Human umbilical artery smooth muscle exhibits a 2-APB-sensitive capacitative contractile response evoked by vasoactive substances and expresses mRNAs for STIM , Orai and TRPC channels

After depletion of intracellular Ca stores the capacitative response triggers an extracellular Ca influx through store-operated channels (SOCs) which refills these stores. Our objective was to explore if human umbilical artery smooth muscle presented this response and if it was involved in the mechanism of serotoninand histamine-induced contractions. Intracellular Ca depletion by a Ca-free extracellular solution followed by Ca readdition produced a contraction in artery rings which was inhibited by the blocker of Orai and TRPC channels 2-aminoethoxydiphenyl borate (2-APB), suggesting a capacitative response. In presence of 2-APB the magnitude of a second paired contraction by serotonin or histamine was significantly less than a first one, likely because 2-APB inhibited store refilling by capacitative Ca entry. 2-APB inhibition of sarcoplasmic reticulum Ca release was excluded because this blocker did not affect serotonin force development in a Ca-free solution. The PCR technique showed the presence of mRNAs for STIM proteins (1 and 2), for Orai proteins (1, 2 and 3) and for TRPC channels (subtypes 1, 3, 4 and 6) in the smooth muscle of the human umbilical artery. Hence, this artery presents a capacitative contractile response triggered by stimulation with physiological vasoconstrictors and expresses mRNAs for proteins and channels previously identified as SOCs.


Introduction
The human umbilical artery is a vessel of fundamental importance for fetal-placental blood flow.Since the human umbilical arteries are branches from the iliac arteries of the fetus, the properties of these blood vessels could offer information of the circulatory system of the newborn.Hence, the knowledge of the mechanisms implicated in the regulation of cytosolic Ca 2+ concentration and therefore, contraction of their vascular smooth muscle cells (VSMC) is very important.
In VSMC Ca 2+ influx through voltage-activated Ca 2+ channels in response to membrane depolarization or to vasoactive agonists represents the most studied pathway.However, an increasing number of investigations in blood vessels have demonstrated that depletion of intracellular Ca 2+ stores triggers a Ca 2+ influx from the extracellular space through ionic channels present in the cellular membrane.This phenomenon is known as "capacitative" or "store-operated" Ca 2+ entry and it is likely involved in the refilling of intracellular Ca 2+ stores (Parekh and Penner, 1997;Putney et al., 2001;Bolotina and Csutora, 2005;Leung et al., 2008).This mechanism has not been previously studied in the human umbilical artery nor in other vessels of feto-placental circulation, and hence, its physiological relevance in agonist-induced contractions in this system is unknown.
Although there is now much information about the structures involved in the capacitative response, further research is necessary to completely understand how these structures interact in order to produce SR refilling and, mainly, what is the physiological importance of this mechanism for each cellular type.Different kinds of experimental protocols have been used for this purpose.For instance, inducing SR depletion with Ca 2+ chelating agents like BAPTA or EGTA in extracellular solutions followed by Ca 2+ re-addition (Putney et al., 2001;Bird et al., 2008), blocking Ca 2+ entry to the SR with sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibitors like thapsigargin or cyclopiazonic acid (CPA) (Putney et al., 2001;Bird et al., 2008), or directly inducing Ca 2+ release from the SR by increasing intracellular inositol 1,4,5-trisphosphate concentration (Fasolato et al., 1993;Fasolato and Nilius, 1998;Parekh and Penner, 1995).
However, in vascular smooth muscle, SR Ca 2+ release and depletion are physiologically produced by mechanisms involving activation of membrane receptors.Hence, in the present work we demonstrate using classical protocols that the human umbilical artery presents a capacitative response, and then that it can be activated by stimulation with serotonin (5-HT) and histamine, both of which produce tonic contractions by coupling to membrane receptors, phospholipase C (PLC) signaling and SR Ca 2+ release and depletion.To strengthen these data, we demonstrate by the polymerase chain reaction (PCR) technique that the mRNAs for STIM, Orai and for the different subtypes of cationic TRPC channels involved in capacitative entry mechanisms are present in the smooth muscle cells of this artery.

Sample collection
Umbilical cords were obtained after vaginal and caesarean deliveries performed in the Instituto Central de Medicina of La Plata, Argentina.They were placed in a transport solution of the following composition (in mM): 130 NaCl, 4.7 KCl, 1.17 KH 2 PO 4 , 1.16 SO 4 Mg, 24 CO 3 HNa, 2.5 Cl 2 Ca, pH 7.4 at 4ºC and immediately taken to our laboratory where they were stored at 4ºC and used within 24 hrs.All the vascular preparations were classified as surgical discard specimens and thus they were exempted from patient consent requirements.

Isometric force measurements
The vessels were placed in a Petri dish filled with Krebs Ringer bicarbonate solution (KRB, see composition later), cleaned of adherent connective tissue, and cut into 3-4 mm wide rings.Special care was taken in not damaging the inner endothelial layer or overdistending the vessel.The ring was gently suspended between two stainless steel wires in a water-jacketed organ bath kept at 37ºC and filled with a KRB with the following composition (mM): 130 NaCl, 4.7 KCl, 1.17 Na 2 HPO 4 , 1.16 MgSO 4 , 24.0 HCO 3 Na, 2.5 CaCl 2 , and 6.0 glucose, constantly bubbled with a mixture of 5% CO 2 and 95% O 2 , giving a pH of 7.40.The lower wire was fixed to a vertical plastic rod immersed in the organ bath, while the upper one was rigidly attached to a force transducer (Letica TRI-201).The preparations were then stretched to obtain a passive force of ≈ 2 grams.In some experiments we used as bath solutions a Ca 2+ -free KRB solution (KRB without CaCl 2 ) or a Ca 2+ -free KRB with the addition of 1 mM of EGTA (see results for details).

Total RNA isolation and Reverse Transciptase Polymerase chain reaction (PCR)
Samples from seven umbilical arteries were used for this analysis.The endothelial layer was carefully eliminated by gently rubbing the inner surface with a cotton swab.Total RNA was obtained from homogenates using Trizol Reagent (Gibco-BRL, Rockville, MD, USA) (Chomczynski and Sacchi, 1987).The integrity of isolated RNA was checked by 1% agarose-formaldehyde gel electrophoresis.Possible contamination with protein or phenol was controlled by measuring the 260/ 280 nm absorbance ratio, while DNA contamination was avoided using 1 U/μl DNAase I (Gibco-BRL).RT-reaction was performed using the Super Script III reverse transcriptase (200 U/μl; Gibco-BRL) and total RNA (50 ng) as template.For PCR, specific primers were used based on the human cDNA sequence to detect TRPC 1, 3, 4, 5, 6 and 7 (TRPC 2 was not analyzed since it is a pseudogene (Parekh and Putney, 2005), STIM (1 and 2) and Orai (1, 2 and 3), as well as the von Willebrand factor (endothelial marker used to assure that endothelial cells were not present in our samples) and β-actin (positive control of the PCR).GenBank accession numbers, primer sequences used and expected size of PCR products are shown in Table 1.Possible contamination with genomic DNA was checked by performing PCR with or without the SuperScript III RT.
The cycling profile was the following: 3 min at 95ºC followed by 30 sec at 95ºC, 30 sec at 58ºC, and 1 min at 72ºC for 35 cycles and 10 min at 72ºC.PCR products were separated by electrophoresis on a 1.5% (w/v) agarose gel and stained with ethidium bromide.

Statistical analysis
Results were expressed as means ± SEM.Paired or unpaired Student's t-tests were used to evaluate statistically significant differences between two groups.ANOVA followed by the Tukey's test was used for multiple group comparisons.The level of statistical significancy was set at p<0.05.

Gene of GeneBank
Sequence of forward primer Sequence of reverse primer Product interest accession number size (bps)

Presence of a capacitative response
We first used a classical protocol, such as exposition to a Ca 2+ -free extracellular medium and then Ca 2+ readdition, to identify the presence of a capacitative response in the human umbilical artery, where this mechanism had not been described before.Hence, vessel rings were first incubated during 15 min at room temperature in a Ca 2+ -free Krebs Ringer bicarbonate (Ca 2+ -free KRB) solution containing 5 mM EGTA to eliminate all the extracellular Ca 2+ that could remain attached to connective tissue.Then, they were mounted in the force transducer and exposed during 15 minutes to a KRB solution without Ca 2+ (at 37ºC) in order to stabilize the temperature.Then, they were stretched to 2 grams and left for another 15 min in this Ca 2+ -free solution.Interestingly, during this time 32% of the vessel rings developed a small spontaneous and transient contraction of 42 ± 7 gF/gW (n=13), probably caused by SR release or leak, induced by the extracellular Ca 2+ -free solution.After that, Ca 2+ was restituted to the bath reaching a final concentration of 2.5 mM.Immediately, the rings developed a rapid contraction which reached a maxi-mal force (peak) and then stabilized at a minor force value (Fig. 1A).To see if all, or part, of this contraction could be inhibited by a blocker of capacitative Ca 2+ entry, the same protocol was repeated in presence of 100 μM 2-aminoethoxydiphenyl borate (2-APB), which is widely used to study this mechanism (Bootman et al., 2002) (Fig. 1B).The peak force induced by addition of Ca 2+ now decreased significantly respect to the control contraction, and it did not fall to a minor value.So, these results suggest that the exposition of artery rings to a Ca 2+ -free solution, during almost 45 minutes, is able to produce SR depletion and SOC activation.
Afterwards, we tested if the remaining force could be inhibited by blocking other Ca 2+ entry pathways that we know are present in smooth muscle cells of the human umbilical artery.In fact, if the artery rings were incubated with 100 μM 2-APB plus 5 μM nifedipine (a voltage-operated Ca 2+ channel inhibitor), 200 μM Gd 3+ (a non selective cationic channels blocker) and 5 μM KB-R7943(at this concentration, an inhibitor of the Na + / Ca 2+ exchanger reverse mode), the contraction induced by Ca 2+ restitution was abolished (Fig. 1C).When these were tested separately, all of them had a significant inhibitory effect but none produced a complete inhibition (Fig. 1E, F and G). Figure 1H shows results obtained using a combination of nifedipine, Gd 3+ and KB-R7943 but not 2-APB, which would leave only the 2-APB-sensitive capacitative response as a Ca 2+ entry mechanism to induce the artery ring contraction.

Vascular agonists and capacitative response
In human umbilical artery both 5-HT and histamine are potent contractile agonists acting through G-coupled 5-HT 2 receptors and H1 receptors, respectively (Bertrand and St-Louis, 1999;Lovren et al., 1999).It is known that they produce activation of inositol 1,4,5-trisphosphate sensitive Ca 2+ channels (InsP 3 receptors) which allow Ca 2+ release from sarcoplasmic reticulum (SR) and its consequent depletion (Tufan et al., 2003).Since this will cause a reduction in SR Ca 2+ content, we hypothesized that it could trigger a capacitative Ca 2+ entry.Therefore, we applied two consecutive stimuli with 1 μM 5-HT or 10 μM histamine separated by a 25 minutes period of agonist wash-out.The obtained results presented in Figure 2A and 2D show that for both vasoconstrictor agents, the second contraction had the same magnitude as the first one.However, when the same protocol was repeated in the presence of 100 μM 2-APB, the second contractions were significantly lower than the first ones (Fig. 2B and  2E).Also, the bars in Figure 2 are showing the mean force relationships between the force values of the first and second contraction for 5-HT (2C) and for (2F) in both conditions.
It is known that 2-APB can also inhibit InsP 3 receptors (Bootman et al., 2002;Maruyama et al., 1997), so the results presented in Figure 2B and 2E may be interpreted as a late effect of 2-APB on InsP 3 receptors.In order to test this possibility, we performed a control experiment where 2-APB was tested on a contraction evoked by 5-HT on an arterial ring exposed to a Ca 2+free extracellular solution (with 1 mM EGTA), so only the contraction due to SR Ca 2+ release would be ob-   served.In this setting, stimulation with 1 μM 5-HT produced a significant transient contraction whose magnitude was not modified by 2-APB.Moreover, when extracellular Ca 2+ was restored, a force development was observed which was now significantly diminished by the presence of 2-APB.Figure 3 shows typical recordings and mean values for these experiments.

Expression of mRNAs for structures implicated in capacitative response
STIM and Orai proteins and TRPC channels, are proposed in the literature to be involved in the capacitative response.Hence we explored the presence of the mRNA for these proteins in the human umbilical artery by the PCR technique.Although samples from 7 different arteries were processed in the same way (see Methods), the endothelial marker (von Willebrand factor) was not expressed by only two of them, so only the results from these samples were considered for conclusions about TRPC channels, STIM and Orai present in vascular smooth muscle (each of these two samples was analyzed twice).As shown in Figure 4, we found expression of mRNA for STIM 1 and 2, and for Orai 1, 2 and 3.The TRPC1, 3, 4 and 6 were also present in smooth muscle cells from the human umbilical artery, while we were not able to demonstrate the expression of TRPC5 and 7.The β-actin control was positive for all the PCRs performed.

Discussion
Our work presents results suggesting that the vasoconstrictors 5-HT and histamine produce an activation of the capacitative response in the human umbilical artery, which seems useful to allow a quick restoration of SR Ca 2+ content.This conclusion is based on the results presented in Figure 2, where it can be seen that the vessels stimulated twice with these agonists evoked two similar contractions in control conditions, while in the presence of 2-APB the second contractions were significantly lower than the first ones.Our interpretation of these data is that during the first stimulation with the vasoconstrictor, the SR depletion caused by the agonist activated a capacitative response which would contribute to the SR refilling.When SOCs were inhibited by 2-APB the Ca 2+ entry was diminished or inhibited and the SR did not completely refill after the first agonist addition, producing then a second contraction of smaller magnitude.
An alternative explanation for our results would be that the second 5-HT or histamine contraction was smaller than the first one because 2-APB had a late inhibitory effect on InsP 3 receptors.However, we can exclude this possibility because when the stimulus on the SR was performed without extracellular Ca 2+ , 2-APB had no effect on the 5-HT induced contraction, but instead significantly diminished the force produced by Ca 2+ re-addition.
Additionally, although we cannot completely exclude the possibility that the endothelium could have modified these responses, we have previously observed (unpublished data) that endothelial NO and protacyclins inhibition does not modify the contractile force induce by 5-HT.This suggests that the observed reduction of contractile force produced by 2-APB may be mostly due to inhibition of the smooth muscle capacitative response.
Our results are in accordance with the data presented by Gregory et al. (2001) in freshly isolated hepatocytes, where 2-APB (up to 100 μM) did not block InsP 3 receptors but was able to inhibit the Ca 2+ inflow when Ca 2+ was restituted after vasopressin-induced SR depletion in a free-Ca 2+ extracellular medium.Moreover, in cells incubated with normal extracellular Ca 2+ , they showed that 2-APB decreased Ca 2+ entry once the SOC response was activated with vasopressin.
The degree of involvement of capacitative Ca 2+ entry in the response to vasoconstrictors seems to be variable in different vessels and species.For instance, Moneer et al. (2005) propose that in A7r5 cells 5-HT produces extracellular Ca 2+ entry only through a capacitative response.On the other hand, Guibert et al. (2004) showed the presence of a capacitative Ca 2+ entry in small intrapulmonary arteries of the rat but provide evidence that it is independent of 5-HT-induced changes in cytosolic Ca 2+ concentration and vessel diameter.However, the same researchers, showed that in human pulmonary artery a part of 5-HT-induced contractions was due to voltage-independent Ca 2+ entry which could be mediated by SOCs (Rodat-Despoix et al., 2009).These results in human pulmonary artery are very similar to our findings in the umbilical artery, and it is interesting to note that both are human vessels in which 5-HT plays an important physiological role.
In order to further characterize the capacitative response in the human umbilical artery we also used a classical protocol of SR depletion induced by extracellular Ca 2+ deprivation, and subsequent development of a contraction by Ca 2+ readdition.This force development was partly inhibited by 2-APB, hence suggesting that part of this response was produced via SOCs due to the SR depletion.Similar results were shown by Potocnick et al. (2001) in skeletal muscle arterioles, where a capacitative Ca 2+ influx produced by re-addition of extracellular Ca 2+ following pre-treatment with an extracellular Ca 2+ -free solution (plus nifedipine) could be inhibited by 2-APB.They also demonstrated that 2-APB diminished the contractile response and (Ca 2+ ) i transient evoked by phenylephrine but not by KCl.In vascular smooth muscle, Ca 2+ -permeable nonselective cationic channels able to be activated by Ca 2+ store-depletion were also described in rabbit portal vein (Albert and Large, 2002), in human pulmonary artery (Golovina et al., 2001), in rabbit pial arterioles (Xu et al., 2006), in mouse and rabbit aorta (Trepakova et al., 2001) and in mouse pulmonary artery (Ng et al., 2009), among others.
It is interesting to note that in the human umbilical artery, 2-APB did not block all the force development induced by Ca 2+ readdition.Instead, this was only eliminated by a combination of 2-APB and other antagonists of Ca 2+ entry pathways (nifedipine, Gd 3+ and KB-R7943).All of these blockers partially inhibited force development when added independently, meaning that there are multiple pathways which act in a concerted way to let Ca 2+ into the cell after a period of extracellular Ca 2+ deprivation.Some of these routes are related to the capacitative response, as evidenced by 2-APB effects (assuming that 2-APB is more selective for SOCs).
Our results suggest that smooth muscle cells in this artery express all the structures which many bibliographical data point as necessary for the production of a capacitative response.Namely, we present evidences that smooth muscle cells of the human umbilical artery express the mRNAs for STIM 1 and 2, for Orai 1, 2 and 3 and also for some of the members of the TRPC channels family, namely the 1, 3, 4 and 6 subtypes.The mRNAs for TRPC5 and 7 were found not to be present in these cells.According to the literature, the TRPC channel most likely to be a SOC in cells presenting capacitative response is the TRPC1 subtype, and to a lesser extent TRPC3, TRPC4 and TRPC6 (Leung et al., 2008;Parekh and Putney, 2005;Dietrich et al., 2006;Ng et al., 2010).Meng et al. (2007) report expression of mRNAs for TRPC 1, 3, 4 and 6 in cultured smooth muscle cells derived from the human umbilical artery.Our results may be considered a step further of those presented by these authors, since we used native cells, while they worked with cultured cells, where the pressure induced by growth factors in the culture media may have altered channel expression (Sandow and Hilton Grayson, 2009).STIM 1 and 2, as well as Orai 1 and 3, seem to be broadly expressed in different tissues (Frischauf et al., 2008).However, Orai 2 is more expressed in the brain and less in other tissues (Frischauf et al., 2008).Both STIM 1 and 2, are able to activate the plasma membrane Orai proteins (Frischauf et al., 2008) and there are some recent reports that STIM1 could also interact with TRPC channels (Soboloff et al., 2006).Moreover, Liao et al. (2008) have reported that a STIM 1/Orai 1/ TRPC 1 interaction is necessary to produce a capacitative response.
All these structures are expressed in the human umbilical artery smooth muscle, but it is beyond the scope of this work to establish if in these cells TRPCs, Orai, or a combination of both, are the target of STIM proteins.However, this subject merits further research in order to clarify the physiological roles of these structures in the human umbilical artery and other human vessels.This may provide more data for the understanding of vascular pathological processes affecting fetoplacental blood flow.
In this work we have shown for the first time that smooth muscle of the human umbilical artery is able to evoke a capacitative contractile response inhibited by 2-APB, that stimulation with physiological vasoactive agonists which release Ca 2+ from the SR is enough to induce this response, and that this artery expresses mRNA for STIM 1-2, Orai 1-3 and TRPC 1, 3, 4 and 6 channels which in the literature have been demonstrated to be involved in this mechanism.

FIGURE 2 .
FIGURE 2. The capacitative contractile response participates in the mechanism of 5-HT and histamine force development in smooth muscle of the human umbilical artery.A: Typical recording of two 1 μM 5-HT-induced contractions separated by 25 min without 5-HT (gF/gW: grams of developed force / weight of wet tissue).B: Typical recording of an experiment similar to that shown in A but in the presence of 100 μM 2-APB.C: mean ± SEM values of experiments shown in A (n=10) and B (n=12).D: Typical recording of two 10 μM histamine-induced contractions separated by 25 min without histamine (gF/gW: grams of developed force / weight of wet tissue).E: Typical recording of an experiment similar to that shown in D but performed in the presence of 100 μM 2-APB.F: mean ± SEM values of experiments shown in D (n=10) and E (n=10).In all cases, * indicates statistically significant differences from controls (p<0.05).

FIGURE 3 .
FIGURE 3. 2-APB does not interfere with 5-HT-induced Ca 2+ release from the sarcoplasmic reticulum but it inhibits the capacitative contractile response in the human umbilical artery.A: Typical recording of 1 μM 5-HT contraction of artery rings in a Ca 2+ -free KRB followed by extracellular Ca 2+ readdition.The chamber had an appropriate amount of the vehicle of 2-APB (DMSO) (gF/gW: grams of developed force / weight of wet tissue).B: Typical recording of an experiment similar to that shown in A but performed in the presence of 100 μM 2-APB.C: mean ± SEM values of peak 5-HT force of experiments shown in A (n=9) and B (n=10).D: mean ± SEM values of stable force after Ca 2+ readdition as shown in A (n=9) and B (n=10).

FIGURE 4 .
FIGURE 4. The PCR technique shows that smooth muscle cells of the human umbilical artery express mRNA for several structures implicated in the capacitative response.A: photograph of a typical PCR experiment showing the presence of mRNAs for STIM 1 and 2, and Orai 1, 2 and 3. Also shown are positive (with primer) and negative (without primer) results for β-actin.B: photograph of a typical PCR experiment showing the presence of mRNAs for TRPC 1, 3, 4 and 6, and the absence of TRPC 5 and 7 (TRPC 2 was not tested, see text for details).A negative result for von Willebrand factor (vW) indicates that there is no contamination of the sample with endothelial cells.A positive controls for βactin was performed but is not shown.