Inhibition of cardiac slow action potentials by 8-bromo-cyclic GMP occurs independent of changes in cyclic AMP levels

Cyclic GMP inhibits the slow inward Ca current of cardiac cells. This effect could be due to a cyclic GMP-mediated phosphorylation of the Ca channel (or some protein modifying Ca channel activity), or alternatively, to enhanced degradation of cyclic AMP owing to stimulation of a phosphodiesterase by cyclic GMP. To test the latter possibility, we examined the effect of extracellular 8-bromo-cyclic GMP on cyclic AMP levels in guinea pig papillary muscles, in parallel with electrophysiological experiments. Isoproterenol (10(-6) M) significantly increased the cyclic AMP levels and induced Ca-dependent slow action potentials. Superfusion with 8-bromo-cyclic GMP (10(-3) M) inhibited the slow action potentials induced by isoproterenol. However, muscles superfused with 8-bromo-cyclic GMP had cyclic AMP levels identical to those of muscles superfused with isoproterenol alone. Similarly, 8-bromo-cyclic GMP had no effect on the increase in cyclic AMP levels of muscles treated with forskolin (10(-6) M) or histamine (10(-6) M). We conclude that the inhibitory effect of cyclic GMP on slow Ca channels in guinea pig ventricular cells is not due to a decrease in the cyclic AMP levels. We hypothesize that a cyclic GMP-mediated phosphorylation is the most likely explanation for the Ca channel inhibition observed in this preparation.     

Effects of 8-bromo-cyclic GMP on slow channel mediated action potentials of 3-days-old embryonic chick ventricle.

The role of cyclic GMP in modulation of cardiac slow channel activity was investigated by observing the effects of 8-bromo-cyclic GMP (8-Br-cGMP) on action potentials of isolated ventricle of 3-days-old chick embryo, which exhibit upstroke primarily due to slow channels. 8-Br-cGMP (0.5 & 1 mM) reduced the maximum diastolic potential, maximal upstroke velocity (+Vmax) and overshoot in 30-60 min. 8-Bromo-cyclic AMP (8 Br-cAMP, 0.5 & 1 mM), isoproterenol (Iso, 0.5-5 microM) and forskolin (0.5-2 microM) caused an increase in +Vmax and overshoot. 8-Br-cGMP antagonised this enhancement of +Vmax. Increase in +Vmax and overshoot by Bay-K-8644 (1 microM) was also blocked by 8-Br-cGMP. These findings show that 8-Br-cGMP inhibited the early embryonic cardiac slow channel activity, which contributes significantly to the upstroke of action potential, under basal conditions as well as after its accentuation by elevation of cyclic AMP levels (by 8-Br-cAMP, Iso & Forskolin) or by direct stimulation of the channel activity (by Bay-K-8644). It is suggested on the basis of these findings that cyclic GMP plays a key role in down modulation of the cardiac slow channel activity in early embryonic chick heart.     

Use of the cell-attached patch clamp technique to examine regulation of single cardiac K channels by cyclic GMP

Cyclic nucleotides play a central role in the modulation of ion channels in a variety of tissues, including the heart. In order to determine the possible role of cyclic GMP (cGMP) in the regulation of the background K channel activity of cardiac cells, the effect of 8-Br-cGMP on the inwardly-rectifying K channels of cultured ventricular myocytes from embryonic chick hearts was examined. 8-Br-cGMP (10(-4) to 10(-3) M) inhibited these single channel currents within 3 to 10 min. Spontaneous recovery of the currents occurred with prolonged (greater than or equal to 15 min) exposure to 8-Br-cGMP, but this recovery was accompanied by altered channel behavior. Thus, a new long-lasting open state of the channel appeared, in addition to the open state observed prior to 8-Br-cGMP addition. Superfusion of the cells with the muscarinic agonist carbamylcholine (10(-5) M) also resulted in inhibition of the currents, which suggests that the cGMP-mediated inhibition of these channels may occur under physiological conditions. Thus, it appears that cGMP may be an important modulator of the background K conductance (and excitability) of cardiac cells.     

Use of the cell-attached patch clamp technique to examine regulation of single cardiac K channels by cyclic GMP

Cyclic nucleotides play a central role in the modulation of ion channels in a variety of tissues, including the heart. In order to determine the possible role of cyclic GMP (cGMP) in the regulation of the background K channel activity of cardiac cells, the effect of 8-Br-cGMP on the inwardly-rectifying K channels of cultured ventricular myocytes from embryonic chick hearts was examined. 8-Br-cGMP (10^-4 to 10^-3 M) inhibited these single channel currents within 3 to 10 min. Spontaneous recovery of the currents occurred with prolonged ( 15 min) exposure to 8-Br-cGMP, but this recovery was accompanied by altered channel behavior. Thus, a new long-lasting open state of the channel appeared, in addition to the open state observed prior to 8-Br-cGMP addition. Superfusion of the cells with the muscarinic agonist carbamylcholine (10^-5 M) also resulted in inhibition of the currents, which suggests that the cGMP-mediated inhibition of these channels may occur under physiological conditions. Thus, it appears that cGMP may be an important modulator of the background K conductance (and excitability) of cardiac cells.     

Effects of parathyroid hormone on cyclic AMP, cyclic GMP, and efflux of calcium in isolated renal tubules.

The effects of parathyroid hormone (PTH) on concentrations of cyclic AMP and cyclic GMP were investigated in isolated renal cortical tubules from hamsters. Efflux of 45Ca from tubules was compared to temporal changes in both cyclic nucleotide concentrations. A rapid increase in cyclic AMP occurred following addition of PTH which was maximal by 1 min but decreased over the next 4 min period. Cyclic GMP concentrations were not significantly altered at 1 min but increased between 1 and 5 min from basal levels. Concentrations of both nucleotides remained significantly elevated from basal levels between 5 and 15 min following PTH. Efflux of 45Ca was increased by PTH with time-course changes closely paralleling changes in cyclic GMP concentrations. Changes in both cyclic AMP and cyclic GMP were related to PTH concentrations of the incubation media and were increased by addition of theophylline. Increasing the calcium concentration from 1 to 3 mM did not significantly alter the effect of PTH on cyclic AMP, however, cyclic GMP concentrations were further increased.     

Neurotensin regulation of TSH secretion in the rat

The ionophore A23187 (6.7 microM) increased the rates of formation of prostaglandins and cyclic AMP in suspensions of thioglycollate-elicited rat peritoneal macrophages. Both effects were inhibited by the calmodulin blocker trifluoperazine (50 microM) and the calcium channel blocker verapamil (500 microM). Inhibitors of phospholipase A2 and cyclo-oxygenase also blocked both actions of A23187. The stimulated prostaglandin formation was markedly reduced when the cells were preincubated with 8-bromo-cyclic AMP (1mM), dibutyryl cyclic AMP (1mM) or cholera toxin (500ng/ml). Addition of exogenous arachidonic acid (30 microM) alleviated this inhibition. We propose that the effect of A23187 on macrophages includes a 'self-limiting' mechanism whereby newly-synthesized prostaglandins can inhibit, via cyclic AMP, a step(s) prior to the transformation of arachidonic acid and thus modulate their own production.     

Direct inhibition of testosterone synthesis in rat testis interstitial cells by ethanol: possible sites of action

The present in vitro studies using interstitial cells of adult rat testes demonstrated that ethanol inhibits LH- and 8-bromo-cyclic AMP-stimulated testosterone synthesis, pregnenolone- and progesterone-stimulated testosterone synthesis, and basal testosterone synthesis. However, the patterns of inhibition following exposure to 0.22 to 880 or 1100 mM ethanol were different. In general, the inhibition curves for LH-, 8-bromo-cyclic AMP-, pregnenolone- and progesterone-stimulated testosterone synthesis were biphasic, with a gradual slope from 0.22 to 220 mM ethanol, and a sharper slope with concentrations of ethanol greater than 220 mM. Basal testosterone synthesis was reduced only to 74% of control with ethanol concentrations up to 44 mM, and higher concentrations of ethanol reduced testosterone synthesis no further. The effect of ethanol on Lh-stimulated cyclic AMP accumulation showed an even different pattern: some of the lower concentrations of ethanol inhibited cyclic AMP accumulation, while higher levels of ethanol progressively increased cyclic AMP accumulation. These studies demonstrate that isolated interstitial cells are highly sensitive to the direct effects of ethanol; they also suggest that the principle site of ethanol inhibition may be at the level of the smooth endoplasmic reticulum where progesterone is converted to testosterone.     

Cyclic AMP regulates the life time of acetylcholine-activated channels in cultured myotubes

'Giga-seal' patch-clamp recording was performed in embryonic chick myotubes at day 3 to 4 of culture. Myotubes were exposed to agents that enhance the concentration of cytosolic cyclic AMP (cAMPi) and their action on acetylcholine- (ACh) activated channels was investigated. While the conductance and the closed time was unaffected by forsokolin, cholera toxin, dibutyryl cyclic AMP and 8-bromo-cyclic AMP, these agents lengthened the ACh-activated channel life time with efficacy that paralleled with their capability to increase the cAMPi.     

Relaxation of Mytilus catch muscle by 8-bromo-cyclic GMP and related compounds

Relaxation of catch tension by 8-bromo-cyclic GMP in the ABRM of Mytilus was blocked in the presence of mersalyl and was markedly reduced after treatment of the muscle with alpha-methyldopa. In the muscle depolarized by 540 mM KCl + 5 mM EGTA solution, 8-bromo-cyclic GMP could not relax Ca-contracture. Hexylamine and phenylethylamine, which are assumed to relax the catch acting on relaxing nerve terminals, could not relax the contracture either. Serotonin and dopamine, which are known to relax the catch acting directly on the muscle fibre membrane, could relax it. In the muscle depolarized by 250 mM KCl + 5 mM EGTA solution, all of the cyclic nucleotides tested (cyclic AMP, cyclic GMP and their analogues), serotonin and dopamine relaxed Ca-contracture, but hexylamine and phenylethylamine did not relax the contracture. The possibilities of the involvement of cyclic GMP in the presynaptic and postsynaptic relaxing mechanisms in the ABRM are discussed.     

Modulation of calcium channels by norepinephrine in internally dialyzed avian sensory neurons

Modulation of voltage-dependent Ca channels by norepinephrine (NE) was studied in chick dorsal root ganglion cells using the whole-cell configuration of the patch-clamp technique. Cells dialyzed with K+ and 2-10 mM EGTA exhibited Ca action potentials that were reversibly decreased in duration and amplitude by NE. Ca channel currents were isolated from other channel contributions by using: (a) tetrodotoxin (TTX) to block gNa, (b) internal K channel impermeant ions (Cs or Na/N-methylglucamine mixtures) as K substitutes, (c) external tetraethylammonium (TEA) to block K channels, (d) internal EGTA to reduce possible current contribution from Ca-activated channels. A marked decline (rundown) of Ca conductance was observed during continual dialysis, which obscured reversible NE effects. The addition of 2-5 mM MgATP to the intracellular solutions greatly retarded Ca channel rundown and permitted a clear assessment of modulatory drug effects. The inclusion of an intracellular creatine phosphate/creatine phosphokinase nucleotide regeneration system further stabilized Ca channels, which permitted recording of Ca currents for up to 3 h. NE reversibly decreased both steady state Ca currents and Ca tail currents in Cs/EGTA/MgATP-dialyzed cells. A possible role of several putative intracellular second messengers in NE receptor-Ca channel coupling was investigated. Cyclic AMP or cyclic GMP added to the intracellular solutions at concentrations several orders of magnitude higher than the Kd for activation of cyclic nucleotide-dependent protein kinases did not block or mask the expression of the NE-mediated decrease in gCa. Addition of internal EGTA to a final concentration of 10 mM also did not affect the expression of the NE response. These results suggest that neither cyclic AMP nor cyclic GMP nor Ca is acting as a second messenger coupling the NE receptor to the down-modulated Ca channel population.     

In vitro studies of the testis: the effect of LH on cyclic nucleotides.

This study evaluated the relationship between LH, cyclic AMP, cyclic GMP, and testosterone using in vitro incubation of decapsulated rat testes and sampling incubation medium. With added LH (1.0, 5.0, 100, and 500 mIU/ml) there were statistically significant increases in cyclic AMP at 5 mIU/ml or more LH, and progressively greater titers of this nucleotide were produced as LH was increased. For cyclic GMP all levels of added LH caused significant increments in titers of nucleotide; however, peak cyclic GMP concentrations occurred with 5 mIU/ml of LH. The addition of 10(-3) and 10-(4)M 8-bromo-cyclic AMP caused significant increases in testosterone production, while no changes in production of this androgen were found with 10(-3), 10(-4), or 10(-5)M 8-bromo-cyclic GMP. Neither cyclic AMP nor cyclic GMP titers were altered by the addition of 1 to 50 micrograms/ml of testosterone to medium bathing the rat testes. The dose response curves of cyclic AMP and cyclic GMP to LH are different. Progressive increments in added LH cause parallel increases of cyclic AMP and a biphasic change of cyclic GMP, 8-bromo-cyclic GMP does not cause testosterone generation, suggesting that cyclic GMP does not result in androgen synthesis. However, cyclic GMP may be involved in other Leydig cell functions.     

Amitriptyline-Mediated Inhibition of Neurite Outgrowth from Chick Embryonic Cerebral Explants Involves a Reduction in Adenylate Cyclase Activity

We have previously shown that amitriptyline, a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants, and that dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, or theophylline can enhance neurite outgrowth from embryonic olfactory explants. In the present study, we examined the mechanism(s) underlying amitriptyline-mediated inhibition of neurite outgrowth by studying the effects of amitriptyline on adenylate cyclase activity and cyclic AMP levels. In cultured chick embryonic cerebral explants, dibutyryl cyclic AMP or theophylline, but not dibutyryl cyclic GMP, enhanced neurite outgrowth and partially reduced the inhibitory effects of amitriptyline on neurite outgrowth. Explants treated with amitriptyline for 2 days showed decreased cyclic AMP levels that significantly correlated with the degree of neurite outgrowth. Amitriptyline inhibited both basal and forskolin-stimulated adenylate cyclase activity in vitro, but only in the presence of GTP. Taken together, these data suggest that amitriptyline inhibits the activity of adenylate cyclase via a GTP-dependent mechanism, and that the subsequent decrease in cyclic AMP level may be involved in amitriptyline-mediated inhibition of neurite outgrowth.     

Cholinergic inhibition of catecholamine-stimulable cyclic AMP accumulation in murine atria.

Carbachol antagonizes isoproterenol-stimulable cyclic AMP accumulation in mouse atria by direct activation of cardiac muscarinic receptors. Inhibition by carbachol occurs rapidly and is completely reversed when the drug is removed. Neither nitroprusside nor 8-bromo-cyclic GMP mimics the actions of carbachol and low concentrations of carbachol block cyclic AMP accumulation without increasing the intracellular cyclic GMP content. Carbachol does not block cyclic AMP accumulation by activating phosphodiesterase since it is fully effective in the face of marked phosphodiesterase inhibition, nor does it appear to inhibit the catalytic activity of adenylate cyclase since it does not decrease either basal or cholera toxin-stimulated cyclic AMP accumulation. The interaction between carbachol and isoproterenol is not competitive, since cholinergic inhibition cannot be surmounted by increasing concentrations of isoproterenol. The site of muscarinic action therefore appears to involve the mechanisms coupling the hormone-receptor complex to adenylate cyclase. This site is distinct from that of cholera toxin action since there is no antagonism between the effects of cholera toxin and carbachol on cyclic AMP metabolism in the atrium.     

Kinetics of the Hydrolysis of 8-Bromo-Cyclic GMP by the Light-Activated Phosphodiesterase of Toad Rods

The hypothesis that cyclic GMP is the internal transmitter of retinal rod phototransduction, when combined with the observations that 8-bromo-cyclic GMP opens the cyclic GMP-dependent outer segment conductance and that rods into which 8-bromo-cyclic GMP has been injected still respond to light, predicts that the light-activated phosphodiesterase (EC 3.1.4.17) must catalyze the hydrolysis of 8-bromo-cyclic GMP. This hypothesis was tested by measuring light-activated toad rod disk membrane phosphodiesterase with a pH assay technique. Phosphodiesterase-catalyzed hydrolysis of 8-bromo-cyclic GMP was confirmed: at pH 8.0, total proton production after flash activation was identical to total amount of 8-bromo-cyclic GMP added as substrate. Photoactivated phosphodiesterase was remarkably less efficient in catalyzing the hydrolysis of 8-bromo-cyclic GMP than of cyclic GMP: Vmax for 8-bromo-cyclic GMP was 0.063 M/M rhodopsin/s, whereas that for cyclic GMP was 11 M/M rhodopsin/s--170 times greater. The Km for 8-bromo-cyclic GMP was 160 microM, and for cyclic GMP, 590 microM. 8-bromo-cyclic GMP competitively inhibited phosphodiesterase-catalyzed hydrolysis of cyclic GMP with a Ki of 1.2 mM. Complete reaction progress curves were analyzed for obedience to Michaelis-Menten kinetics: cyclic GMP hydrolysis, 8-bromo-cyclic GMP hydrolysis, and cyclic GMP hydrolysis in the presence of 8-bromo-cyclic GMP as competitive inhibitor were found to follow the integrated form of the Michaelis-Menten equation over the time course of the reactions, assuming phosphodiesterase was activated as a step. The kinetic parameters extracted from reaction progress curves were consistent with those derived from analysis of the initial velocity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP increases the concentration of insulin receptors in cultured fibroblasts and lymphocytes.

Incubation of SV40 transformed fibroblasts with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or 1-methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, produced a two-fold increase in insulin receptor concentration without an effect on receptor affinity. The increase was dose-dependent, was observed after 8 hrs of treatment, and reached a maximum level by 12 to 24 hours. Upon removal of the nucleotide, receptor number decreased towards basal level.Incubation of cultured human lymphocytes (IM-9 line) with cyclic AMP derivatives or MIX also increased the number of insulin receptors without an alteration in receptor affinity. This effect was partially blocked by inhibition of protein synthesis and was independent of changes in cell cycle. The increase in insulin receptors was a specific response to cyclic AMP as the number of receptors for human growth hormone was unaltered. Incubation with 8-bromo-cyclic GMP did not alter the level of insulin binding.     

In vitro immune response of human peripheral lymphocytes. III. Effect of anti-mu or anti-delta antibody on PWM-induced increase of cyclic nucleotides in human B lymphocytes.

Stimulation of human peripheral blood lymphocytes (PBL) with pokeweed mitogen (PWM) induced consistent increases of intracellular levels of cyclic AMP and cyclic GMP within 15 min. Increases of cyclic AMP were observed in both B and T lymphocyte populations, but increase of cyclic GMP was observed only in the B lymphocyte population. The addition of anti-mu antibody to B cells abolished PWM-induced increase of cyclic GMP without any effect on cyclic AMP response. Anti-delta antibody did not show any inhibitory or stimulatory effect on PWM-induced increase of cyclic GMP or cyclic AMP. Pretreatment of B cells with anti-mu antibody at 37 degrees C for 1 hr inhibited PWM-induced increase of cyclic GMP, whereas pretreatment with anti-mu antibody at 4 degrees C did not show any inhibitory effect on PWM-induced increase of cyclic GMP. The effect of anti-mu-pretreatment was reversible and pretreated cells were recovered from the inhibitory effect of anti-mu antibody after 36 hr culture.     

The effects of parathyroid hormone on L-type voltage-dependent calcium channel currents in vascular smooth muscle cells and ventricular myocytes are mediated by a cyclic AMP dependent mechanism.

The present study demonstrated that L channel currents were decreased in smooth muscle cells, and increased in ventricular myocytes by both bovine parathyroid hormone, (bPTH-(1-34)), and dibutyryl cyclic AMP (db-cAMP), using the whole cell version of the patch clamp technique with Ba2+ as the charge carrier. The effects of bPTH-(1-34) and db-cAMP on L channel currents were additive but not synergistic. Furthermore, the effects of bPTH-(1-34) on L channel currents in these 2 cell preparations were abolished in the presence of a cAMP antagonist. These results suggest that the effects of bPTH-(1-34) on L channel currents in vascular smooth muscle cells and ventricular myocytes are mediated by a cAMP-dependent mechanism.     

Regulation by Dibutyryl Cyclic AMP of Carnosine Synthesis in Astroglia-Rich Primary Cultures Kept in Serum-Free Medium

The synthesis of carnosine (beta-Ala-His) by astroglia-rich primary cultures was much higher if the cells were cultivated in Ham's nutrient mixture F-12 than if they were grown in Dulbecco's modified Eagle's medium. Carnosine synthesis was not affected by the presence of insulin, transferrin, phorbol myristate acetate, or dexamethasone. However, dibutyryl cyclic AMP and other agents that can, directly or indirectly, activate cyclic AMP-dependent protein kinases strongly lower the rate of carnosine synthesis. The depression of carnosine synthesis was dependent on the concentration of dibutyryl cyclic AMP. The effect was maximal (approximately 80% inhibition) in cultures preincubated with 1 mM dibutyryl cyclic AMP for 4 days. The adenylate cyclase activator forskolin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and 8-bromo-cyclic AMP caused the same depression as dibutyryl cyclic AMP, whereas neither butyrate nor dibutyryl cyclic GMP elicited any effect.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.