Characterization of opioid receptors on smooth muscle cells from guinea pig stomach

1992 ◽  
Vol 262 (3) ◽  
pp. G461-G469 ◽  
Author(s):  
L. Zhang ◽  
Z. F. Gu ◽  
T. Pradhan ◽  
R. T. Jensen ◽  
P. N. Maton
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Opioid Receptors ◽  
Muscle Cells ◽  
Receptor Subtypes ◽  
Selective Antagonist ◽  
Kappa Agonist ◽  
Gastric Smooth Muscle ◽  
Selective Ligand ◽  
Guinea Pig Stomach

On the basis of opioid-stimulated contraction of dispersed gastric smooth muscle cells it has been suggested that these cells possess opioid receptors of three subtypes: kappa (kappa), mu (mu), and delta (delta). We have used selective peptidase-resistant radioligands, agonists and antagonists, to examine receptor subtypes on dispersed gastric smooth muscle cells from guinea pigs prepared by collagenase digestion. The kappa-agonist U-50488H, the mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO), and the delta-agonist [D-Pen2,Pen5]enkephalin (DPDPE) each caused muscle contraction. The concentrations required to caused half-maximal contraction were U50488H (6 pM) greater than DAGO (13 pM) greater than DPDPE (6 nM). The abilities of these agonists to inhibit binding of [3H]U-69593 (kappa-preferring) by 50% were U50488H (43 nM) greater than DAGO (43 microM) greater than DPDPE (200 microM). Their abilities to inhibit binding of [3H]naloxone (mu-preferring) by 50% were DAGO (0.2 microM) greater than U50488H (10 microM) greater than DPDPE (greater than 100 microM). No binding could be detected with the delta-selective ligand [3H]DPDPE. The kappa-preferring antagonist Mr2266 (10 nM) preferentially inhibited contraction stimulated by the kappa-agonist U50488H, and naltrexone (10 nM) (mu-selective antagonist) preferentially inhibited contraction stimulated by the mu-agonist DAGO. ICI 174864 (200 microM; delta-selective antagonist) had no effect on contraction stimulated by mu-, kappa-, or delta-agonists. Contraction stimulated by the delta-agonist DPDPE was inhibited by both kappa- and mu-receptor antagonists. Studies on the effect of the antagonists on binding of [3H]naloxone and [3H]U69593 also provided evidence for kappa- and mu-sites but nor for delta-sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Different receptors mediate the action of bombesin-related peptides on gastric smooth muscle cells

1991 ◽  
Vol 260 (5) ◽  
pp. G683-G690 ◽  
Author(s):  
C. Severi ◽  
R. T. Jensen ◽  
V. Erspamer ◽  
L. D'Arpino ◽  
D. H. Coy ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Contractile Response ◽  
Muscle Cells ◽  
Receptor Subtypes ◽  
Gastric Smooth Muscle ◽  
Neuromedin B ◽  
Specific Receptors ◽  
Selective Preservation ◽  
Guinea Pig Stomach

Recent studies suggest that different subtypes of receptors may mediate the action of various bombesin-related peptides in different tissues. In the present study the ability of bombesin and its structurally related peptides [litorin, gastrin-releasing peptide (GRP), GRP18-27, neuromedin B, [Leu8]litorin, and bombesin nonapeptide BN(6-14)] to interact with smooth muscle cells isolated from guinea pig stomach was investigated. Each peptide induced a specific contractile response with potencies (D50 in pM) of [Leu8]litorin (0.7) greater than bombesin (1.2) greater than litorin (3) greater than neuromedin B (3.5) = GRP (3.8) = GRP18-27 (3.9) greater than BN(6-14) (70.9). The specific bombesin receptor antagonist psi 13,14-bombesin differed in its potency for inhibiting equipotent concentrations of bombesin, GRP, or neuromedin B, was equipotent for bombesin or GRP (IC50 12.7 and 22.1 nM), and was 11 times less potent for neuromedin B (IC50 234.5 nM), suggesting the presence of subtypes of receptors mediating the action of bombesin-related peptides. To further investigate this possibility, a technique of receptor protection that enables selective preservation of one receptor type was used. GRP or bombesin protected completely the response to GRP or bombesin but abolished the subsequent contractile response to neuromedin B. Neuromedin B, instead, protected only the response to neuromedin B. These results demonstrate that gastric smooth muscle cells possess specific receptors that interact with bombesin-related peptides and that two receptor subtypes mediate the contractile response to these peptides: one subtype is selective for bombesin or GRP, the other for neuromedin B.

Human esophageal smooth muscle cells express muscarinic receptor subtypes M1 through M5

2000 ◽  
Vol 279 (5) ◽  
pp. G1059-G1069 ◽  
Author(s):  
Jian Wang ◽  
Pawel S. Krysiak ◽  
Lisanne G. Laurier ◽  
Stephen M. Sims ◽  
Harold G. Preiksaitis
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscarinic Receptor ◽  
Muscle Cells ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Functional Responses ◽  
Selective Antagonist ◽  
Esophageal Physiology ◽  
Esophageal Smooth Muscle Cells

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10–14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific α-actin. mRNA encoding muscarinic receptor subtypes M1–M5 was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca2+ concentration ([Ca2+]i) using fura 2 fluorescence. Basal [Ca2+]i, which was 135 ± 22 nM, increased transiently to 543 ± 29 nM in response to 10 μM ACh in CM cells ( n = 8). This response was decreased <95% by 0.01 μM 4-diphenylacetoxy- N-methylpiperidine, a M1/M3-selective antagonist, whereas 0.1 μM methoctramine, a M2/M4-selective antagonist, and 0.1 μM pirenzepine, a M1-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca2+]i mediated primarily by the M3 receptor and involving release of Ca2+ from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.

Smooth muscle cells from guinea pig stomach possess high-affinity galanin receptors that mediate relaxation

1994 ◽  
Vol 266 (5) ◽  
pp. G839-G845 ◽  
Author(s):  
Z. F. Gu ◽  
T. K. Pradhan ◽  
D. H. Coy ◽  
R. T. Jensen
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Maximal Effect ◽  
Dependent Manner ◽  
High Affinity ◽  
Gastric Smooth Muscle ◽  
Guinea Pig Stomach ◽  
Galanin Receptors

Galanin-like immunoactivity occurs in nerves and plexi in muscle layers throughout gastrointestinal tract including the stomach. Galanin can affect gastric emptying and contraction or relaxation of gastric muscle in different species. The aim of this study was to investigate the direct effect of galanin on dispersed gastric smooth muscle cells and to characterize any galanin receptors that mediated any effect. Dispersed gastric smooth muscle cells were prepared from guinea pig stomach by collagenase digestion. Porcine galanin (p-galanin; 1 microM) did not stimulate contraction when present alone; however, p-galanin (1 microM) inhibited carbachol-induced contraction with a half-maximal effect at 7 nM. p-Galanin (1 microM) increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content by 10 s and caused a maximal increase of 80% over basal. 125I-galanin (porcine) bound to dispersed cells in a time- and temperature-dependent manner. Binding was saturable, reversible, and specific. Binding of 125I-galanin was inhibited almost equally by porcine and rat galanin (Ki = 6-8 nM) but was not inhibited by the galanin-associated peptide [preprogalanin-(108-123)]. The fragment galanin-(1-16) was equally potent to rat galanin; however, the fragment galanin-(9-29) was 56-fold less potent (Ki = 370 nM). Computer analysis demonstrated there were two binding sites for p-galanin on gastric smooth muscle cells, a high-affinity site (Kd = 2.6 nM) with low capacity (Bmax = 175 fmol/mg protein) and a low-affinity site (Kd = 150 nM) with large capacity (Bmax = 3,611 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of SR-49059, a vasopressin V1a antagonist, on human vascular smooth muscle cells

1995 ◽  
Vol 268 (1) ◽  
pp. H404-H410 ◽  
Author(s):  
C. Serradeil-Le Gal ◽  
J. M. Herbert ◽  
C. Delisee ◽  
P. Schaeffer ◽  
D. Raufaste ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Specific Binding ◽  
Muscle Cells ◽  
Maximal Response ◽  
Receptor Subtypes ◽  
Functional Studies ◽  
Antagonist Binding

The effects of SR-49059, a new nonpeptide and selective arginine vasopressin (AVP) V1a antagonist, were investigated in binding and functional studies on cultured human aortic vascular smooth muscle cells (VSMC). Characterization of human vascular V1a receptors, using a specific V1a radioiodinated ligand, showed that [125I]-linear AVP antagonist binding to human VSMC membranes was time dependent, reversible, and saturable. A single population of high-affinity binding sites (apparent equilibrium dissociation constant = 15 +/- 6 pM; maximum binding density = 36 +/- 5 fmol/mg protein, i.e., approximately 3,000 sites/cell) with the expected V1a profile was identified. Exposure of these cells to AVP dose-dependently produced cytosolic free [Ca2+] increase [AVP concentration required to obtain a half-maximal response (EC50) = 23 +/- 9 nM] and proliferation (EC50 = 3.2 +/- 0.5 nM). SR-49059 strongly and stereospecifically inhibited [125I]-linear AVP antagonist binding to VSMC V1a receptors [inhibition constant (Ki) = 1.4 +/- 0.3 nM], AVP-evoked Ca2+ increase [concentration of inhibitor required to obtain 50% inhibition of specific binding (IC50) = 0.41 +/- 0.06 nM], and the mitogenic effects induced by 100 nM AVP (IC50 = 0.83 +/- 0.04 nM). OPC-21268, another nonpeptide V1a antagonist, was more than two orders of magnitude less potent than SR-49059 in these models. However, the consistent affinity (Ki = 138 +/- 21 nM) and activity found with OPC-21268 on human VSMC in comparison with the inactivity already observed for other human V1a receptors (liver, platelets, adrenals, and uterus) strongly suggested the existence of human AVP V1a-receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway

2013 ◽  
Vol 304 (5) ◽  
pp. G527-G535 ◽  
Author(s):  
Senthilkumar Rajagopal ◽  
Divya P. Kumar ◽  
Sunila Mahavadi ◽  
Sayak Bhattacharya ◽  
Ruizhe Zhou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Kinase Activity ◽  
Muscle Relaxation ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Receptor Activation ◽  
Gastric Smooth Muscle ◽  
Selective Ligand ◽  
Gastric Muscle ◽  
Kinase Pathway

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5−/− mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2′-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

2018 ◽  
Vol 54 (5) ◽  
pp. 400-407 ◽  
Author(s):  
I. F. Shaidullov ◽  
M. U. Shafigullin ◽  
L. M. Gabitova ◽  
F. G. Sitdikov ◽  
A. L. Zefirov ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Hydrogen Sulfide ◽  
Potassium Channels ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Gastric Smooth Muscle

Muscarinic receptor size on smooth muscle cells and membranes

1986 ◽  
Vol 251 (2) ◽  
pp. G195-G200
Author(s):  
S. M. Collins ◽  
C. Y. Jung ◽  
A. K. Grover
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscarinic Receptor ◽  
Plasma Membranes ◽  
Muscle Cells ◽  
High Energy ◽  
Membrane Preparation ◽  
Galactose Oxidase ◽  
Radiation Inactivation ◽  
Gastric Smooth Muscle

The loss of [3H]quinuclidinyl benzilate ([3H]QNB) binding following high-energy radiation was used to compare the muscarinic receptor size on single smooth muscle cells isolated by collagenase digestion from the canine stomach and on plasma membranes derived from intact gastric smooth muscle without exposure to exogenous proteolysis. Radiation inactivation of galactose oxidase (68 kdaltons), yeast alcohol dehydrogenase (160 kdaltons), and pyruvate kinase (224 kdaltons) activities were used as molecular-weight standards. Radiation inactivation of [3H]QNB binding to rat brain membranes, which gave a target size of 86 kdaltons, served as an additional control. In isolated smooth muscle cells, the calculated size of the muscarinic receptor was 80 +/- 8 kdaltons. In contrast, in a smooth muscle enriched plasma membrane preparation, muscarinic receptor size was significantly smaller at 45 +/- 3 kdaltons. Larger molecular sizes were obtained either in the presence of protease inhibitors (62 +/- 4 kdaltons) or by using a crude membrane preparation of gastric smooth muscle 86 +/- 7 kdaltons).

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