Rat Mesenteric Arterial Smooth Muscle Cells Research Articles

Allicin relaxes isolated mesenteric arteries through activation of PKA-KATP channel in rat

Allicin is a natural effective organosulfur compound isolated from garlic, which possesses many beneficial properties, such as antibacterial, anti-inflammatory, antimicrobial, hypotensive and hypolipidemic. In the present study, we investigated the effects and the underlying mechanisms of allicin on isolated mesenteric arteries (MAs). We examined MAs relaxation induced by allicin on rat-isolated mesenteric artery (MA) rings, the KATP channels with patch, and the expression of Kir6.1 and SUR2B with western blotting and NO production with Diaminofluorescein-FM diacetate (DAF-FMDA) in rat mesenteric artery smooth muscle cells (MASMCs). The results showed that allicin elicited the dose-dependent vasorelaxation effect with phenylephrine (PE) precontracted rat MA rings. The vasorelaxation effect was endothelium and NO independent but could be diminished by inhibition of PKA and KATP channels in the vascular smooth muscle. Allicin activated KATP channels in rat MASMCs, and the activation of KATP channels was inhibited by the inhibitors of PKA and KATP channels. But allicin had no effect on the expression of KATP subtypes Kir6.1 and SUR2B. These observations suggest that allicin exerts vasorelaxation effect through activation of PKA-KATP-signaling pathway.

Blockade of voltage-gated K+ currents in rat mesenteric arterial smooth muscle cells by MK801

MK801 (dizocilpine), a phencyclidine (PCP) derivative, is a potent noncompetitive antagonist of the N-Methyl-D-aspartate receptor (NMDAr). Another PCP derivative, ketamine, was reported to block voltage-gated K+ (Kv) channels, which was independent of NMDAr function. Kv currents are major regulators of the membrane potential (Em) and excitability of muscles and neurons. Here, we investigated the effect of MK801 on the Kv channels and Em in rat mesenteric arterial smooth muscle cells (RMASMCs). We used the whole-cell patch clamp technique to analyze the effect of MK801 enantiomers on Kv channels and Em. (+)MK801 inhibited Kv channels in a concentration-dependent manner (IC50 of 89.1 ± 13.1 μM, Hill coefficient of 1.05 ± 0.08). The inhibition was voltage- and state- independent. (+)MK801 didn't influence steady-state activation and inactivation of Kv channels. (+)MK801 treatment depolarized Em in a concentration-dependent manner and concomitantly decreased membrane conductance. (−)MK801 also similarly inhibited the Kv channels (IC50 of 134.0 ± 17.5 μM, Hill coefficient of 0.87 ± 0.09). These results indicate that MK801 directly inhibits the Kv channel in a state-independent manner in RMASMCs. This MK801-mediated inhibition of Kv channels should be considered when assessing the various pharmacological effects produced by MK801, such as schizophrenia, neuroprotection, and hypertension.

Eukaryotic elongation factor 2 kinase controls proliferation and migration of vascular smooth muscle cells.

Eukaryotic elongation factor 2 kinase (eEF2K), also known as calmodulin (CaM)-dependent protein kinase (CaMK) III, is a unique member of CaMK family protein. We have recently found that expression of eEF2K protein increased in mesenteric artery from spontaneously hypertensive rats. As pathogenesis of hypertension is in part regulated by vascular structural remodelling via proliferation and migration of vascular smooth muscle cells (SMCs), we tested the hypothesis that eEF2K controls SMCs proliferation and migration. In rat mesenteric arterial SMCs, an eEF2K inhibitor, A-484954 (10 μm), significantly inhibited platelet-derived growth factor (PDGF)-BB (10 ng mL(-1) )-induced SMCs proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay. PDGF-BB (10 ng mL(-1) )-induced SMCs migration was significantly inhibited by A-484954 (10 μm) as determined by a Boyden chamber assay. A-484954 (10 μm) significantly inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K, extracellular signal-regulated kinase (ERK), Akt, p38 and heat-shock protein (HSP) 27 as determined by Western blotting. It was confirmed that a CaM inhibitor, W-7 (50 μm), inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K. In an ex vivo mesenteric arterial ring assay, 10% foetal bovine serum-induced SMCs outgrowth was significantly inhibited by A-484954 (10 μm). We for the first time revealed that eEF2K mediates PDGF-BB-induced SMCs proliferation and migration through activating ERK, Akt, p38 and HSP27 signals in a CaM-dependent manner. Our results suggest eEF2K as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.

Histone Deacetylase 4 Controls Neointimal Hyperplasia via Stimulating Proliferation and Migration of Vascular Smooth Muscle Cells

Histone deacetylases (HDACs) are transcriptional coregulators. Recently, we demonstrated that HDAC4, one of class IIa family members, promotes reactive oxygen species-dependent vascular smooth muscle inflammation and mediates development of hypertension in spontaneously hypertensive rats. Pathogenesis of hypertension is, in part, modulated by vascular structural remodeling via proliferation and migration of vascular smooth muscle cells (SMCs). Thus, we examined whether HDAC4 controls SMC proliferation and migration. In rat mesenteric arterial SMCs, small interfering RNA against HDAC4 inhibited platelet-derived growth factor (PDGF)-BB-induced SMC proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay as well as migration as determined by Boyden chamber assay. Expression and activity of HDAC4 were increased by PDGF-BB. HDAC4 small interfering RNA inhibited phosphorylation of p38 mitogen-activated protein kinase and heat shock protein 27 and expression of cyclin D1 as measured by Western blotting. HDAC4 small interfering RNA also inhibited PDGF-BB-induced reactive oxygen species production as measured fluorometrically using 2', 7'-dichlorofluorescein diacetate and nicotinamide adenine dinucleotide phosphate oxidase activity as measured by lucigenin assay. A Ca(2+)/calmodulin-dependent protein kinase II inhibitor, KN93, inhibited PDGF-BB-induced SMC proliferation and migration as well as phosphorylation of HDAC4. In vivo, a class IIa HDACs inhibitor, MC1568 prevented neointimal hyperplasia in mice carotid ligation model. MC1568 also prevented increased activation of HDAC4 in the neointimal lesions. The present results for the first time demonstrate that HDAC4 controls PDGF-BB-induced SMC proliferation and migration through activation of p38 mitogen-activated protein kinase/heat shock protein 27 signals via reactive oxygen species generation in a Ca(2+)/calmodulin-dependent protein kinase-dependent manner, which may lead to the neointimal hyperplasia in vivo.

Abstract 22: Histone Deacetylase (HDAC) 4 Controls Neointimal Hyperplasia via Stimulating Proliferation and Migration of Vascular Smooth Muscle Cells

Histone deacetylases (HDACs) are transcriptional co-regulators. We have recently demonstrated that a class IIa HDAC, HDAC4 promotes reactive oxygen species (ROS)-dependent vascular smooth muscle inflammation and mediates the development of hypertension in spontaneously hypertensive rats. Pathogenesis of hypertension is in part modulated by vascular structural remodeling via proliferation and migration of vascular smooth muscle cells (SMCs). We thus examined whether HDAC4 controls SMCs proliferation and migration. In rat mesenteric arterial SMCs, small interfering RNA (siRNA) against HDAC4 inhibited platelet-derived growth factor (PDGF)-BB-induced SMCs proliferation as determined by a cell counting (51% inhibition, n=7) or bromodeoxyuridine incorporation assay (95% inhibition, n=6) and migration as determined by Boyden chamber assay (71% inhibition, n=3). Expression and activity of HDAC4 were increased by PDGF-BB (30% increase, n=5 and 170% increase, n=4, respectively). HDAC4 siRNA inhibited phosphorylation of p38 (69% inhibition, n=5) and heat shock protein (HSP) 27 (91% inhibition, n=5) and expression of cyclin D1 (58% inhibition, n=5) as measured by Western blotting. HDAC4 siRNA also inhibited PDGF-BB-induce ROS production as measured fluorometrically using 2’ 7’-dichlorofluorescein diacetate (77% inhibition, n=4) and nicotinamide adenine dinucleotide phosphate oxidase activity as measured by lucigenin assay (61% inhibition, n=4). A Ca 2+ /calmodulin (CaM)-dependent protein kinase (CaMK) II inhibitor, KN93 inhibited PDGF-BB-induced SMCs proliferation (58% inhibition, n=4) and migration (75% inhibition, n=3) as well as phosphorylation of HDAC4 (84% inhibition, n=4). In vivo, a class IIa HDACs inhibitor, MC1568 prevented neointimal hyperplasia in mice carotid ligation model (54% inhibition, n=6). MC1568 also inhibited increased activity of HDAC4 in the neointimal lesions. The present results for the first time demonstrate that HDAC4 controls PDGF-BB-induced SMCs proliferation and migration through activation of p38/HSP27 signals via ROS generation in a CaMKII-dependent manner, which may lead to the neointima hyperplasia in vivo.

Abstract 303: A Novel Adipocytokine, Omentin Prevents Vascular Smooth Muscle Cell Migration by Inhibiting O 2 -/p38/HSP27 Pathway

(Background) Omentin, a novel adipocytokine, is mainly expressed in the visceral rather than subcutaneous adipose tissue. Secretion and plasma concentration of omentin decrease in the obese subjects. We previously demonstrated that omentin is anti-inflammatory in vascular smooth muscle cells (SMCs). While vascular remodeling via proliferation, migration and apoptosis of SMCs is also important for hypertension development, it remains to be clarified whether omentin affects those processes. (Methods and Results) Cultured rat mesenteric arterial SMCs were stimulated with platelet-derived growth factor (PDGF)-BB (10 ng/ml) in the absence or presence of omentin (300 ng/ml). Omentin pretreatment (2 h) inhibited PDGF (6 h)-induced SMCs migration (30 % inhibition, n=11, P<0.05) as determined by Boyden camber assay. Omentin pretreatment (2 h) inhibited NADPH oxidase -derived O2- generation as determined by dihydroethidium staining and lucigenin assay (30 % inhibition, n=5, P<0.05) as well as phosphorylation of p38 (20 % inhibition, n=19-23, P<0.01) and heat shock protein (HSP) 27 (20 % inhibition, n=14-17, P<0.01) as determined by Western blotting. Omentin pretreatment (2 h) inhibited PDGF (6 h)-induced lamellipodia formation as determined by rhodamine-phalloidin staining. In ex vivo, omentin pretreatment (6 h) also inhibited fetal bovine serum (FBS) (10%)-induced SMCs out-growth from rat isolated mesenteric artery. (Conclusion) We for the first time demonstrate that omentin prevented SMCs migration by preventing the activation of ROS-dependent p38/HSP27 signals. It is suggested that omentin is anti-hypertensive ‘good adipocytokine’.

Effects of different concentrations of dexmedetomidine on BKCa channels in rat mesenteric arterial smooth muscle cells

Objective To evaluate the effects of different concentrations of dexmedetomidine on large-con-ductance Ca2+-activated K+ (BKca) channels in the rat mesenteric arterial smooth muscle (MASM) cells.Methods Sprague-Dawley rats of both sexes,weighing 180-220 g,were used in this study.Single MASM cell was freshly isolated from mesenteric arteries in two steps.Ten cells were chosen and studied.When holding potential was 40 mV and the concentration of free calcium ions was 10-7 mol/L,inside-out patch-clamp technique was used to record the single BKCa channel current before and after application of different concentrations of dexmedetomidine (0,10-9,10-8,10-7,10-6,10-5 mol/L).Total open probability (NPo),amplitude (Am),mean open time (To) and mean close time (To) of single BKca channel were observed and recorded.Results Compared with the baseline value,dexmedetomidine 10-7,10-6 and 10-5 mol/L increased NPo in a concentration-dependent man-ner,and dexmedetomidine 10-9,10-8,10-7,10-6,10-5mol/L shortened Tc (P ＜0.05 or 0.01).Compared with the value obtained when the concentration of dexmedetomidine was 10-9 mol/L,Tc was significantly shortened when the concentration of dexmedetomidine was 10-8 mol/L (P ＜ 0.05),and no significant change was found in Am and To obtained when different concentrations of dexmedetomidine were applied (P ＞ 0.05).Conclusion Dexmedetomidine 10-7,10-6 and 10-5 mol/L activate BKca channels in rat MASM cells in a concentration-depen-dent manner,which is one of the mechanisms of decrease in blood pressure by dexmedetomidine. Key words: Dexmedetomidine ; Large-conductance Ca2 +-activated potassium channels ; Mesenteric arteries; Muscle, smooth, vascular

Endosomal proteolysis regulates calcitonin gene‐related peptide responses in mesenteric arteries

Calcitonin gene-related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR●RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin-converting enzyme-1 (ECE-1). However, it is not known if ECE-1 regulates the resensitization of CGRP-induced responses in functional arterial tissue. CLR, ECE-1a-d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA-SMCs) and mesenteric arteries was analysed by RT-PCR and by immunofluorescence and confocal microscopy. CGRP-induced signalling in cells was examined by measuring cAMP production and ERK activation. CGRP-induced relaxation of arteries was measured by isometric wire myography. ECE-1 was inhibited using the specific inhibitor, SM-19712. RMA-SMCs and arteries contained mRNA for CLR, ECE-1a-d and RAMP1. ECE-1 was present in early endosomes of RMA-SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium-independent relaxation of arteries. ECE-1 inhibition had no effect on initial CGRP-induced responses but reduced cAMP generation in RMA-SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. ECE-1 regulated the resensitization of responses to CGRP in RMA-SMCs and mesenteric arteries. CGRP-induced relaxation did not involve endothelium-derived pathways. This is the first report of ECE-1 regulating CGRP responses in SMCs and arteries. ECE-1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine.

ARSENIC TRIOXIDE INHIBITS THE VOLTAGE-GATED K+ CHANNELS IN RAT MESENTERIC ARTERIAL SMOOTH MUSCLE CELLS

ARSENIC TRIOXIDE INHIBITS THE VOLTAGE-GATED K+ CHANNELS IN RAT MESENTERIC ARTERIAL SMOOTH MUSCLE CELLS

A novel adipocytokine, vaspin inhibits platelet-derived growth factor-BB-induced migration of vascular smooth muscle cells

Vaspin is a novel adipocytokine originally identified in visceral white adipose tissues of Otsuka Long-Evans Tokushima fatty rats, an animal model of type 2 diabetes. We have previously shown that vaspin has anti-inflammatory effects in vascular smooth muscle cells (SMCs). SMCs migration is an important process for development atherosclerosis. However, effects of vaspin on SMCs migration remain to be clarified. Rat mesenteric arterial SMCs were treated with platelet-derived growth factor (PDGF)-BB (10ng/ml, 90min) in the absence or presence of vaspin (0.01–10ng/ml, pretreatment for 2h). SMCs migration was evaluated by a Boyden chamber assay. Western blotting was performed to analyze cellular signals. Reactive oxygen species (ROS) generation was fluorometrically measured using 2′,7′-dichlorofluorescein diacetate. Vaspin significantly inhibited PDGF-BB-induced SMCs migration. Vaspin significantly inhibited PDGF-BB-induced phosphorylation of p38 and heat shock protein (HSP) 27 as well as ROS generation. SMCs migration was blocked by an inhibitor of p38 or an anti-oxidant drug, N-acetyl-l-cysteine (NAC). NAC significantly inhibited the PDGF-BB-induced phosphorylation of p38 and HSP27. In addition, vaspin inhibited PDGF-BB-induced actin cytoskeletal reorganization (lamellipodia formation) as revealed by a rhodamine phalloidin staining. The present study for the first time revealed that vaspin inhibits PDGF-BB-induced SMCs migration through inhibiting p38/HSP27 signals via preventing the ROS generation.

Abstract 9389: Histone Deacetylase 4 Causes Vascular Inflammation and Mediates Development of Hypertension in Spontaneous Hypertensive Rats

Histone deacetylases (HDAC)s are transcription factors which play important roles on structural remodeling of chromatin. It is recently clarified that HDAC4 mediates heart failure and vascular smooth muscle cells (SMCs) hypertrophy. Moreover our recent study demonstrated that HDAC4 protein increases in mesenteric artery from spontaneous hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). Vascular inflammation is essential for pathogenesis of hypertension. We therefore examined whether HDAC4 affects on vascular inflammatory responses and mediates hypertensive vascular diseases. Protein expression (n=6-7, P<0.01) and activity (n=3-5, P<0.01) of HDAC4 in rat mesenteric arterial SMCs were increased by TNF-α (10 ng/ml, 1-3 h). An HDACs inhibitor, trichostatin A (TSA) (10 μM, pretreatment for 30 min), inhibited TNF (10 ng/ml, 24 h)-induced vascular cell adhesion molecule (VCAM)-1 expression (n=4, P<0.01). TSA also inhibited TNF (20 min)-induced phosphorylation of NF-κB (n=4, P<0.01). HDAC4 siRNA inhibited TNF-induced VCAM-1 (n=3-6, P<0.01), monocyte adhesion (n=6, P<0.05), phosphorylation (n=3-6, P<0.05) and transcriptional activity (n=4-5, P<0.05) of NF-κB. Furthermore, HDAC4 siRNA inhibited TNF-induced reactive oxygen species (ROS) production (n=4, P<0.01). In vivo study, blood pressure (n=4, P<0.05), ROS production (n=2) and VCAM-1 expression (n=4) in isolated mesenteric artery were increased in SHR compared with WKY, which were prevented by a long-term TSA treatment to SHR (100, 500 μg/kg/day, 3 weeks). In isolated mesenteric artery, the increased angiotensin II-induced contraction in SHR was reversed by a TSA treatment (n=4-6, P<0.01). The endothelium-dependent relaxation induced by acetylcholine in SHR was augmented by TSA (n=6-8, P<0.01). The present results indicate that in vascular SMCs TNF-induced HDAC4 mediates vascular inflammation via VCAM-1 induction through ROS-dependent NF-κB activation. It is also suggested that pro-inflammatory effects as well as hypercontractile effects of HDAC4 may mediate the development of hypertension in SHR. Our results may imply that HDAC4 is a promising important target for drug therapy against essential hypertension.

Serotonin depolarizes the membrane potential in rat mesenteric artery myocytes by decreasing voltage-gated K + currents

We hypothesized that voltage-gated K + (K v) currents regulate the resting membrane potential ( E m), and that serotonin (5-HT) causes E m depolarization by reducing K v currents in rat mesenteric artery smooth muscle cells (MASMCs). The resting E m was about −40 mV in the nystatin-perforated patch configuration, and the inhibition of K v currents by 4-aminopyridine caused marked E m depolarization. The inhibition of Ca 2+-activated K + (K Ca) currents had no effect on E m. 5-HT (1 μM) depolarized E m by ∼11 mV and reduced the K v currents to ∼63% of the control at −20 mV. Similar 5-HT effects were observed with the conventional whole-cell configuration with a weak Ca 2+ buffer in the pipette solution, but not with a strong Ca 2+ buffer. In the presence of tetraethylammonium (1 mM), 5-HT caused E m depolarization similar to the control condition. These results indicate that the resting E m is largely under the regulation of K v currents in rat MASMCs, and that 5-HT depolarizes E m by reducing K v currents in a [Ca 2+] i-dependent manner.

Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K + current in rat mesenteric artery smooth muscle cells

A large array of voltage-gated K + channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 μg/ml) inhibited the Kv currents by 29.2 ± 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 μg/ml) by 24.5 ± 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4–20 μg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 μg/ml each) induced an additive inhibition of Kv currents by 42.3 ± 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 μg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.

Modulation of voltage-dependent K+ channel current in vascular smooth muscle cells from rat mesenteric arteries.

Voltage-dependent K+ (Kv) channels were studied in smooth muscle cells (SMCs) freshly isolated from rat mesenteric arteries. A delayed outward rectifier Kv current (IK) with a weak voltage dependence was identified. The amplitude of IK, but not its inactivation kinetics, was inhibited by 4-aminopyridine (4-AP) (IC50, 5.1 +/- 0.9 mM). The inhibitory effect of 4-AP was not use-dependent, and the unbinding of 4-AP from IK channels was complete in the absence of depolarization stimuli, suggesting the binding of 4-AP to the closed state of Kv channels. There was no change in the steady-state inactivation, but the steady-state activation curve of IK was shifted in the presence of 4-AP by +6 mV. Including 4-AP in pipette solution instantly inhibited IK upon the rupture of cell membrane, indicating that 4-AP bound to the inner mouth of Kv channel pores. Several Kv channel proteins encoding the native IK-type Kv channels, but not the transient outward A-type Kv channels, were identified. Among the identified IK-encoding gene transcripts, the expression of Kv1.5 was the most abundant. Our results elucidate the modulating mechanisms for the 4-AP-induced IK inhibition in rat mesenteric artery SMCs and suggest that the unique properties of Kv channels in these cells might be related to the heteromeric expression of the IK-encoding genes with Kv1.5 subunit playing an important role.

Superoxide Anion-Induced Formation of Inositol Phosphates Involves Tyrosine Kinase Activation in Smooth Muscle Cells from Rat Mesenteric Artery

Our previous studies have demonstrated an enhanced production of inositol phosphates (IPs) induced by superoxide in smooth muscle cells (SMCs). The mechanisms for this effect, however, remained largely unknown. In the present study, it was found that superoxide increased IP production in SMCs from rat mesenteric arteries in a time-dependent manner. The effect of superoxide on IP formation was significantly inhibited by the antioxidants n-acetylcysteine or α-lipoic acid. Genistein and tyrphostin A25, two tyrosine kinase inhibitors, also inhibited the superoxide-induced IP formation. The application of monoclonal antibody against phospholipase Cγ (PLCγ) significantly inhibited the superoxide-induced IP formation. Finally, the expression level of PLCγ proteins was increased 6 hrs after exposing SMCs to superoxide. The present findings demonstrate that superoxide activates the tyrosine kinase pathway and suggest that the tyrosine kinase-mediated IP formation may represent a novel mechanism underlying the signalling role of superoxide in rat mesenteric artery SMCs.

Vasopressin (V1) receptor characteristics in rat aortic smooth muscle cells

We report saturable, high-affinity, specific, reversible binding sites for both [3H]arginine vasopressin ([3H]AVP) and d(CH2)5Tyr(Me)-[3H]AVP, a V1-selective antagonist, in cultured smooth muscle cells obtained from rat aorta (RA) and rat mesenteric artery (RMA). Specific binding of [3H]AVP had the following characteristics in adherent monolayers of RA and RMA smooth muscle cells: dissociation constant (KD) = 1.42 and 1.23 nM and maximal binding capacity (Bmax) = 9,500 and 29,910 sites/cell, respectively. Lower KD and higher Bmax values were detected for 3H-labeled V1 antagonist binding to both types of cells. Complete dissociation of [3H]-AVP binding to RA cells occurred in a biphasic manner with two rate constants of dissociation, suggesting high- and low-affinity states of the binding site for the agonist interaction. Partial dissociation of the antagonist-specific binding occurred, and it was monophasic, suggesting interaction of the 3H-labeled V1 antagonist radioligand to the high-affinity binding state. Inhibition constant (Ki) values determined by competitive inhibition of [3H]AVP binding to RA cells by a series of AVP-related peptide analogues and antagonists were consistent with the saturation data. AVP in a concentration-dependent manner induced the accumulation of inositol phosphates [mean effective concentration (EC50) 1 nM] in the adherent RA cells. The free cytosolic Ca2+ level in the dispersed RA smooth muscle cells was increased by AVP (EC50 8.1 nM). Pretreatment with the V1 antagonist abolished these AVP-evoked responses. The data support the conclusion that the agonist binding occurs at a homogeneous population of V1-subtype receptors in the high-affinity (KD = approximately 1 nM) state and that these receptors are functionally coupled to phospholipase C.