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¹ Department of Asthma, Allergy and Respiratory Science, King's College London School of Medicine, King's College London, London SE1 1UL, UK

	Abstract

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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.

(Received 7 November 2005; accepted after revision 12 December 2005; first published online 22 December 2005)
Corresponding author V. Snetkov: Department of Asthma, Allergy and Respiratory Science, New Hunt's House, Guy's Hospital Campus, King's College London, London SE1 1UL, UK. Email: vladimir.snetkov{at}kcl.ac.uk

	Introduction

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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).

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 G_q 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 K_V 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.

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).

	Methods

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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).

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 R_340/380 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).

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 R_340/380 caused by this agonist was expressed as a fraction of the response to KPSS, which was recorded in each artery.

Membrane permeabilization

-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).

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.

	Results

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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.

View larger version (15K): [in this window] [in a new window]   Figure 1.  Developed tension and [Ca2+]i of intrapulmonary arteries in response to PGF2 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.

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.

Figure 2 demonstrates that removal of the endothelium, confirmed by the lack of any relaxation of the artery to 1 µM carbachol, did not prevent the rise of [Ca²⁺]_i evoked by 0.1 µM PGF₂, thus indicating that it was unlikely to be of endothelial origin. The lack of any significant contribution of the endothelium to the overall Fura PE-3 signal in this preparation was also demonstrated by the observation that treatment of intact arteries with carbachol to raise endothelial cell [Ca²⁺]_i did not cause any change in R_340/380 (not shown) (see also Ward & Snetkov, 2004).

View larger version (11K): [in this window] [in a new window]   Figure 2.  Responses to 0.1 and 10 µM PGF2 in an artery before (left) and after (right) denudation of the endothelium 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).

View larger version (9K): [in this window] [in a new window]   Figure 3.  Effect of Ca2+ removal and La3+ on the rise in [Ca2+]i induced by 0.1 µM PGF2 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.

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View larger version (16K): [in this window] [in a new window]   Figure 4.  The role of Ca2+ release and store operated Ca2+ entry in the response to 0.1 µM PGF2 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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View larger version (10K): [in this window] [in a new window]   Figure 5.  Response of intrapulmonary arteries to the FP receptor agonist fluprostenol 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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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

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).

View larger version (11K): [in this window] [in a new window]   Figure 6.  Effect of L-type channel blockade on the response to PGF2 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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View larger version (15K): [in this window] [in a new window]   Figure 7.  Effect of 2-APB and La3+ on the rise in [Ca2+]i induced by 10 µM PGF2 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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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.

View larger version (13K): [in this window] [in a new window]   Figure 8.  Effect of diltiazem and 2-APB on the response to the TP receptor agonist U-46619 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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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).

View larger version (12K): [in this window] [in a new window]   Figure 9.  Sr2+ permeability of 0.1 µM and 10 µM PGF2 induced Ca2+ influx pathways 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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View larger version (13K): [in this window] [in a new window]   Figure 10.  Role of PLC/IP3-induced Ca2+ release in the response to PGF2 and U-46619 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).

View larger version (11K): [in this window] [in a new window]   Figure 11.  Tension responses to 100 µM PGF2 (A), 1 µM U-46619 (B) and 1 and 10 µM fluprostenol (C) in -toxin permeabilized arteries at pCa 7.0 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

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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 G_q-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.

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.

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.

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).

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.

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).

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 I_CAT (Large, 2002) of which TRPC6 is thought to be a crucial component (Inoue et al. 2001). One apparent difference between I_CAT and the ROC in IPA is that activation of I_CAT 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.

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.

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.

	References

Top Abstract Introduction Methods Results Discussion References

 
Aaronson PI, Robertson TP, Knock GA, Becker S, Snetkov V & Ward JPT (2006). Hypoxic pulmonary vasoconstriction: mechanisms and controversies. Journal of Physiology 570, 53–58.[Abstract/Free Full Text]

Abrenica B, Pierce GN & Gilchrist JSC (2003). Nucleoplasmic calcium regulation in rabbit aortic vascular smooth muscle cells. Can J Physiol Pharmacol 81, 301–310.[CrossRef][Medline]

Albert AP & Large WA (2003). Store-operated Ca²⁺-permeable non-selective cation channels in smooth muscle cells. Cell Calcium 33, 345–356.[CrossRef][Medline]

Balwierczak JL (1991). The relationship of KCl- and prostaglandin F₂-mediated increases in tension of the porcine coronary artery with changes in intracellular Ca²⁺ measured with Fura-2. Br J Pharmacol 104, 373–378.[Medline]

Boersma JI, Janzen KM, Oliveira L & Crankshaw DJ (1999). Characterization of excitatory prostanoid receptors in the human umbilical artery in vitro. Br J Pharmacol 128, 1505–1512.[CrossRef][Medline]

Bradley AB & Morgan KG (1987). Alterations in cytoplasmic calcium sensitivity during porcine coronary artery contractions as detected by aequorin. J Physiol 385, 437–448.[Abstract/Free Full Text]

Breyer RM, Bagdassarian CK, Myers SA & Breyer MD (2001). Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol 41, 661–690.[CrossRef][Medline]

Broad LM, Cannon TR & Taylor CW (1999). A non-capacitative pathway activated by arachidonic acid is the major Ca²⁺ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin. J Physiol 517, 121–134.[Abstract/Free Full Text]

Byron K & Taylor CW (1995). Vasopressin stimulation of Ca²⁺ mobilization, two bivalent cation entry pathways and Ca²⁺ efflux in A7r5 rat smooth muscle cells. J Physiol 485, 455–468.[Abstract/Free Full Text]

Cogolludo A, Moreno L, Bosca L, Tamargo J & Perez-Vizcaino F (2003). Thromboxane A₂-induced inhibition of voltage-gated K⁺ channels and pulmonary vasoconstriction: role of protein kinase C. Circ Res 93, 656–663.[Abstract/Free Full Text]

Daray FM, Minvielle AI, Puppo S & Rothlin RP (2003). Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein. Br J Pharmacol 139, 1409–1416.[CrossRef][Medline]

Ding X & Murray PA (2005). Cellular mechanisms of thromboxane A2-mediated contraction in pulmonary veins. Am J Physiol 289, L825–L833.

Dorn GW, Becker MW & Davis MG (1992). Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle. J Biol Chem 267, 24897–24905.[Abstract/Free Full Text]

Evans AM, Cobban HJ & Nixon GF (1999). ET_A receptors are the primary mediators of myofilament calcium sensitization induced by ET-1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway. Br J Pharmacol 127, 153–160.[CrossRef][Medline]

Flemming R, Cheong A, Dedman AM & Beech DJ (2002). Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle. J Physiol 543, 455–464.[Abstract/Free Full Text]

Furutani H, Zhang XF, Iwamuro Y, Lee K, Okamoto Y, Takikawa O, Fukao M, Masaki T & Miwa S (2002). Ca²⁺ entry channels involved in contractions of rat aorta induced by endothelin-1, noradrenaline, and vasopressin. J Cardiovasc Pharmacol 40, 265–276.[CrossRef][Medline]

Goff CD, Corbin RS, Theiss SD, Frierson HF Jr, Cephas GA, Tribble CG, Kron IL & Young JS (1997). Postinjury thromboxane receptor blockade ameliorates acute lung injury. Ann Thorac Surg 64, 826–829.[Abstract/Free Full Text]

Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ & Yuan JX (2001). Upregulated TRP and enhanced capacitative Ca²⁺ entry in human pulmonary artery myocytes during proliferation. Am J Physiol 280, H746–H755.

Gordienko DV, Greenwood IA & Bolton TB (2001). Direct visualization of sarcoplasmic reticulum regions discharging Ca²⁺ sparks in vascular myocytes. Cell Calcium 29, 13–28.[CrossRef][Medline]

Guibert C, Marthan R & Savineau JP (2004). 5-HT induces an arachidonic acid-sensitive calcium influx in rat small intrapulmonary artery. Am J Physiol 286, L1228–L1236.

Hansen M, Boitano S, Dirksen ER & Sanderson MJ (1995). A role for phospholipase C activity but not ryanodine receptors in the initiation and propagation of intercellular calcium waves. J Cell Sci 108, 2583–2590.[Abstract]

Helliwell RM & Large WA (1997). Alpha 1-adrenoceptor activation of a non-selective cation current in rabbit portal vein by 1,2-diacyl-sn-glycerol. J Physiol 499, 417–428.[Abstract/Free Full Text]

Himpens B, Kitazawa T & Somlyo AP (1990). Agonist-dependent modulation of Ca²⁺ sensitivity in rabbit pulmonary artery smooth muscle. Pflugers Arch 417, 21–28.[CrossRef][Medline]

Hisayama T, Takayanagi I & Okamoto Y (1990). Ryanodine reveals multiple contractile and relaxant mechanisms in vascular smooth muscle: simultaneous measurements of mechanical activity and of cytoplasmic free Ca²⁺ level with Fura-2. Br J Pharmacol 100, 677–684.[Medline]

Hori M, Sato K, Sakata K, Ozaki H, Takano-Ohmuro H, Tsuchiya T, Sugi H, Kato I & Karaki H (1992). Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle. J Pharmacol Exp Ther 261, 506–512.[Abstract/Free Full Text]

Horiuti K (1986). Some properties of the contractile system and sarcoplasmic reticulum of skinned slow fibres from Xenopus muscle. J Physiol 373, 1–23.[Abstract/Free Full Text]

Hyvelin JM, O'Connor C & McLoughlin P (2004). Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway. Am J Physiol 287, L673–L684.[CrossRef]

Inoue R, Okada T, Onoue H, Hara Y, Shimizu S, Naitoh S, Ito Y & Mori Y (2001). The transient receptor potential protein homologue TRP6 is the essential component of vascular ₁adrenoceptor-activated Ca²⁺-permeable cation channel. Circ Res 88, 325–333.[Abstract/Free Full Text]

Ito K, Shimomura E, Iwanaga T, Shiraishi M, Shindo K, Nakamura J, Nagumo H, Seto M, Sasaki Y & Takuwa Y (2003). Essential role of rho kinase in the Ca²⁺ sensitization of prostaglandin F₂-induced contraction of rabbit aortae. J Physiol 546, 823–836.[Abstract/Free Full Text]

Iwamuro Y, Miwa S, Zhang XF, Minowa T, Enoki T, Okamoto Y, Hasegawa H, Furutani H, Okazawa M, Ishikawa M, Hashimoto N & M