Biomedical subjects

J R Grider

Publications and source records attributed to J R Grider.

At least 55 records · Page 3Linked to original sources

PubMed1993

Expression of functional receptors for vasoactive intestinal peptide in freshly isolated and cultured gastric muscle cells.

Vasoactive intestinal peptide (VIP) receptors were characterized in freshly isolated and cultured smooth muscle cells from guinea pig stomach by radioligand binding and by measurement of relaxation in single isolated and cultured cells. 125I-VIP bound to both freshly isolated and cultured muscle cells: binding was rapid, specific, saturable and temperature-dependent, and was inhibited in a concentration-dependent fashion by VIP, VIP10-28, PHI and secretin, in this order. Competition curves for VIP could be resolved into high- and low-affinity components, yielding similar binding constants in freshly isolated and cultured cells (high-affinity Kd 0.11 and 0.22 nM; low-affinity Kd 59 and 37 nM; high-affinity binding sites: 1183 and 1021 per cell, representing about 1% of total binding sites). VIP10-28 inhibited 125I-VIP binding completely and acted as potent competitive antagonist of VIP-induced relaxation (Ki 0.5 nM). PHI and secretin, however, inhibited partly 125I-VIP binding: the pattern of inhibition implied that VIP interacts with VIP-preferring receptors that are recognized by PHI and secretin as well as with VIP-specific receptors. The pattern of binding is consistent with recent evidence indicating that VIP activates two signalling pathways, a VIP-specific, nitric oxide/cGMP-dependent pathway and a common cAMP-dependent pathway shared by all three peptides. PHI and secretin were relatively more potent as relaxant agents than as inhibitors of 125I-VIP binding raising the possibility that PHI and secretin could interact additionally with PHI- and secretin-preferring receptors in mediating relaxation.

Animals↗

PubMed1993

Vasoactive intestinal peptide release and L-citrulline production from isolated ganglia of the myenteric plexus: evidence for regulation of vasoactive intestinal peptide release by nitric oxide.

Vasoactive intestinal peptide release and L-[3H]citrulline production were examined in ganglia isolated from the myenteric plexus of guinea-pig intestine. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperizinium stimulated vasoactive intestinal peptide release and L-[3H]citrulline production; the latter was considered an index of nitric oxide production. Both vasoactive intestinal peptide release and L-[3H]citrulline production were abolished by tetrodotoxin, hexamethonium, and the nitric oxide synthase inhibitor, NG-nitro-L-arginine. Inhibition of vasoactive intestinal peptide release by NG-nitro-L-arginine was reversed by L-arginine but not by D-arginine. Exogenous nitric oxide stimulated vasoactive intestinal peptide release whereas exogenous vasoactive intestinal peptide had no effect on L-[3H]citrulline production. The pattern of stimulation by nitric oxide and inhibition by NG-nitro-L-arginine implied that vasoactive intestinal peptide release is facilitated by and may be dependent on nitric oxide production. Consistent with this notion, vasoactive intestinal peptide release in response to either 1,1-dimethyl-4-phenylpiperizinium or nitric oxide was abolished by KT 5823, an inhibitor of cyclic GMP-dependent protein kinase activity and by LY83583, an inhibitor of soluble guanylate cyclase activity. The study provides the first direct evidence of nitric oxide production from enteric ganglia.

Alkaloids↗

PubMed1993

Interplay of VIP and nitric oxide in regulation of the descending relaxation phase of peristalsis.

Involvement of vasoactive intestinal peptide (VIP) and nitric oxide (NO) in neurally induced relaxation was examined in smooth muscle from rat colon. Relaxation induced by field stimulation or radial stretch (i.e., descending relaxation phase of the peristaltic reflex) was accompanied by VIP release and NO production. NG-nitro-L-arginine (L-NNA) abolished NO production in both preparations but only partly inhibited VIP release (45 +/- 8% at 8 Hz and 59 +/- 10% at 10 g stretch) and relaxation (62 +/- 5% and 35 +/- 6%); the effect of L-NNA was reversed by L-arginine but not D-arginine. The pattern implied that NO production normally acts to enhance VIP release. In addition, VIP induced relaxation and stimulated NO production in muscle strips and isolated colonic muscle cells: L-NNA abolished NO production but only partly inhibited relaxation (58 +/- 6%); oxyhemoglobin had no effect. The effect of L-NNA on relaxation was reversed by L-arginine but not by D-arginine. The protein kinase A inhibitor (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS] and the protein kinase G inhibitor KT5823 inhibited VIP-induced relaxation by 76 +/- 5 and 35 +/- 4%, respectively; a combination of the two inhibitors abolished relaxation. (R)-p-cAMPS blocked the direct relaxant effect of VIP, whereas KT5823 blocked the indirect effect of VIP mediated by NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Activation of distinct cAMP- and cGMP-dependent pathways by relaxant agents in isolated gastric muscle cells.

The mechanism of action of vasoactive intestinal peptide (VIP) was examined in isolated gastric and taenia coli muscle cells and compared with that of nitric oxide (NO), sodium nitroprusside (SNP), and isoproterenol. In gastric muscle cells, VIP stimulated NO production, increased adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels, and induced relaxation in a concentration-dependent fashion. The NO synthase inhibitor NG-nitro-L-arginine abolished NO and cGMP production and partly inhibited relaxation. The soluble guanylate cyclase inhibitor LY 83583 abolished cGMP production and partly inhibited relaxation. (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], a preferential inhibitor of cAMP-dependent protein kinase (cAK), and KT5823, a preferential inhibitor of cGMP-dependent protein kinase (cGK), partly inhibited relaxation separately and abolished relaxation in combination. The pattern implied that VIP induced relaxation by activation of cAK and by NO-mediated stimulation of cGMP and activation of cGK. In taenia coli muscle cells, VIP did not increase NO production or cGMP levels: relaxation was accompanied by an increase in cAMP and was partly inhibited by (R)-p-cAMPS and KT5823 and abolished by a combination of both inhibitors. Isoproterenol increased only cAMP levels in both cell types, which induced relaxation by activating cAK at low concentrations of agonist and both cAK and cGK at high concentrations in a pattern identical to that observed with VIP in taenia coli muscle cells. SNP and NO increased only cGMP levels in both cell types, which induced relaxation by activating cGK only. We conclude that cAK and cGK can be activated separately and mediate relaxation independently.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoquinolines↗

PubMed1993

Functional difference between SP and NKA: relaxation of gastric muscle by SP is mediated by VIP and NO.

The mechanism of action of endogenous tachykinins [substance P (SP) and neurokinin A and B (NKA and NKB)] and of receptor-specific tachykinin analogues (SP methyl ester (SPME), [beta-Ala8]NKA-(4-10), and senktide) was examined in circular muscle of guinea pig stomach. Cross-desensitization studies confirmed that SPME and SP interacted with NK-1 receptors, [beta-Ala8]NKA-(4-10) and NKA with NK-2 receptors, and senktide and NKB with NK-3 receptors. NK-1 and NK-3-receptor agonists induced relaxation and stimulated vasoactive intestinal peptide (VIP) release and nitric oxide (NO) production: tetrodotoxin abolished VIP release, NO production, and relaxation, converting the response to NK-1-receptor agonists to contraction; the NO synthase inhibitor NG-nitro-L-arginine (L-NNA) abolished NO production, partly inhibited VIP release (56-64%, P < 0.01), and abolished relaxation; the VIP antagonist VIP-(10-28) partly inhibited NO production (73-74%, P < 0.001) and relaxation (56-58%, P < 0.01); and atropine augmented relaxation by 28-35% (P < 0.01). The pattern of inhibition implied that: 1) relaxation was mediated by VIP and NO; 2) VIP release was partly dependent on NO production, since it was strongly inhibited by L-NNA; and 3) NO was largely produced by the action of VIP on muscle cells, since it was strongly inhibited by VIP-(10-28). NK-2-receptor agonists elicited only contraction that was not affected by tetrodotoxin; these agonists also inhibited VIP release, NO production, and relaxation induced by NK-1- and NK-3-receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Inhibition of IP3 and IP3-dependent Ca2+ mobilization by cyclic nucleotides in isolated gastric muscle cells.

The mechanisms by which cAMP and cGMP and agents that stimulate one (isoproterenol and nitroprusside) or both cyclic nucleotides (VIP) decrease cytosolic free Ca2+ ([Ca2+]i) and inhibit contraction were examined in dispersed, intact, and saponin-permeabilized gastric muscle cells. In these cells, the [Ca2+]i transient responsible for initial contraction is mediated by inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release (K. N. Bitar, P. G. Bradford, J. W. Putney, Jr., and G. M. Makhlouf, Science Wash. DC 232: 1143-1145, 1986, and J. Biol. Chem. 261: 16591-16596, 1986). In intact muscle cells, dibutyryl cAMP and all three relaxant agents inhibited contraction, [Ca2+]i, and net Ca2+ efflux (i.e., Ca2+ release) in a concentration-dependent fashion. In permeabilized muscle cells, cAMP, cGMP, and all three relaxant agents 1) inhibited cholecystokinin (CCK)-induced IP3 production (maximal 38-48%), 2) inhibited CCK- and IP3-induced Ca2+ efflux (maximal 55-59%) and contraction (maximal 59-66%), and 3) stimulated Ca2+ uptake (maximal 25-30%), in a concentration-dependent fashion. cAMP and cGMP were equipotent inhibitors of IP3 production and of CCK- and IP3-induced Ca2+ efflux and contraction, whereas cGMP was distinctly more potent as a stimulant of Ca2+ uptake. For all functions, maximal effects induced by cAMP and cGMP were similar to those induced by the three relaxant agents. Inhibition of Ca2+ release was the main determinant of inhibition of contraction; stimulation of Ca2+ uptake was relatively minor (< 5% of Ca2+ efflux). Decrease in IP3 production did not contribute to inhibition of Ca2+ efflux and contraction since inhibition of IP3-induced Ca2+ efflux was similar to inhibition of CCK-induced IP3-dependent Ca2+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

VIP-mediated G protein-coupled Ca2+ influx activates a constitutive NOS in dispersed gastric muscle cells.

Vasoactive intestinal peptide (VIP) and peptide histidine-isoleucine (PHI) receptors and the signaling pathways to which they are coupled were characterized in dispersed gastric smooth muscle cells. Radioligand binding using 125I-labeled VIP and PHI identified 4 classes of receptors: VIP-preferring and PHI-preferring receptors recognized by both ligands and readily desensitized by the preferred ligand, and VIP-specific and PHI-specific receptors recognized by only 1 ligand and resistant to desensitization. All except VIP-specific receptors were coupled to adenylate cyclase. VIP-specific receptors mediated a G protein-coupled Ca2+ influx that led to activation of NO synthase (NOS), NO-dependent activation of soluble guanylate cyclase, and activation of guanosine 3',5'-cyclic monophosphate (cGMP) kinase resulting in muscle relaxation. The entire cascade was blocked by Ca2+ channel and/or calmodulin antagonists. The NOS inhibitor NG-nitro-L-arginine abolished L-[3H]citrulline (coproduct of NO synthesis) and cGMP generation and partly inhibited (52 +/- 4%) relaxation. The components of response mediated by VIP-specific receptors (increase in [Ca2+]i, L-[3H]citrulline, and cGMP) were preserved after desensitization. Insertion of guanosine 5'-O-(beta-thio)diphosphate into reversibly permeabilized muscle cells abolished responses mediated by VIP-preferring and VIP-specific receptors. VIP stimulated both adenosine 3',5'-cyclic monophosphate (cAMP)-kinase and cGMP-kinase activities consistent with stimulation of cAMP and cGMP. Both kinases contributed to relaxation that was partly inhibited by cAMP-kinase [H-89 and (R)-p-adenosine 3',5'-cyclic monophosphorothioate] and cGMP-kinase (KT-5823) inhibitors and abolished by a combination of the 2 types of inhibitors. We conclude that VIP-specific receptors mediate a G protein-coupled Ca2+ influx leading to activation of a constitutive Ca2+/calmodulin-dependent NOS and generation of NO, which is partly responsible for relaxation in smooth muscle.

Amino Acid Oxidoreductases↗

PubMed1993

Coexistence of histamine H1 and H2 receptors coupled to distinct signal transduction pathways in isolated intestinal muscle cells.

Histamine receptors were characterized in muscle cells isolated from the longitudinal muscle layer of guinea pig intestine. Histamine caused concentration-dependent contraction and stimulated significant increases in [Ca++]i (141 +/- 16 nM, P < .001) and cyclic AMP (cAMP) (10.3 +/- 0.5 pmol/10(6) cells, P < .001). The H1 receptor antagonist, mepyramine, inhibited contraction and the increase in [Ca++]i, whereas the H2 receptor antagonist, cimetidine, augmented contraction and the increase in [Ca++]i (277 +/- 31 nM, P < .01) and abolished the increase in cAMP. In cells maximally contracted with a nonhistamine agonist [cholecystokinin octapeptide (CCK)], histamine caused concentration-dependent relaxation when the contractile effect mediated by H1 receptors was blocked with mepyramine; relaxation was inhibited by cimetidine in a concentration-dependent fashion. The effects mediated by H1 and H2 receptors were characterized further in cells in which only one receptor type was preserved by selective receptor protection. In cells in which only H1 receptors were preserved, histamine caused contraction that was inhibited by mepyramine; no relaxation of CCK-induced contraction could be elicited by histamine in the presence of mepyramine. Conversely, in cells in which only H2 receptors were preserved, histamine had little or no contractile effect by itself but caused relaxation of CCK-induced contraction in the absence of mepyramine. We conclude that in, intestinal muscle cells, H1 receptors mediating Ca(++)-dependent contraction coexist with H2 receptors mediating cAMP-dependent relaxation. The effect of histamine reflects activation of dominant H1 receptors as well as of H2 receptors which act to attenuate [Ca++]i and contraction by stimulating an increase in intracellular cAMP.

Animals↗

PubMed1993

Characterization of distinct receptors for the peptidyl leukotrienes LTC4 and LTD4/LTE4 coupled to the same signaling pathway in isolated gastric muscle cells.

Receptors for the peptidyl leukotrienes, (LT)C4, LTD4 and LTE4, and the signaling pathways to which they are coupled were characterized in isolated guinea pig gastric muscle cells. The three LTs were equipotent contractile agonists (EC50 values = 0.10-0.12 nM), but they elicited their responses by interacting with distinct receptors. The contractile responses to LTD4 and LTE4, but not LTC4, were inhibited by the LTD4 antagonist, SKF 104353 [2-(S)-hydroxy-3-(R)-[(2-carboxyethyl)thiol]-3-[2-(8- phenyloctyl)phenyl]-propanoic acid]. Similar Ki estimates for SKF 104353 suggested interaction of LTD4 and LTE4 with a common receptor. Decisive evidence for distinct LTC4 and LTD4/LTE4 receptors was obtained by applying a receptor protection technique. Cells in which LTC4 was used as a receptor protective agent while other receptors were inactivated by N-ethylmaleimide retained their responses to LTC4 only. Cells in which LTD4, LTE4 or SKF 104353 were used as a receptor protective agent retained their responses to LTD4 and LTE4 only. Both LTC4 and LTD4/LTE4 receptors were coupled to Pl hydrolysis: all three LTs stimulated similar increases in inositol 1,4,5-trisphosphate (IP3) levels (3.9-4.3 pmol/10(6) cells), protein kinase C activity (85-94 pmol/mg/min) and cytosolic-free Ca++ ([Ca++]i) (278-306 nM). Contractile responses were abolished: 1) when Pl hydrolysis was inhibited by neomycin and 2) when Ca++ stores were depleted by pretreatment of muscle cells with caffeine in Ca(++)-free medium, but not when muscle cells were incubated in Ca(++)-free medium or with Ca++ channel blockers, suggesting that contraction and [Ca++]i were mediated by IP3-dependent Ca++ release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Collagen production by human smooth muscle cells isolated during intestinal organogenesis.

The extracellular matrix influences organogenesis by modulating cell behavior. In humans, collagen is the major matrix constituent of the adult intestinal wall and is synthesized by smooth muscle cells. The objective of the current study was to examine collagen production by fetal human intestinal smooth muscle cells isolated during intestinal morphogenesis. Techniques were developed for the isolation and culture of human fetal intestinal smooth muscle cells. The cultured cells were confirmed as muscle by immunohistochemical stains for cytoskeletal filaments and documentation of contractile behavior. In culture, these cells stained for mesenchymal and muscle cytoskeletal proteins: vimentin, actin, and desmin, and did not stain for neural or epithelial markers. The muscle cells contracted in response to acetylcholine, in contrast to human fetal dermal fibroblasts which did not contract appreciably. Collagen production was assayed by the uptake of [3H]-proline into collagenase-digestible protein. Collagen production was greatest at 11 weeks gestation, the youngest age studied. By 20 weeks gestation, collagen production had decreased to adult levels. However, when compared to another matrix-producing fetal mesenchymal cell, the dermal fibroblast, intestinal smooth muscle cells produced twice as much collagen. Collagen types were determined by polyacrylamide slab gel electrophoresis. Smooth muscle cells predominantly produced types I and III collagen alpha chains. Therefore, collagen production is a significant function of human fetal intestinal smooth muscle cells, and probably plays a major role in the development of intestinal structure. The in vitro model presented here provides a means of studying the regulation of this collagen production throughout intestinal organogenesis.

Collagen↗

PubMed1992

Enteric GABA: mode of action and role in the regulation of the peristaltic reflex.

The mode of action of gamma-aminobutyric acid (GABA) and the role of myenteric GABA neurons in the regulation of peristalsis were examined in various preparations of rat colonic muscle. GABA had no contractile, relaxant, or modulatory effect on smooth muscle cells isolated from the circular muscle layer. In innervated circular muscle strips, GABA elicited concentration-dependent relaxation accompanied by release of vasoactive intestinal peptide (VIP). Relaxation and VIP release were inhibited by tetrodotoxin and by the GABAA receptor antagonist bicuculline but not by the GABAB receptor antagonist phaclofen. Relaxation was inhibited by the VIP receptor antagonist VIP-(10-28) implying that VIP release was coupled to muscle relaxation. Relaxation was augmented by atropine implying that GABA also activated cholinergic neurons causing release of acetylcholine that attenuated the relaxant response. This pharmacological profile was evident when GABA was released from intrinsic GABA neurons during peristalsis induced by radial stretch. Blockade of GABAA receptors with bicuculline inhibited the descending relaxation mediated by VIP motor neurons and the ascending contraction mediated by cholinergic motor neurons. Stimulation of these receptors with exogenous GABA had the opposite effect. We conclude that on release from myenteric neurons, GABA acts via GABAA receptors on cholinergic and VIP motor neurons responsible for the two components of the peristaltic reflex.

Animals↗

PubMed1992

Stimulation of nitric oxide from muscle cells by VIP: prejunctional enhancement of VIP release.

The source of nitric oxide (NO) and its role in neurally induced relaxation was examined in smooth muscle of the stomach and tenia coli. Field stimulation of gastric muscle strips was accompanied by frequency-dependent relaxation, vasoactive intestinal peptide (VIP) release, and NO production: the NO synthase inhibitor, NG-nitro-L-arginine (L-NNA) completely inhibited NO production and partly inhibited VIP release (52-54%) and relaxation (58-88%); inhibition of all three functions was reversed by L-arginine but not by D-arginine. In isolated gastric muscle cells, VIP caused relaxation and stimulated NO production: L-NNA completely inhibited NO production and partly inhibited relaxation; the inhibition was reversed by L-arginine but not by D-arginine. Abolition of NO production with only partial inhibition of relaxation implied that NO production from muscle cells induced by the action of VIP was partly responsible for relaxation. By contrast, field stimulation of tenia coli was accompanied by relaxation and VIP release but not by NO production. Neither VIP release nor relaxation was affected by L-NNA. In isolated muscle cells of tenia coli, VIP caused relaxation but did not stimulate NO production; relaxation in these cells was not affected by L-NNA. We conclude that 1) VIP is the primary relaxant transmitter in both gastric muscle and tenia coli, 2) the release of VIP in gastric muscle but not in tenia coli stimulates NO production from target muscle cells, and 3) NO amplifies the relaxant effect of VIP in muscle cells and acts presynaptically to enhance the release of VIP.

Animals↗

PubMed1992

Receptor-coupled G proteins mediate contraction and Ca++ mobilization in isolated intestinal muscle cells.

Guanosine 5'-O-(gamma-thio)triphosphate (GTP[S]), NaF and cholecystokinin-octapeptide (CCK-8) were used to examine the participation of G proteins in agonist-induced contraction of smooth muscle cells isolated separately from circular and longitudinal muscle layer of guinea pig intestine. All three agents stimulated inositol 1,4,5-triphosphate (InsP3) production and protein kinase C activity to the same extent in permeabilized (GTP[S] and CCK-8) and nonpermeabilized (NaF and CCK-8) muscle cells. InsP3 production was 9 to 13 times higher in circular muscle cells consistent with preferential hydrolysis of phosphatidylinositol 4,5-biphosphate in this cell type. InsP3 production and protein kinase C activation in permeabilized muscle cells were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM). Maximal concentrations of GTP[S] (100 microM), CCK-8 (1 nM) and InsP3 (1 microM) elicited similar increases in [Ca++]i, net 45Ca++ efflux and contraction in permeabilized circular, but not longitudinal, muscle cells [( Ca++]i: 224 +/- 35 nM, 279 +/- 29 nM and 288 +/- 45 nM increase above basal level; 45Ca++ efflux: 35 +/- 2%, 34 +/- 3% and 37 +/- 3% decrease in cell Ca++ content; contraction: 26 +/- 2%, 24 +/- 2% and 25 +/- 2% decrease in cell length). The responses to GTP[S] and CCK-8 were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM) and heparin (10 micrograms/ml), whereas the response to InsP3 was abolished by heparin only. Maximal concentrations of NaF and CCK-8 elicited similar increases in [Ca++]i and contraction in nonpermeabilized circular and longitudinal muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Coexistence of contractile and relaxant 5-hydroxytryptamine receptors coupled to distinct signaling pathways in intestinal muscle cells: convergence of the pathways on Ca2+ mobilization.

Muscle cells were dispersed separately from circular and longitudinal muscle layers of guinea pig intestine, and 5-hydroxytryptamine (5-HT) receptors were characterized in naive cells and in cells in which one receptor type was preserved by selective receptor protection. In naive cells from both regions, 5-HT caused contraction and stimulated increases in cytosolic free calcium concentration ([Ca2+]i) (3-fold; p < 0.01) and cAMP levels (40-60%; p < 0.01) that were inhibited, respectively, by the 5-HT2 antagonist ketanserin and the 5-HT1p antagonist N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP). In circular muscle cells, where agonist-induced increase in [Ca2+]i is mediated by Ca2+ release from inositol (1,4,5)trisphosphate-sensitive stores, 5-HT caused an increase in inositol (1,4,5)trisphosphate levels that was inhibited by ketanserin. In cells maximally contracted with a non-5-HT agonist (cholecystokinin octapeptide), 5-HT caused relaxation when the contractile effect mediated by 5-HT2 receptors was blocked with ketanserin; relaxation and the concomitant increase in cAMP were inhibited by 5-HTP-DP. The singular contributions of the Ca2+ and cAMP signaling pathways were identified in cells where only one receptor type was preserved. In cells with only 5-HT2 receptors, 5-HT caused contraction and an increase in [Ca2+]i but not in cAMP levels; contraction and the increase in [Ca2+]i were inhibited by ketanserin. Conversely, in cells with only 5-HT1p receptors, 5-HT caused relaxation and an increase in cAMP levels but not in [Ca2+]i; relaxation and the increase in cAMP levels were inhibited by 5-HTP-DP. The two signaling pathways were functionally linked, converging to regulate the level of [Ca2+]i. Thus, the increase in [Ca2+]i was augmented 1) when cAMP production was inhibited by 5-HTP-DP in naive cells or 2) when cAMP production was suppressed in cells where 5-HT1p receptors were inactivated and only 5-HT2 receptors were preserved. The results imply that the increase in cAMP levels mediated by 5-HT1p receptors acted to attenuate the increase in [Ca2+]i mediated by 5-HT2 receptors. We conclude that the response to 5-HT in muscle cells is a compound effect involving activation of two receptor types coupled to distinct signaling pathways that converge on [Ca2+]i as the determinant of mechanical activity.

5-Hydroxytryptophan↗

PubMed1991

Identification of opioid receptors on gastric muscle cells by selective receptor protection.

Opioid receptors on isolated gastric smooth muscle cells were characterized pharmacologically by a technique in which synthetic selective opioid agonists and antagonists were used to protect and thus enrich a specific receptor type while all other receptors were inactivated by N-ethylamaleimide. Treatment of the cells with the selective mu-receptor agonist DAGO or antagonist CTAP preserved only the response to DAGO; treatment with the selective delta-receptor agonist DPDPE or antagonist naltrindole preserved only the response to DPPE; and treatment with the selective kappa-receptor agonist U50,488H or antagonist nor-binaltorphimine preserved only the response to U50,488H. The results established the presence of distinct kappa-, delta-, and mu-opioid receptors capable of mediating contraction of isolated gastric muscle cells. The pattern of interaction of endogenous opioid peptides with protected receptors implied that dynorphin-(1-13) and Met-enkephalin were selective agonists for kappa- and delta-opioid receptors, respectively, and Leu-enkephalin a preferential agonist of mu-opioid receptors. The results were confirmed by a reverse approach in which opioid receptors were inactivated by site-directed irreversible antagonists. beta-Funaltrexamine, a mu-selective antagonist, abolished the response to mu-receptor agonists, whereas beta-chlornaltrexamine, a mu- and kappa-selective antagonist, abolished the response to mu-receptor agonists and partially inhibited the response to kappa-receptor agonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1991

Inhibition of muscle cell relaxation by somatostatin: tissue-specific, cAMP-dependent, pertussis toxin-sensitive.

The direct action of somatostatin on smooth muscle was examined in muscle cells isolated from the stomach and intestine of human and guinea pig. Somatostatin inhibited relaxation in gastric but not intestinal muscle cells of the two species, and its mechanism of action was explored in more detail in gastric muscle cells of the guinea pig. Somatostatin inhibited relaxation induced by vasoactive intestinal peptide (VIP, 83 +/- 7%, P less than 0.001) and isoproterenol (85 +/- 5%, P less than 0.001), as well as the concomitant increase in adenosine 3',5'-cyclic monophosphate (cAMP) production [81 +/- 25% inhibition with VIP (P less than 0.02) and 68 +/- 12% inhibition with isoproterenol (P less than 0.01)]. Inhibition of relaxation and cAMP production was abolished by pretreatment of the cells with pertussis toxin. Relaxation induced by the permeant derivative of cAMP, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate, by sodium nitroprusside, which acts by increasing levels of guanosine 3',5'-cyclic monophosphate, or by ATP, which acts by opening of K+ channels, was not affected by somatostatin. The fact that inhibition by somatostatin and its reversal by pertussis toxin was confined to agonists that stimulate an increase in the levels of cAMP implied that somatostatin acts by inhibiting the generation and not the action of cAMP. It is concluded that somatostatin receptors on gastric muscle cells mediate inhibition via a GTP-binding, pertussis-sensitive regulatory protein, Gi, coupled to adenylate cyclase.

Adenosine Triphosphate↗

PubMed1991

InsP3-dependent Ca2+ mobilization in circular but not longitudinal muscle cells of intestine.

The presence and functional coupling of inositol 1,4,5-trisphosphate (InsP3) receptors was examined in permeabilized muscle cells and microsomes isolated separately from the circular and longitudinal muscle layers of guinea pig intestine. Specific binding of [3H]InsP3 occurred only in circular muscle cells and microsomes; binding to longitudinal muscle cells or microsomes was minimal or absent. Binding to circular muscle cell microsomes was rapid (steady state in 2 min), saturable, reversible, and temperature independent, and was inhibited in a concentration-dependent manner by unlabeled InsP3 (IC50 10 nM) and by the InsP3 receptor antagonist heparin (EC50 4 micrograms/ml); inhibition by InsP4 and InsP2 was minimal. Consistent with the preferential location of InsP3 receptors, InsP3 elicited concentration-dependent contraction (EC50 3 nM), 45Ca2+ efflux (EC50 55 nM), and increase in [Ca2+]i in circular muscle cells and 45Ca2+ efflux in microsomes; all these events were blocked by heparin (10 micrograms/ml). Although ATP-dependent Ca2+ stores were present in longitudinal muscle cells and microsomes, they were not sensitive to 1 microM InsP3, which was maximally effective in circular muscle cells; 100 microM InsP3 elicited partial Ca2+ release (30-50%) and contraction (47%) that could only be blocked by high concentrations of heparin (100 micrograms/ml). We conclude that specific, high-affinity, heparin-sensitive InsP3 receptors capable of mobilizing intracellular Ca2+ are present predominantly in circular muscle cells of the intestine.

Animals↗

PubMed1990

Receptor type for cholecystokinin on isolated intestinal muscle cells of the guinea pig.

Smooth muscle cells isolated from the longitudinal muscle layer of guinea pig ileum were used to determine the presence and type of cholecystokinin/gastrin receptor mediating contraction. This was accomplished with a series of cholecystokinin and gastrin agonists (CCK-8, des(SO3)CCK-8, gastrin-17, des(SO3)gastrin-17 and pentagastrin) and antagonists (glutaramic acid derivatives CR 1392, CR 1409, CR 1505 and proglumide). The order of potency of agonists based on EC50 values derived from concentration-response curves was: CCK-8 greater than des(SO3)CCK-8 greater than gastrin-17 greater than des(SO3)gastrin-17. The inhibitory dissociation constant (Ki) for the antagonist CR 1505 derived from Schild plots was the same whether sulfated CCK-8 or desulfated gastrin-17 was used as agonist (4.47 +/- 0.76 versus 4.68 +/- 0.78 nM). Pentagastrin acted as a partial agonist and inhibited partially the response to CCK-8. The Ki values determined for all antagonists with pentagastrin as agonist were similar to those with CCK-8 as agonist. The order of potency of agonists and the independence of Ki values from the type of agonist used implied that CCK and gastrin interact with one receptor type; the receptor is more sensitive to CCK-8 but is minimally influenced by sulfation of the tyrosine residue. In this respect, the receptor appears to be distinct from the CCK receptor on gallbladder muscle cells and pancreatic acinar cells.

Animals↗