 μM PGF2, but only attenuated the [Ca2+]i response to 10 μM PGF2.
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The results shown in Figs 6 and 7 indicated that the [Ca2+]i rise in response to 10 μM PGF2 could be distinguished from that caused by 0.1 μM PGF2 in that it demonstrated diltiazem-sensitive and -insensitive components, the latter of which was relatively La3+ resistant compared to the SOCE elicited by 0.1 μM PGF2. Moreover, the temporal profile of the increase in [Ca2+]i caused by 10 μM PGF2 was different from that of the FP response (Fig. 1A). Taking into account the reported high affinity of PGF2 for the FP receptor (Narumiya et al. 1999) and our evidence that the rise in [Ca2+]i to 0.1 μM PGF2 represented the effect of maximal simulation of the FP receptor (Fig. 1B), it seemed evident that it must be activation of TP receptors which was responsible for activation of these additional components of Ca2+ entry by 10 μM PGF2. In order to focus specifically on the components of the response coupled to the TP receptor in the absence of the complicating factor introduced by the coactivation of FP receptors and therefore SOCE, we next examined the response to the TXA2 analogue and selective TP agonist U-46619.
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As expected, force development elicited by 0.1 μM U-46619 was abolished by the TP receptor antagonist SQ-29548 (1 μM) in all five arteries tested. Moreover, the rise in [Ca2+]i in response to 50 and 100 nM U-46619 was abolished by 1 μM SQ-29548 in 2 of 2 and 4 of 5 arteries examined, respectively, and fell by 40% in the remaining artery tested at 0.1 μM U-46619.
U-46619 caused a concentration-dependent contraction which was increasingly attenuated by 10 μM diltiazem at progressively higher concentrations of U-46619 (Fig. 8A). As shown in Fig. 8B, diltiazem also partially inhibited the rise in [Ca2+]i evoked by U-46619 (0.05 μM); in six arteries this was reduced by 44 ± 11%. These results echo those observed with 10 μM PGF2, suggesting that while a fraction of the U-46619 contraction was dependent upon the opening of L-type voltage-gated Ca2+ channels, another Ca2+ influx pathway was making an important contribution to this response. In support of this possibility, both 30 and 75 μM 2-APB, when applied in the presence of 10 μM diltiazem, strongly inhibited contraction over a range of U-46619 concentrations (Fig. 8C). 2-APB at 75 μM also reduced the rise in [Ca2+]i caused by 0.05 μM U-46619 by 76 ± 5% (n= 4) in the presence of diltiazem, a value similar to that found for its effect on the [Ca2+]i response to 10 μM PGF2.
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A, the effect of diltiazem on the contraction caused by a range of concentrations of U-46619 (n= 13). In each experiment, responses to ascending concentrations of U-46619 applied cumulatively were recorded before and after application of diltiazem (10 μM). B, an example of the effect of 10 μM diltiazem on the rise in [Ca2+]i to 0.05 μM U-46619. C, both 30 and 75 μM 2-APB (n= 7 and 8, respectively) strongly suppressed the contractions caused by a range of concentrations of U-46619 when applied in the presence of diltiazem.
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As described above, 10 μM La3+ abolished the sustained rise in [Ca2+]i in response to 0.1 μM PGF2 but reduced the diltiazem-insensitive increase in [Ca2+]i in response to 10 μM PGF2 by only 50%. Moreover, 75 μM 2-APB caused a greater suppression of the increase in [Ca2+]i caused by 0.1 μM PGF2 than it did of the diltiazem-insensitive rises in [Ca2+]i caused by 10 μM PGF2 and 50 nM U-46619. This difference, although not as pronounced as that for La3+, was highly significant (P < 0.01). These results suggested that TP receptors could activate a non-voltage-gated Ca2+ influx pathway different from the SOCE induced by FP receptor stimulation.
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More direct evidence for this possibility was obtained in experiments in which Ca2+ was replaced by Sr2+ (Fig. 9). Under these conditions, whereas 0.1 μM PGF2 still caused an initial transient increase in the Fura PE-3 signal, the sustained increase in signal present in Ca2+ was absent (Fig. 9A). This result is in accordance with previous reports that Sr2+ does not permeate SOCE very well (Broad et al. 1999; Ma et al. 2000). On the other hand, application of either 10 μM PGF2 (Fig. 9B) or 50 nM U-46619 (Fig. 9C) led to a sustained increase in the Fura PE-3 signal which was not greatly different from that observed in Ca2+-containing solution. The results of these experiments, which are summarized in Fig. 9D, show that TP receptor stimulation was activating a Sr2+ permeable pathway different from SOCE. This pathway was not due to L-type Ca2+ channels, since it was not significantly affected by 10 μM diltiazem (n= 5) or, in one experiment, by 10 μM verapamil (not shown).
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A, substitution of 4 mM Sr2+ for 1.8 mM Ca2+ abolished the sustained increase in the Fura PE-3 signal caused by 0.1 μM PGF2, but had a much smaller effect on the increase in this ratio to either 10 μM PGF2 (B) or 0.05 μM U-46619 (C). D, mean ±S.E.M. results from 5 experiments with each agonist, shown as the sustained (i.e. after 10 min) increase in the Fura PE-3 signal in the presence of Sr2+ expressed as a percentage of that observed in Ca2+. In panels A–C, bars beneath the traces show application of agonist. Sr2+ was used at a concentration of 4 mM because the high K+ contraction under these conditions was similar to that observed in normal PSS.
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Additional evidence that the response to the TP receptor was occurring via a non-SOCE pathway was obtained in experiments utilizing the phospholipase C inhibitor U-73122. As shown in Fig. 10A, both the transient and sustained rises in [Ca2+]i induced by 0.1 μM PGF2 were very markedly suppressed by pretreatment with 3 μM U-73122; in eight experiments these responses were inhibited by 87 ± 3% and 92 ± 4%, respectively. On the other hand, the additional rise in [Ca2+]i caused by 10 μM PGF2 (i.e. when 10 μM PGF2 was applied directly after 0.1 μM PGF2 as shown in Fig. 10A) was suppressed to a significantly smaller extent by U-73122 (29 ± 5% inhibition; n= 5), and the accompanying contraction was not significantly affected (5 ± 11% increase, n= 7, not shown). U-73122 had similar effects on the U-46619-induced rise in [Ca2+]i, which was reduced by 27 ± 12% in five experiments (Fig. 10B) and on the U-46619-induced contraction (7 ± 14% increase; n= 6, n.s., not shown). These results were consistent with the concept that the lower concentration of PGF2 was activating SOCE by causing IP3-mediated Ca2+ release, whilst both U-46619 and the higher concentration of PGF2 were increasing [Ca2+]i mainly by stimulating a separate pathway which was largely independent of both IP3 and SOCE. In accordance with this idea, pretreatment of arteries with thapsigargin (1 μM), which had virtually eliminated the response to 0.1 μM PGF2 and fluprostenol (Figs 4A and 5C), caused only a partial (38 ± 8%, n= 9) inhibition of the response to 0.1 μM U-46619. Figure 10B illustrates a typical experiment of this type.
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A, U-73122 (3 μM) suppressed the rise in [Ca2+]i to 0.1 μM PGF2 to a much greater extent than it did the increase in [Ca2+]i to 10 μM PGF2 or 0.01 μM U-46619. B, thapsigargin (1 μM) caused a partial inhibition of the rise in [Ca2+]i elicited by 0.01 μM U-46619.
Contractions in -toxin permeabilized IPA
Both PGF2 and U-46619 caused contractions in -toxin permeabilized IPA, and in both cases these contractions were reversed by SQ-29548. Figure 11A and B illustrates an example of the effect of 1 μM SQ-29548 on the contraction to 100 μM PGF2 and 1 μM U-46619, respectively, recorded in the presence of 10 μM cyclopiazonic acid (to block any effects on Ca2+ stores) at pCa 7.0. Conversely, in four arteries tested, fluprostenol (1 and 10 μM) did not cause a contraction in permeabilized IPA either in absence or presence of 1 μM GTP (Fig. 11C).
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PGF2 (30 μM) was then added to confirm that the artery was responsive to TP receptor stimulation. The TP receptor antagonist SQ-29548 (1 μM) reversed the responses to PGF2 and U-46619.
Discussion
As far as we are aware, these results represent the first characterization in intact arteries of distinct store- and receptor-activated [Ca2+]i influx pathways activated by a single vasoconstricting agonist. The data are consistent with previous reports in isolated smooth muscle cells and smooth muscle-derived cell lines that vasoconstrictors stimulate the opening of separate SOCs and ROCs (Byron & Taylor, 1995; Broad et al. 1999; Iwamuro et al. 1999; Furutani et al. 2002; Large, 2002; Moneer & Taylor, 2002; Albert & Large, 2003), and the recent demonstration by Jernigan et al. (2006) that cyclopiazonic acid and UTP activate a SOC and ROC, respectively, in rat IPA.
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The FP response
At a concentration of 0.1 μM, PGF2 caused a marked transient and sustained rise in [Ca2+]i which was mimicked by the FP receptor agonist fluprostenol. The rises in [Ca2+]i caused by both agonists at this concentration were also similar in that neither was associated with a contraction, and neither was affected by the TP receptor antagonist SQ-29548 or the L-type Ca2+ channel antagonist diltiazem. On the other hand, both transient and sustained rises in [Ca2+]i to 0.1 μM PGF2 were strongly suppressed by the phospholipase C antagonist U-73122 and virtually abolished by pretreatment with thapsigargin. The initial [Ca2+]i transient persisted in Ca2+-free solution or in the presence of 10 μM La3+, but the sustained rise in [Ca2+]i was abolished by both. These results indicate that at a concentration which was subthreshold for contraction, PGF2 nonetheless caused a rise in [Ca2+]ivia the activation of FP receptors. This rise in [Ca2+]i was likely initiated by an IP3-mediated Ca2+ release from the sarcoplasmic reticulum, and the effects of La3+ and 2-APB suggest that it was sustained by SOCE, consistent with the known coupling of the FP receptor to Gq-protein and phospholipase C (Narumiya et al. 1999). Interestingly, 75 μM 2-APB had little effect on the initial Ca2+ transient (Fig. 4B), suggesting that at this concentration it was not strongly antagonizing the IP3 receptor.
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We have previously found that SOCE stimulated by thapsigargin caused some tension development in IPA (Snetkov et al. 2003), although thapsigargin-induced SOCE causes large tension-independent rises in [Ca2+]i in a number of other resistance arteries and arterioles (Flemming et al. 2002; Snetkov et al. 2003). Experiments in which [Ca2+]i was elevated to varying degrees using progressively higher concentrations of [K+] suggested that the amplitude of the rise in [Ca2+]i caused by 0.1 μM PGF2 would have been expected to cause some contraction in IPA. There are a number of possible explanations for the lack of contraction associated with the FP response, one being that the rise in [Ca2+]i due to SOCE is somehow localized primarily to regions of the cell which lack elements of the contractile apparatus (Flemming et al. 2002). It is interesting in this regard that Gordienko et al. (2001) and Pucovsky & Bolton (2006) have shown that the primary Ca2+ discharge region of the sarcoplasmic reticulum both basally and during stimulation by agonists is located in close apposition to the nucleus, and that Abrenica et al. (2003) have reported that thapsigargin causes a rise in [Ca2+]i which is mainly restricted to the nucleus and the perinuclear cytoplasm. SOCE is enhanced in proliferating as compared to quiescent pulmonary artery myocytes, and its inhibition suppresses proliferation (Golovina et al. 2001). In addition, Dorn et al. (1992) reported that PGF2 caused SQ-29548-resistant (i.e. presumably FP receptor-mediated) increases in [Ca2+]i and phosphatidylinositol turnover in cultured aorta smooth muscle cells with EC50 values for both responses of < 0.05 μM, while in intact aorta the threshold and EC50 for PGF2-induced contraction were > 0.1 and 5 μM, respectively. These results are consistent with the possibility that nanomolar concentrations of PGF2 may act via the FP receptor and SOCE to cause a rise in cytoplasmic [Ca2+] which is specifically funnelled into the nucleus, allowing activation of transcription factors and promoting proliferation. It is also possible that the inability of SOCE stimulated by FP receptor activation to cause contraction in part reflects an absence of Ca2+ sensitization (Fig. 11), especially since contraction in IPA seems to be particularly dependent on the rhoA pathway (Hyvelin et al. 2004). This would also concur with our observation that rises in [Ca2+]i to U-46619, which caused Ca2+ sensitization, were often no larger than those to 0.1 μM PGF2, yet were always associated with contraction. A resolution of these issues will require further study using methods capable of assessing compartmentalization of [Ca2+]i during stimulation with FP- and TP-receptor selective agonists.
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The TP response
At a higher concentration (10 μM) PGF2 caused a contraction which was abolished by SQ-29548, indicating that it resulted from TP receptor stimulation. Although our experiments in -toxin skinned IPA and previous studies in other arteries (Bradley & Morgan, 1987; Himpens et al. 1990; Ito et al. 2003; Ding & Murray, 2005) showed that this contraction is partially dependent on Ca2+ sensitization, it also clearly requires the presence of Ca2+ for its full development, and is associated with a rise in [Ca2+]i which involves multiple mechanisms.
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Although 10 μM PGF2 would be expected to stimulate SOCE, the properties of the rise in [Ca2+]i caused by this concentration of agonist showed that it involved two additional pathways of Ca2+ influx. For example, when 10 μM PGF2 was applied directly after 0.1 μM PGF2 the temporal profile of the increment in [Ca2+]i caused by the higher concentration of agonist differed from that caused by the lower concentration. This increment in [Ca2+]i typically lacked a, or had a much smaller, initial transient component, and developed gradually over several minutes, unlike SOCE, which began immediately and remained essentially constant (Figs 1 and 2). This increment in [Ca2+]i was relatively insensitive to U-73122, suggesting that it did not require IP3-mediated Ca2+ release. Similar properties were demonstrated for the response to U-46619, again indicating that TP receptors were involved. Although diltiazem partially inhibited the [Ca2+]i increases elicited by 10 μM PGF2 and U-46619, the greater part of both responses was diltiazem insensitive. Moreover, this diltiazem-resistant rise in [Ca2+]i was mainly independent of SOCE, since it was comparatively insensitive to 10 μM La3+, which abolished SOCE (Fig. 3B, Robertson et al. 2000).
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More direct evidence that TP receptor stimulation by either U-46619 or 10 μM PGF2 was activating a Ca2+ entry mechanism which was not mediated by either SOCE or L-type Ca2+ channels came from experiments in which Ca2+ was replaced by Sr2+, which has been shown to permeate receptor- but not store-operated channels in A7r5 and other types of cell lines (Broad et al. 1999; Ma et al. 2000). SOCE caused by 0.l μM PGF2 did not cause a sustained rise in the Fura PE-3 signal when Ca2+ was replaced with Sr2+. In contrast, both U-46619 and 10 μM PGF2 induced large diltiazem-insensitive responses with Sr2+ in the solution. Also, the rise in [Ca2+]i to U-46619 was only partially prevented by pretreatment with thapsigargin, whereas those caused by 0.1 μM PGF2 or fluprostenol were abolished.
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The results therefore show that 10 μM PGF2, in addition to stimulating SOCE via FP receptors, also acted on TP receptors to activate Ca2+ influx through both L-type Ca2+ channels and another Ca2+ pathway which was Sr2+ permeable, relatively La3+ resistant, and which seemed to be mostly independent of IP3-mediated Ca2+ release, since it was little affected by the phospholipase C antagonist U-73122, which has previously been shown to inhibit Ca2+ release (Hansen et al. 1995).
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Evidence that vasoconstrictors acting on G-protein coupled receptors may activate receptor operated non-selective cation channels (ROCs) different from those opened by store depletion has emerged from electrophysiological and Ca2+ imaging studies in isolated or cultured smooth muscle cells (Byron & Taylor, 1995; Broad et al. 1999; Iwamuro et al. 1999; Large, 2002), and from pharmacological studies in intact arteries (Furutani et al. 2002; Jernigan et al. 2006). Vascular smooth muscle demonstrates a number of ROCs with diverse properties (Large, 2002) which may coexist in single types of cells (Iwamuro et al. 1999). The best-characterized ROC in vascular smooth muscle is the noradrenaline-activated ICAT (Large, 2002) of which TRPC6 is thought to be a crucial component (Inoue et al. 2001). One apparent difference between ICAT and the ROC in IPA is that activation of ICAT is potently inhibited by U-37122 (Helliwell & Large, 1997), which, however, had little effect on the TP-receptor-stimulated ROC at a concentration (3 μM) which almost abolished Ca2+ release and SOCE, indicating almost complete suppression of phospholipase C activity (Fig. 10A). Whether this implies that the channels involved are different, or alternatively that the 1 and TP receptors are coupled to the same channel through separate pathways, remains to be determined.
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Several non-SOCE pathways, as defined by their insensitivity to both L-type Ca2+ channel antagonists and prior treatment with Ca2+ store releasing agents, have recently been found to be activated in small pulmonary arteries by other vasoconstrictors. Ca2+ influx stimulated by 5-HT was observed to be insensitive to nitrendipine, to the lanthanide Gd3+, and to prior Ca2+ store release with cyclopiazonic acid (Guibert et al. 2004). This Ca2+ influx was also blocked by the DAG lipase inhibitor RHC-80267, suggesting that arachidonic acid was involved as a second messenger (Guibert et al. 2004), much as it is thought to be important in vasopressin-induced activation of a ROC in A7r5 cells (Broad et al. 1999). We did not explore whether arachidonic acid plays a similar role during TP receptor-associated ROC activation, although the lack of effect of U-73122 suggests that if this fatty acid is involved, it is not coming from DAG produced by phospholipase C. We also found that a rise in [Ca2+]i induced by sphingosylphosphorylcholine in IPA was insensitive to diltiazem, U-73122, and to prior Ca2+ store release with thapsigargin (Thomas et al. 2006), and was also Sr2+ permeable (Thomas et al., unpublished observation). UTP-stimulated Ca2+ entry in these arteries persisted in the presence of diltiazem, cyclopiazonic and 10 mM Ni2+, but was abolished by 50 μM SKF-96365. Conversely, SOCE activated by cyclopiazonic acid was completely blocked by Ni2+ but was unaffected by SKF-96365 (Jernigan et al. 2006). Whether these various receptor-operated Ca2+ influx pathways can be ascribed to the same ROC remains unresolved. It is noteworthy that a reciprocal relationship between SOC- and ROC-mediated Ca2+ entry has previously been described in a number of types of cells, including A7r5 (Moneer & Taylor, 2002). Intriguingly, the initial Ca2+ transient caused by PGF2 fell significantly at the highest concentration of this agonist (Fig. 1), suggesting that a reciprocal relationship could also exist between ROC-mediated Ca2+ entry and Ca2+ release, although confirmation of this possibility will require additional study.
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In summary, our results show that PGF2 raises [Ca2+]ivia multiple pathways. At a low concentration, this agonist activates FP receptors to cause an IP3-mediated release of intracellular Ca2+ stores. This in turn leads to SOCE. However, neither Ca2+ release nor SOCE results in contraction. At higher concentrations, which cause contraction, PGF2 also acts on TP receptors to activate a separate Ca2+ entry pathway which can be distinguished from SOCE in that it is relatively insensitive to U-73122, La3+ and thapsigargin, and is permeable to Sr2+. This pathway, which would appear to represent a ROC, seems to closely resemble those shown to be activated by 5-HT, and by sphingosylphosphorylcholine in these arteries (Guibert et al. 2004; Thomas et al. 2006). At the same time, TP receptor stimulation causes Ca2+ influx through L-type Ca2+ channels, which are presumably opened by depolarization arising as a result of Na+ influx through the ROC and/or K+ channel inhibition. The effect of the resulting rise in [Ca2+]i on contraction is amplified by TP receptor-dependent Ca2+ sensitization.
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