Cardiac beta-adrenoceptors and adenylyl cyclase activity in human left ventricular hypertrophy due to pressure overload

The effect of left ventricular hypertrophy (LVH) due to chronic pressure overload on right atrial (RA) and left ventricular (LV) myocardial beta-adrenergic receptor (beta-AR) density and subtypes, adenylyl cyclase (AC) activity and ADP-pertussis toxin ribosylated proteins was investigated in humans with LVH due to aortic stenosis and in patients without LVH undergoing heart surgery for mitral stenosis or coronary artery disease taken as controls. Both groups presented normal systolic function or plasma catecholamine levels. In LVH and controls, beta-AR density was similar in RA (62 +/- 6 vs 77 +/- 12 fmol.mg-1 protein) and LV (39 +/- 7 vs 32 +/- 2 fmol.mg-1 protein). In LVH, beta 1-AR percentage was < than in controls in LV (35 +/- 11 vs 73 +/- 5%, P < 0.05) but not in RA (79 +/- 5 vs 73 +/- 8%). Basal AC activity in RA (19 +/- 4 vs 21 +/- 6 pmol.mg-1 protein) and LV (22 +/- 5 vs 27 +/- 3 pmol.mg-1 protein) was similar in LVH and in controls. Isoprenaline-induced stimulation of AC in RA was similar in LVH and in controls (51 +/- 18 vs 36 +/- 18%) but < in LV of LVH (7 +/- 6 vs 45 +/- 6%, P < 0.05). In the presence of ICI-118,551 (a beta 2-adrenoceptor antagonist), isoprenaline failed to induce any increase in cAMP in LVH. The quantification of ADP-pertussis toxin ribosylated proteins indicated a lower concentration of substrates in LV myocardial membranes from LVH. These data indicate that in LVH due to pressure overload, there is a down-regulation of beta 1-AR and an increase in beta 2-AR density. This is associated with alterations of the transmembrane signalling marked by a decreased capacity of isoprenaline to stimulate AC and an impaired expression of Gi proteins.     

Effect of clenbuterol on beta-adrenoceptors and adenylate cyclase activity in smooth muscle and epithelium of the trachea of calves

A potent beta-agonist (clenbuterol) was administered perorally to young calves for 50 days. After this period the animals were slaughtered and beta-adrenoceptor density, ligand affinity, and basal and stimulated adenylate cyclase activities were studied in smooth muscle and epithelium of the trachea. Although the density of lung beta-adrenoceptors was down regulated by clenbuterol, cAMP production remained constant (epithelium) or even increased (smooth muscle). Therefore desensitization of beta-adrenoceptors in the trachea was not observed. This might be a reason for the effectiveness of long-term treatment with beta-agonists.     

Cardiac hypertrophy alters myocardial response to ischaemia and reperfusion in vivo

The impact of cardiac hypertrophy on myocardial biochemical and physiological responses to ischaemia-reperfusion (I-R) was investigated in vivo. Hypertrophy was produced by aortic constriction (PH) or swimming training (TH). Open-chest rat hearts in PH, TH and a sedentary control group (SC) were subjected: (1) to ischaemia, by surgical occlusion of the main descending branch of the left coronary artery for 30 min; (2) to I-R, by releasing the occluded blood vessel for 15 min; or (3) to a sham operation. Ischaemia per se had little effect on heart oxidative and antioxidant status, or lipid peroxidation. However, I-R significantly decreased glutathione (GSH) content, increased glutathione disulfide (GSSG) content, and reduced GSH/GSSG ratio in the SC hearts. These alterations were associated with decreased activities of GSH peroxidase and GSSG reductase, and an increase in lipid peroxidation. Myocardial ATP, total adenine nucleotide content and energy charge in SC were significantly decreased after ischaemia, whereas levels of purine nucleotide derivatives, particularly adenosine, were elevated. No significant alteration of GSH status of adenine nucleotide metabolism occurred after ischaemia or I-R in hypertrophied hearts. In both PH and TH, glutathione content was significantly higher than in SC, whereas activities of GSH peroxidase and GSSG reductases were lower. TH rats maintained a higher heart rate (HR), peak systolic pressure, and energy charge during I-R. These data indicate that hypertrophied but well-functioned hearts may be more resistant to I-R induced disturbances of myocardial oxidative and antioxidant functions.     

Endothelin-induced inositol phosphate formation in rat kidney. Studies on receptor subtypes, G-proteins and regulation during ontogenesis

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12-16 weeks old) ET's concentration-dependently increased IP-formation with an order of potency ET-1 > ET-3. While the non-selective ET-receptor antagonist bosentan (10 microM) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 microM) inhibited it only by 70%, the ETB-receptor antagonist IRL 1038 (1 microM) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal sites from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET-receptor number and the amount of immunodetectable Gq/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET-receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ETA-receptors declined, while that of ETB-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ETA- and ETB-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of Gq/11.     

Functional activity and expression of the myocardial postreceptor adenylyl cyclase system in pressure overload hypertrophy in rat

OBJECTIVE: The aim of the present study was to investigate the functional regulation of the myocardial postreceptor adenylyl cyclase (AC) system in compensated left ventricular hypertrophy (LVH) and the effect of long-term angiotensin converting enzyme (ACE) inhibition. METHODS: Pressure overload LVH was induced in rats by supravalvular aortic banding for 12 weeks. At 12 weeks left ventricular function and inner diameters were analyzed by echocardiography of anesthetized animals, and responsiveness to forskolin (systolic developed pressure) was determined in isolated perfused hearts. Functional activities of AC and the stimulatory G protein Gs were measured as well as mRNA expression (quantitative slot blot analyses) of AC type V, isoforms of Gs alpha and Gi alpha 2. G protein alpha-subunits were also quantified by immunoblotting. Rats were treated with ramipril (Ram, 10 mg/kg per day p.o.) during weeks 7 to 12 to induce regression of LVH or with vehicle (Veh, tap water). RESULTS: Pressure overload induced severe LVH (3.2 +/- 0.09 g/kg in Veh vs. 1.8 +/- 0.03 in sham; P < 0.05) which was significantly reduced by ramipril (2.7 +/- 0.09; P < 0.05 vs. Veh). In-vivo left ventricular function and diameters were unchanged in LVH. In contrast, in hearts with LVH, responsiveness of left ventricles to forskolin was attenuated and basal, GTP gamma S and forskolin as well as manganese chloride-stimulated adenylyl cyclase activity was significantly downregulated by approximately 40% (basal 20.8 +/- 1.9 pmol cAMP/mg per min vs. 34.0 +/- 2.2 in sham; P < 0.01). However, no significant changes of AC type V mRNA were found in hypertrophied left ventricles. Functional activity of the stimulatory G protein Gs was reduced in LVH (48 +/- 7 pmol cAMP/mg per min in Veh vs. 68 +/- 3 in sham), whereas mRNA expression of long and short Gs alpha-isoforms was not altered and that of Gi alpha 2 was only slightly increased in ramipril-treated animals. Western analysis showed no significant differences of Gs alpha or Gi alpha 2 subunits. Long-term blockade of the renin-angiotensin system had no effect on the activity of the adenylyl cyclase system. CONCLUSIONS: Functional desensitization of adenylyl cyclase and stimulatory G protein occurred in rat adaptive LVH prior to the onset of severe left ventricular dysfunction which was not restored by ACE-inhibitor treatment. The desensitization seems not to be mediated by significant changes of mRNA expression of AC type V or abundance of regulatory G proteins.     

Regional myocyte hypertrophy parallels regional myocardial dysfunction during post-infarct remodeling

The molecular genetic events involved in the etiology of human granulosa cell (GC) tumors, which represent approximately 7% of all malignant ovarian neoplasms, are unknown. Amplification and/or overexpression of the ERBB genes are a feature of many cancer types, and overexpression of erbB2 correlates with poor prognosis in epithelial ovarian cancer. In the present study, we used immunohistochemistry to determine the level and frequency of expression of different erbB receptors in GC tumors. Ten of 12 tumors expressed erbB4 at moderate to high levels in >50% of cancer cells, whereas erbB2 (6 of 12) and erbB3 (2 of 12) were expressed less frequently. Western blot experiments showed that the only available GC tumor cell line, COV434, also expressed erbB receptors. Heregulin (HRG)-beta2, a ligand for erbB3 and erbB4 receptors, stimulated tyrosine phosphorylation of the erbB receptors, which was accompanied by activation of Erk1 and Erk2, two mitogen-activated protein kinases with a functional role in mitogenesis. Importantly, HRG increased cell proliferation in COV434 cells, and treatment with HRG/PE40, a ligand toxin shown previously to be cytotoxic against human breast cancer cells overexpressing erbB receptors, led to a dramatic and irreversible decrease in cell number. These results indicate that erbB receptor signaling pathways may be critical in the control of GC tumor cell proliferation and that HRG/PE40 is a potential therapeutic agent for the treatment of GC tumors.     

Dynamic regulation of calcium influx by G-proteins, action potential waveform, and neuronal firing frequency

The time course of Ca2+ channel activation and the amplitude and rate of change of Ca2+ influx are primarily controlled by membrane voltage. G-protein-coupled signaling pathways, however, modulate the efficacy of membrane voltage on channel gating. To study the interactions of membrane potential and G-proteins on Ca2+ influx in a physiological context, we have measured N-type Ca2+ currents evoked by action potential waveforms in voltage-clamped chick dorsal root ganglion neurons. We have quantified the effect of varying action potential waveforms and frequency on the shape of Ca2+ current in the presence and absence of transmitters (GABA or norepinephrine) that inhibit N current. Our results demonstrate that both the profile of Ca2+ entry and the time course and magnitude of its transmitter-induced inhibition are sensitive functions of action potential waveform and frequency. Increases in action potential duration enhance total Ca2+ entry, but they also prolong and blunt Ca2+ signals by slowing influx rate and reducing peak amplitude. Transmitter-mediated inhibition of Ca2+ entry is most robust with short-duration action potentials and decreases exponentially with increasing duration. Increases in action potential frequency promote a voltage-dependent inactivation of Ca2+ influx. In channels exposed to GABA or norepinephrine, however, this inactivation is counteracted by a time- and frequency-dependent relief of modulation. Thus, multiple stimuli are integrated by Ca2+ channels, tuning the profile of influx in a changing physiological environment. Such variations are likely to be significant for the control of Ca2+-dependent cellular responses in all tissues.     

Stable cholinergic-muscarinic and alpha-adrenergic inhibition of rat parotid adenylate cyclase

(-) Isoproterenol-stimulated adenylate cyclase activity of washed at parotid membrane preparations from gland slices previously treated with the alpha-adrenergic agonist, methoxamine, was inhibited approximately 30%. The action of methoxamine required exogenous Ca2+ and was blocked by the alpha-adrenergic blocking agent, phentolamine. Incubation of gland slices with carbamylcholine resulted in a dose-dependent inhibition (50%) of (-) isoproterenol-stimulated adenylate cyclase activity in washed membranes. The action of carbamylcholine was independent of exogenous Ca2+ and was blocked by preincubation with atropine. Cholinergic inhibition of parotid adenylate cyclase was compared to the cholinergic inhibition of adenylate cyclase in dog heart homogenates. While carbamylcholine caused a limited (20-30%) inhibition of basal and (-) isoproterenol-stimulated activities when added to dog heart homogenates, it failed to produce any effect in parotid homogenates prepared in an identical manner. Cholinergic inhibition of parotid adenylate cyclase activity was stable and persisted in washed particulate fractions while the inhibition in dog heart homogenates was reversible by washing. Cholinergic inhibition of adenylate cyclase was markedly dependent on the presence of GTP and was abolished when Mn2+ was subsituted for Mg2+ in both systems. Guanyl-5'-yl imidodiphosphate had little effect on the inhibition in parotid preparations but abolished the inhibition in heart homogenates. It is concluded that, in contrast to the cholinergic action in dog heart homogenates, the exposure of parotid slices to carbamylcholine results in a stable lesion in the adenylate cyclase activity.     

Synergistic activation of adenylate cyclase by guanylyl imidophosphate and epinephrine

A kinetic analysis of the synergistic activation of turkey erythrocyte adenylate cyclase by 1-catecholamines and guanylyl imidodiphosphate (Gpp(NH)p) is described. We have found that the role of the catecholamine hormone is to facilitate the activation of the enzyme by the guanyl nucleotide according to the following mechanism: R-E+G=R-EG R-EG+H=HR-EG leads to HR-E'G where R is the receptor, E the enzyme, G the guanyl nucleotide effector, and H the hormone. The binding steps are fast and reversible but the conversion of the inactive enzyme E to its active stable form (E') occurs with a rate constant of k=0.7 min-1. This step is essentially irreversible in the presence of high Gpp(NH)p concentrations. In the absence of beta-agonist (1-catecholamine) and at low free Mg2+ concentrations, the activation of the enzyme is insignificant. At high Mg2+ concentration the conversion of E to E' occurs slowly in the absence of hormone, probably by another pathway. Thus, the presence of a guanyl nucleotide at the allosteric site is obligatory but not sufficient to induce the conversion of the inactive enzyme to its active form. The process of enzyme activation requires both Gpp(NH)p and hormone and under these conditions is essentially irreversible. The permanently active enzyme is stable in the absence of hormone and Gpp(NH)p and its high catalytic activity is stable for many hours. However, hormone and ATP induce a conversion of the high activity to the low activity form. Thus, it seems that both the process of enzyme activation by Gpp(NH)p and its reversal are hormone dependent. Both processes are blocked by the beta-blocker propranolol.     

ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.     

Distribution of myocardial beta-adrenoceptor subtypes and coupling to the adenylate cyclase in children with congenital heart disease and implications for treatment

In congestive heart failure, down-regulation of myocardial beta-adrenoceptors (beta-AR) due to an elevated sympathetic tone is well known. In infancy and childhood, heart failure is usually related to congenital heart disease (CHD). Therefore, 71 samples of right atrial tissue of infants and children with CHD undergoing cardiac surgery were studied for beta-adrenoceptor density and distribution of the beta 1-/beta 2-AR subtypes. In 49 cases, the coupling of the beta-AR to the adenylate cyclase (AC) was examined. In a further study of 19 myocardial samples, AC was selectively stimulated with beta 1- or beta 2-AR whereas the other subtype was blocked by an antagonist. The following results were obtained: (1) Infants and children with severe acyanotic or cyanotic CHD had severely reduced beta-AR densities. (2) In most of the cases, the beta-AR down regulation is beta 1-subtype selective, but in critically ill newborns with congenital aortic valve stenosis or transposition of the great arteries, there is additional significant beta 2-AR down-regulation. In Fallot patients treated with the beta-antagonist propranolol, a significant increased beta-AR number compared with untreated Fallot patients was found. (3) beta-Adrenoceptor reduction in CHD is correlated with elevated noradrenaline plasma levels, thus proving a sympathetic dysregulation. (4) In CHD with moderate hemodynamic load, beta 2-AR coupling to AC was markedly more efficient than beta 1-AR coupling. The small number of myocardial beta 2-AR produced most of the cyclic adenosine monophosphate. (5) In severe acyanotic and cyanotic CHD, a partial decoupling of the beta 2-AR to the AC occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemosignal transduction in the vomeronasal organ of garter snakes: Ca(2+)-dependent regulation of adenylate cyclase

Earthworm shock secretion contains a 20-kDa vomeronasally mediated chemoattractive protein for garter snakes. Both the ligand-receptor binding and the chemoattractivity of ES20 are Ca(2+)-dependent. When ES20 binds to its G-protein-coupled receptors in the vomeronasal epithelium, the inositol 1,4,5-trisphosphate (IP3) level is increased, but the level of cAMP is reduced. Furthermore, forskolin-stimulated levels of cAMP are completely blocked by ES20-receptor binding or by Ca2+ alone and the effect of calcium ions can be nullified by EGTA. Previously, we hypothesized that the decrease in cAMP was due to activation of a Ca(2+)-dependent phosphodiesterase. In the present study, we provide evidence that the decrease in cAMP is due mainly to the regulation of adenylate cyclase (AC) activity by Ca2+ or is indirectly mediated by ES20. Results obtained with intact vomeronasal sensory epithelium suggest that the binding of ES20 to its receptors facilitates generation of IP3 which mobilizes intracellularly sequestered Ca2+, resulting in an increase of cystosolic Ca2+. A further increase in cytosolic Ca2+ occurs through Ca2+ influx from extracellular sources. Garter snake vomeronasal AC does not require calmodulin for its activity and shows a biphasic response to increasing concentrations of Ca2+; its activity is modulated both positively and negatively by this bivalent cation.     

Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro

The results reported here show some characteristics of adenylate cylase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F- under conditions where cyclic nucleotide degradative pathways are effectively inhibited. Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 X g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E1 and E2, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F1 alpha and F2 alpha. Stimulation by prostaglandin E1 and E2 is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E1 is equal to that caused by optimal fluoride concentrations. Stimulation caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.     

Structure of adenylate cyclase and the coupling with the receptor-G protein system

Adenylate cyclase is a key enzyme that couples with both the stimulatory and inhibitory G proteins (Gs and Gi). The cyclase has been purified and shown to be a glycoprotein of molecular weight 115,000-180,000. Cloning of cDNAs for adenylate cyclase showed that the cyclase is a member of a large family consisting of a variety of subtypes of the enzyme. These subtypes show different responses to calmodulin and G protein beta gamma subunits, and their distributions in tissues and organs are also different. This suggests that each subtype is involved in a particular physiological function. The general structure of adenylate cyclase is composed of two cytoplasmic domains and two membrane-spanning domains, each of which contains 6 transmembrane spans (12 spans in a molecule). The amino acid sequence of each cytoplasmic domain, which is thought to contain a nucleotide (ATP) binding site, is well-conserved among the various subtypes. This review also focuses on the regulation of adenylate cyclase activity by G protein subunits, particularly on several models for adenylate cyclase inhibition by Gi. As one of these mechanisms, direct inhibition of adenylate cyclase by the beta gamma subunits recently demonstrated by us will be discussed.     

Temperature-dependent stimulation and inhibition of adenylate cyclase by Aplysia bag cell peptides

The bag cell peptides (alpha-, beta-, and gamma-BCP) are secreted by the neuroendocrine bag cells of Aplysia, and provide feedback modulation of bag cell excitability and cAMP levels. We report here that if 200-500 mM NaCl is included in the assay buffer, the BCPs alter adenylate cyclase activity in a manner consistent with their effects on cAMP levels in intact bag cells. Specifically, beta-BCP and the related peptide A from the atrial gland stimulate the enzyme, while the effects of alpha-BCP(1-7) and gamma-BCP are temperature-dependent, stimulating at 30 degrees C and inhibiting at 15 degrees C. Both stimulation and inhibition require GTP, suggesting mediation by Gs and Gi. The ionic requirements of stimulation and inhibition differ: Cl- is necessary to support stimulation, but not inhibition. Moreover, pertussis toxin blocks inhibition, but does not affect stimulation. These results suggest that the temperature-sensitive mechanism lies upstream from the G-proteins in the signal transduction pathway.     

5-Hydroxytryptamine-induced tachyphylaxis of the molluscan heart and concomitant desensitization of adenylate cyclase

High concentrations of 5-hydroxytryptamine (5HT) first excite the isolated ventricle of Mercenaria mercenaria and then specifically desensitize it to further additions of the neuropeptide. This 5HT-induced tachyphylaxis is paralleled by a 5HT-specific desensitization of the myocardial adenylate cyclase and a decrease in intracellular cyclic AMP. However, FMRF-NH2, a cardioexcitatory tetrapeptide, can still increase contractility, cyclic AMP, and the adenylate cyclase activity of a tachyphylactic ventricle. These results are consistent with the hypothesis that 5HT augments molluscan myocardial contractility by elevating intracellular cyclic AMP.     

Localization of G-proteins in macrophages and E. coli during phagocytosis

Heterotrimeric GTP-binding proteins (G-proteins) have been shown to play an important role in cellular signalling. However, G-protein involvement in the intracellular spreading of bacterial pathogens is still poorly understood. In this study, antibodies, that recognize G-protein alpha-subunits (anti-G alpha), were used to investigate the localization of G-proteins in the macrophage-like cell line P388D1 and E. coli, also in their L-forms, during phagocytosis. In E. coli, anti-G alpha-binding sites were detected preferably in the cell wall and septa of the whole bacterial forms as well as in the cytoplasm of L-forms. Western blotting of bacterial lysates demonstrated protein bands with positive immunoreaction to antibodies against Gs alpha, Gi alpha, and Gcommon alpha with a higher affinity to the antibody against Gs alpha. Immunoreaction with the anti-Gs alpha-antibody was markedly higher in pathogenic strains of E. coli. Because of the conserved structure in all GTP-binding proteins which seem to derive from a single primordial protein involved in signal transduction mechanisms, it is reasonable to assume that some anti-Ga-positive proteins in E. coli might be related to G-proteins of higher organisms. A putative candidate for bacterial G-proteins seems to be a 36 kDa protein. Enhancement in G-protein immunostaining in the cytoplasm of macrophages around the internalized bacteria testifies to the involvement of G-proteins in mediation of endocytosis responses of phagocytes.     

Differential regulation of N- and Q-type Ca2+ channels by cyclic nucleotides and G-proteins

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway delimited to the membrane. In addition, modulation of Ca2+ channel activity by protein kinases also contributes to the dynamic regulation of neuronal physiology. Recently, differences in these modulations between Ca2+ channel subtypes have been shown in several neuronal preparations. Here we show that two types of presynaptic Ca2+ channel (N-type and Q-type) are differentially regulated by cAMP and G-proteins using a Xenopus oocyte expression system. Treatment to increase cytosolic cAMP concentration with forskolin and 3-isobutyl-1-methylxanthine (IBMX) markedly potentiated Q-type channel current, and the enhancement was reversed by protein kinase A inhibitors. Much smaller enhancement was observed in N-type channel current after the cAMP elevation. When large depolarizing prepulse was applied to the oocytes for evaluation of the tonic inhibition of Ca2+ channels by intrinsic G-protein activity, N-type channel current elicited a large prepulse facilitation but Q-type channels did not. The tonic inhibition of N-type channels was abolished by an intracellular perfusion with a 'cut-open' recording configuration, or by co-expression with G(alpha o). When kappa opioid receptors were co-expressed and stimulated with agonists, depolarization-resistant inhibition was more apparent in Q-type channels than in N-type channels. These results suggest that Q-type channels are more susceptible to the protein kinase A-mediated facilitation than N-type channels, and that activity of N-type channels can be more highly regulated in a voltage-dependent manner by G(betagamma) than that of Q-type channels. These differences may account for the selective regulation of neurotransmitter release by these Ca2+ channels.