M2 and M3 muscarinic receptor activation of urinary bladder contractile signal transduction. II. Denervated rat bladder

Normal rat bladder contractions are mediated by the M(3) muscarinic receptor subtype. The M(2) receptor subtype mediates contractions of the denervated, hypertrophied bladder. This study determined signal transduction mechanisms mediating contraction of the denervated rat bladder. Denervated bladder muscle strips were exposed to inhibitors of enzymes thought to be involved in signal transduction in vitro followed by a cumulative carbachol concentration-response curve. Outcome measures were the maximal contraction, the potency of carbachol, and the affinity of darifenacin for inhibition of contraction. Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) with 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH(3)) has no effect on denervated bladder contractions, whereas inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) with O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609) attenuates the carbachol maximum and potency. Inhibition of rho kinase with (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632) reduces carbachol maximum, carbachol potency, and increases darifenacin affinity. Inhibition of rho kinase, protein kinase A (PKA), and protein kinase G (PKG) with 1-(5-isoquinolinesulfonyl)-homopiperazine.HCl (HA-1077) reduces the carbachol maximum and potency. Inhibition of PKC with chelerythrine increases darifenacin affinity, whereas inhibition of rho kinase, PKA, PKG, and protein kinase C (PKC) with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine.2HCl (H7) reduces the carbachol potency while increasing darifenacin affinity. Inhibition of rho kinase, PKA, and PKG with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H89) increases darifenacin affinity. This study demonstrates that different signal transduction mechanisms mediate the contractile response in the denervated rat bladder than in normal rat bladder. In normal rat bladder, PI-PLC and PC-PLC mediate the contraction, but in denervated bladder only PC-PLC is involved. In the denervated bladder, the rho kinase pathway is more dominant than in normal bladders. PKA seems to mediate a contractile response in normal bladders, whereas it seems to inhibit contraction in denervated bladders.     

Signal transduction underlying carbachol-induced contraction of human urinary bladder

The present study was designed to reexamine the muscarinic acetylcholine receptor subtype mediating carbachol-induced contraction of human urinary bladder and to investigate the underlying signal transduction. Based upon the nonselective tolterodine, the highly M(2)-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M(3)-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M(3) receptors. The phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122) (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. The phospholipase D inhibitor butan-1-ol relative to its negative control butan-2-ol (0.3% each) caused small but detectable inhibition of carbachol-induced bladder contraction. The Ca(2+) entry blocker nifedipine (10-100 nM) strongly inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SK&F 96,365) (1-10 microM), an inhibitor of store-operated Ca(2+) channels, caused little inhibition. The protein kinase C inhibitor bisindolylmaleimide I (1-10 microM) did not significantly affected carbachol-induced bladder contraction. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 microM), an inhibitor of rho-associated kinases, concentration dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of human urinary bladder via M(3) receptors largely depends on Ca(2+) entry through nifedipine-sensitive channels and activation of a rho kinase, whereas phospholipase D and store-operated Ca(2+) channels contribute only in a minor way. Surprisingly, phospholipase C or protein kinase C do not seem to be involved to a relevant extent.     

Alterations of glucose-dependent and -independent bladder smooth muscle contraction in spontaneously hypertensive and hyperlipidemic rat

Alteration of bladder contractility was examined in the spontaneously hypertensive and hyperlipidemic rat (SHHR; age, 9 months; systolic blood pressure, >150 mm Hg; plasma cholesterol, >150 mg/dl). Carbachol (CCh) induced time- and dose-dependent contractions in Sprague-Dawley (age-matched control) rats and SHHR; however, maximal levels differed significantly (13.3 +/- 2.2 and 5.4 +/- 1.9 microN/mm(2) following 10 microM CCh treatment, respectively; n = 5). This difference, which was maintained in calcium-replaced physiological salt solution (PSS), was suppressed by pretreatment with rho kinase inhibitor, 1 microM Y27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide]; moreover, total activity of rho kinase was also reduced in SHHR bladder. Pretreatment of bladders under high-glucose (HG) conditions (22.2 mM glucose-contained PSS for 30 min) led to enhancement of CCh-induced contraction solely in control animals. Under HG conditions, both protein kinase C (PKC) activity and production of diacylglycerol (DG) derived from incorporated glucose declined in SHHR bladder; however, sustained elevation of plasma glucose level was not detected in SHHR. These results suggested that bladder contractility dysfunction in SHHR is attributable to alteration of rho kinase activity and the DG-PKC pathway. This dysfunction may occur prior to chronic hyperglycemia onset in progressive hypertension and hyperlipidemia.     

Functional selectivity of muscarinic receptor antagonists for inhibition of M3-mediated phosphoinositide responses in guinea pig urinary bladder and submandibular salivary gland

Binding and functional affinities of the muscarinic acetylcholine (mACh) receptor antagonists darifenacin, tolterodine, oxybutynin, and atropine were assessed in Chinese hamster ovary (CHO) cells expressing the human recombinant M2 (CHO-m2) or M3 (CHO-m3) receptors, and in guinea pig bladder and submandibular gland. In [N-methyl-3H]scopolamine methyl chloride binding studies in CHO cells, darifenacin displayed selectivity (14.8-fold) for the M3 versus M2 mACh receptor subtype. Oxybutynin was nonselective, whereas atropine and tolterodine were weakly M2-selective (5.1- and 6.6-fold, respectively). Antagonist functional affinity estimates were determined by the inhibition of agonist-induced [3H]inositol phosphate accumulation in CHO-m3 cells and antagonism of the agonist-induced inhibition of forskolin-stimulated cyclic AMP accumulation in CHO-m2 cells. Darifenacin was the most M3-selective antagonist (32.4-fold), whereas oxybutynin, atropine, and tolterodine exhibited lesser selectivity. Functional affinity estimates in guinea pig urinary bladder and submandibular salivary gland using indices of phosphoinositide turnover revealed that oxybutynin, darifenacin, and tolterodine each displayed selectivity for the response in the bladder, relative to that seen in the submandibular gland (9.3-, 7.9-, and 7.4-fold, respectively). In contrast, atropine displayed a similar affinity in both tissues. These data demonstrate that in bladder, compared with submandibular gland from a single species, the mACh receptor antagonists darifenacin, tolterodine, and oxybutynin display selectivity to inhibit agonist-mediated phosphoinositide responses. It is proposed that both responses are mediated via M3 mACh receptor activation and that differential functional affinities displayed by some, but not all, antagonists are indicative of the influence of cell background upon the pharmacology of the M3 mACh receptor.     

Signal transduction underlying carbachol-induced contraction of rat urinary bladder. II. Protein kinases

We have investigated the role of several protein kinases in carbachol-stimulated, M(3) muscarinic receptor-mediated contraction of rat urinary bladder. Concentration-response curves for the muscarinic receptor agonist carbachol were generated in the presence of multiple concentrations of inhibitors of various protein kinases, their inactive controls, or their vehicles. Bladder contraction was not significantly inhibited by three protein kinase C inhibitors (chelerythrine, 1-10 microM; calphostin C, 0.1-1 microM; and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (G? 6850), 1-10 microM), by the tyrosine kinase inhibitor genistein or its inactive control daidzein (3-30 microM each), or by two inhibitors of activation of mitogen-activated protein kinase [10-100 microM 2'-amino-3'-methoxyflavone (PD 98,059) and 3-30 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 124)] or their negative control 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 126) (3-30 microM). Although high concentrations of wortmannin (3-30 microM) inhibited bladder contraction, this was not mimicked by another inhibitor of phosphatidylinositol-3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294,002) (3-30 microM) and, hence, was more likely due to direct inhibition of myosin light chain kinase by wortmannin than to an involvement of phosphatidylinositol-3-kinase. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 microM), an inhibitor of rho-associated kinases, concentration-dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of rat urinary bladder does not involve protein kinase C, phosphatidylinositol-3-kinase, tyrosine kinases, or extracellular signal-regulated kinases; in contrast, rho-associated kinases appear to play an important role in the regulation of bladder contraction.     

The role of several kinases in mice tolerant to delta 9-tetrahydrocannabinol

It has been suggested that the cannabinoid receptor type 1 (CB1), a G protein-coupled receptor, is internalized after agonist binding and activation of the second messenger pathways. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor, thus contributing to tolerance. Alterations in phosphorylation of proteins in the signal transduction cascade after CB1receptor activation could also alter tolerance to cannabinoids. We addressed our hypothesis by evaluating the role of several kinases in antinociceptive tolerance to Delta(9)-tetrahydrocannabinol (THC). We evaluated cAMP-dependent protein kinase (PKA) using KT5720, a PKA inhibitor; protein kinase C (PKC) using bisindolylmaleimide I, HCl (bis), a PKC inhibitor; cGMP-dependent protein kinase (PKG) using KT5823, a PKG inhibitor; beta-adrenergic receptor kinase (beta-ARK) using low molecular weight heparin (LMWH), a beta-ARK inhibitor; and phosphatidylinositol-3 kinase (PI3-K) using 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a PI3-K inhibitor and PP1, a Src family tyrosine kinase inhibitor. The cAMP analog used was dibutyryl-cAMP and the cGMP analog used was dibutyryl-cGMP. Our data indicate that selective kinases may be involved in cannabinoid tolerance. Mice and rats were rendered tolerant to Delta(9)-THC. The PKG inhibitor KT5823, the beta-ARK inhibitor LMWH, the PI3-K inhibitor LY294002, and inhibition of PKC by bis had no effect on tolerance. At a higher dose, bis attenuated the antinociceptive effect of delta(9)-THC in nontolerant mice. PP1, the Src family tyrosine kinase inhibitor, and KT5720, the PKA inhibitor, reversed THC-induced tolerance. In addition, inhibition of PKA reversed a decrease in dynorphin release shown to accompany THC tolerance in rats. These data support a role for PKA and Src tyrosine kinase in phosphorylation events in delta(9)-THC-tolerant mice.     

Signal transduction underlying carbachol-induced contraction of rat urinary bladder. I. Phospholipases and Ca2+ sources

We have reexamined the muscarinic receptor subtype mediating carbachol-induced contraction of rat urinary bladder and investigated the role of phospholipase (PL)C, D, and A2 and of intra- and extracellular Ca2+ sources in this effect. Based on the nonsubtype-selective tolterodine, the highly M2 receptor-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]-piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M3 receptor-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M3 receptors. Carbachol stimulated inositol phosphate formation in rat bladder slices, and this was abolished by the phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122; 10 microM). Nevertheless, U 73,122 (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. Carbachol had only little effect on PLD activity in bladder slices, but the PLD inhibitor butan-1-ol, relative to its negative control butan-2-ol (0.3% each), caused detectable inhibition of carbachol-induced bladder contraction. The cytosolic PLA2 inhibitor arachidonyltrifluoromethyl ketone weakly inhibited carbachol-induced contraction at a concentration of 300 microM, but the cyclooxygenase inhibitor indomethacin (1-10 microM) remained without effect. The Ca2+ entry blocker nifedipine (10-100 nM) almost completely inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96,365; 10 microM), an inhibitor of store-operated Ca2+ channels, caused little inhibition. We conclude that carbachol-induced contraction of rat bladder largely depends on Ca2+ entry through nifedipine-sensitive channels and, perhaps, PLD, PLA2, and store-operated Ca2+ channels, whereas cyclooxygenase and, surprisingly, also PLC are not involved to a relevant extent.     

The M2 muscarinic receptor mediates contraction through indirect mechanisms in mouse urinary bladder

We investigated the contractile role of M2 muscarinic receptors in mouse urinary bladder. When measured in the absence of other agents, contractions elicited to the muscarinic agonist oxotremorine-M exhibited properties consistent with that expected for an M3 response in urinary bladder from wild-type and M2 knockout (KO) mice. Evidence for a minor M2 receptor-mediated contraction was revealed by a comparison of responses in M3 knockout and M2/M3 double knockout mice. Treatment of wild-type and M2 knockout urinary bladder with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard) caused a large inhibition of the muscarinic contractile response. The residual contractions were much smaller in M2 knockout bladder compared with wild type, suggesting that M2 receptors rescue the muscarinic contractile response in wild-type bladder following inactivation of M3 receptors with 4-DAMP mustard. When measured in the presence of prostaglandin F2alpha and isoproterenol or forskolin, oxotremorine-M mediated a potent contractile response in urinary bladder from M3 KO mice. This response exhibited an M2 profile in competitive antagonism studies and was completely absent in M2/M3 KO mice. Following 4-DAMP mustard treatment, oxotremorine-M elicited a contractile response in wild-type urinary bladder in the presence of KCl and isoproterenol or forskolin, and this response was diminished in M2 KO mice. Our results show that the M2 receptor mediates contractions indirectly in the urinary bladder by enhancing M3 receptor-mediated contractions and inhibiting relaxation. We also show that it is difficult to detect M2 receptor function in competitive antagonism studies under conditions where a simultaneous activation of M2 and M3 receptors occurs.     

Pharmacological evidence for distinct muscarinic receptor subtypes coupled to the inhibition of adenylate cyclase and to the increased generation of inositol phosphates in the guinea pig myometrium

In the guinea pig myometrium, muscarinic receptor activation leads to contraction and elicits two biochemical responses viz. an increased formation of inositol phosphates (via a guanine nucleotide regulatory protein, distinct from the stimulatory and inhibitory G proteins of the adenylate cyclase system and a decreased synthesis of cyclic AMP involving inhibitory G protein activation. We now describe two major differences in the effects of muscarinic agonists. First, the greater potency of carbachol in inhibiting cyclic AMP formation (EC50 = 8 nM) than in stimulating the accumulation of inositol phosphates and tension (EC50 = 15 and 2 microM, respectively). Second, carbachol, oxotremorine and pilocarpine were equally effective in eliciting cyclic AMP inhibition but the order of potency for inositol phosphate formation was carbachol greater than oxotremorine and pilocarpine was without effect. The partial agonists, pilocarpine and oxotremorine, inhibited carbachol-mediated inositol phosphate formation. Pirenzepine, selective for muscarinic M1 receptor subtype, displayed a low affinity for antagonizing cyclic AMP inhibition, inositol phosphate generation and tension due to carbachol (Ki = 286, 92 and 110 nM, respectively). AF-DX116 (11-[( 2-[(diethylamino)methyl]-1- piperidinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine- 6-one), selective for cardiac M2 receptors blocked cyclic AMP inhibition with high affinity (Ki = 1.14 nM) while it antagonized inositol phosphate formation with low affinity (Ki = 346 nM). Both high (Ki = 1 nM) and low (Ki = 100 nM) affinities were displayed by AF-DX116 in antagonizing contractions due to carbachol (24 and 76% inhibition, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase A- and C-dependent modulation of murine inducible nitric oxide synthase

Effects of pharmacological modulation of protein kinase A, C and G (PKA, PKC and PKG) were examined on inducible form of nitric oxide synthase (iNOS) expressed in COS cells to elucidate regulatory mechanism of iNOS by protein kinases. Formation of nitric oxide (NO), as an index of NOS activity, was assessed by measurement of nitrite in incubation medium in long term observation and by hemoglobin assay method in kinetic study. In long term observation (18 hours), activation of PKA by 8-Br-cAMP increased NO formation that was inhibited by N-(2-[p-bromocinnamylamino] ethyl)-5-isoquinolinesulfonamide (H89). Though activation of PKC by 12-O-tetradecanoyl phorbol-13-acetate (TPA) decreased NO formation, PKC inhibitor, chelerythrine, failed to inhibit the decrease. Activation of PKG with 8-Br-cGMP and inhibition with KT5823 resulted in no change in NO formation. Western blot analysis revealed that neither 8-Br-cAMP nor TPA affect iNOS expression. In kinetic study (short term perfusion study), no change in NO formation was observed by 8-Br-cAMP and TPA. These results indicate that, in living cells, PKG does not play a regulatory role in iNOS activity and that PKA and PKC do not directly modulate iNOS activity. However, PKA and PKC would possibly modify NOS activity indirectly via cofactors necessary for NO formation.     

Neuronally released acetylcholine acts on the M2 muscarinic receptor to oppose the relaxant effect of isoproterenol on cholinergic contractions in mouse urinary bladder

We investigated whether M(2) muscarinic receptor activation opposes isoproterenol-induced relaxation in mouse urinary bladder and whether endogenous acetylcholine acts through a similar M(2) mechanism. When measured in urinary bladder from M(3) receptor knockout mice, the muscarinic agonist oxotremorine-M elicited only very weak contractions. In the presence of alpha,beta-methylene ATP (30 microM) and isoproterenol (1 microM), however, oxotremorine-M elicited a robust contractile response. This response was completely absent in bladder from M(2)/M(3) double knockout mice, indicating that activation of the M(2) receptor inhibits the relaxant effect of isoproterenol on the contraction to alpha,beta-methylene ATP. Similar results were obtained when prostaglandin F(2alpha) (5 microM) was used as the contractile agent but not when serotonin was used. Electrical field stimulation of the urinary bladder from wild-type mouse elicited contractions that were inhibited 20% by atropine and 40% by desensitization with alpha,beta-methylene ATP. When measured in the presence of alpha,beta-methylene ATP to desensitize the purinergic component of contraction, isoproterenol exhibited moderately greater relaxant activity in field-stimulated bladder from the M(2) knockout mouse compared with that observed in wild-type bladder. This differential relaxant effect of isoproterenol was greatly increased in the presence of physostigmine. In contrast, no differential effects were noted for isoproterenol in similar experiments on bladders from M(3) knockout and M(2)/M(3) double knockout mice in the presence of physostigmine. Our results suggest that neuronally released acetylcholine acts on the M(2) muscarinic receptor to inhibit the relaxant effect of isoproterenol on the minor, cholinergic component of contraction in the field-stimulated mouse urinary bladder.     

Cyclic nucleotide-dependent protein kinases.

The actions of several hormones and neurotransmitters evoke signal transduction pathways which rapidly elevate the cytosolic concentrations of the intracellular messengers, cAMP and cGMP. The cyclic-nucleotide dependent protein kinases, cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), are the major intracellular receptors of cAMP and cGMP. These enzymes become active upon binding respective cyclic nucleotides and modulate a diverse array of biochemical events through the phosphorylation of specific substrate proteins. The focus of this review is to describe the progress made in understanding the structure and function of both PKA and PKG.     

Role of protein kinase C in parathyroid hormone stimulation of renal 1,25-dihydroxyvitamin D3 secretion.

PTH is a major regulator of renal proximal tubule 1,25(OH)2D3 biosynthesis. However, the intracellular pathways involved in PTH activation of the mitochondrial 25-hydroxyvitamin D3-1 alpha-hydroxylase (1-OHase) remain unknown. PTH can activate both the adenylate cyclase/protein kinase A (PKA) and the plasma membrane phospholipase C/protein kinase C (PKC) pathways. The present study was undertaken to determine whether PKC may mediate PTH activation of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase activity. Rat PTH 1-34 fragment in vitro translocated PKC activity from cytosolic to soluble membrane fraction from freshly prepared rat proximal tubules. Physiologic concentrations (10(-11)-10(-10) M) of rat PTH 1-34 fragment increased PKC translocation three- to fourfold while PKA activity ratio increased at PTH 10(-7) M. PTH stimulation of PKC and PKA was reduced in the presence of staurosporine (10 nM) by 41 and 29%, respectively. Sangivamycin (10 and 50 microM) also reduced PTH-stimulated PKC translocation, but did not alter PKA activity ratio. In vitro perifusion of renal proximal tubules with PTH (10(-11) M) increased 1,25(OH)2D3 steady-state secretion two- to fourfold. Sangivamycin at the same concentration that inhibited PKC translocation by 52% completely inhibited PTH-stimulated 1,25(OH)2D3 secretion. The present studies indicate that the phospholipase C/PKC pathway may mediate PTH stimulation of mammalian renal proximal tubule 1,25(OH)2D3 secretion.     

蛋白激酶C和蛋白激酶G对失血性休克大鼠血管平滑肌细胞钙敏感性的调节作用

目的探讨蛋白激酶C(PKC)、蛋白激酶G(PKG)对失血性休克大鼠血管平滑肌细胞钙敏感性的调节作用。方法经股动脉放血使平均动脉压维持在40mmHg(1mmHg=0.133kPa)2h制备失血性休克大鼠模型。采用离体血管环张力测定技术,测定在失血性休克大鼠肠系膜上动脉(SMA)血管环去极化状态下(120mmol/LK+)对梯度浓度Ca2+的收缩反应性(钙敏感性)变化;同时观察PKC、PKG活性调节剂对钙敏感性的影响。测定大鼠SMA的PKC、PKG活性变化,分析PKC、PKG活性变化与血管钙敏感性变化间的关系。结果休克2hSMA血管环对钙敏感性明显降低,其量-效曲线明显右移,最大收缩力(Emax)明显降低(P均<0.01)。PKC激动剂PMA1×10-7mol/L可明显提高休克血管环对钙敏感性,PKC拮抗剂staurosporine1×10-7mol/L可降低休克血管环对钙敏感性(P<0.05或P<0.01);PKG激动剂8Br-cGMP1×10-4mol/L可使休克血管钙敏感性降低,其拮抗剂KT-58231×10-6mol/L可提高休克血管钙敏感性(P均<0.05)。休克2hSMA的PKC活性明显降低,PKG活性明显升高(P<0.05和P<0.01),分别与休克血管钙敏感性变化呈正相关和负相关(P均<0.01)。结论PKC和PKG参与了失血性休克血管平滑肌细胞钙敏感性的调控,PKC可上调血管平滑肌细胞的钙敏感性,PKG可下调其钙敏感性。     

Protein kinase C and vascular smooth muscle contractility: effects of inhibitors and down-regulation

We have compared two different methods of attenuating protein kinase C (PKC) activity in vascular smooth muscle. First, the effects of two purported PKC inhibitors, staurosporine (stauro) and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], were examined on contractility of isolated, intact canine femoral artery. In arterial rings stauro was equipotent in relaxing contractions induced by phenylephrine (PE), phorbol-12,13-dibutyrate (PDBu) and KCl (IC50, 0.31 +/- 0.19; 0.35 +/- 0.2; and 0.34 +/- 0.16 microM). H-7, in comparison, was markedly less potent than stauro (IC50, 0.67 +/- 0.2, 2.33 +/- 0.24; and 6.5 +/- 5.5 microM for PE, PDBu and KCl, respectively). Pretreatment of tissues with 1 microM stauro suppressed tension development almost completely when PE and PDBu were the contractile agonists, and partially in K(+)-depolarized rings. H-7, in contrast, had no inhibitory effect on agonist-induced contraction. Neither basal nor K(+)-stimulated calcium influx was affected by 10 microM stauro. Second, prolonged exposure of canine carotid arterial rings to PDBu (1-100 nM for 24 hr), a means of depleting PKC from the tissue, caused dose-dependent attenuation of agonist-induced contractions. Preincubation with 100 nM PDBu caused complete inhibition of tension induced by norepinephrine (NE) and serotonin and partial inhibition of PDBu- and KCl-induced contractions. Lowering the concentration of PDBu during preincubation to 30, 10 or 1 nM reduced markedly the inhibitory effects. The inactive phorbolester 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD) had no effect on agonist-induced contractions. PKC activity was determined in rings contracted isometrically with PDBu or NE after prolonged exposure to vehicle, 4 alpha-PDD or PDBu.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased 45Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on 45Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.     

An analysis of the action of phorbol 12, 13-dibutyrate on mechanical activity in rat uterine smooth muscle

The effects of phorbol 12, 13-dibutyrate (PDB) on mechanical activity in the pregnant rat uterus were investigated in isolated strips. In Ca(++)-containing solution, PDB (2.5 x 10(-8) to 10(-6) M) increased in a concentration-dependent manner the amplitude of the electrically induced contraction, but had no effect on the resting tension. PDB (10(-7) M) had a dual action, stimulatory then inhibitory, on contractions evoked by K(+)-rich (40 mM K+) solution or oxytocin. The inhibitory effect appeared more rapidly and the percentage of inhibition was increased for 10(-6) M PDB, which in addition abolished completely oxytocin-induced contraction after 20 min of application. PDB also reduced the amplitude of transient contraction evoked by oxytocin in Ca(++)-free solution. In saponin-skinned strips, 10(-7) M PDB increased the contraction induced by pCa ranging from 7 to 6, whereas 10(-6) M PDB reduced all Ca(++)-activated contractions from pCa = 7 to pCa = 5. PDB had no significant effect on the Ca(++)-uptake and the Ca(++)-release mechanisms of the intracellular Ca(++)-store. All of the effects of PDB were antagonized by the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (2 x 10(-5) M). In addition, the inactive phorbol 13,20-diacetate (10(-8) to 10(-6) M) had no effect on the mechanical activity in uterus. These results suggest the existence of different sites of action of PDB in rat uterus, via the activation of protein kinase C: 1) contractile machinery; 2) potential-dependent Ca channels; and 3) phospholipase C.     

Constitutive activity and inverse agonism at the M2 muscarinic acetylcholine receptor

Introduction of a single-point mutation (Asn to Tyr) at position 410 at the junction between transmembrane domain 6 and the third extracellular loop of the human M(2) muscarinic acetylcholine (mACh) receptor generated a mutant receptor (N410Y) that possesses many of the hallmark features of a constitutively active mutant receptor. These included enhanced agonist binding affinity and potency, in addition to agonist-independent accumulation of [(3)H]inositol phosphates in cells coexpressing the chimeric Galpha(qi5) protein and the N410Y mutant M(2) mACh receptor. Constitutive activity was sensitive to inhibition by a range of muscarinic ligands, including those used clinically in the management of overactive bladder (oxybutynin, tolterodine, and darifenacin), indicating that these ligands behave as inverse agonists at the M(2) mACh receptor. Long-term (24-h) treatment of Chinese hamster ovary cells expressing the N410Y mutant M(2) mACh receptor with certain mACh receptor inverse agonists (atropine, darifenacin, and pirenzepine) elicited a concentration-dependent up-regulation of cell surface receptor expression. However, not all ligands possessing negative efficacy in the [(3)H]inositol phosphate accumulation assays were capable of significantly up-regulating receptor expression, perhaps indicating a spectrum of negative efficacies among ligands traditionally defined as mACh receptor antagonists. Finally, structurally distinct agonists exhibited differences in their relative potencies for the activation of Galpha(i/o) versus Galpha(s), consistent with agonist-directed trafficking of signaling at the N410Y mutant, but not at the wild-type M(2) mACh receptor. This indicates that the N410Y mutation of the M(2) mACh receptor alters receptor-G-protein coupling in an agonist-dependent manner, in addition to generating a constitutively active receptor phenotype.     

The use of occupation isoboles for analysis of a response mediated by two receptors: M2 and M3 muscarinic receptor subtype-induced mouse stomach contractions

Smooth muscle contains multiple muscarinic receptor subtypes, including M2 and M3. M2 receptors outnumber M3 receptors. Based on the potency of subtype selective anticholinergics, contraction is mediated by the M3 subtype. However, results from knockout (KO) mice show that the M2 receptor mediates approximately 45% of the contractile response produced by the M3 receptor. The traditional theory of one receptor mediating a response does not allow assessment of interactions between receptors when more than one receptor participates in a response. Our study was performed using a novel analysis method based on dual receptor occupancy to determine how M2 and M3 receptor subtypes interact to mediate contraction in mouse stomach. Cumulative carbachol concentration contractile responses were determined for wild-type, M2-KO, and M3-KO stomach body smooth muscle. Using affinity constants for carbachol at M2 and M3 cholinergic receptors, the concentration values were converted to fractional receptor occupation. The resulting occupation-effect relations showed maximum effects for the M2 and M3 subtypes, respectively. These occupation-effect relations allow determination of the additive (expected) isobole based on this dual occupancy, thereby providing a curve (mathematically derived) for comparison against the experimentally derived value in wild type. The actual values determined experimentally in the wild type were not statistically significantly different from that predicted by the isobole. This confirms that the interaction between these mutually occupied receptors is additive. The new method of analysis also expands the traditional Schild theory that was based on a single receptor type to which the agonist and antagonist bind.     

Comparison of functional antagonism between isoproterenol and M2 muscarinic receptors in guinea pig ileum and trachea

The ability of the M2 muscarinic receptor to mediate an inhibition of the relaxant effects of forskolin and isoproterenol was investigated in guinea pig ileum and trachea. In some experiments, trachea was first treated with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) mustard to inactivate M3 receptors. The contractile response to oxotremorine-M was measured subsequently in the presence of both histamine (10 microM) and isoproterenol (10 nM). Under these conditions, [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3b]-[1,4]benzodiazepine-6-one (AF-DX 116) antagonized the contractile response to oxotremorine-M in a manner consistent with an M3 mechanism. However, when the same experiment was repeated using forskolin (4 microM) instead of isoproterenol, the response to oxotremorine-M exhibited greater potency and was antagonized by AF-DX 116 in a manner consistent with an M2 mechanism. We also measured the effects of pertussis toxin treatment on the ability of isoproterenol to inhibit the contraction elicited by a single concentration of either histamine (0.3 microM) or oxotremorine-M (40 nM) in both the ileum and trachea. Pertussis toxin treatment had no significant effect on the potency of isoproterenol for inhibiting histamine-induced contractions in the ileum and trachea. In contrast, pertussis toxin treatment enhanced the relaxant potency of isoproterenol against oxotremorine-M-induced contractions in the ileum but not in the trachea. Also, pertussis toxin treatment enhanced the relaxant potency of forskolin against oxotremorine-M-induced contractions in the ileum and trachea. We investigated the relaxant potency of isoproterenol when very low, equi-effective (i.e., 20-34% of maximal response) concentrations of either histamine or oxotremorine-M were used to elicit contraction. Under these conditions, isoproterenol exhibited greater relaxant potency against histamine in the ileum but exhibited similar relaxant potencies against histamine and oxotremorine-M in the trachea. Following 4-DAMP mustard treatment, a low concentration of oxotremorine-M (10 nM) had no contractile effect in either the ileum or trachea. Nevertheless, in 4-DAMP mustard-treated tissue, oxotremorine-M (10 nM) reduced the relaxant potency of isoproterenol against histamine-induced contractions in the ileum, but not in the trachea. We conclude that in the trachea the M2 receptor mediates an inhibition of the relaxant effects of forskolin, but not isoproterenol, and the decreased relaxant potency of isoproterenol against contractions elicited by a muscarinic agonist relative to histamine is not due to activation of M2 receptors but rather to the greater contractile stimulus mediated by the M3 receptor compared with the H1 histamine receptor.