Mechanism of the vascular angiotensin II/alpha2-adrenoceptor interaction

alpha(2)-Adrenoceptors potentiate vascular responses to angiotensin II. The goal of this study was to test the hypothesis that the phospholipase C (PLC)/protein kinase C (PKC)/c-src/phosphatidylinositol 3-kinase (PI3K) pathway contributes to the vascular angiotensin II/alpha(2)-adrenoceptor interaction. In rats in vivo, intrarenal infusions of angiotensin II (10 ng/kg/min) increased renal vascular resistance by 5.8 +/- 0.5 units, and this response was enhanced (p < 0.05) to 9.1 +/- 1.2 units by UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; 3 microg/kg/min; alpha(2)-adrenoceptor agonist]. Intrarenal infusions of U-73122 [1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione; 3 microg/min; PLC inhibitor], GF109203X [bisindolylmaleimide I; 10 microg/min; PKC inhibitor], CGP77675 [1-(2-{4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenyl}ethyl)piperidin-4-ol; 5 microg/min; c-src inhibitor], and wortmannin (1 microg/min; PI3K inhibitor) abolished the angiotensin II/alpha(2)-adrenoceptor interaction. In isolated perfused rat kidneys, angiotensin II (0.3, 1, and 3 nM) increased perfusion pressure (by 15 +/- 8, 39 +/- 4, and 93 +/- 9 mm Hg, respectively), and UK-14,304 (1 microM) potentiated these responses (to 36 +/- 4, 67 +/- 7, and 135 +/- 17 mm Hg, respectively). This angiotensin II/alpha(2)-adrenoceptor interaction was abolished by U-73122 (10 microM), GF109203X (3 microM), CGP77675 (5 microM), and wortmannin (0.2 microM). Preglomerular microvascular smooth muscle cells expressed phospholipase (PLC)-beta(2), PLC-beta(3), c-src, phospho(tyrosine 416)-c-src, and PI3K. In these cells, angiotensin II (0.1 microM) and UK-14,304 (1 microM) per se did not increase phospho-c-src; however, the combination of angiotensin II plus UK-14,304 doubled phospho-c-src, and this interaction was abolished by U-73122 (10 microM) and GF109203X (3 microM). In conclusion, the PLC/PKC/c-src/PI3K pathway may contribute importantly to the interaction between alpha(2)-adrenoceptors and angiotensin II on renal vascular resistance.     

Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL.

The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.     

Phorbol ester modulation of cyclic AMP accumulation in a primary culture of rat aortic smooth muscle cells

Over the past few years, the importance of calcium and cyclic AMP in the regulation of vascular smooth muscle tone has been well documented. We used a primary culture of rat aortic myocytes to study the effect of protein kinase C on isoproterenol- and forskolin-stimulated cyclic AMP production. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) to these cells, but not an inactive analog, increased the stimulation of cyclic AMP production induced with isoproterenol or forskolin without changes in the apparent affinity of these compounds but did not affect the basal cAMP level. TPA also enhanced the cholera toxin-stimulated cyclic AMP accumulation. Isoproterenol and cholera toxin increased the forskolin apparent potency suggesting that interaction of activatory GTP-dependent protein with the catalytic subunit of adenylate cyclase facilitates forskolin interaction to the catalytic subunit. Treatment of myocytes with pertussis toxin had no effect on the basal level of cyclic AMP production and did not significantly modify isoproterenol- and forskolin-induced stimulation. Pertussis toxin treatment of cells did not affect the TPA-enhanced isoproterenol or forskolin stimulations suggesting that pertussis toxin and TPA actions would not share a common target of myocyte adenylate cyclase system. Our data would be in agreement with a possible direct interaction of protein kinase C with the catalytic subunit of adenylate cyclase system.     

Inhibition of protein phosphorylation by opioid-inhibited adenylyl cyclase in rat brain membranes.

Opioid inhibition of adenylyl cyclase is a major second messenger system associated with opioid receptors in brain. To identify membrane phosphoproteins whose phosphorylation state is modulated by opioid inhibition of adenylyl cyclase, rat striatal membranes were preincubated with opioid agonists in the presence of 500 microM 5'-adenylyl-imidodiphosphate (which acted as a substrate for adenylyl cyclase, but not for protein kinase) before addition of [gamma-32P]ATP. Under these conditions, adenylyl cyclase in the membranes formed cyclic AMP, which stimulated cyclic AMP-dependent protein kinase. This process was confirmed by observing forskolin-stimulated phosphorylation of two bands of MW 85 and 63 kDa, which were also stimulated directly by cyclic AMP. Forskolin-stimulated phosphorylation of these two bands was inhibited by 15 to 30% by opioid agonists such as D-Ala2-Met5-enkephalinamide. This inhibition of phosphorylation was mediated by opioid receptors, because it required both sodium and GTP, and was blocked by naloxone. These results suggest that these two proteins may be primary targets of opioid-inhibited adenylyl cyclase in striatal membranes.     

Activation of protein kinase A increases phospholipase D activity and inhibits phospholipase D activation by acetylcholine in tracheal smooth muscle

Increased cAMP by stimulation of adenylyl cyclase with forskolin or by beta-adrenoceptor activation with isoproterenol increased phospholipase D (PLD) activity in tracheal smooth muscle strips. PLD activity was measured by the accumulation of phosphatidylethanol. A linear increase in the concentration of phosphatidylethanol was observed over 20 min in muscle strips treated with either forskolin or isoproterenol. Cholinergic stimulation with acetylcholine (ACh), by contrast, caused a rapid increase in phosphatidylethanol followed by a slow decline in the concentration of phosphatidylethanol from 5 to 20 min in the continued presence of ACh. Concomitant treatment with ACh and either forskolin or isoproterenol eliminated the rapid increases in phosphatidylethanol associated with ACh treatment. The response to forskolin or isoproterenol was not influenced by ACh. Inhibition of protein kinase C with calphostin C or bisindolylmaleimide I had no effect on isoproterenol- or forskolin-stimulated PLD activity but inhibited ACh-activated PLD activity. Protein kinase A (PKA) inhibitors H-89 and KT5720 significantly decreased forskolin- and isoproterenol-mediated activation of PLD activity. PKA inhibition also eliminated inhibition of ACh-stimulated PLD activity by forskolin or isoproterenol. Activation of adenylyl cyclase by forskolin or by isoproterenol caused increased phosphorylation of phospholipase C-beta(2) isoform and reduced the formation of inositol phosphates after ACh stimulation of muscarinic receptors. These results suggest that increasing the concentration of cAMP activates PLD via activation of PKA and that the increased activity of PKA also inhibits cholinergic stimulation of PLD, in part at least by inhibiting the activation of phospholipase C by ACh.     

Signal transduction pathways involved in the mechanical responses to protease-activated receptors in rat colon

Recording simultaneously in vitro the changes of endoluminal pressure (index of circular muscle activity) and isometric tension (index of longitudinal muscle activity), we examined the mechanisms responsible for the apamin-sensitive relaxant and contractile responses induced by protease-activated receptor (PAR)-1 and PAR-2 activating peptides, SFLLRN-NH2 and SLIGRL-NH2, respectively, in rat colon. In the circular muscle, the inhibitory effects of SFLLRN-NH2 and SLIGRL-NH2 were significantly reduced by ryanodine, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, but unaffected by 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X), a protein kinase C (PKC) inhibitor, or genistein, a tyrosine kinase inhibitor. In the longitudinal muscle, the contractile responses to SFLLRN-NH2 and SLIGRL-NH2 were significantly reduced by nifedipine, an L-type calcium channel blocker, ryanodine, GF109203X, genistein, and abolished by U73122. The effects of genistein were additive with GF109203X but not with nifedipine. In the longitudinal muscle, the relaxant responses to the highest concentrations of SFLLRN-NH2 and SLIGRL-NH2 were abolished by nifedipine, reduced by genistein, and unaffected by ryanodine or GF109203X. In conclusion, influx of extracellular Ca2+ through L-type voltage-dependent channels or release of Ca2+ from intracellular stores are determining for the opening of the apamin-sensitive K+ channels responsible for longitudinal muscle relaxation or circular muscle inhibitory response, respectively, in rat colon. The longitudinal muscle contraction is mediated by activation of PLC; PKC and tyrosine kinase are involved in the cascade process, playing a parallel role. Indeed, tyrosine kinase and L-type Ca2+ channels would act sequentially. The influx of Ca2+ in turn would cause release of Ca2+ from sarcoplasmic reticulum.     

The phospholipase C inhibitor U73122 inhibits phorbol ester-induced platelet activation

Activation of phospholipase C (PLC) is a central component of the signal transduction process in numerous cells, including platelets. U73122 has been widely used as a selective PLC inhibitor. In the present study, the effects of U73122 on platelet function have been further examined. Platelets were stimulated with collagen (via PLC-gamma), the stable thromboxane mimetic U46619 (via PLC-beta), or phorbol myristate acetate (PMA) via protein kinase C (PKC). Consistent with inhibition of PLC, U73122 inhibited platelet aggregation and [3H]-serotonin release in response to collagen and U46619 in a concentration-dependent manner. Similarly, U73122 blocked collagen-induced release of thromboxane A2. U73122 also inhibited U46619-induced [32P]phosphatidic acid production and phosphorylation of the major PKC substrate, pleckstrin. U73122 had no effect on PMA-induced pleckstrin phosphorylation, [3H]-serotonin release, or intracellular vacuole formation. However, U73122 did inhibit PMA-induced platelet aggregation and fibrinogen binding. Overall, these results suggest that U73122, in addition to its inhibition of PLC, also affects PKC-independent events that interfere with platelet aggregation.     

Cellular signaling by the potent bronchoconstrictor endothelin-1 in airway smooth muscle

OBJECTIVE: The objective of this study was to determine whether the potent bronchoconstrictor endothelin-1 was coupled to the activation of the inositol phosphate and/or inhibition of the cyclic adenine monophosphate second messenger pathways in porcine airway smooth muscle. DESIGN: Prospective, controlled, in vitro, nonblinded study. SETTING: University biochemical and molecular biological research laboratory. SUBJECTS: Pigs of both genders. INTERVENTIONS: Airway smooth muscle was dissected from the trachea of pigs exsanguinated under anesthesia. Airway smooth muscle from six animals preloaded with 3H-myoinositol was exposed to endothelin-1, carbachol (positive control) or vehicle for 30 mins. Some tissues were pretreated with antagonists selective for the ET(A) (BQ-485) and ET(B) (BQ-788) endothelin receptor subtypes. Newly synthesized 3H-inositol phosphates were recovered by column chromatography. Airway smooth muscle from an additional 7 pigs was homogenized and incubated in the presence of 32P-alpha-adenosine triphosphate, guanosine triphosphate (GTP) and either carbachol or endothelin to measure the inhibitory influence of carbachol (positive control) or endothelin on GTP-stimulated adenylyl cyclase activity. Newly synthesized 32P-cyclic adenosine monophosphate was isolated by sequential column chromatography over Dowex and alumina. MEASUREMENTS AND MAIN RESULTS: Total inositol phosphates increased in porcine airway smooth muscle in response to either carbachol or endothelin. The endothelin receptor antagonist BQ-485 (ET(A) selective) but not BQ-788 (ET(B) selective) dose-dependently inhibited endothelin-1 induced inositol phosphate accumulation. In adenylyl cyclase assays, carbachol (positive control), but not endothelin-1, significantly inhibited GTP-stimulated adenylyl cyclase activity. CONCLUSION: Endothelin-1 couples to the activation of the inositol phosphate pathway via the ET(A) receptor subtype but does not couple to inhibition of the adenylyl cyclase pathway in porcine airway smooth muscle. The potent bronchoconstrictive effects of endothelin likely involve the acute activation of the inositol phosphate pathway in airway smooth muscle.     

Tonic inhibitory role for cAMP in alpha(1a)-adrenergic receptor coupling to extracellular signal-regulated kinases 1/2

alpha(1a)-Adrenergic receptors (ARs) couple to phosphoinositide hydrolysis, adenylyl cyclase, and mitogen-activated protein kinase (MAPK) pathways. However, the interaction among these signaling pathways in activating extracellular signal-regulated kinase 1/2 (ERK1/2) is not well understood. We investigated the coupling of alpha(1a)-ARs to ERK1/2 in Chinese hamster ovary (CHO)-K1 cells stably transfected with mouse alpha(1a)-ARs, as well as the interaction between ERK1/2 and norepinephrine-induced cAMP accumulation. alpha(1a)-AR activation by norepinephrine increased the cytosolic Ca(2+) concentration and phosphorylated ERK1/2 in a time- and concentration-dependent manner. ERK1/2 phosphorylation was blocked by the MAPK kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD 98059) and the alpha(1)-AR antagonist prazosin. A transient elevation in intracellular Ca(2+) was required for the phosphorylation of ERK1/2; however, activation of protein kinase C did not seem to be required for ERK1/2 phosphorylation. Norepinephrine also stimulated cAMP accumulation in transfected CHO-K1 cells in a concentration-dependent manner via alpha(1a)-ARs, which was blocked by the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Norepinephrine-induced ERK1/2 phosphorylation was inhibited by the adenylyl cyclase activator forskolin and was enhanced by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ 22536) and the protein kinase A inhibitor 4-cyano-3-methylisoquinoline. In conclusion, in transfected CHO-K1 cells, alpha(1a)-AR activation activates both phospholipase C and adenylyl cyclase-mediated signaling pathways. alpha(1a)-AR-mediated ERK1/2 phosphorylation was dependent on a rise in intracellular Ca(2+), and this pathway was reciprocally regulated by the concomitant activation of adenylyl cyclase, which inhibits ERK1/2 phosphorylation. Thus, alpha(1a)-AR stimulation of cAMP production may play an important role in regulating ERK1/2 phosphorylation in cell lines and native tissues.     

Cyclic AMP and mechanisms of vasodilation

Cyclic AMP and the mechanism of vasodilation have been reviewed by first discussing the enzymes involved (adenyl cyclase, cyclic nucleotide phosphodiesterases, cyclic AMP-dependent protein kinase) and then agents that increase cAMP in smooth muscle. Two mechanisms of vasodilation are described: (i) effects on contractile proteins; (ii) effects on Ca2+ levels. Evidence for compartments of cAMP is also presented.     

In vivo activity of a phospholipase C inhibitor, 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), in acute and chronic inflammatory reactions

To investigate the role of phospholipase C (PLC) in inflammatory processes, we tested 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), a widely used PLC inhibitor, in several in vitro and in vivo assays. We first examined the effects of U73122 on human phospholipase C-beta (PLC-beta) isozymes and found that U73122 significantly inhibited recombinant human PLC-beta2, with an IC(50) of approximately 6 microM. U73122 had little effect on PLC-beta1, PLC-beta3, or PLC-beta4. Consistent with its ability to inhibit PLC-beta2 enzymatic activity, U73122 reduced interleukin-8 and leukotriene B(4)-induced Ca(2+) flux and chemotaxis in human neutrophils in a concentration-dependent manner. In vivo, U73122 blocked carrageenan-induced hind paw edema in rats, carrageenan-induced macrophage and lymphocyte accumulation into subcutaneous chambers in dogs, lipopolysaccharide-induced macrophage, lymphocyte infiltration and prostaglandin E(2) production in a mouse peritonitis model, and 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in mice. These results implicate PLC-dependent signaling pathways in the development of acute and chronic inflammatory responses in vivo.     

Ca2+-independent, inhibitory effects of cyclic adenosine 5'-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2alpha, Rho, and myosin phosphatase in vascular smooth muscle

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca(2+)-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca(2+)-induced Ca(2+)-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca(2+)-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca(2+) mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca(2+)-mediated Rho activation is dependent on class II alpha-isoform of phosphoinositide 3-kinase (PI3K-C2alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) in manners dependent on both PI3K and Rho kinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca(2+)-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2alpha, Rho, and Rho kinase in a manner independent of Ca(2+) and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.     

Involvement of P2Y receptors in pyridoxal-5'-phosphate-induced cardiac preconditioning

Using an isolated non‐working rat heart model, this study investigated the mechanisms of pharmacological pre‐conditioning (PC) induced by P2Y receptor stimulation with pyridoxal‐5′‐phosphate (PLP). After 6‐hydroxydopamine pretreatment and a 15‐min stabilization period, isolated rat hearts were perfused for 25 min then subjected to 40 min of global ischemia and 30 min of reperfusion (I/R); exposed for 15 min to 0.05 μm PLP bracketed for 25 min with broad‐spectrum P2 antagonists (suramin or PPADS) or with more specific P2Y antagonists (AMPαS or MRS2578), 1 μm each, followed by a 5‐min PLP‐free perfusion before I/R; treated during 25 min with either glybenclamide (GLY, 1 μm ), 5‐hydroxydecanoic acid (5‐HD, 100 μm ), U73122 (0.5 μm ), H89 (1 μm ), or KN93 (1 μm ), with an infusion starting 5 min before PLP. The main endpoints were the rate–pressure product (RPP), creatine kinase (CK) release and area necrosis. Recovery of RPP, measured 5 min after reperfusion, was rapidly improved by PLP, blocked by the P2 antagonists, and decreased with the different inhibitors. Fifteen minutes after the end of ischemia, CK release reached maximal values in all groups. PLP provided significant protection, whereas the P2 antagonists, 5‐HD, a mitochondrial selective K_ATP antagonist and GLY a non‐selective K_ATP channel blocker, suppressed the protective effect on myocardial injury. The suppression of the cardioprotective effects of PLP by AMPαS, the PKA inhibitor (H89), and phospholipase C blocker (U73122) is in agreement with the P2Y11 receptor as a receptor for PLP‐induced PC. The suppression of the cardioprotective effects of PLP by MRS2578 and U73122 is in agreement with the P2Y6 receptor as a receptor for PLP‐induced PC. Pre‐ischemic exposure to nanomolar concentrations of PLP is protective against I/R . P2Y11 and P2Y6 represents the most likely candidate receptors for PLP‐induced cardiac PC.     

The Impact of Streptozotocin-induced Diabetes Mellitus on Cyclic Nucleotide Regulation of Skeletal Muscle Amino Acid Metabolism in the Rat

The impact of diabetes on cyclic nucleotide-associated mechanisms regulating skeletal muscle protein and amino acid metabolism was assessed using epitrochlaris preparations from streptozotocin-induced diabetic rats. 1 nM epinephrine inhibited alanine and glutamine release from control preparations, but no inhibition was observed from diabetic preparations with <0.1 mM. 10 nM epinephrine stimulated lactate production from control muscle but stimulation in diabetic preparations was observed only at 0.1 mM. Serotonin inhibited amino acid release and stimulated lactate production equally in control and diabetic muscle. 0.1 mM epinephrine increased cyclic (c)AMP levels by 360% in control muscles, but these levels were increased only 83% in diabetic muscle. Basal-, fluoride-, and serotonin-stimulated adenylyl cyclase activities were equal in membrane preparations of diabetic and control muscle, but epinephrine-stimulated adenylyl cyclase was reduced by 60% in diabetic muscle. Carbamylcholine stimulation of alanine and glutamine release was blunted in diabetic preparations. Carbamylcholine increased cGMP levels in control but not in diabetic muscle. In diabetic muscle, guanylyl cyclase activity was 65% of control and the stimulation of cyclase activity by sodium azide was less in diabetic than control preparations. Added cGMP stimulated alanine and glutamine release from control, but not from diabetic muscle. These data suggest a loss of adrenergic and cholinergic responsiveness in diabetic muscle. Because amino acid release also showed a decreased responsiveness to added cAMP and cGMP, the presence of other derangements in the mechanism(s) of cyclic nucleotide regulation of muscle amino acid metabolism also seems likely.     

Amplification of the cyclic AMP response to forskolin in pheochromocytoma PC12 cells through adenosine A(2A) purinoceptors.

In this study, we present evidence on the ability of endogenous adenosine to modulate adenylyl cyclase activity in intact PC12 cells. The adenosine receptor antagonists PD 115199, xanthine amine congener, 8-cyclopentyl-1,3-dipropylxanthine, 8-(p-sulfophenyl)theophylline, and 3,7-dimethyl-1-propargylxanthine inhibited 10 microM forskolin-induced cyclic AMP (cAMP) accumulation, with IC(50) values of 2.76 +/- 1.16 nM, 17.4 +/- 1.08 nM, 443 +/- 1. 03 nM, 2.00 +/- 1.01 microM, and 2.25 +/- 1.05 microM, respectively. Inhibition by 2.5 nM PD 115199 was only partially reversed by increasing forskolin concentrations up to 100 microM. The addition of PD 115199 with or 60 min after forskolin caused a comparable inhibition of forskolin effect over the next hour. Both exogenous adenosine (0.1 microM) and its precursor, AMP (10 and 100 microM), significantly enhanced forskolin-induced cAMP accumulation, whereas inosine was ineffective. Forskolin activity was also potentiated by the hydrolysis-resistant adenosine receptor agonists 5'-N-ethylcarboxamido adenosine and CGS 21680 (8.9- and 12.2-fold increase, respectively). Adenosine deaminase (1 U/ml) and 8-SPT (25 microM), which nearly abolished the response to 1 microM adenosine, also reduced cAMP accumulation caused by AMP (-78 and -54%, respectively). These results demonstrate that in PC12 cells, activation of adenylyl cyclase by forskolin is highly dependent on the occupancy of A(2A) adenosine receptors and that AMP potentially contributes to the amplification of forskolin response.     

Influence of stimulators and inhibitors of cyclic nucleotides on lower esophageal sphincter

These studies were performed in vitro to investigate the nature of the second messenger for lower esophageal sphincter (LES) smooth muscle relaxation in response to electrical field stimulation (EFS) and vasoactive intestinal polypeptide (VIP). It was seen that VIP, permeant derivatives of the cyclic nucleotide 8-bromo cyclic GMP (BrcGMP) and 8-bromo cyclic AMP (8-BrcAMP), the guanylate cyclase stimulant sodium nitroprusside (SNP), the adenylate cyclase stimulant forskolin, M&B 22,948 (cGMP phosphodiesterase inhibitor) and SK&F 94,120 (cAMP phosphodiesterase inhibitor) caused dose-dependent and tetrocotoxin resistant fall in LES tension. Guanylate cyclase inhibitor methylene blue (MB) (3 x 10(-5) M), caused significant antagonism of fall in LES tension by SNP without modifying the inhibitory response of forskolin. The possible adenylate cyclase inhibitor N-ethylmaleimide (NEM) (1 x 10(-4) M), on the other hand, caused significant antagonism of fall in LES tension by forskolin without any effect on that caused by SNP. The inhibitory responses of 8-BrcGMP and 8-BrcAMP were not modified by MB or NEM. NEM (1 x 10(-4) M) and MB (3 x 10(-5) M) caused significant inhibition of the fall in LES tension with EFS. NEM also caused inhibition of fall in LES tension by VIP. Furthermore, SK&F 94,120 and not M&B 22,948 caused significant potentiation of fall in LES tension by EFS. From these results we conclude that: 1) cAMP and cGMP may act as second messengers for LES relaxation with EFS and VIP, and 2) VIP may act primarily via cAMP system and remains a strong possibility for one of the inhibitory neurotransmitters in the LES.     

Does cyclic AMP mediate rat urinary bladder relaxation by isoproterenol?

Cyclic AMP is the prototypical second messenger of beta-adrenergic receptors, but recent findings have questioned its role in mediating smooth muscle relaxation upon beta-adrenergic receptor stimulation. We have investigated the signaling mechanisms underlying beta-adrenergic receptor-mediated relaxation of rat urinary bladder. Concentration-response curves for isoproterenol-induced bladder relaxation were generated in the presence or absence of inhibitors, with concomitant experiments using passive tension and KCl-induced precontraction. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536; 1 microM), the protein kinase A inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 10 microM), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 1 microM), and Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 30 microM), and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 3 microM) produced only minor if any inhibition of relaxation against passive tension or KCl-induced precontraction. Among various potassium channel inhibitors, BaCl2 (10 microM), tetraethylammonium (3 microM), apamin (300 nM), and glibenclamide (10 microM) did not inhibit isoproterenol-induced relaxation. Some inhibition of the isoproterenol effects against KCl-induced tone but not against passive tension was seen with inhibitors of calcium-dependent potassium channels such as charybdotoxin and iberiotoxin (30 nM each). A combination of SQ 22,536 and ODQ significantly inhibited relaxation against passive tension by about half, but not that against KCl-induced tone. Moreover, the combination failed to enhance inhibition by charybdotoxin against KCl-induced tone. We conclude that cAMP and cGMP each play a minor role in beta-adrenergic receptor-mediated relaxation against passive tension, and calcium-dependent potassium channels play a minor role against active tension.     

The signal transduction of endothelin-1-induced circular smooth muscle cell contraction in cat esophagus

It has been known that endothelin-1 (ET-1) exerts important actions in gastrointestinal smooth muscle motility, but its precise mechanism remains unsolved. We investigated the intracellular mechanism of ET-1-induced circular smooth muscle cell contraction in cat esophagus. ET-1 produced contraction of smooth muscle cells isolated by enzymatic digestion. The contraction in response to ET-1 was concentration-dependent. Pertussis toxin (PTX) blocked contraction induced by ET-1 in intact cells. To identify the specific G protein involved in the contraction, muscle cells were permeabilized with saponin. The G(i3) or G(beta) protein antibody inhibited the contraction. Neomycin phospholipase C (PLC) inhibitor inhibited the contraction, but 7,7-dimethyleicosadienoic acid (phospholipase A(2) inhibitor) and p-chloromercuribenzoic acid (phospholipase D inhibitor) had no effects. Incubation of permeabilized cells with PLC-beta(3) isozyme antibody inhibited the contraction. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, chelerythrine [protein kinase C (PKC) inhibitor], or genistein (protein tyrosine kinase inhibitor) inhibited the contraction, but not by diacylglycerol (DAG) kinase inhibitor, R59949. To test whether the contraction may be PKC isozyme-specific, we examined the effect of PKC isozymes antibodies on the contraction. PKC-epsilon antibody inhibited the contraction. To characterize further the specific PKC isozymes that mediate the contraction, we used, as an inhibitor, N-myristoylated peptides (myr-PKC) derived from the pseudosubstrate sequences of PKC-alphabetagamma, -alpha, -delta, or -epsilon. myr-PKC-epsilon inhibited the contraction, confirming that PKC-epsilon isozyme is involved in the contraction. To examine whether mitogen-activated protein kinases (MAPKs) mediate the contraction, specific MAPK inhibitors [MAPK kinase inhibitor, PD98059, (2'-amino-3'-methoxy-flavone), and p38 MAPK inhibitor, SB202190 (4-4-fluorophenyl) 2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole)] were used. PD98059 or SB202190 blocked the contraction. ET-1 increased the intensity of the detection bands identified by immunological methods as MAPK monoclonal p44/p42 peptides. PD98059 decreased the intensity of the detection bands compared with ET-1. In conclusion, ET-1-induced contraction in cat esophageal circular muscle cells depends on PTX-sensitive G(i3) protein and PLC-beta(3) isozyme, resulting in the activation of PKC-epsilon- or protein-tyrosine kinase-dependent pathway, subsequently mediating the activation of p44/p42 MAPK or p38 MAPK pathway.     

Specific inhibition of isoproterenol-stimulated cyclic AMP accumulation by M2 muscarinic receptors in rat intestinal smooth muscle.

The ability of oxotremorine-M to inhibit cyclic AMP accumulation in the presence of a variety of adenylate cyclase activators was studied in slices from the longitudinal muscle of the rat ileum. Oxotremorine-M was found to inhibit forskolin- and isoproterenol-stimulated cyclic AMP accumulation maximally by 17 and 32%, respectively, but not the stimulation due to other activators of adenylate cyclase. Inhibition of cyclic AMP accumulation by oxotremorine-M was unaffected by tetrodotoxin and was completely reversed by atropine. AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidynyl]acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one) an M2-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant (KB) of 0.20 microM, consistent with the dissociation constants for binding at the M2 muscarinic receptor site (KD = 0.092 microM) and inhibition of adenylate cyclase activity (KB = 0.13 microM). Hexahydrosiladifenidol, an M3-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant of 0.67 microM, again consistent with the dissociation constant for binding at the M2 site (KD = 0.83 microM). The agreement between the estimates of the dissociation constants of muscarinic antagonists for binding and for inhibition of cyclic AMP accumulation suggest that oxotremorine-M inhibition of isoproterenol-stimulated cyclic AMP accumulation in slices of rat intestinal smooth muscle is mediated by the M2 receptor.     

Comparison of signal transduction mechanisms of alpha-2C and alpha-1A adrenergic receptor-stimulated prostaglandin synthesis.

Prostaglandin (PG) synthesis elicited by adrenergic transmitter in the vascular smooth muscle cells (VSMC) of rabbit aorta is primarily mediated through activation of alpha-2C and alpha-1A adrenergic receptors (ARs). We have now investigated and compared the signal transduction mechanisms involved in alpha-2C and alpha-1A AR-stimulated prostacyclin (PGI2) production, measured as 6-keto-PGF1 alpha, in vascular smooth muscle cells. Norepinephrine, methoxamine (an alpha-1 AR agonist) and UK-14304 (an alpha-2 AR agonist) enhanced 6-keto-PGF1 alpha production. UK-14304 and norepinephrine (in the presence of propranolol), but not methoxamine, reduced basal adenosine 2':3'-cyclic monophosphate (cyclic AMP) as well as forskolin- and isoproterenol-stimulated cyclic AMP accumulation. Forskolin and isoproterenol did not alter basal 6-keto-PGF1 alpha production and alpha AR agonist-induced 6-keto-PGF1 alpha production. Alpha-2C and alpha-1A AR-stimulated 6-keto-PGF1 alpha production was independent of cyclic AMP levels in vascular smooth muscle cells. Both alpha-2C and alpha-1A AR-stimulated 6-keto-PGF1 alpha production required extracellular Ca++. Pertussis toxin prevented inhibition of cyclic AMP accumulation and reduced 6-keto-PGF1 alpha production in response to AR agonists. Guanosine 5'-O-(3-thiotriphosphate) potentiated 6-keto-PGF1 alpha production induced by norepinephrine and UK-14304 but not by methoxamine, whereas at a higher Mg++ concentration (4 mM), guanosine 5'-O-(3-thiotriphosphate) potentiated 6-keto-PGF1 alpha production by all three agonists. In contrast, the effect of UK-14304 on cyclic AMP was prevented in the presence of 4 mM Mg++. These data suggest that the pertussis toxin-sensitive G protein(s) mediated the stimulation of PG synthesis by alpha-1A and alpha-2C AR activation and the decrease in cyclic AMP accumulation by alpha-2C AR activation.