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Am J Physiol Heart Circ Physiol 289: H960-H967, 2005; doi:10.1152/ajpheart.00059.2005
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Ca2+ signaling in human fetoplacental vasculature: effect of CGRP on umbilical vein smooth muscle cytosolic Ca2+ concentration

Yuan-Lin Dong, Sujatha Vegiraju, and Chandrasekhar Yallampalli

Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas

Submitted 21 January 2005 ; accepted in final form 23 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CGRP is a potent vasodilator with increased levels in fetoplacental circulation during late pregnancy. We have recently demonstrated that acute CGRP exposure to fetoplacental vessels in vitro induced vascular relaxation, but the signaling pathway of CGRP in fetoplacental vasculature remains unclear. We hypothesized that CGRP relaxes fetoplacental vasculature via regulating smooth muscle cytosolic Ca2+ concentrations. In the present study, by using human umbilical vein smooth muscle (HUVS) cells (HUVS-112D), we examined CGRP receptors, cAMP generation, and changes in cellular Ca2+ concentrations on CGRP treatment. These cells express mRNA for CGRP receptor components, calcitonin receptor-like receptor, and receptor activity-modifying protein-1. Direct saturation binding for 125I-labeled CGRP to HUVS cells and Scatchard analysis indicate specificity of the receptors for CGRP [dissociation constant (KD) = 67 nM, maximum binding capcity (Bmax) = 2.7 pmol/million cells]. Exposure of HUVS cells to CGRP leads to a dose-dependent increase in intracellular cAMP accumulation, and this increase is prevented by CGRP antagonist CGRP8–37. Using fura-2-loaded HUVS cells, we monitored the effects of CGRP on intracellular Ca2+ concentration ([Ca2+]i). In the presence of extracellular Ca2+, bradykinin (10–6 M), a fetoplacental vasoconstrictor, increases HUVS cells [Ca2+]i concentration. CGRP (10–8 M) abolishes bradykinin-induced [Ca2+]i elevation. When the cells were pretreated with glibenclamide, an ATP-sensitive potassium channel blocker, the CGRP actions on bradykinin-induced Ca2+ influx were profoundly inhibited. In the absence of extracellular Ca2+, CGRP (10–8 M) attenuated the increase of [Ca2+]i induced by a sarcoplasmic reticulum Ca2+ pump ATPase inhibitor thapsigargin (10–5 M). Furthermore, Rp-cAMPS, a cAMP-dependent protein kinase A inhibitor, blocks CGRP actions on thapsigargin-induced Ca2+ release from sarcoplasmic reticulum. Our results suggested that CGRP relaxes human fetoplacental vessels by not only inhibiting the influx of extracellular Ca2+ but also attenuating the release of intracellular Ca2+ from the sarcoplasmic reticulum, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.

vascular smooth muscle cells; fetoplacental circulation; calcitonin gene-related peptide; cytosolic calcium mobilization

CALCITONIN GENE-RELATED PEPTIDE (CGRP) is one of the most potent endogenous vasodilators known (33). CGRP is primarily synthesized in the sensory neurons of dorsal root ganglia, which extend axons centrally to the spinal cord and peripherally to various organs, including blood vessels (5), and is present in the bloodstream (2). In the pregnant woman, the serum levels of CGRP are significantly increased in both maternal and fetal circulation (31). The magnitude of increases in fetal serum CGRP is related to the fetal weight and gestational age (25), suggesting that CGRP may be involved in fetal growth and development. Recently, we have demonstrated that CGRP-receptor components calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein-1 (RAMP-1) are abundantly expressed by the vascular endothelium and underlying smooth muscle cells in the umbilical artery and vein, chorionic artery and vein, and stem villous vessels of the human placental villi (10). Furthermore, an in vitro isometric force measurement showed that CGRP dose dependently relaxes umbilical artery and vein, chorionic artery and vein, and stem villous vessels (10), implying that CGRP present in fetoplacental circulation may play a role in the control of local vascular tone. However, the mechanisms of CGRP actions on human fetoplacental vessels remain under investigation.

The increase in the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells plays an important role in tension development in smooth muscle (4). Vasocontractors mobilize Ca2+ at both the plasma membrane level and the intracellular level through different mechanisms and finally increase the [Ca2+]i and contract the muscle (7). Vasodilators could affect these mechanisms to decrease the [Ca2+]i and then relax the vessels (7). Recently, several mechanisms have been suggested for the muscle-relaxing effects of CGRP, including the stimulation of the adenylyl cyclase pathway in the human osteosarcoma cells (11), the activation of guanylate cyclase in the neonatal rat spinal cord (26), the modulation of phospholipase expression in skeletal muscle cells (20), and the regulation of the [Ca2+]i in osteoblastic UMR-106 cells (16). However, the effects of CGRP on [Ca2+]i in fetoplacental vasculature have not yet been clarified. On the other hand, CGRP causes hyperpolarization of the smooth muscle cells in mesenteric arteries by activating an outward potassium channel current (22). Membrane hyperpolarization in response to CGRP was inhibited by glibenclamide, a blocker of ATP-sensitive potassium channels, in mesenteric arteries of rats. However, whether the activation of ATP-sensitive potassium channels was involved in CGRP action on [Ca2+]i in human umbilical vein smooth muscle (HUVS) cells has not yet been established. We hypothesized that cytosolic Ca2+ mobilization is involved in CGRP-induced fetoplacental vascular relaxation. Thus the aims of the present study are to investigate the effect of CGRP on [Ca2+]i and the role of cAMP in [Ca2+]i regulation by using fura-2, a fluorescent Ca2+ indicator, in HUVS cell line, HUVS-112D.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. HUVS-112D cell line (American Type Culture Collection; Manassas, VA) was grown in Kaighn's F12K medium containing 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, and supplemented with 0.1 mg/ml heparin, 0.03 mg/ml endothelial cell growth supplement, and 10% fetal bovine serum, and maintained according to the manufacturer's instructions. The cells were serum starved for at least 12 h before stimulation.

Immunofluorescent staining. HUVS cells were cultured in Lab-Tek two-well chamber slides (Nunc; Naperville, IL) at 1 x 105 cells/ml for 48 h. The cells fixed by 70% ice-cold acetone on chamber slides were exposed to anti-{alpha}-smooth muscle actin-FITC (mouse monoclonal, 1:200, Santa Cruz Biotechnology; Santa Cruz, CA) and incubated at room temperature for 4 h. The slides were rinsed with PBS (0.1 M, pH 7.4) for 30 min and then mounted with coverslips using 4'6-diamidino-2-phenylindole (Vector Laboratories; Burlingame, CA). The slides were viewed with an Olympus microscope with Image-ProPlus Software (Olympus Optical; Tokyo, Japan).

Isolation of RNA and RT-PCR analysis. Total RNA was isolated from the cells using TRIzol reagent (GIBCO-BRL; Grand Island, NY), and first-strain cDNA was synthesized by RT as previously described (8). Briefly, 2 µg of RNA were added to the reaction mixture containing 3.5 mM MgCl2; 50 mM KCl; 10 mM Tris·HCl; 0.5 mM dGTP, dATP, dTTP, and dCTP; 5 µl RNAse inhibitor; 10 units murine leukemia virus RT; and 0.05 µg random primer. For RT, samples were placed into a Progene thermal cycler (Techne; Princeton, NJ) for one cycle at 25°C for 10 min, 42°C for 40 min, and 94°C for 2 min. PCR was initiated by the specific primer sets for CRLR and RAMP-1 (CRLR: 5'-CAGAATTGCTTGAACCTCTC-3' and 5'-CAGAATTGCTTGAACCTCTC-3'; RAMP-1: 5'-GAGACGCTGTGGTGTGACTG-3'27 and 5'-TCGGCTACTCTGGACTCCTG-3'). Primer sequences for CRLR and RAMP-1 were derived from published sequences (23). PCR was carried out according to the following conditions: an initial denaturation step at 95°C for 7 min, followed by 35 cycles of 30 s at 95°C, 1 min at 60°C, and 30 s at 72°C. Reactions were terminated by a 7-min elongation step at 72°C. PCR products were loaded onto a 1.8% agarose gel containing 0.5 µg/ml ethidium bromide and run in 0.5% Tris-borate-EDTA buffer at 100 V for 2 h. Gels were placed on a UV light box and imaged. The identity of the amplified sequences has been verified by sequencing the gel-extracted PCR product.

CGRP binding assay. Membranes were prepared from HUVS cells, and radiolabeled CGRP binding assay was performed as previously described (9). Cell membrane preparations were incubated with 1 x 10–11 M of 125I-labeled CGRP with or without varying concentrations of unlabeled CGRP (10–14 to 10–7 M) in a total volume of 300 µl assay buffer with 0.5% heat-inactivated BSA for 150 min at 4°C. After incubation, 600 µl of assay buffer were added to each tube and centrifuged at 12,000 g for 5 min at 4°C. The bound radioactivity remaining in the pellets was counted with a gamma counter. Specific binding was calculated by subtracting the labeled CGRP bound in the presence of 0.5 µM unlabeled CGRP from the total amount of labeled CGRP bound, and the receptor density in the cells was expressed as CGRP femtomole bound per milligram of protein. The data were analyzed with the Scatchard method.

RIA measurement of intracellular cAMP. Cells cultured in 35-mm well plates were initially treated with four doses of CGRP (10–10 to 10–7 M) in the presence of 100 µM phosphodiesterase inhibitor IBMX (Sigma; St. Louis, MO) for 5 min. Cells treated with IBMX alone served as the control. Reactions were terminated by replacing the medium with ice-cold ethanol and freezing the cells at –80°C. Supernatant obtained after brief sonication and centrifugation of cells was concentrated in a speed vacuum pump and reconstituted in a 500-µl assay buffer. cAMP was quantified using cAMP 125I assay kit (Amersham Biosciences UK) as described by the manufacturer. The cAMP standards (2–128 fmol/tube) and samples were acetylated by adding triethylamine/acetic anhydride (2:1 vol/vol, 5 µl/tube). Labeled cAMP bound to its antibodies was recovered by using magnetic beads coated with goat anti-rabbit IgG. The radioactivity was quantified with a gamma counter, and the results were expressed as femtomole per million cells.

Measurement of [Ca2+]i. Real-time recordings of [Ca2+]i were performed on single cells as previously described (15). Cells were plated onto glass coverslips at a density of 1 x 105/ml in Kaighn's F12K medium and kept in culture until the cells became confluent. Cells were washed with a physiological salt solution (PSS) containing (in mM) 125 NaCl, 5 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.25 CaCl2, 6 glucose, and 25 HEPES (pH 7.4). Cells were then incubated with 2 µM fura-2 AM (Molecular Probes; Eugene, OR) at 25°C for 60 min in a dark compartment. After being washed with the PSS buffer, the coverslip with attached cells was placed in a Sykes-Moore chamber of 1 ml volume on the stage of a EPI200-TE200-IUC Quantitative Fluorescence Live-Cell and Multidimensional Imaging System (Nikon). Fluorescence was excited at 340 nm (F340) and 380 nm (F380), and the emission was measured at 510 nm. The ratio of F340/F380 (R340/380) was considered to be an index of [Ca2+]i in the smooth muscle cells (24). Background fluorescence, obtained by quenching the fura-2 fluorescence with MnCl2 (1 mM), was subtracted. Cells showing a lack of basal responsiveness to KCl (30 mmol/l, defined as an increase of [Ca2+]i ≥ 50% of basal) were excluded from further study. No differences in KCl responsiveness were noted in cells used from passages 4 to 5 (the majority used) compared with those of later passages.

To investigate whether the effects of CGRP on [Ca2+]i were due to a block of an influx of Ca2+ from the extracellular milieu, we prepared the cells in the medium containing Ca2+ (1.25 mM) and stimulated the Ca2+ influx by bradykinin, a vasoconstrictor in human umbilical arteries and veins. To determine whether the effects of CGRP on [Ca2+]i were due to an inhibition of Ca2+ mobilization from intracellular stores, we prepared the cells in a Ca2+-free medium and induced the Ca2+ release from intracellular stores by thapsigargin, a selective antagonist of the sarcoplasmic reticulum Ca2+ pump. The changes in R340/380 induced by bradykinin (10–6 M) and thapsigargin (10–5 M) in the cell preparations served as the control.

Statistics. Results are expressed as means ± SE. Data were analyzed for statistical differences with one-way ANOVA, followed by post hoc corrections to verify differences between individual groups. A P value of <0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Demonstration of vascular smooth muscle cell property by HUVS cells. By using monoclonal anti-{alpha}-smooth muscle actin-FITC, we demonstrated the abundant expression of the smooth muscle cell marker {alpha}-actin in the HUVS cells, confirming the smooth muscle cell nature of this cell line (Fig. 1).



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  		Fig. 1. Demonstration of {alpha}-actin in human umbilical vein smooth muscle (HUVS) cells in culture by immunofluorescent staining using monoclonal actin-{alpha}-smooth muscle actin-FITC is shown. {alpha}-Actin (green) was identified around the nuclei (orange) in the HUVS cells. Original magnification x200.

 
Existence of CGRP receptors in HUVS cells. RT-PCR analysis with specific primers showed that both CRLR and RAMP-1 mRNA are abundantly expressed by the HUVS cells, implying the existence of CGRP receptors in these cells (passages 4 and 5) (Fig. 2). Specific binding of CGRP on HUVS cells was examined to determine the binding function of CGRP receptors on these cells and their specificity. The direct saturation-binding curve for 125I-labeled CGRP to HUVS cells was obtained. As shown in Fig. 3, the Scatchard analysis for this binding indicated specificity of the receptors for CGRP in this cell line. CGRP binding in these cells revealed a dissociation constant (KD) of 67 nM, with a maximum binding capacity (Bmax) value of 2.7 pmol/million cells.



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  		Fig. 2. RT-PCR analysis of mRNA expression for the CGRP receptor components calcitonin receptor-like receptor (CRLR, 497 bp), receptor activity modifying protein-1 (RAMP-1, 220 bp), and 18S (320 bp) mRNA in HUVS cells (passages 4 and 5) is shown.

 


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  		Fig. 3. Scatchard analysis of 125I-labeled CGRP binding to HUVS cells. Cell membranes (1 mg/ml membrane protein) prepared from cells were incubated with 2.5 fmol of 125I-labeled human CGRP per tube as described in MATERIALS AND METHODS. Each point represents mean of triplicate incubations of the cells; dissociation constant (KD) = 67nM, maximum binding capacity (Bmax) = 2.7 pmol/million cells.

 
Effects of CGRP on cAMP production. To determine whether CGRP is coupled to adenylate cyclase in these muscle cells, we treated the cells with increasing concentrations of CGRP. As shown in Fig. 4, in the presence of IBMX, a cAMP phosphodiesterase inhibitor, CGRP (1 x 10–10 to 10–7 M) caused a dose-dependent increase in cAMP production in the HUVS cells, suggesting that adenylate cyclase activation mediated CGRP actions in HUVS cells. Furthermore, CGRP8–37, a CGRP receptor antagonist, completely blocks the elevation in cAMP in cultured cells (Fig. 5), implying that the actions of CGRP on HUVS cell cAMP production were mediated through CGRP receptors.



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  		Fig. 4. Effect of CGRP on the cAMP production in the HUVS cells. cAMP content was measured in the cells incubated with CGRP at 1 x 10–10 M, 1 x 10–9 M, 1 x 10–8 M, and 1 x 10–7 M in the presence of IBMX (1 x 10–4 M). Values are means ± SE (n = 6 experiments). Bars with different letters (a, b, c, d, e) at the top vary significantly (P < 0.05).

 


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  		Fig. 5. Effect of CGRP8–37 on CGRP-induced cAMP production in the HUVS cells. cAMP content was measured in the cells incubated with CGRP at 1 x 10–8 M and CGRP8–37 (1 x 10–7 M) plus CGRP (1 x 10–8 M) in the presence of IBMX (1 x 10–4 M). CTL, control. Values are means ± SE (n = 6 experiments). Bars with different letters (a, b) at the top vary significantly (P ≤ 0.01).

 
Effects of CGRP on [Ca2+]i induced by bradykinin in the presence of extracellular Ca2+. In fura-2-loaded HUVS cell monolayers, the application of bradykinin, a vasocontractor in the fetoplacental vasculature, induced a transient increase in [Ca2+]i in normal PSS containing 1.25 mM Ca2+. This increase in [Ca2+]i formed a sharp peak, which fell rapidly after ~10 s but remained above the basal levels (Fig. 6A). However, when bradykinin was added at 200 s after incubation with CGRP, the [Ca2+]i increase was completely abolished (Fig. 6B). When the cells were pretreated with glibenclamide, an ATP-sensitive potassium channel blocker, the CGRP actions on bradykinin-induced [Ca2+]i influx were profoundly inhibited (Fig. 6C). Summarized data in Fig. 7 demonstrated that bradykinin stimulates Ca2+ influx, which is blocked by CGRP, suggesting that CGRP relaxes fetoplacental vasculature via, at least in part, inhibiting the influx of extracellular Ca2+. Furthermore, glibenclamide abolished CGRP action on bradykinin-induced Ca2+ influx, indicating that CGRP inhibits extracellular Ca2+ influx via activation of ATP-sensitive potassium channels.



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  		Fig. 6. Representative recordings showing the effects of CGRP on the extracellular Ca2+ influx induced by bradykinin (BK) in HUVS cells. A: recordings of intracellular calcium concentration ([Ca2+]i) in a monolayer of HUVS cells after application of BK (10–6 M); B: effects of CGRP (10–8 M) on [Ca2+]i induced by BK (10–6 M) in HUVS cells; C: effects of glibenclamide (Gli, 10–6 M) on [Ca2+]i inhibited by CGRP (10–8 M). Measurements were made in the presence of extracellular Ca2+ (1.25 mM). Each tracing is made in 7–10 individual cells. R340/380, ratio of fluorescence at 340 nm and 380 nm.

 


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  		Fig. 7. Summarized data showing percentage changes in [Ca2+]i after treatment by BK, CGRP, and Gli. Measurements were made in the presence of extracellular Ca2+ (1.25 mM). Each group consisted of 6 experiments. Each experiment was done in 7–10 individual cells. Values are means ± SE. Bars with different letters (a, b, c) at the top vary significantly (P < 0.05).

 
To determine whether bradykinin-induced elevation of [Ca2+]i is an influx of extracellular Ca2+ or a release from its intracellular stores, we examined the effect of CGRP on [Ca2+]i in the HUVS cells in the Ca2+-free medium containing 2 mM EGTA. As shown in Fig. 8A, bradykinin (10–6 M) failed to change [Ca2+]i in the HUVS cells. Taken together with the findings in Fig. 6A, in which bradykinin increases [Ca2+]i in the presence of extracellular Ca2+, these results suggested that in the HUVS cells, bradykinin increases [Ca2+]i primarily through an influx of extracellular Ca2+ rather than the release of Ca2+ from intracellular stores.



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  		Fig. 8. Representative recordings showing the effects of CGRP on the intracellular Ca2+ release induced by thapsigargin (Thap) in HUVS cells. A: recordings of [Ca2+]i in a monolayer of HUVS cells after application of BK (1 x 10–6 M) or Thap (1 x 10–5 M); B: effects of CGRP (1 x 10–8 M) on intracellular Ca2+ release-induced by Thap (1 x 10–5 M); C: effects of Rp-cAMPS (1 x 10–7 M) on CGRP actions in [Ca2+]i release. Measurements were made in the Ca2+-free medium supplemented with 2 mM EGTA. Each tracing is made in 7–10 individual cells.

 
Effects of CGRP on the [Ca2+]i induced by thapsigargin in the absence of extracellular Ca2+. To observe the effect of CGRP on [Ca2+]i release from intracellular stores, we cultured the cells in Ca2+-free medium. Figure 8A shows the representative recordings of changes in [Ca2+]i induced by thapsigargin in Ca2+-free medium containing 2 mM EGTA. Thapsigargin, a sarcoplasmic reticulum Ca2+ pump ATPase inhibitor, caused transient elevation of [Ca2+]i in the HUVS cells. This increase in [Ca2+]i formed a sharp peak, which fell rapidly after ~10 s but remained above the basal levels. However, the application of CGRP in Ca2+-free medium substantially attenuated [Ca2+]i release from sarcoplasmic reticulum (Fig. 8B). Furthermore, as shown in Fig. 8C, Rp-cAMPS, a cAMP-dependent protein kinase A inhibitor, blocks CGRP actions, suggesting that CGRP attenuates sarcoplasmic reticulum Ca2+ release via activation of adenylate cyclase. Summarized data shown in Fig. 9 confirmed the findings that CGRP relaxes fetoplacental vascular smooth muscle cells by not only inhibiting the influx of extracellular Ca2+ but also attenuating the release of intracellular Ca2+, and these actions might be attributed to cAMP accumulation induced by CGRP.



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  		Fig. 9. Summarized data showing percentage changes in [Ca2+]i after treatments by Thap, CGRP, and Rp-cAMPS. Measurements were made in the Ca2+-free medium supplemented with 2 mM EGTA. Each group consisted of 6 experiments. Each experiment was done in 7–10 individual cells. Values are means ± SE. Bars with different letters (a, b) at the top vary significantly (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Regulation of placental blood flow in both maternal and fetal compartments affects placental transport of oxygen and nutrients, which ultimately determines fetal growth and well being. Unlike other vascular systems, the fetoplacental unit lacks innervation and therefore depends on humoral substances for the control of vascular tone (6). CGRP is a powerful vasodilator circulating in fetal placental units (25). We have recently reported that CGRP dose dependently relaxes the umbilical and chorionic artery and vein in the human placenta, suggesting that CGRP may play a role in human fetoplacental vascular relaxation. However, the mechanisms of CGRP actions on the fetoplacental vasculature have not yet been fully documented. In the present study, we demonstrated the existence of CGRP receptors on HUVS cells by RT-PCR analysis and competitive binding assay. We also found that CGRP acts on HUVS cells to cause intracellular cAMP accumulation. Furthermore, CGRP was found to decrease [Ca2+]i via two mechanisms. First, it blocked the influx of Ca2+ from the extracellular milieu via activation of ATP-sensitive potassium channels as indicated by the experiments with glibenclamide. Second, CGRP inhibits the release of Ca2+ from the sarcoplasmic reticulum in HUVS cells as indicated by the experiments with thapsigargin. Therefore, CGRP may act as a circulating hormone in the fetoplacental circulation and may play an important role in the control of fetoplacental vascular tone.

Involvement of ATP-sensitive potassium channels in CGRP actions. Intracellular Ca2+ homeostasis has been thought to be a primary factor in regulating contractility of all kinds of muscles, including vascular smooth muscles. Previous studies (22, 30) have suggested that CGRP activates, in addition to adenylate cyclase, ATP-sensitive potassium channels to produce membrane hyperpolarization. The resultant inhibition of Ca2+ uptake through voltage-dependent Ca2+ channels is thought to be central to the muscle relaxation (22, 30). It has been reported that CGRP decreases [Ca2+]i and Ca2+ sensitivity of contractile elements in intact and {alpha}-toxin-permeabilized smooth muscle strips in the porcine coronary artery (12), but the potential disadvantage in using a muscle strip for the measurement of [Ca2+]i could be the heterogeneity of cell types. In that case, the fluorescence signal from cell types other than smooth muscle cells could interfere with the measurement and the interpretation of the results. The present study, by using fura-2-loaded HUVS cells, demonstrates that bradykinin, a powerful vasoconstrictor in the human fetoplacental circulation (1, 13), induces a substantial increase in [Ca2+]i in normal PSS containing 1.25 mM Ca2+. Application of CGRP on the cells completely abolishes [Ca2+]i increase induced by bradykinin. However, when the cells were pretreated with glibenclamide, an ATP-sensitive potassium channel blocker, the CGRP action on [Ca2+]i influx was significantly attenuated. Because the ability of CGRP to decrease bradykinin-induced [Ca2+]i could be blocked by glibenclamide, we suggested that opening of K+ channels and resultant plasma membrane hyperpolarization might mediate such an action. Our present results support the hypothesis that CGRP relaxes fetoplacental vasculature via, at least in part, inhibiting the net uptake of extracellular Ca2+ into smooth muscle cells.

Regulation of intracellular Ca2+ by CGRP. Alternatively, the release of Ca2+ from the intracellular stores is another key step in excitation-contraction coupling events in vascular smooth muscle. Thapsigargin, a selective antagonist of the sarcoplasmic reticulum Ca2+ pump, increases cytosolic Ca2+ by emptying intracellular Ca2+ stores (14). The present study showed that CGRP attenuated thapsigargin-induced [Ca2+]i increase in Ca2+-free PSS. These results strongly indicate that CGRP functions as a vasodilator in fetoplacental circulation by inhibiting both the influx of extracellular Ca2+ and the release of intracellular Ca2+ from sarcoplasmic reticulum of the smooth muscle cells. To the best of our knowledge, this is the first report demonstrating the regulation of CGRP on intracellular Ca2+ mobilization in HUVS cells.

It has been reported that the release of Ca2+ from sarcoplasmic reticulum is mediated by activation of the phospholipase C-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] cascade, which couples to receptors by G proteins (18). Inhibitors of phospholipase C (neomycin) and Ins(1,4,5)P3 (heparin) block the release of intracellular Ca2+ (34). Further studies (17, 28) have shown that cAMP stimulates Ca2+ uptake into the sarcoplasmic reticulum through activation of Ca2+ pump ATPase. Our study showed that Rp-cAMPS, a cAMP-dependent protein kinase A inhibitor, attenuated the action of CGRP. It is likely that CGRP-induced cAMP accumulation in the HUVS cells activates Ca2+ pump ATPase on the sarcoplasmic reticulum membrane, and the activation of Ca2+ pump stimulates Ca2+ uptake into the sarcoplasmic reticulum and results in decreased cytosolic free Ca2+ concentration.

Role of cAMP in CGRP signaling pathway. In many vascular preparations, there is a strong correlation between CGRP-induced relaxation and the elevation in intracellular cAMP. CGRP stimulates the accumulation of cAMP in cultured vascular smooth muscle cells from rat thoracic aortas (19). CGRP-induced dilation of the rat middle cerebral artery was accompanied by an increase in adenylate cyclase activity (32). We (10) have previously demonstrated that CGRP-induced relaxation of human chorionic arteries was inhibited by cAMP-dependent protein kinase A inhibitor Rp-cAMPS, implying that cAMP accumulation is involved in the smooth muscle relaxation in fetoplacental circulation. It has been proposed that cAMP induces vasodilation through three distinct ways: 1) an activation of Ca2+-activated potassium channels, 2) an inhibition of myosin phosphorylation through phosphorylation of myosin light chain kinase, and 3) a stimulation of Ca2+-ATPase (Ca2+ pump) in the sarcoplasmic reticulum. The present study showed that CGRP dose dependently increases HUVS cells cAMP production, and inhibition of cAMP-dependent protein kinase A by Rp-cAMPS blocks CGRP actions, suggesting that CGRP may attenuate sarcoplasmic reticulum Ca2+ release via activation of Ca2+-ATPase (Ca2+ pump) on the sarcoplasmic reticulum.

CRLR and RAMP-1 mediated CGRP action. CGRP exerts its biological effect through a 7TM G protein-coupled receptor CRLR in the presence of accessory proteins. It has been reported that CRLR functions as a receptor for three ligands: CGRP, adrenomedullin, and intermedin in the presence of its RAMPs. Coexpression of CRLR with RAMP-1 forms a CGRP receptor, whereas RAMP2 or RAMP3 produces an adrenomedullin receptor, and coexpression of CRLR with any of the three RAMPs mediates intermedin signaling (29). The present study demonstrated that both CRLR and RAMP-1 were expressed by HUVS cells, and CGRP8–37, a CGRP receptor antagonist, completely blocks the elevation in cAMP in HUVS cells, implying that the action of CGRP on HUVS cell cAMP production was mediated through CGRP receptor component CRLR and RAMP-1.

Bradykinin in fetoplacental circulation. Bradykinin is a systemic vasodilator but has been shown to cause vasoconstriction in isolated human umbilical arteries and veins (1, 13). The mechanism by which bradykinin induces directionally opposite effects on different vascular beds is unknown. Two mammalian bradykinin receptor subtypes B1 and B2 receptors have been pharmacologically defined. Both bradykinin receptor genes have been cloned from human tissues. It has been demonstrated that the pharmacological action of bradykinin in the human umbilical vein is mediated by the bradykinin B2 receptor (21). Bradykinin-induced vasoactive response is associated with changes in intracellular Ca2+ levels. In human and rabbit aortic vascular smooth muscle cells (3), bradykinin activates R-, T-, and L-type Ca2+ channels and induces a sustained increase of nuclear Ca2+. In the rat afferent and efferent glomerular arterials microdissected from the juxtamedullary renal cortex, bradykinin increases [Ca2+]i through activation of B2 receptors located on the endothelium, opens voltage-independent channels, and mobilizes intracellular Ca2+ (27). The present study demonstrated that bradykinin increases [Ca2+]i in HUVS cells in normal PSS containing 1.25 mM Ca2+ but failed to change the [Ca2+]i levels in the cells in the Ca2+-free medium. These results suggested that in the HUVS cells, bradykinin increases [Ca2+]i mainly through an influx of extracellular Ca2+ rather than the mobilization of Ca2+ in intracellular stores. Further study is apparently warranted to determine the type of bradykinin receptor on the HUVS cells and the Ca2+ channels on the cell membrane that was involved in the extracellular Ca2+ mobilization.

In summary, the present study supports the notion that the direct Ca2+-dependent, vascular-relaxant effects of CGRP play a central role in the control of the fetoplacental vascular tone. These direct vascular actions of CGRP may explain the extremely low vascular resistance in fetoplacental circulation in normal pregnancy. Further studies are needed to examine the direct effects of CGRP on the L-type Ca2+ channel and on potassium chloride-induced increases in Ca2+ and to explore the possible link between CGRP inhibited [Ca2+]i mobilization in HUVS cells and increased vascular resistance in complicated pregnancies, such as intrauterine fetal growth restriction and preeclampsia.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The National Heart, Lung, and Blood Institute Grants HL-70883 (to Y. L. Dong) and HL-58144 (to C. Yallampalli) supported this study.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Leoncio A. Vergara for technical assistance in the digital imaging processing and analysis of [Ca2+]i measurements and Cheryl R. Welch for administrative support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. L. Dong, Dept. of Obstetrics and Gynecology, Univ. of Texas Medical Branch, 301 Univ. Blvd., MRB 11.138, Galveston, TX 77555-1062 (e-mail: ydong{at}utmb.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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