Biomedical subjects

K S Murthy

Publications and source records attributed to K S Murthy.

At least 55 records · Page 3Linked to original sources

PubMed1995

Coexpression of 5-HT2A and 5-HT4 receptors coupled to distinct signaling pathways in human intestinal muscle cells.

BACKGROUND & AIMS: The type and function of 5-hydroxytryptamine (5HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. METHODS: Contraction, relaxation, inositol 1,4,5-triphosphate (IP3) and adenosine 3',5'-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. RESULTS: 5-HT binding was completely inhibited by 5-HT and partially by 5-HT2A (ketanserin), 5-HT4 (SDZ-205,557), and 5-HT1p (N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP3, which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only 5-HT4/5-HT1p receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. CONCLUSIONS: 5-HT2A receptors mediating contraction and 5-HT4 receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT4 receptors are closely similar or identical to 5-HT1p receptors.

4-Aminobenzoic Acid↗

PubMed1995

Surgical treatment of transposition of great arteries with interrupted pulmonary artery: a rare association of embryopathies.

The first case of TGA with VSD, ASD, and interrupted LPA and its successful surgical treatment is being reported along with a review of the relevant literature. The dominant presenting clinical features have been explained and an attempt has been made to explain the paucity of records in regard to this embryopathy. An arterial switch for TGA and a glutaraldehyde pretreated autologous pericardial roll for repair of interrupted LPA were carried out. Pre- and postoperative hemodynamics and angiography data are also presented.

Ductus Arteriosus, Patent↗

PubMed1995

Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells.

The signaling pathways mediating relaxation by vasoactive intestinal peptide (VIP), peptide histidine-isoleucine amide (PHI), isoproterenol (ISO), and sodium nitroprusside (SNP) were examined in dispersed rabbit and guinea pig gastric muscle cells. In rabbit muscle cells, SNP stimulated only guanosine 3',5'-cyclic monophosphate (cGMP) and cGMP-dependent protein kinase (cG-kinase) activity; VIP stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP, and both cG-kinase and cAMP-dependent protein kinase (cA-kinase) activities; PHI and ISO stimulated only cAMP and cA-kinase activity, and at higher concentrations, cross-activated cG-kinase. All four agents elicited concentration-dependent relaxation. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1 microM) selectively inhibited cA-kinase activity and abolished relaxation when only cA-kinase was elevated. 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c,d,e)- trinden-1-one (KT-5823; 1 microM) selectively inhibited cG-kinase activity and abolished relaxation when only cG-kinase was elevated. When both kinases were elevated, H-89 and KT-5823 partially inhibited relaxation and abolished relaxation in combination. In permeabilized guinea pig and rabbit muscle cells, all agents elicited relaxation and inhibited inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Both functions were inhibited in parallel fashion by protein kinase inhibitor PKI(6-22) and by KT-5823. We conclude that cA-kinase and cG-kinase act separately and in concert to inhibit IP3-dependent Ca2+ release and induce relaxation.

Animals↗

PubMed1995

Functional characterization of phosphoinositide-specific phospholipase C-beta 1 and -beta 3 in intestinal smooth muscle.

Soluble and membrane phosphoinositide-specific phospholipases obtained separately from dispersed circular and longitudinal intestinal muscle cells were characterized for substrate specificity and G protein dependence using selective antibodies to various isoforms of phospholipase C (PLC) and G protein subunits. Western blot analysis disclosed the presence of the main PLC isozymes, PLC-gamma 1, PLC-delta 1, and PLC-beta 1. Soluble PLC from circular and longitudinal muscle was stimulated by guanosine 5'-O-(3-thiophosphate) and inhibited by PLC-beta 1 antibody (80-90%) and PLC-beta 3 antibody (approximately 25%) but not by G protein antibodies. Membrane PLC from circular and longitudinal muscle was stimulated by cholecystokinin octapeptide (CCK-8) and inhibited selectively by PLC-beta 1 antibody (85%), PLC-beta 3 antibody (15%), and G alpha q/11 antibody (90%). CCK-8-induced contraction in permeabilized circular muscle cells was also selectively inhibited by PLC-beta 1 antibody (76%), PLC-beta 3 antibody (24%), and G alpha q/11 antibody (86%). The combined effects of PLC-beta 1 and PLC-beta 3 antibodies on PLC activity and muscle contraction were additive, causing complete inhibition. Soluble and membrane PLC from circular and longitudinal muscle were immunologically similar but functionally different. The enzymes from circular muscle preferentially hydrolyzed endogenous and exogenous phosphatidylinositol 4,5-biphosphate (PIP2), confirming previous findings of preferential hydrolysis of PIP2 in dispersed intestinal circular muscle cells.

Animals↗

PubMed1995

Agonist-mediated activation of PLA2 initiates Ca2+ mobilization in intestinal longitudinal smooth muscle.

Recent studies have shown that Ca2+ mobilization in longitudinal muscle is initiated by inositol 1,4,5-trisphosphate (IP3)-independent Ca2+ influx that acts as a trigger for Ca(2+)-induced Ca2R release. The present study examined whether arachidonic acid (AA) acts as mediator of the initial Ca2+ influx. Cholecystokinin octapeptide caused transient concentration-dependent increase in AA release in dispersed intestinal longitudinal but not circular muscle cells followed by sustained increase in both muscle cell types. The initial increase in AA release coincided with the initial Ca2+ transient and muscle contraction: all three events were abolished by guanosine 5'-O-(2-thiodiphosphate), pertussis toxin (PTX), and the phospholipase A2 (PLA2) inhibitor, dimethyleicosadienoic acid, but were not affected by calphostin C or neomycin. Exogenous AA caused concentration-dependent contraction and increase in cytosolic free Ca2+ ([Ca2+]i) in longitudinal but not circular muscle cells; both events were abolished by Ca2+ channel blockers. Depletion of Ca2+ stores with thapsigargin attenuated with thapsigargin attenuated agonist- and AA-mediated increase in [Ca2+]i and contraction in longitudinal muscle cells: the residual [Ca2+]i increase (35%) and contraction (25%) reflected the component of Ca2+ influx. We conclude that AA released by agonist-mediated G protein-dependent PTX-sensitive activation of PLA2 mediates Ca2+ influx, which then triggers Ca(2+)-induced Ca2+ release. The process is independent of phosphatidylinositol hydrolysis and occurs exclusively in longitudinal smooth muscle, in which Ca2+ release channels are highly sensitive to Ca2+, ryanodine, and cyclic ADP-ribose and insensitive to IP3.

Animals↗

PubMed1995

Adenosine A1 receptor-mediated activation of phospholipase C-beta 3 in intestinal muscle: dual requirement for alpha and beta gamma subunits of Gi3.

Four native and cloned adenosine receptors (ARs), designated A1AR, A2aAR, A2bAR, and A3AR, have been characterized functionally and by radioligand binding. In the present study, we have used selective antibodies to identify the G protein subunits and phospholipase C (PLC)-beta isoform coupled to A1ARs in smooth muscle membranes and permeabilized muscle cells from rabbit intestine. Immunoblot analysis disclosed the presence of a full complement of G proteins. Adenosine caused contraction of dispersed muscle cells and increases in D-myo-inositol-1,4,5-trisphosphate, intracellular calcium, and cAMP levels. Contraction and the increases in D-myo-inositol-1,4,5-trisphosphate and intracellular calcium levels were abolished by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine and augmented by the A2 antagonist CGS-15943; the reverse occurred with cAMP. A selective A1AR agonist, cyclopentyladenosine, inhibited forskolin-stimulated cAMP accumulation; the inhibition was reversed by treatment of the cells with pertussis toxin or a G alpha i3-specific antibody. The pattern of inhibition implied coexistence of A1ARs and A2ARs coupled to interactive signaling pathways, with A2ARs mediating activation of adenylyl cyclase and A1ARs mediating activation of PLC and inhibition of adenylyl cyclase. Adenosine-stimulated PLC activity in muscle membranes was selectively blocked by G alpha i3- and G beta-specific antibodies, as well as by a PLC-beta 3-specific antibody, but not by antibodies to other PLC-beta isoforms or G proteins. A combination of maximally effective concentrations of G alpha i3- and G beta-specific antibodies did not elicit greater inhibition than did either alone. In contrast, cholecystokinin-stimulated PLC activity was selectively blocked by PLC-beta 1- and G alpha q/11-specific antibodies. Adenosine-stimulated contraction and 45Ca2+ efflux in permeabilized muscle cells were also selectively blocked by G alpha i3-, G beta-, and PLC-beta 3-specific antibodies, whereas cholecystokinin-stimulated contraction was selectively blocked by PLC-beta 1- and G alpha q/11-specific antibodies. The results indicate that A1ARs are coupled to PLC-beta 3 via both alpha and beta gamma subunits of Gi3.

Adenylyl Cyclase Inhibitors↗

PubMed1995

Adenosine A1 and A2b receptors coupled to distinct interactive signaling pathways in intestinal muscle cells.

Adenosine receptors and the signaling pathways to which they are coupled were examined in dispersed intestinal muscle cells. The receptors were characterized by their ability to induce contraction or relaxation, mobilize Ca2+ and stimulate or inhibit cAMP, in naive cells and in cells where only one receptor type was preserved by selective receptor protection. Adenosine elicited contraction and increased [Ca2+]i and cAMP; the contraction was mimicked by the A1 selective agonist, cyclopentyladenosine. A selective A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine, and pertussis toxin abolished contraction and the increase in [Ca2+]i and augmented the increase in cAMP. Conversely, a preferential A2 antagonist, 9-chloro-2-(2-furyl) [1,2,4]triazolo[1,5-c]quinazolin-5-amine augmented contraction and the increase in [Ca2+]i and abolished the increase in cAMP; a cAMP-kinase inhibitor, Rp-cAMP[S], had a similar effect, augmenting contraction and the increase in [Ca2+]i. Adenosine elicited also relaxation of maximally contracted cells that increased or decreased in parallel with cAMP. The selective A2a agonist, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine, was a very weak relaxant agent, and the selective A2a antagonist, 8-(3-chlorostyryl)caffeine, had no effect on adenosine-induced relaxation. In cells where only A1 receptors were preserved, the cAMP response to adenosine was abolished, although contraction and [Ca2+]i were increased to the same extent as when naive cells were treated with the A2 antagonist. Conversely, in cells where only A2 receptors were preserved, contraction and the increase in [Ca2+]i were abolished and the increase in cAMP was augmented to the same level as when naive cells were treated with the A1 antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1995

Agonist-mediated activation of phosphatidylcholine-specific phospholipase C and D in intestinal smooth muscle.

The contributions of phosphoinositide (PI)- and phosphatidylcholine (PC)-specific phospholipases [PI-specific phospholipase C (PI-PLC), PC-specific phospholipase C (PC-PLC), and phospholipase D (PLD)] to diacylglycerol (DAG) formation and regulation of the enzymes by G proteins, Ca2+, and protein kinase C (PKC) were examined in dispersed intestinal circular and longitudinal muscle cells. DAG formation induced by cholecystokinin was biphasic and paralleled by PKC activity. The initial phase (approximately 1 min) was mediated by PI-PLC in circular muscle cells and by both PI- and PC-PLC in longitudinal muscle cells, whereas the sustained phase was mediated by PC-PLC and PLD in both cell types. PC-PLC activity during the initial phase was identified by rapid formation of the initial products [3H]phosphocholine (5 sec) and [3H]myristate-labeled DAG (approximately 15 sec). PLD activity did not contribute to DAG formation during the initial phase, and PI hydrolysis had no effect on PC-PLC or PLD activity during the initial or sustained phases. PLD activity during the sustained phase was evident by the formation of [3H]phosphatidylethanol, a PLD-specific transphosphatidylation product. Dephosphorylation of phosphatidic acid (PA) by phosphatidate phosphohydrolase (PPH) accounted for about 50% of DAG formation; inhibition of PPH activity by propranolol or suppression of PA formation by ethanol inhibited DAG formation by 59-69% and 57-62%, respectively. Residual DAG in the presence of ethanol was augmented 55-57% by DAG kinase inhibitor, whereas residual PA was inhibited by 60-67%, implying that PA was derived from DAG, and DAG from PLC-mediated PC hydrolysis. In the presence of ethanol, calphostin C inhibited phosphatidylethanol formation but had no effect on PA or DAG levels, implying that only PLD activity was modulated by PKC. Maintenance of resting intracellular Ca2+ concentrations, rather than an agonist-induced increase in the intracellular Ca2+ concentration, was required for optimal PC-PLC and PLD activity. Guanosine-5'-O-(beta-thio)diphosphate abolished DAG and PA formation in reversibly permeabilized muscle cells. We conclude that DAG formation in intestinal muscle is mediated by time-dependent activation of three phospholipases (PI-PLC, PC-PLC, and PLD) and two converting enzymes (DAG kinase and PPH). PC-PLC and PLD are Ca2+ dependent and appear to be G protein coupled; only PLD is PKC sensitive.

Animals↗

PubMed1994

Fluoride activates G protein-dependent and -independent pathways in dispersed intestinal smooth muscle cells.

The existence of G protein-dependent and -independent mechanisms activated by sodium fluoride was examined in muscle cells isolated separately from the circular and longitudinal layers of guinea pig intestine. The cells were transiently permeabilized by incubation with Trans. Port Reagent in the presence or absence of GDP beta S (100 microM) and then re-sealed. In the absence of GDP beta S, NaF (1 mM) induced contraction and caused an increase in [Ca2+]i, IP3 and diacylglycerol levels and in protein kinase C (PKC) activity in both cell types. In the presence of GDP beta S, the increases in IP3, DAG and PKC were abolished whereas contraction and the increase in [Ca2+]i were partly inhibited. Residual contraction and [Ca2+]i were abolished by the Ca2+ channel blocker, methoxyverapamil. We conclude that contraction and Ca2+ mobilization induced by NaF is mediated by G protein activation as well as by a G protein-independent mechanism involving activation of plasmalemmal Ca2+ channels.

Animals↗

PubMed1994

Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide-dependent activation of membrane-bound NO synthase in smooth muscle mediated by pertussis toxin-sensitive Gi1-2.

Plasma membranes isolated from dispersed gastric muscle cells exhibited calmodulin-dependent NOS activity that was stimulated by Ca2+ in the range 0.1-1 mM (maximum 10 microM). Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) (in the presence of GTP), and GTP gamma S (guanosine 5'-O-(gamma-thio)triphosphate) stimulated NOS activity in a concentration-dependent fashion above that maximally stimulated by Ca2+. The increase in NOS activity induced by VIP, PACAP, and GTP gamma S was abolished by GDP beta S (guanosine 5'-O-(beta-thio)diphosphate), which had no effect on NOS activity stimulated by Ca2+. The NOS inhibitor NG-nitro-L-arginine and the calmodulin antagonist calmidazolium abolished NOS activity stimulated by all agents including Ca2+. NOS activity stimulated by GTP gamma S, VIP, and PACAP was inhibited by Gi alpha 1-2 antibody but not by Gq alpha, Gs alpha, and Gi alpha 3 antibodies. NOS activity stimulated by VIP and PACAP was inhibited by 80-83% in membranes derived from pertussis toxin-treated cells. We conclude that a Ca2+/calmodulin-dependent NOS present in plasma membranes of gastric muscle cells is activated by two homologous peptide transmitters, VIP and PACAP, via a common receptor coupled to pertussis toxin (PTx)-sensitive Gi1-2. The study provides the first evidence of receptor-mediated G protein activation of NOS in smooth muscle cells.

Adenylate Cyclase Toxin↗

PubMed1994

Agonist-activated, ryanodine-sensitive, IP3-insensitive Ca2+ release channels in longitudinal muscle of intestine.

We have previously shown that Ca2+ mobilization in longitudinal muscle is not mediated by inositol 1,4,5-trisphosphate (IP3) and depends on an obligatory influx of Ca2+. The present study examined whether Ca2+ influx activates ryanodine-sensitive Ca2+ channels to cause Ca(2+)-induced Ca2+ release. Ryanodine bound with high affinity to longitudinal muscle cells [dissociation constant (Kd) 7.3 +/- 0.3 nM] and microsomes (Kd 7.5 +/- 0.4 nM) and induced concentration-dependent 45Ca2+ efflux [50% effective concentration (EC50) 1.3 +/- 0.5 nM], increase in cytosolic free Ca2+ (EC50 2.0 +/- 0.7 nM), and contraction (EC50 0.9 +/- 0.2 nM) but had no effect in circular muscle cells. Ryanodine binding and ryanodine-induced Ca2+ release were enhanced by caffeine and inhibited by dantrolene and ruthenium red but were not affected by IP3 or heparin. Changes in Ca2+ concentration (50-500 nM) caused Ca2+ release from permeabilized longitudinal but not circular muscle cells loaded with 45Ca2+. The contractile agonist cholecystokinin-8 elicited 45Ca2+ efflux in both circular and longitudinal muscle cells; efflux in longitudinal muscle cells was abolished by Ca2+ channel blockers and by pretreatment of the cells with ryanodine. Pretreatment with thapsigargin abolished agonist-induced 45Ca2+ efflux in both cell types. We conclude that ryanodine-sensitive IP3-insensitive Ca2+ release channels with properties similar to those in cardiac muscle are present in longitudinal but not circular muscle cells of intestine and that agonist-mediated Ca2+ influx activates these channels, leading to Ca(2+)-induced Ca2+ release.

Animals↗

PubMed1994

Inhibition of nitric oxide synthase activity in dispersed gastric muscle cells by protein kinase C.

The present study examined whether NO synthase (NOS) activity in gastric muscle cells was inhibited by protein kinase C (PKC). Vasoactive intestinal peptide (VIP) increased L-[3H]citrulline production (a coproduct and index of NO synthesis) in muscle strips (81.9 +/- 11.6%) and dispersed muscle cells (80.9 +/- 4.6%) of rabbit stomach. Cholecystokinin octapeptide (CCK-8), carbachol, and phorbol 12-myristate 13-acetate (PMA) inhibited VIP-induced L-[3H]citrulline production in muscle cells and muscle strips; the inhibition was reversed by pretreatment with the PKC inhibitor, calphostin C. The Ca(2+)-mobilizing agents, CCK-8, acetylcholine, ionomycin, and KCl, all of which increased PKC activity in dispersed muscle cells, did not increase L-[3H]citrulline production. After treatment of the cells with calphostin C, all four agents stimulated L-[3H]citrulline production, although to a lesser extent than VIP (approximately 50%). VIP-induced relaxation of basal but not carbachol-stimulated tension was accompanied by increase in L-[3H]citrulline production and was inhibited by the NOS inhibitor NG-nitro-L-arginine (L-NNA). Preincubation of carbachol-treated muscle strips with calphostin C restored the ability of VIP to stimulate L-[3H]citrulline production and the ability of L-NNA to inhibit VIP-induced relaxation. We conclude that 1) VIP-stimulated NOS activity is inhibited by agents that increase PKC activity in gastric smooth muscle cells, and 2) agents that increase both cytosolic free Ca2+ concentration and PKC activity stimulate NOS activity only when PKC activity is suppressed.

Amino Acid Oxidoreductases↗

PubMed1994

Coexistence of three tachykinin receptors coupled to Ca++ signaling pathways in intestinal muscle cells.

Receptors for tachykinins and the signaling pathway to which they are coupled were characterized in dispersed muscle cells from the longitudinal muscle layer of the rat intestine. A technique of receptor protection whereby selective agonists and antagonists were used to protect one receptor while other receptors were inactivated with N-ethylmaleimide enabled each tachykinin receptor type to be identified separately. Protection of neurokinin (NK)-1 receptors with the selective NK-1 agonist, substance P methylester, or antagonist, GR-82,334 (Glp-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-D-Pro[spiro-gamma-lactam]Leu-Trp-NH2), preserved the contractile response and increase in cytosolic-free Ca++ ([Ca++]i) induced by substance P methylester only; protection of NK-2 receptors with the selective NK-2 agonist, beta-[Ala8]NKA(4-10), or the selective NK-2b antagonist, L-659,877 [cyclo(Leu-Met-Gln-Trp-Phe-Gly)], preserved the contractile response and increase in [Ca++]i induced by beta-[Ala8]NKA(4-10) only; and protection of NK-3 receptors with the selective NK-3 agonist, senktide succinyl-[Asp6,MePhe8]substance P(6-11), preserved the contractile response and increase in [Ca++]i induced by succinyl-[Asp6,MePhe8]substance P(6-11) only. When used as a protective agent, the NK-2a antagonist, MEN-10,376 (H-Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Lys-NH2), did not preserve the response to any tachykinin agonist. Protection of NK-1, NK-2 and NK-3 receptors preserved fully the responses to the preferential endogenous agonists, substance P, NKA and NKB, respectively, but they also preserved in part (30-40%) the responses to the nonpreferential agonists. Because substance P and NKA are coreleased from the same precursor in intestinal muscle tissue, the pattern implied the existence of considerable spareness in the contractile response of muscle cells to tachykinins. Studies on dispersed circular muscle cells using selective tachykinin agonists as protective agents confirmed the presence of three tachykinin receptor types. The results demonstrate the coexistence of NK-1, NK-2b and NK-3 receptors on muscle cells of rat intestine that are preferentially activated by substance P, NKA and NKB, respectively, and are coupled separately to one signaling pathway mediating contraction.

Amino Acid Sequence↗

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

Current perspectives on the dissolution stability of solid oral dosage forms.

Dissolution stability (i.e., retention of the dissolution characteristics of a solid oral dosage form from the time of manufacture up to its expiration date) is a critical parameter from the standpoint of quality control, regulatory compliance, and impact on the bioavailability of the product. Significant changes in the in vitro release profiles of a drug product during storage may alter its bioavailability. Factors that affect the dissolution stability of a product during aging include formulation components (active drug, excipients, and coating materials), processing factors, storage conditions, and packaging. The role of each of these factors in promoting changes in dissolution in both immediate-release and modified-release products is dependent on the product and has to be evaluated on a case-by-case basis. Although data obtained under accelerated conditions of storage are not useful in predicting the dissolution shelf-life of the product under ambient conditions, they are of value in assessing the "ruggedness" of the product and its ability to withstand the varied climatic conditions during transport, shipping, and storage. The clinical significance of alterations in the in vitro dissolution profiles that may occur during aging and strategies to avert and counteract such changes are discussed in the article.

Administration, Oral↗

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

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↗