Effects of phenothiazines on the membrane-bound guanylate and adenylate cyclase in tetrahymena pyriformis

The particulate-bound guanylate cyclase activity of Tetrahymena pyriformis was shown previously to be Ca²⁺-dependent and to be activated by an endogenous calmodulin-like protein (Tetrahymena Ca²⁺-binding protein, TCBP) [S. Nagao, Y. Suzuki, Y. Watanabe and Y. Nozawa, Biochem. biophys. Res. Commum.90, 261 (1979)]. Phenothiazine derivatives, such as chlorpromazine and trifluoperazine, that interact with calmodulin were found to inhibit the Ca²⁺-dependent guanylate cyclase activity and the TCBP-induced activation of the guanylate cyclase activity. Ethylene glycol-bis (β-aminoethyl ether)-N, N'-tetraacetic acid (EGTA), a Ca²⁺ chelator, also inhibited the activation of guanylate cyclase. However, the mechanisms by which EGTA and trifluoperazine act were different. The EGTA-induced inhibition could not be overcome by increasing the concentration of TCBP, whereas the trifluoperazine-induced inhibition could be overcome by increasing the concentration of TCBP, but not by increasing the concentration of Ca²⁺. These findings suggest that the mechanism by which trifluoperazine inhibits the activation of guanylate cyclase involves competition with TCBP.     

Inhibitory effects of calmodulin antagonists on plasma membrane cyclases in Tetrahymena: calmodulin-dependent guanylate cyclase and calmodulin-independent adenylate cyclase

Trifluoperazine was shown previously to inhibit the activation of Tetrahymena guanylate cyclase activity by calmodulin [S. Nagao, S. Kudo and Y Nozawa, Biochem. Pharmac. 19, 2709 (1981)]. The present paper reports that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), another representative calmodulin inhibitor, inhibited the calmodulin-induced activation of the guanylate cyclase, and that trifluoperazine and W-7 also inhibited Tetrahymena adenylate cyclase. The adenylate cyclase activity was found to be present in a membrane-bound form and not to be influenced by calmodulin. The inhibitions of the adenylate cyclase activity by these agents were dose-dependent and not Ca2+-dependent. These findings suggest that the inhibitory actions of these drugs may not necessarily be specific for calmodulin-dependent enzymes in T. pyriformis.     

Activation of hepatic guanylate cyclase by nitrosyl-heme complexes: Comparison of unpurified and partially purified enzyme

The objective of this study was to evaluate the effects of several agents on activation of both unpurified and partially purified hepatic soluble guanylate cyclase by performed NO (nitric oxide or nitrosyl)-heme complexes. Guanylate cyclase was activated by NO complexes of the heme compounds, hematin, hemoglobin, myoglobin, catalase and cytochrome c, and also by the reaction product of NO and ferredoxin, a non-heme, iron sulfur electron transfer protein. NO-lipoxygenase, which contains non-heme iron, did not activate guanylate cyclase. NO-heme complexes activated unpurified enzyme almost equally well in the presence of either Mg²⁺ or Mn²⁺. However, activation of purified (350- to 750-fold) guanylate cyclase was markedly greater with Mg²⁺ than with Mn²⁺. At concentrations that did not alter basal enzymatic activity, Ca²⁺ markedly inhibited guanylate cyclase activation in the presence of Mg²⁺ but not of Mn²⁺. Hemoproteins inhibited activation of unpurified and purified enzyme by NO-heme complexes, and increasing the concentrations of the latter overcame the inhibition. Gel filtration studies indicated that uncomplexed and NO-complexed hematin bind to common or adjacent sites on guanylate cyclase. Whereas dl-dithiothreitol enhanced activation, ferricyanide, cystine, o-iodosobenzoic acid and ethacrynic acid inhibited activation of guanylate cyclase by NO-heme complexes. The data indicate that the effects of these diverse agents on guanylate cyclase activation by preformed NO-heme are similar to their effects on enzyme activation by NO and nitroso compounds, both of which readily form NO-heme complexes. Therefore, the effects of these diverse agents may be on guanylate cyclase rather than on NO-heme formation.     

Cetiedil inhibition of calmodulin-stimulated enzyme activity

Cetiedil, an in vitro anti-sickling agent, inhibited calmodulin-stimulated cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) and Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activities. The drug had no effect on basal enzyme activities in the absence of calmodulin. The inhibition of phosphodiesterase was competitive with respect to the concentrations of both cAMP and calmodulin. Cetiedil did not inhibit calmodulin-stimulated enzyme activities by acting as a calcium chelator, since increasing the concentration of calcium did not reverse the inhibitory effect.     

Ethanol-induced changes in cyclic guanosine 3',5'-phosphate metabolism in mouse vestibular region

The content of cyclic guanosine 3',5'-phosphate (cGMP) in the region of the vestibular nuclei of mice increased after 10–14 days of ethanol intake. The increase was preceded by an increase in guanylate cyclase activity. A second increase was noted after 4 weeks on an ethanol-containing diet at the same time as decreased cGMP-phosphodiesterase activity was noted. The early increases were less pronounced in the alcohol-rejecting DBA/2J strain but were reproduced by preincubation or addition of arachidonic acid. Cerebellectomy had no effect on early or late rises in cGMP levels.     

INCREASE IN REACTIVITY OF HUMAN PLATELET GUANYLATE CYCLASE DURING AGGREGATION POTENTIATES THE DISAGGREGATING CAPACITY OF SODIUM NITROPRUSSIDE

1. Basal and stimulated guanylate cyclase activity during ADP-induced human platelet aggregation in comparison with the actions of sodium nitroprusside (SNP) on platelets was investigated. 2. Sodium nitroprusside exhibited both ex vivo and in vitro antiplatelet effects, as assessed by inhibition of subsequent ADP-induced aggregation in platelet-rich plasma. A strong correlation between decrease in aggregation and increase in platelet guanylate cyclase activity in the presence of SNP was obtained. 3. When SNP was administered after the induction of aggregation, it caused acceleration of disaggregation (in reversible aggregation) and produced disaggregation (under conditions of otherwise irreversible aggregation) which was time-dependent. 4. Platelet aggregation was accompanied by a transient increase in platelet cyclic GMP content and guanylate cyclase activation by the nitric oxide (NO) donor SNP. Changes in guanylate cyclase activity were haem-associated and probably reflected saturation of enzyme by haem. 5. Maximal SNP disaggregating effect coincided with peak guanylate cyclase responsiveness to SNP. 6. The present investigation provides evidence that increased responsiveness of platelet guanylate cyclase to NO during aggregation facilitates disaggregation in the presence of SNP. Thus, availability of NO (endogenous or exogenous) at sites of incipient platelet aggregation in vivo may play a pivotal role regarding limitation of this process.     

Ambroxol as an inhibitor of nitric oxide-dependent activation of soluble guanylate cyclase

The influence of ambroxol on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase was investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes and slightly enhanced it at 50 and 100 microM. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of both enzymes. The IC(50) values for inhibition by ambroxol of sodium nitroprusside-stimulated human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase were 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of soluble guanylate cyclase by protoporphyrin 1X. Thus, it is possible that the molecular mechanism of the therapeutic action of ambroxol involves the inhibition of nitric oxide (NO)-dependent activation of soluble guanylate cyclase.     

Age related changes in cardiac and aortic phosphodiesterase activities in normotensive and hypertensive rats.

The activities of cAMP¹ and cGMP phosphodiesterase were studied in the aorta (freed of adventitia layer) and in the heart (ventricles) of normotensive and mincralocorticoid hypertensive rats of 8 or 16 weeks of age. The enzyme activities were determined at low (1 μM) and high (100 μM) substrate concentrations. The changes in activity were compared to the changes in organ weight, protein and DNA content. The increase in organ weight that occurred with both age and hypertensive treatment corresponded mostly to a marked elevation in protein content in the aorta, but not in the heart, where the DNA content increased without any significant variation in protein content. In both tissues. eGMP phosphodiesterase activity measured at low substrate concentration was sensitive to endogenous Ca²⁺-dependent activation and markedly increased with age. This increase was proportionally larger than the variations in DNA content of the tissues, but lower than those of total protein in the aorta. It could not be ascribed to an increase in the activator content of the tissues, which was in excess. By contrast. cGMP phosphodiesterase activity measured at high substrate concentration and cAMP phosphodiesterase activity, measured at either substrate concentration, were not sensitive to the Ca²⁺-dependent activation and did not undergo large changes with age except for a significant decrease in cAMP phosphodiesterase activity at high substrate concentration per mg heart cytosol protein. No relationship could be found between the elevation of blood pressure, due to age or to the influence of the mineralocorticoid treatment, and phosphodiesterase activities, which varied in a similar manner in control and hypertensive rats. The results are consistent with the view that a cGMP phosphodiesterase. which is sensitive to Ca²⁺-dependent endogenous activation, increases in aorta and heart cells with the age of the rat.     

Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor-inotropic response.

1. In this paper we have determined the different signalling pathways involved in muscarinic acetylcholine receptor (AChR)-dependent inhibition of contractility in rat isolated atria. 2. Carbachol stimulation of M2 muscarinic AChRs exerts a negative inotropic response, activation of phosphoinositide turnover, stimulation of nitric oxide synthase and increased production of cyclic GMP. 3. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase, shifted the dose-response curve of carbachol on contractility to the right. These inhibitors also attenuated the muscarinic receptor-dependent increase in cyclic GMP and activation of nitric oxide synthase. In addition, sodium nitroprusside, isosorbide, or 8-bromo cyclic GMP, induced a negative inotropic effect, increased cyclic GMP and activated nitric oxide synthase. 4. These results suggest that carbachol activation of M2 AChRs, exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This in turn, triggers cascade reactions involving calcium/calmodulin and protein kinase C, leading to activation of nitric oxide synthase and soluble guanylate cyclase.     

Antagonism of serotonin-activated adenylate cyclase in the liver fluke Fasciola hepatica by levorphanol and dextrorphan

A highly active serotonin (5-HT)¹ -stimulated adenylate cyclase is present in particles from the liver fluke $\text{Fasciola hepatica}$ [1]. This enzyme is activated through a single class of receptors by indoleamines and LSD derivatives. Several recent reports showed that opiates can interact with adenylate cyclase from various tissues [2–4]. In this report we examine the effects of morphine-like drugs upon adenylate cyclase activity in cell free particles from $\text{F}$. hepatica. We report a non-stereospecific inhibition of both basal and serotonin-stimulated cyclase activity and discuss the possible mechanism of this inhibition.     

Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor function

1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.     

Calmodulin activates adenylate cyclase from rat anterior pituitary

Bovine brain calmodulin activated adenylate cyclase in calmodulin-deficient rat anterior pituitary membranes. This activation appeared to be specific by the following criteria: 1) calmodulin activation was Ca2+ dependent and responded biphasically to calcium, displaying activation at low and inhibition at higher concentrations; 2) calmidazolium, a potent calmodulin antagonist, inhibited calmodulin activation of adenylate cyclase; 3) activation of the enzyme occurred in a dose-dependent manner, at calmodulin concentrations normally found in most cells (1- to 20-microM range). However, this response was not saturated using calmodulin concentrations as high as 50 microM. The data suggest that endogenous calmodulin can be dissociated from normal anterior pituitary adenylate cyclase, that the enzyme can be subsequently stimulated by addition of micromolar concentrations of calmodulin, and that this enzyme appears to be at least 50-fold less sensitive to calmodulin than is the brain adenylate cyclase.     

The alpha-adrenergic control of rabbit liver glycogenolysis.

We have confirmed that the electrical stimulation of the splanchnic nerve in the rabbit causes glycogenolysis m a cyclic AMP-independent way as found by Shimazu and Amakawa [1]; glycogen phosphorylase (1,4-α-d-Glucan: orthophosphate α-glucosyltransferase, EC 2.4.1.1) was activated, but phosphorylase b kinase (ATP: phosphorylase b phosphotransferase, EC 2.7.1.38) was not. We could, however, not confirm the observation of a decrease in phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) activity. Pretreatment of the rabbits with the α-adrenergic blocking agent phentolamine prevented the splanchnic nerve stimulation from activating glycogen phosphorylase.The addition of norepinephrine (10^?7 M) to isolated rabbit hepatocytes activated glycogen phosphorylase without an activation of phosphorylase b kinase. At 10^?6 M, norepinephrine activated both enzymes. Phentolamine blocked the activation of glycogen phosphorylase by norepinephrine at 10^?7M but not at 10^?6M. Absence of Ca²⁺ from the incubation medium prevented norepinephrine (10^?7 M) from activating glycogen phosphorylase. The ionophore A 23187 also caused an activation of phosphorylase (but not of phosphorylase b kinase) provided that Ca²⁺ was present in the incubation medium. These data indicate that sympathetic nervous control of liver glycogenolysis is achieved, via α-adrenergic receptors, by an increased concentration of cytosolic Ca²⁺ ions which stimulate rather than activate phosphorylase b kinase. The neurotransmitter involved is most probably norepinephrine.     

Adenylate cyclase from various dopaminergic areas of the brain and the action of antipsychotic drugs.

The present report is a comparative study of adenylate cyclase activity in various areas of the brain identified as dopaminergic. Low levels of dopamine were found to stimulate adenylate cyclase from the striatum, median eminence, olfactory tubercle, nucleus accumbens and amygdala. Apomorphine, known to mimic the pharmacological and physiological effects of dopamine, stimulated adenylate cyclase from the median eminence. Several different classes of drugs effective in the treatment of schizophrenia were potent inhibitors of the stimulation by dopamine of the enzyme from these various regions. The drugs studied included representatives of the phenothiazine, butyrophenone, dibenzodiazepine and dibenzoxazepine classes. The inhibition by the dibenzoxazepine, loxapine, which is structurally very similar to the dibenzodiazepine, clozapine, was competitive with respect to dopamine. The calculated inhibition constant (K_i) for loxapine of about 15 nM was similar to that observed for some of the more potent phenothiazines. The results, considered together with previously published data, support the possibility that the therapeutic effects, as well as the extrapyramidal and endocrinological side effects, of these antipsychotic agents may be attributable to their ability to block the activation of adenylate cyclase in various select areas of the brain.     

Different effects of various beta-adrenoceptor antagonists on adenylate cyclase, guanylate cyclase and calmodulin-dependent phosphodiesterase in heart

A series of six beta-adrenergic blocking drugs including propranolol, bufetolol, bunitrolol, pindolol, labetalol and acebutolol were examined for effects on adenylate cyclase, guanylate cyclase and calmodulin-dependent phosphodiesterase from heart. The adrenergic blocking agents had no apparent effects on basal activities of adenylate cyclase, guanylate cyclase and phosphodiesterase. The drugs blocked the enhancement of adenylate cyclase activity by isoproterenol, but not by guanine nucleotide or fluoride. The inhibitory effects of beta-antagonists were overcome by sufficiently large doses of isoproterenol. Sodium azide specifically required catalase whereas NaNO2 required cysteine to activate myocardial guanylate cyclase. Among beta-adrenergic blocking drugs tested, both pindolol and acebutolol inhibited the stimulation of guanylate cyclase by NaNo2 or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). However, other beta-blocking drugs did not significantly affect the activation by NaN3, NaNO2 and MNNG. Several beta-antagonists, such as labetalol, bunitrolol, pindolol and acebutolol were also effective in blocking the activation of phosphodiesterase by calmodulin. The inhibitory effects of beta-adrenergic blocking drugs, i.e. pindolol and acebutolol upon either nitroso compound-stimulated guanylate cyclase or calmodulin-activated phosphodiesterase display little correlation with their potency as beta-adrenergic blocking agents. These data suggest that beta-antagonists may have another site of action which is not directly related to the control of catecholamine metabolism.     

Role of nitric oxide/cyclic GMP in myocardial adenosine A1 receptor-inotropic response

In this study we have determined the different signalling pathways involved in adenosine A(1)-receptor (A(1)-receptor)-dependent inhibition of contractility in rat isolated atria. N-cyclopentyladenosine (CPA) stimulation of A(1)-receptor exerts: negative inotropic response, inositol phosphates accumulation, stimulation of nitric oxide synthase (NOS), increased production of nitric oxide (NO) and cyclic GMP. Inhibitors of phospholipase C (PLC), protein kinase C (PKC), calcium/calmodulin, NOS and guanylate cyclase shifted the dose-response curve of CPA on contractility to the right. Those inhibitors also attenuated the A(1)-receptor-dependent increase in cyclic GMP and activation of NOS. These results suggest that CPA activation of A(1)-receptors exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving calcium/calmodulin and PKC, leading to activation of NOS and soluble guanylate cyclase.     

Stimulatory effect of ionophores on adenosine 3',5'-monophosphate content in human mononuclear leukocytes

Ionophores A23187 and bromo-lasalocid ethanolate enhanced the cyclic AMP content in human mononuclear leukocytes. The maximum effect of A23187 with a 10-min incubation was found with 0.3–1.0μM concentrations with or without l-isoproterenol (1 μM) or prostaglandin E ₁ (pge ₁) (0.3 μM). The maximum effect after 5 min of incubation at 37° was observed with 0.05, 0.2 and 1 μm A23187. The effect of ionophore A23187 was enhanced by both aminophylline (1 mM) and isobutyl-methylxanthine (1 mM). Calcium (1 mM). aspirin (1 mM) and indomethacin (100 μM) decreased the stimulatory action of A23187. Bromo-lasalocid ethanolate increased cyclic AMP content in cells maximally at a 3 μM concentration with or without 0.3 μM pge ₁.     

Inhibition of Escherichia coli heat-stable enterotoxin effects on intestinal guanylate cyclase and fluid secretion by quinacrine

Enterotoxigenic Escherichia coli may produce a heat-stable enterotoxin (ST) that causes diarrheal disease in humans and in animals ST activates particulate guanylate cyclase in intestinal mucosal cells and causes intestinal fluid secretion. In this study, we examined the effects of quinacrine on ST activation of guanylate cyclase and ST-mediated intestinal fluid secretion. Quinacrine significantly reduced ST activation of particulate guanylate cyclase in rat intestinal tissue. Additionally, quinacrine reduced ST-mediated fluid secretion in a rat intestinal loop assay (P less than 0.05). In the suckling mouse model, subcutaneous quinacrine (0.1 mumole/mouse) reduced ST-induced fluid secretion at a submaximally effective dose of the toxin, but it did not reduce ST-mediated fluid secretion at a near maximally effective dose. Quinacrine (0.1 mumole/mouse) did not significantly reduce intestinal fluid secretion induced by the analog of cyclic GMP, 8-bromo cyclic GMP. However, at a higher concentration of quinacrine (1 mumole/mouse), significant inhibition of 8-bromo cyclic GMP-induced secretion was observed. Inhibition by the antimalarial agent quinacrine of ST-induced fluid secretion, by a block prior to guanylate cyclase activation, suggests a possible role for a phospholipase early in the sequence of events of ST activation of guanylate cyclase. The results suggest that ST may activate membrane phospholipases prior to ST activation of guanylate cyclase.     

Haem-dependent activation of guanylate cyclase and cyclic GMP formation by endogenous nitric oxide: a unique transduction mechanism for transcellular signaling

The interaction between nitric oxide (NO) synthesized in one cell and the haem group of cytosolic guanylate cyclase located in target cells to form NO-haem-guanylate cyclase represents a unique signal transduction mechanism that links extracellular stimuli to the synthesis of cyclic GMP in nearby target cells. Autacoids, neurotransmitters, and macrophage- and neutrophil-activating factors interact with selective extracellular receptors to trigger formation of NO from L-arginine. NO may be viewed as a second messenger. The NO diffuses into adjacent target cells and causes haem-dependent activation of guanylate cyclase, thereby stimulating cyclic GMP accumulation. Guanylate cyclase-bound haem serves as a transducer in transferring the signal from NO to guanylate cyclase. Cyclic GMP acts as a third messenger in causing vascular smooth muscle relaxation, inhibition of platelet aggregation and adhesion, and modulation of macrophage, neutrophil, and other phagocytic cell functions. The unique physical and chemical properties of NO allow it to function as an intercellular modulator within a localized environment. This intercellular or transcellular signaling mechanism involving a common signal transduction mechanism permits the rapid initiation of localized complementary cellular functions leading to increased local blood flow, inhibition of local thrombosis, and modulation of phagocytosis and cytotoxicity.     

Protective effect of NG-nitro-L-arginine (N5-[imino(nitroamino)methyl]-L-ornithine) against cyanide-induced convulsions in mice.

The effects of NG-nitro-L-arginine (NNA; an inhibitor of the oxidative L-arginine pathway) on convulsions induced by cyanide were investigated in mice. NNA prevented cyanide-induced convulsions in a dose-dependent manner. Furthermore, the inhibitory effect against convulsions induced by cyanide with NNA was abolished by pretreatment of L-arginine. However, NNA did not change blood cyanide levels in mice 5 min after injection of potassium cyanide. Since NNA prevents against oxidative L-arginine pathway-dependent guanylate cyclase activation in intact cells, it was suggested that the protection against cyanide-induced convulsions with NNA may be due to the inhibition of guanylate cyclase. In support, methylene blue, an inhibitor of guanylate cyclase, elicited a similar inhibition against convulsions induced by cyanide.