©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Adrenomedullin Stimulates Two Signal Transduction Pathways, cAMP Accumulation and Ca Mobilization, in Bovine Aortic Endothelial Cells (*)

(Received for publication, September 6, 1994; and in revised form, November 14, 1994)

Yoshiyuki Shimekake Kiyoshi Nagata (§) Shigeki Ohta Yoshikazu Kambayashi Hiroshi Teraoka Kazuo Kitamura (1) Tanenao Eto (1) Kenji Kangawa (2) Hisayuki Matsuo (2)

From the  (1)Shionogi Research Laboratories, Shionogi & Company, Ltd., 5-12-4 Sagisu, Fukushima-ku, Osaka, Osaka 553, Japan, the First Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki, Miyazaki 889-16, Japan, and the (2)National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 564, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The biological action of adrenomedullin, a novel hypotensive peptide, on bovine aortic endothelial cells, was examined. The specific binding of adrenomedullin to these cells was observed, and adrenomedullin was found to induce intracellular cAMP accumulation in a dose-dependent manner. EC for the cAMP accumulation was about 100 times lower than the apparent IC for the binding assay. Adrenomedullin also induced increase of intracellular free Ca in endothelial cells in a dose-dependent manner. The Ca response to adrenomedullin was biphasic with an initial transient increase due to the release from thapsigargin-sensitive intracellular Ca storage and a prolonged increase by influx through the ion channel on the plasma membrane. This intracellular free Ca increase resulted from phospholipase C activation and inositol 1,4,5-trisphosphate formation, and seemed to cause nitric oxide synthase activation by monitoring intracellular cGMP accumulation. Both cAMP accumulation and Ca increased responses to adrenomedullin were mediated by cholera toxin-sensitive G protein, but the two signal transduction pathways were independent. Thus, the results suggest that adrenomedullin elicits the hypotensive effect through at least two mechanisms, a direct action on vascular smooth muscle cells to increase intracellular cAMP and an action on endothelial cells to stimulate nitric oxide release, with both leading to vascular relaxation.


INTRODUCTION

Adrenomedullin is a novel hypotensive peptide isolated from human pheochromocytoma(1) . It consists of 52 amino acid residues with one intracellular disulfide bond and is classified into the calcitonin gene-related peptide (CGRP) (^1)family. It has a highly conserved structure among mammals such as porcine (2) and rat (3) . Only a few pharmacological activities of adrenomedullin have been revealed, such as a potent hypotensive effect in anesthetized rat and cAMP accumulation in rat platelets and aortic smooth muscle cells (RASMC)(1, 4) . The mechanism of the strong hypotensive effect and other physiological roles are still unclear. Adrenomedullin may act primarily on vascular endothelial cells because it exists in plasma at comparable levels (5) with atrial natriuretic peptide, another circulating hypotensive hormone secreted by the heart. To investigate the physiological role(s) and mechanism of pharmacological effects, we examined the response of bovine aortic endothelial cells (BAEC) to adrenomedullin. Here we report the action of adrenomedullin on BAEC through two independent signal transduction pathways, cAMP accumulation and Ca mobilization.


EXPERIMENTAL PROCEDURES

Materials

Human adrenomedullin, bradykinin, and CGRP were obtained from Peptide Institute Inc. (Osaka, Japan). Bovine serum albumin, EGTA, 3-isobutyl-1-methylxanthine, HEPES, and N^G-nitro-L-arginine methyl ester were from Sigma. N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) was from Seikagaku Kogyo Co. (Tokyo, Japan). U-73122, U-73343, pertussis toxin (PTX) and cholera toxin (CTX) were from Funakoshi (Tokyo, Japan). Fura-2/AM was from Nacalai Tesque (Kyoto, Japan). I-Adrenomedullin was synthesized by the lactoperoxidase method and purified by high performance liquid chromatography (specific activity: 80 TBq/mmol) as in (6) .

Cell Culture

Primary culture of BAEC was prepared as reported previously (7) and maintained in 199 medium (Flow Laboratories, Irvine, Scotland) supplemented with fetal calf serum (20%), HEPES (20 mM, pH 7.3), heparin (12,000 units/liter, NOVO, Denmark), and bovine endothelial mitogen (100 mg/liter, Biomedical Technologies Inc., Stoughton, MA). The cells at the third or fourth passage were used for all experiments. RASMC were prepared as in (8) and cultured in Dulbecco's modified Eagle's medium plus 10% fetal calf serum. The cells at the seventh passage were used. All cultures were kept at 37 °C under 5% CO(2) atmosphere.

Competitive Binding Assay

According to the procedure in the previous report(9) , confluent cells were incubated in Hanks' solution, pH 7.4, containing protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml soybean trypsin inhibitor, 250 µg/ml bacitracin, and 0.1 mM amastatin) and 20 pMI-adrenomedullin in the presence or absence of cold peptides for competition for 2 h at 20 °C. After washing twice with ice-cold Hanks' solution containing inhibitors, cells were lysed in 10% SDS, 1 N NaOH and counted for radioactivity in a counter.

Measurements of cAMP and cGMP

Cellular cAMP and cGMP were measured as reported previously(10) . After washing twice with Hanks', 20 mM HEPES, 0.1% bovine serum albumin, 0.5 mM 3-isobutyl-1-methylxanthine, pH 7.4, cells were incubated for 15 min in the same solution in the presence or absence of adrenomedullin. Cells were lysed in 6% trichloroacetic acid, and the acid was removed by water-saturated diethyl ether extraction. After lyophilization of the aqueous phase, cyclic nucleotides were determined by radioimmunoassay (Yamasa, Tokyo, Japan).

Determination of Cytoplasmic Free Ca Concentration ([Ca](i))

Cells were harvested with 0.1% trypsin and 0.01% EDTA and then washed once with growth medium. Next, the cells were counted, resuspended in Hanks', 20 mM HEPES, pH 7.4, and incubated with 2 µM Fura-2/AM for 30 min at 37 °C. The Fura-2-loaded cells were washed with HEPES-buffered Hanks' and resuspended in the same solution at 1 times 10^5 cells/ml in a cuvette with continuous stirring. Fluorescence was measured with a CAF-100 spectrofluorometer (Japan Spectroscopy Inc., Tokyo, Japan). The level of Ca mobilization was determined from the excitation ratio of 340 nm and 380 nm (F/F). The ratio of 0.1 corresponded to about 34 nM in Delta[Ca](i).

Measurement of Inositol 1,4,5-Trisphosphate

Cells were washed twice with Hanks', 20 mM HEPES, 10 mM LiCl, pH 7.3, and incubated in the same solution for 10 min. Next, the cells were exposed to 0.1 µM adrenomedullin in the same solution at 37 °C for an appropriate time. The cells were then lysed in 15% trichloroacetic acid and processed as described above for the cAMP assay up to lyophilization. Inositol 1,4,5-trisphosphate (Ins-1,4,5-P(3)) was determined by the protein binding assay (Amersham Corp.) as described in (11) .


RESULTS

Binding Assay

Specific binding of adrenomedullin to BAEC was observed as shown in Fig. 1. Because cold adrenomedullin at 5 µM did not show complete displacement of binding [I]-adrenomedullin, the exact IC value could not be calculated, but it seemed to be around 10 nM. Some reported that CGRP and CGRP (8-37), a CGRP antagonist, inhibit adrenomedullin-induced actions in certain systems (4, 12) . In BAEC, CGRP at 1 µM did not compete with I-adrenomedullin binding at all, suggesting that adrenomedullin does not share the same binding site (or receptor) with CGRP in BAEC.


Figure 1: Competitive binding assay of I-adrenomedullin. Procedures are described in detail under ``Experimental Procedures.'' Briefly, BAEC were incubated with 20 pMI-adrenomedullin in the presence of 0-5 µM cold adrenomedullin (opencircles) or CGRP (closedcircles) for 2 h at 20 °C. After washing twice with ice-cold Hanks' solution, cells were lysed and counted for radioactivity of cell lysates. The values are expressed as means ± S.E. (n = 4).



Effect of Adrenomedullin on Intracellular cAMP Accumulation

A recent report showed that adrenomedullin causes accumulation of intracellular cAMP in smooth muscle cells(4) . Next, we examined alteration of the intracellular cAMP level in BAEC responding to adrenomedullin. Adrenomedullin increased the cAMP level in a dose-dependent manner as shown in Fig. 2A. An effective response was observed at concentrations higher than 10 pM, and EC was 0.175 ± 0.017 nM (n = 4). This EC value was about 100 times lower than the apparent IC in the binding assay, suggesting that the high affinity binding sites for cAMP response were hidden among the large number of binding sites with lower affinity. The maximal effect was observed at 1 nM and higher. Similar increase of cellular cAMP level was observed in RASMC, as shown in the previous report(4) , but EC (1.41 ± 0.49 nM, n = 4) was about 10 times higher than that obtained in BAEC. The increase of cAMP is usually mediated by CTX-sensitive G protein. The cAMP increase by adrenomedullin was also diminished when cells were pretreated with CTX (100 ng/ml, 20 h) as observed in dopamine-D1 receptor (13) and epidermal growth factor receptor(14) , while pretreatment with PTX (100 ng/ml, 20 h) had no effect on cAMP response to adrenomedullin (Fig. 2B). The addition of H-89 (10 µM), a selective cAMP-dependent protein kinase inhibitor(15) , at 30 min before CTX treatment (2 µg/ml, 4 h) did not alter the deterioration of cAMP response to adrenomedullin by CTX under the condition that blocked the cAMP-dependent hormone release from rat GH3 cells (16) (Fig. 2C). Therefore, it is unlikely that CTX desensitizes the adrenomedullin receptor through the constitutive cAMP-dependent protein kinase activation.


Figure 2: Response of intracellular cAMP level to adrenomedullin. A, dose response of intracellular cAMP accumulation to adrenomedullin. BAEC (opencircles) or RASMC (closedcircles) were incubated with 0-100 nM adrenomedullin for 15 min and then intracellular cAMP levels were measured by radioimmunoassay.**, significantly different (p < 0.01) from basal level by Dunnett multiple comparison. B, effect of toxins on cAMP response of BAEC to adrenomedullin. After treatment with each toxin (100 ng/ml) for 20 h, BAEC were incubated with (hatchedbars) or without (openbars) 0.1 µM adrenomedullin for 15 min, and then cAMP levels were measured. ##, significantly different (p < 0.01) from each control by Student's multiple range test. C, effect of cAMP-dependent protein kinase inhibition on the cAMP response deteriorated by CTX. After treatment with 10 µM H-89 for 30 min, BAEC were further treated with CTX (2 µg/ml) for 4 h. Then, cells were incubated with (hatchedbars) or without (openbars) 0.1 µM adrenomedullin for 15 min, and cAMP levels were measured. ##, significantly different (p < 0.01) from each control by Student's multiple range test.



Effect of Adrenomedullin on [Ca](i)

Adrenomedullin induced a [Ca](i) increase in BAEC, which consisted of an initial peak within 30 s and a following prolonged plateau phase that was parallel with the basal level (Fig. 3A). Adrenomedullin increased [Ca](i) in a dose-dependent manner (Fig. 3B). The maximal response was observed at 0.1 µM and higher. The EC was 3.07 ± 0.21 nM (n = 7), which was comparable with that of bradykinin (6.7 nM(17) ), and the maximal [Ca](i) level was one-third to one-half of that induced by bradykinin at 1 µM. This [Ca](i) increase was receptor-mediated because the successive addition of adrenomedullin failed to elicit a significant [Ca](i) increase, probably due to receptor desensitization (data not shown). The [Ca](i) increase profile by adrenomedullin was similar to that of bradykinin, which was thought to be mediated by both Ins-1,4,5-P(3) receptor (release from intracellular Ca storage) and Ca channel in the plasma membrane (influx of extracellular Ca).


Figure 3: Dose response of [Ca] increase to adrenomedullin. A, a representative series of traces is shown. AM, adrenomedullin; BK, bradykinin. B, dose-response curve is expressed as means ± S.E. (n = 7) with values relative to the response to 1 µM adrenomedullin in each experiment as 100%. [Ca] increase was measured with Fura-2-loaded cells suspended in Hanks' solution by monitoring fluorescence ratio of 340-380 nm (F/F).



For further investigation of the Ca mobilization mechanism of adrenomedullin in BAEC, we examined the effects of channel blockers and other inhibitory reagents. First, EGTA, SK& (a receptor operating ion channel blocker), and nifedipine (an L-type Ca channel blocker) did not affect the initial peak for [Ca](i) increase by adrenomedullin, but EGTA and SK& caused loss of the prolonged plateau phase (Fig. 4A), suggesting that this prolonged plateau phase was due to Ca influx through the plasma membrane ion channel (with the exception of the L-type channel). Next, thapsigargin (3 µM), which is a microsomal Ca-ATPase inhibitor and known to inhibit the bradykinin-induced Ca mobilization(18) , significantly diminished the initial peak of [Ca](i) increase by adrenomedullin, while the second prolonged phase was retained (Fig. 4B). This retained prolonged phase was lost when cells were successively treated with SK&, suggesting again that this prolonged response was mediated by an ion channel. Ziegelstein et al.(19) have recently reported the existence of a ryanodine-sensitive intracellular Ca storage site in vascular endothelial cells. Ryanodine at 5 µM did not affect the [Ca](i) increase response to adrenomedullin as shown in Fig. 4B. When the cells were pretreated with 10 µM U-73122, an inhibitor of agonist-induced phospholipase C activation(20) , adrenomedullin did not induce either the initial or the prolonged [Ca](i) increase, while pretreatment with 10 µM U-73343, an inactive analog to U-73122(20) , did not affect the [Ca](i) increase response at all (Fig. 4C). Pretreatment of cells with 10 µM H-89 did not affect the [Ca](i) increase response. These results suggest that activation of phospholipase C is required for an adrenomedullin-induced biphasic [Ca](i) increase and that the activation of cAMP-dependent protein kinase is not involved in the [Ca](i) increase as is often the case in hormone-secreting cells(21, 22) . In addition, CGRP at 1 µM did not affect the successive [Ca](i) response to adrenomedullin (Fig. 4C). This also suggests that the receptor responsible for the [Ca](i) increase response to adrenomedullin is not the CGRP receptor, as the adrenomedullin response would have been desensitized by CGRP pretreatment. For further characterization of the G protein involved in the [Ca](i) increase response to adrenomedullin, the effects of PTX and CTX were again examined (Fig. 4D). The adrenomedullin-induced [Ca](i) increase was not affected at all by the PTX treatment (100 ng/ml, 20 h) but was greatly diminished by the CTX treatment (2 µg/ml, 4 h). The addition of H-89 (10 µM) at 30 min before CTX treatment did not alter the deterioration of [Ca](i) response, suggesting again that the desensitization of the adrenomedullin receptor through cAMP-dependent protein kinase constitutively activated by CTX is unlikely. Thus, the G protein in BAEC responsible for the [Ca](i) response to adrenomedullin showed similar toxin sensitivity to that observed in the cAMP response (Fig. 2B).


Figure 4: Effects of reagents possibly influencing on [Ca] increase response to adrenomedullin. A, effects of EGTA and ion channel blockers. AM, adrenomedullin; Ni, nifedipine. B, effects of thapsigargin (TG) and ryanodine. C, effects of inhibitors and CGRP. D, effects of pretreatment with toxins (PTX, 100 ng/ml, 20 h; CTX, 2 µg/ml, 4 h). In the experiments with H-89, cells were preincubated with 10 µM H-89 for 30 min before further treatment. The traces were obtained from one experiment but are representative of three experiments.



Effect of Adrenomedullin on Ins-1,4,5-P(3) Level

Intracellular Ca mobilization is often mediated by Ins-1,4,5-P(3) formation, as observed with some hormones, bradykinin(23, 24) , and angiotensin II(25, 26) . Adrenomedullin at 0.1 µM also increased the intracellular Ins-1,4,5-P(3) level within 10 s (Fig. 5). The maximal level was about one-third of that by bradykinin, corresponding to the difference in maximal [Ca](i) levels induced by bradykinin and adrenomedullin (Fig. 3A). These observations suggest that adrenomedullin activates phospholipase C followed by Ins-1,4,5-P(3) formation and then increases [Ca](i) via Ins-1,4,5-P(3) receptor (a thapsigargin-sensitive and ryanodine-insensitive subtype) of endoplasmic reticulum in BAEC.


Figure 5: Time course of Ins-1,4,5-P(3) response of BAEC to adrenomedullin. After preincubation with Hanks' solution, cells were incubated with the same solution in the presence of 100 nM adrenomedullin, and then intracellular Ins-1,4,5-P(3) levels were determined as described under ``Experimental Procedures.'' Values are expressed as means ± S.E. (n = 4).**, significantly different (p < 0.01) from the level at 0 min by Dunnett multiple comparison.



Effect of Adrenomedullin on Intracellular cGMP Concentration

The mechanism of the hypotensive effect of bradykinin has been suggested as involving activation of NO synthase in endothelial cells through mobilization of intracellular Ca, with the resulting NO permeating into smooth muscle cells to activate soluble guanylate cyclase(27) . Therefore, adrenomedullin may also induce a hypotensive effect through an NO-mediated pathway because it induces [Ca](i) increase as shown above. This led us to examine the intracellular cGMP level in BAEC. Adrenomedullin at 0.1 µM significantly elevated the cGMP level (Fig. 6). The potency was about one-third of that by bradykinin and was parallel to that for Ins-1,4,5-P(3) formation and the [Ca](i) response. Pretreatment of cells with N^G-nitro-L-arginine methyl ester, an NO synthase inhibitor, deteriorated the cGMP accumulation by adrenomedullin, suggesting that the cGMP generation was mediated by NO.


Figure 6: Intracellular cGMP increase response of BAEC to adrenomedullin. After preincubation with or without 100 nMN^G-nitro-L-arginine methyl ester, cells were treated with Hanks' solution in the absence (openbar) or presence (hatchedbar) of 100 nM adrenomedullin for 15 min, and then cGMP levels were measured as described under ``Experimental Procedures.'' Results are expressed as means ± S.E. (n = 4). ##, significantly different (p < 0.01) from each control by Student's multiple range test.




DISCUSSION

Adrenomedullin acts as a hypotensive substance in anesthetized rat, being as effective as CGRP(1) , although its hypotensive mechanism has not been clarified. Recently, adrenomedullin was found to be 10-fold more potent than CGRP for increasing the cAMP level in RASMC, suggesting that adrenomedullin and CGRP may share the same receptor (4) . In this report, we investigated the actions of adrenomedullin on BAEC, because vascular endothelial cells may be the primary target of the circulating hormones (adrenomedullin is present in normal plasma at about 17 pg/ml(3) ), and BAEC showed the most specific binding of I-adrenomedullin among the several cultured cells examined (data not shown). Results from the competitive binding assay and [Ca](i) response indicated the existence of an adrenomedullin-specific receptor in BAEC. We also found that adrenomedullin could activate at least two signal transduction pathways, cAMP accumulation and [Ca](i) mobilization, in BAEC. The cAMP accumulation by adrenomedullin seemed to be mediated by CTX-sensitive PTX-insensitive G protein, probably G, which is involved in many systems of receptor-operated cAMP increase. As the cAMP response to adrenomedullin was not completely diminished by CTX treatment (Fig. 2, B and C), it could be possible that the cAMP increase response to adrenomedullin is mediated in part by other mechanism(s), such as facilitating the interaction between activated G and adenyl cyclase as shown in the case of angiotensin II action in RASMC (28) . The second signal by adrenomedullin, [Ca](i) mobilization, was also mediated by CTX-sensitive and PTX-insensitive subtype of G protein. In relation to this, Gil-Longo et al.(29) have reported that the [Ca](i) and cGMP increase in BAEC by bradykinin was mediated by CTX-sensitive G protein and proposed G and G as candidates for the G protein. Using some inhibitors and blockers, we showed that the [Ca](i) increase response of BAEC to adrenomedullin was biphasic. Adrenomedullin activated phospholipase C through its specific receptor and coupled CTX-sensitive G protein and accelerated Ins-1,4,5-P(3) formation to stimulate Ca release from the intracellular Ca storage, endoplasmic reticulum, through thapsigargin-sensitive Ins-1,4,5-P(3) receptor on the one hand (the initial rapid increase phase), and through the ion channel on the plasma membrane to promote Ca influx on the other hand (the prolonged increase phase).

We also showed the possibility that the [Ca](i) increase response of BAEC to adrenomedullin caused activation of NO synthase and NO release monitored by cGMP formation. It has been reported that bradykinin evokes a PTX-insensitive phosphoinositol response to increase [Ca](i) and cGMP in endothelial cells(29) . Endothelin 3 also elicited a [Ca](i) increase response through the receptor coupled with PTX-sensitive G protein in endothelial cells causing NO release(30) . All of these peptide hormones were thought to stimulate Ins-1,4,5-P(3) formation through phospholipase C activation. The toxin sensitivity was similar in [Ca](i) response to adrenomedullin and to bradykinin but not to endothelin 3, suggesting that at least two subtypes of G protein could be involved in the [Ca](i) increase response. Adrenomedullin was likely to open ion channel(s), because EGTA and SK& diminished the prolonged phase in the [Ca](i) response, while the response was completely diminished by the addition of SK& after treatment with thapsigargin. The activation of phospholipase C was also involved in the ion channel opening, because U-73122 completely extinguished the adrenomedullin-induced biphasic [Ca](i) response. In this respect, Ins-1,4,5-P(3) and inositol 1,3,4,5-tetraphosphate are possible candidates for direct action on the ion channel(31, 32) . The two signal transduction pathways activated by adrenomedullin seemed to be independent, because a strong inhibitor of cAMP-dependent protein kinase, H-89, had no effect on [Ca](i) response to adrenomedullin at a concentration that caused great loss of cAMP-dependent hormone release from GH3 cells(16) . In addition, cAMP-elevating agents, such as forskolin, dibutylyl-cAMP, or 3-isobutyl-1-methylxanthine, did not increase [Ca](i) in endothelial cells (data not shown). It is not clear whether these two adrenomedullin-induced independent signal transduction pathways are mediated by one receptor or not. The big difference between the apparent IC value in binding assay (Fig. 1) and EC value in cAMP increase response (Fig. 2A) suggests the existence of different receptor subtypes. However, there are several reports of one receptor being coupled to two independent signal mechanisms, for example, the prostacyclin receptor (33) and the endothelin receptor(34) .

Our results suggest that the hypotensive effect of adrenomedullin could be explained by at least two mechanisms. First, adrenomedullin can directly act on vascular smooth muscle cells to cause intracellular cAMP increase leading to relaxation, probably through inactivation of myosin light chain kinase. Second, adrenomedullin can act on vascular endothelial cells to cause a [Ca](i) increase leading to the activation of NO synthase and NO release. The released NO could then induce the relaxation of smooth muscle cells. In smooth muscle cells, [Ca](i) increase should lead to contraction, but we could not detect any [Ca](i) increase in RASMC by adrenomedullin even at 1 µM (data not shown), while intracellular cAMP increased greatly (Fig. 2A). Therefore, the signal transduction mechanism by adrenomedullin in smooth muscle cells must differ from that in endothelial cells. Recently, immunoreative adrenomedullin was detected in the conditioned medium of cultured endothelial cells(35) , suggesting the secretion of adrenomedullin from endothelial cells. Therefore, adrenomedullin may act as a local modulator as well as a circulating hormone in the cardiovascular system. The physiological role of intracellular cAMP accumulation in endothelial cells is not clear. This activity is seen to be important because the activity was 15-fold more potent than [Ca](i) increasing activity. Further investigation, for example, by altering the expression of some genes, should be done to solve this problem.


FOOTNOTES

*
This work was supported in part by research grants from the Science and Technology Agency (Encourage System of Center of Excellence), the Ministry of Health and Welfare, and the Human Science Foundation of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondece should be addressed. Tel.: 81-6-458-5861; Fax: 81-6-458-0987.

(^1)
The abbreviations used are: CGRP, calcitonin gene-related peptide; RASMC, rat aortic smooth muscle cells; BAEC, bovine aortic endothelial cells; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; PTX, pertussis toxin; CTX, cholera toxin; Ins-1,4,5-P(3); inositol 1,4,5-trisphosphate.


ACKNOWLEDGEMENTS

We thank Dr. Masafumi Fujimoto for helpful advice and discussion.


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