Genetic manipulation and functional analysis of cAMP signalling in cardiac muscle: implications for a new target of pharmacotherapy.

Adenylate cyclase is a membrane-bound enzyme that catalyses the conversion of ATP into cAMP upon activation of cell-surface G-protein-coupled receptors, such as beta-adrenergic receptors, and initiates a cascade of phosphorylation reactions within the cell. Type 5 adenylate cyclase is a major isoform in the heart as well as in the striatum of the brain. Mice with a disrupted type 5 adenylate cyclase gene exhibited normal cardiac function under basal conditions, but a decreased response to isoprenaline stimulation. When mice were subjected to pressure overload stress with aortic banding, they developed cardiac hypertrophy, but with a significant reduction in the number of apoptotic cardiac myocytes as well as preserved cardiac function. When type 5 adenylate cyclase activity was inhibited pharmacologically, by the use of a novel P-site inhibitor with enhanced selectivity for this isoform, there were no changes in cardiac myocyte contractility, but the development of cardiac myocyte apoptosis induced by isoprenaline stimulation was effectively prevented. These results indicate that type 5 adenylate cyclase may serve as a better target of pharmacotherapy to prevent the development of cardiac myocyte apoptosis and thus failure in response to various cardiac stresses.

Accumulation of molecules involved in alpha1-adrenergic signal within caveolae: caveolin expression and the development of cardiac hypertrophy.

OBJECTIVE: Caveolin, a major protein component of caveolae, is now considered to be an inhibitor of cellular growth and proliferation. In this study, we examined the localization of the molecules involved in alpha1-adrenergic receptor signal relative to that of caveolin in the heart and the changes in caveolin expression during the development of hypertrophy in SHR. METHODS: We purified the caveolar protein fractions from rat cardiac tissues, H9C2 cells, and rat vascular smooth muscle cells. Using radioligand receptor binding assay and immunoblot analysis, we examined the distribution and the amount of alpha1-AR and caveolin. RESULTS: Caveolin-3, the alpha1-adrenergic receptor, Gq and PLC-beta subtypes (PLC-beta1, -beta3) were found exclusively in the caveolar fraction in the above tissues. Caveolin-3 were co-immunoprecipitated with alpha1-adrenergic receptor and Gq from the cardiac tissues. The amount of caveolin subtypes expression (caveolin-1 and -3) and the amount of the alpha1-adrenergic receptor were examined in the hearts of SHR and age-matched WKY (4- and 24-weeks-old). The amount of caveolin-3 expression was significantly smaller in SHR at 24-weeks-old than that in SHR at 4-weeks-old and that in WKY at 24-weeks-old. CONCLUSIONS: The molecules involved in alpha1-adrenergic signaling are confined to the same microdomain as caveolin. A decrease in caveolin-3 expression may play a role in the development of cardiac hypertrophy in SHR, presumably through de-regulating the inhibition of growth signal in the hearts of SHR in the hypertrophic stage.

Central antinociceptive effect of L-ornithine, a metabolite of L-arginine, in rats and mice.

L-Arginine produces central antinociception by acting as a precursor of kyotorphin (L-tyrosyl-L-arginine), a [Met5]enkephalin releaser. This study investigated the antinociceptive activity of L-ornithine, a metabolite of L-arginine. L-Ornithine given s.c. at 300-1000 mg/kg suppressed carrageenin-induced hyperalgesia in rats in a naloxone-reversible manner. L-Ornithine and L-arginine, when given i.c.v. at 10-100 mu g/mouse, elicited antinociception even in intact mice, the effects being abolished by naloxone or naltrindole, and potentiated by bestatin, an inhibitor of aminopeptidase and kyotorphinase. The antinociception induced by i.c.v. L-ornithine was also inhibited by i.c.v. L-leucyl-L-arginine, a kyotorphin receptor antagonist, but was resistant to intracisternal anti-kyotorphin serum. L-Tyrosyl-L-ornithine, a synthetic dipeptide, (1-10 mu g/mouse, i.c.v.), exerted kyotorphin-like antinociception in mice. These findings suggest that L-ornithine produces L-arginine-like antinociception via kyotorphin receptors. However, this effect does not appear to be mediated by kyotorphin itself, but most likely by L-tyrosyl-L-ornithine, a putative dipeptide.

Participation of opiate and serotonergic systems in brain conditioning stimulatory inhibition of the potentials evoked by tooth pulp stimulation in the pars caudalis of the trigeminal sensory nucleus of the rat.

Effects of conditioning stimulations of the nucleus raphe magnus (NRM), nucleus reticularis paragigantocellularis (NRPG), mesencephalic periaqueductal central gray (PAG), nucleus dorsomedialis hypothalami (DMH), corpus striatum (CP), sensory cortex (SCT) and visual cortex (VCT) and actions of morphine, naloxone and metergoline on the potentials recorded from the pars caudalis of the trigeminal sensory nucleus evoked by the electrical stimulation of rat incisor pulps were examined. The spinal potentials evoked by electrical stimulation of the pulp consisted of 3 components. Component 2 was mainly inhibited by morphine and antagonized by naloxone. Conditioning stimulation of NRM, NRPG, PAG, CP, SCT and DMH strongly inhibited component 2. VCT did not show any inhibition. 33-67% of antagonism was observed by naloxone in the NRM, NRPG, PAG, CP, SCT and DMH. On the other hand, 27-44% of antagonism was observed by metergoline, and the antagonism was not enhanced by the additional administration of naloxone. These results conclusively show that the endorphin system as well as the serotonergic system is in the series involved in the descending inhibition for nociception in the trigeminal sensory nucleus.

Effect of neuroactive peptides on labeled 5-hydroxytryptamine release from rat spinal slices in vitro.

Effects of neuroactive peptides on the release of labeled 5-hydroxytryptamine (5-HT) from preloaded rat spinal cord slices were investigated. The 5-HT release was significantly stimulated by somatostatin (10-50 microM) and substance P (10-50 microM), but not by neurotensin (50 microM), beta-endorphin (30 microM) and methionine-enkephalin (met-enk) (100 microM). Somatostatin-stimulated 5-HT release was markedly inhibited by gamma-aminobutyric acid (GABA) (30 microM), but not by baclofen (30 microM) and met-enk (100 microM). Substance P-stimulated 5-HT release was strongly inhibited by GABA (30 microM) and baclofen (30 microM), but not by met-enk (100 microM). High concentrations (20 mM) of potassium also stimulated 5-HT release. The high potassium-stimulated 5-HT release was not affected by GABA (30 microM) and met-enk (100 microM). These results suggested further evidence on the important role of somatostatin and substance P as modulators of serotonergic neurones.

Effects of dopaminergic agonists and antagonists on [3H]apomorphine binding to striatal membranes: sulpiride lack of interactions with positive cooperative [3H]apomorphine binding.

Effects of dopaminergic agonists and antagonists on [3H]apomorphine binding to striatal membranes of rat brain was examined. Haloperidol and spiroperidol exhibited biphasic inhibition of [3H]apomorphine binding; one of which had the Hill coefficient of 0.9, whereas the other had that of 0.4. The former accounted for 65% of [3H]apomorphine binding while the latter consisted of 35% of the binding. Furthermore, the latter disappeared after kainic acid lesions. On the other hand, sulpiride and metoclopramide reduced [3H]apomorphine binding to 31% with the Hill coefficient of 0.9. The inhibition of [3H]apomorphine binding with the Hill coefficient of 0.4 which was shown by haloperidol and spiroperidol was not observed for sulpiride and metoclopramide. Previously, we demonstrated non- and positive-cooperative [3H]apomorphine binding to stiatal membranes. In the present study, it has been also shown that sulpiride inhibits non-cooperative [3H]apomorphine binding leaving that with allosteric properties unaffected. No inhibition of dopamine-sensitive adenylate cyclase was observed by 10(-4) M sulpiride while 90% inhibition was obtained with 10(-5) M haloperidol. From those results, it is suggested that non-cooperative [3H]spomorphine binding is not coupled with dopamine-sensitive adenylate cyclase.

Cortically induced inhibition of neurons of rat substantia nigra (pars compacta).

The effects of electrical stimulation of prefrontal cortex upon neurons of substantia nigra (pars compacta) in anesthetized rats were mostly inhibition without antidromic excitation. By studying nigral neurons in which the inhibition from caudate-putamen was antagonized by iontophoretic bicuculline, it was found in only half of them that the same drug also antagonized the inhibition from prefrontal cortex.

Effects of morphine and haloperidol on the electrical activity of rat nigrostriatal neurons.

The action of opiates on electrical activity in nigrostriatal neurons was compared with the actions of known dopamine receptor agonists and antagonists in order to clarify the effects of opiates on postsynaptic dopamine receptors in the striatum. The systemic administration of either morphine or haloperidol increased the rate of spontaneous firing of neurons in substantia nigra. The injection of either drug directly into the caudate nucleus of the striatum also increased the firing rates of nigral neurons. The administration of the opiate antagonist, naloxone, blocked or reversed the action of systemically or locally applied morphine on firing rates, but not those of haloperidol. The dopamine receptor agonists, dopa and apomorphine, decreased the firing rate in nigral neurons and also reversed the stimulation of firing rates by morphine. The agonists were only partially successful in reversing the effects of haloperidol. The differences in the ability of naloxone and dopa to reverse the stimulated firing rates produced by morphine or haloperidol support the hypothesis that opiates do not act directly on the postsynaptic dopamine receptor in the striatum.

[Pharmacological comparison between enflurane and halothane].

General pharmacological properties of enflurane (E) and halothane (H) were investigated. Maximum blood concentrations of both drugs reached 30 min after inhalation. E showed lower maximum blood concentration, initial velocity of uptake and shorter half life than H. Neither drug had any effect on neuromuscular junction, but E increased N-M effect of succinylcholine. Both drugs decreased tension of uterine and intestine muscles. Poly- and monosynaptic reflexes were inhibited more by H. ED50's of E and H for intestine muscles. Poly- and monosynaptic reflexes were inhibited more by H. ED50's of E and H for righting reflux were 1.25 and 1.40%, respectively. Tonic and clonic convulsions and death induced by electric shock were inhibited more by E. Almost equal anticonvulsive potency was observed for chemoshocks. Death due to electric and chemoshock was remarkably inhibited by both drugs. Spontaneous EEG was altered in a different manner, although flattened with spikes at 4% concentration of both drugs. E altered rhythmicity of recruiting response and both drugs inhibited this response. H inhibited more remarkably the augmenting response and E inhibited the arousal response. E increased negative potentials of the primary response evoked by sensory stimulation, while H decreased these potentials. Both drugs completely inhibited the secondary response.

Effects of narcotic analgesic drugs on the cyclic adenosine 3',5'-monophosphate-adenylate cyclase system in rat brain.

1. The concentrations of cyclic adenosine 3',5'-monophosphate (cyclic AMP), measured in discrete brain areas removed from rats killed by microwave irradiation, rose transiently in most areas after the administration of morphine. The most pronounced changes, however, were found 2 h after doses of either 10 or 60 mg/kg morphine when cyclic AMP levels declined significantly in the hypothalamus, medulla and cerebellum. In morphine-tolerant rat brains there were no decreases in cyclic AMP levels. 2. Basal adenylate cyclase activity in crude nerve-ending fractions from discrete areas of rat brain was unaffected by the addition of active analgesic agonists, antagonists or inactive isomers to the assay medium in vitro, except for a nonspecific inhibition at drug concentrations of 1 mM. 3. The acute administration of morphine or levorphanol, but not dextrorphan produced transient increases in basal cyclase activity of crude nerve-ending preparations from midbrain and striatum. In morphine-tolerant rats, these changes in basal adenylate cyclase activity were no longer seen.