Evidence for a role of protein kinase C in the activation of the pyruvate dehydrogenase complex by insulin in Zajdela hepatoma cells.

The signal transduction pathway involved in the activation of pyruvate dehydrogenase (PDH) by insulin is still unknown. In this study, we have examined the possible involvement of protein kinase C (PKC) in the process. In addressing this question, we examined (1) the insulin-like effects of the PKC activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) on the PDH complex, (2) the effects of various PKC inhibitors on the PDH activation by insulin, and (3) the response of PKC-depleted cells to insulin. We used as an experimental model Zajdela hepatoma cultured (ZHC) cells, which have been demonstrated to be responsive to physiological doses of insulin. Half-maximal and maximal stimulations of the PDH complex by insulin were observed at 0.05 and 5 nmol/L, respectively. Stimulation of PDH activity by insulin (5 nmol/L) occurred within 5 minutes of incubation and was maximal (+70%) at 7.5 minutes. In the presence of PMA (162 nmol/L), enzyme activity increased within 30 seconds, was maximal (+90%) at 5 minutes, and was no longer detectable after 10 minutes. Total PDH activity was unchanged by insulin or PMA treatment. The effects of PMA and insulin on basal PDH activity were not additive. Moreover, various inhibitors of PKC--staurosporine, sphingosine, acridine orange--completely blocked the stimulation of PDH activity induced by insulin or PMA. A 17-hour treatment of ZHC cells with 500 nmol/L PMA efficiently downregulated PKC, as attested by the marked decrease in the enzyme activity and the loss of phorbol 12,13-dibutyrate binding to intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological analysis of the cardiac effects of 5-HT and some 5-HT receptor agonists in the pithed rat.

A pharmacological analysis of the effects of 5-HT on heart rate has been performed in the pithed rat. 5-HT induced a dose-dependent increase in heart rate whereas 5-HT1 receptor agonists--8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 5-methoxy-N,N-dimethyl-tryptamine (5-MeODMT), 5-methoxy 3-(1,2,3,6-tetrahydro-4-piridinyl) 1H indole (RU 24969) and 1-(m-trifluoromethylphenyl)-piperazine (TFMPP)--failed to increase heart rate. The increase in heart rate induced by the selective 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) was not significant. The dose-response curve to 5-HT for its tachycardic effects was shifted two-fold to the right by ketanserin and LY 53857 and nine-fold to the right by methiothepin. The effects of high doses of 5-HT (higher than 100 micrograms/kg iv) were antagonized by methiothepin, (-)propranolol, 2-(2-[4(O-methoxyphenyl)-piperazine-1-yl]-ethyl)4,4-dimethyl-1,3 (2H-4H) isoquinoline-dione (AR-C 239) and by pretreatment with reserpine. The 5-HT1 receptor antagonists, pindolol and spiroxatrine, the 5-HT3 receptor antagonist MDL 72222 and the alpha 2-adrenoceptor blocking agent idazoxan failed to antagonize the tachycardia induced by 5-HT. It is concluded that in the pithed rat, the tachycardia induced by 5-HT remained unexplained (implication of 5-HT2 receptors probably different from the classical vascular 5-HT2 receptor, or implication of 5-HT1C receptors?). Moreover, at high doses (higher than 100 micrograms/kg iv), 5-HT may increase heart rate by releasing catecholamines.

[Hemodynamic profile of 8-OH-DPAT and 5-HT1 agonists in the anesthetized dog]. / Profil hémodynamique du 8-OH-DPAT et des agonistes 5-HT1 chez le chien anesthésié.

In anaesthetized dogs, intravenous administration of 8-OH-DPAT (1-300 micrograms/kg i.v.) induced dose-dependent decrease in blood pressure (BP) and total peripheral resistance (TPR). Heart rate (HR) and cardiac output (CO) changed little. 5-Carboxamidotryptamine (5-CT) (0.3-3 micrograms/kg i.v.) dose-dependently decreased BP and TPR but increased HR, CO, myocardial contractility and pulmonary arterial pressure (PAP). 5-MeODMT and RU 24969 decreased BP only after the highest dose used (300 micrograms/kg i.v. and 1 mg/kg i.v. respectively) but significantly increased PAP at all doses used. These results indicated that in dogs as in other animal species, 8-OH-DPAT decreases BP by systemic vasodilatation without a reflex activation of the myocardium. This lack of a reflex tachycardia suggests a centrally-mediated effects of 8-OH-DPAT. In contrast to 8-OH-DPAT, the vasodilatation induced by 5-CT triggers a reflex tachycardia. The increase in PAP induced by 5-MeODMT, RU 24969 and--to a lesser extent--5-CT, may be due to a direct stimulation of 5-HT2 receptors. However, a 5-HT1 component could not be ruled out.

Comparison between ketanserin and LY 53857 on vascular and cardiac 5-HT2 and alpha 1-adrenergic receptors in the pithed rat.

In normotensive pithed rats, both ketanserin and LY 53857 potently antagonized the increase in blood pressure induced by 5-HT in a noncompetitive manner. However, LY 53857 differs from ketanserin in that it shifts the pressor dose-response curve to 5-HT in a parallel way at low doses but in a noncompetitive way at higher doses. Ketanserin and LY 53857 were weak antagonists of the pressor effects of phenylephrine. LY 53857 was more potent and more selective than ketanserin for 5-HT2 vs alpha 1-adrenergic receptors.

Haemodynamic aspects and serotonin.

Serotonin (5-HT) induced a transient decrease followed by an increase and then a longer-lasting decrease in blood pressure. The initial decrease in blood pressure results from a reduction in cardiac output as a result of the profound bradycardia. The secondary increase in blood pressure is caused by an increase in both cardiac output and total peripheral resistance. The final decrease in blood pressure is ascribed to a reduction in total peripheral resistance. The effects of 5-HT on regional haemodynamics are known to vary according to the vascular bed and even in the same vascular bed. Accordingly, 5-HT induced either vasoconstriction or vasodilatation. Vasoconstriction occurred in most cases in large arteries and is due to stimulation of 5-HT2 receptors. However, 5-HT1-like receptors are probably also implicated. 5-HT-induced vasodilatation is attributed to stimulation of 5-HT1-like receptors. However, limited evidence suggests also an implication of 5-HT3 and even 5-HT2 receptors in the dilator effects of 5-HT. 5-HT1A receptor agonists decreased blood pressure by a reduction in total peripheral resistance. This vasodilatation seems to be widespread. Other 5-HT1-like receptor agonists have differential effects according to the animal species used.

DOI is a mixed agonist-antagonist at postjunctional 5-HT2 receptors in the pithed rat.

The maximal pressor effect induced by DOI in the pithed rat was smaller than that induced by 5-HT, suggesting partial agonistic properties of DOI. DOI shifted the dose-pressor response curve of 5-HT to the right. It is concluded that, in addition to its 5-HT2 agonistic properties, DOI also possesses 5-HT2 antagonistic properties in the pithed rat.

Characterization of DOI, a putative 5-HT2 receptor agonist in the rat.

DOI (1-100 micrograms/kg i.v.) induced an increase in mean blood pressure in the anaesthetized rat. Similarly, in the pithed rat, DOI (1-100 micrograms/kg i.v.) induced a dose-dependent increase in mean blood pressure, as did 5-HT. However, in contrast to 5-HT, DOI did not change the heart rate in either intact or pithed rats. In the pithed rat, the dose-pressor response curves to both 5-HT and DOI were unaffected by MDL 72222 (5-HT3 receptor antagonist), spiroxatrine or (+/-)-pindolol (5-HT1A receptor antagonists), idazoxan (alpha 2-adrenoceptor blocking agent) and AR-C 239 (alpha 1-adrenoceptor blocking agent). Only the selective 5-HT2 receptor antagonist. LY 53857, significantly and dose dependently shifted to the right the dose-response curves to both 5-HT and DOI. These results indicated that DOI possesses 5-HT2 agonistic properties and that the pressor response induced by DOI in the pithed rat is mediated via 5-HT2 receptors.

Ventrolateral medullary pressor area: site of hypotensive and sympatho-inhibitory effects of (+/-)8-OH-DPAT in anaesthetized dogs.

Injections of 8-OH-DPAT (0.1-6 micrograms/kg) into the vertebral artery or into the cisterna magna (5 micrograms/kg) produced a dose-dependent decrease in blood pressure, heart rate and renal sympathetic nerve activity in intact anaesthetized dogs and baroreceptor-denervated dogs. 8-OH-DPAT reduced the renal sympathetic nerve activity without changing the blood pressure or heart rate in catecholamine-depleted animals. Methiothepin (0.2 mg/kg) injected into the vertebral artery reduced the blood pressure without changing the heart rate or renal sympathetic nerve activity in baroreceptor-denervated dogs. The pressor response to i.v. phenylephrine was largely attenuated. Subsequent administration of 8-OH-DPAT (3 micrograms/kg) into the vertebral artery failed to alter the sympathetic discharge. Methiothepin (0.2 mg/kg) injected into the vertebral artery reversed the sympatho-inhibitory effect of 8-OH-DPAT (3 micrograms/kg) injected by the same route without changing the blood pressure. (+/-)Pindolol (0.2 mg/kg) injected into the vertebral artery of baroreceptor-denervated dogs reduced the blood pressure and heart rate without changing renal sympathetic activity. Subsequent administration of 8-OH-DPAT (3 micrograms/kg) failed to alter the sympathetic discharge. Bilateral microinjection of 8-OH-DPAT (1 microgram) into the nucleus tractus solitarii or into the medullary raphe nuclei failed to alter the blood pressure, heart rate or renal sympathetic activity. In contrast, bilateral microinjections of 8-OH-DPAT into the ventrolateral pressor area (VLPA) (0.2 microgram) produced a marked decrease in blood pressure, heart rate and renal sympathetic nerve activity. These effects were prevented or reversed by microinjections of methiothepin (10 micrograms) at the same sites. These results indicate that the central sympatho-inhibitory effects of 8-OH-DPAT were due to the stimulation of 5-HT1A receptors located in the ventrolateral pressor area. 5-HT autoreceptors did not seem to be involved.

Participation of sympathetic and vagal tones in the hypotensive and bradycardic effects of some 5-HT1-like receptor agonists in the rat.

In normotensive anaesthetized rats, 8-OH-DPAT, RU 24969 and 5-MeODMT, three 5-HT1-like receptor agonists, decreased blood pressure and heart rate. The decrease in blood pressure was suppressed by pithing or pretreatment with hexamethonium. Bilateral section of the vagus nerves, pretreatment with a beta-adrenoceptor blocking agent or with atropine, combination of bivagotomy plus beta-adrenoceptor blockade strongly reduced the bradycardia. These results are compatible with a centrally-mediated decrease in sympathetic tone and increase in vagal tone, as the cause of the hypotension and bradycardia induced by these 5-HT1-like receptor agonists in normotensive anaesthetized rats.

Cardiovascular properties of a 5-HT1-like receptor agonist, 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) in normotensive anaesthetized rats.

Intravenous administration of the putative 5-HT1-like receptor agonist RU 24969 (10-1000 micrograms/kg) in anaesthetized rats induced a decrease in heart rate and blood pressure. The bradycardia was reduced, but not suppressed, by tertatolol, bilateral vagotomy or the combination of both treatments. The alpha 1-adrenoceptor blocking agent, AR-C 239, decreased the bradycardia induced by high doses of RU 24969. After treatment with hexamethonium, RU 24969 induced an increase in arterial blood pressure. The hypotensive response induced by RU 24969 was not altered by atropine. The hypotensive and bradycardic effects of RU 24969 were antagonized by methysergide (5-HT1-like receptor antagonist) and in part by spiroxatrine (5-HT1A receptor antagonist). Ketanserin (5-HT2 receptor antagonist) potentiated the effects of low doses of RU 24969. The cardiovascular effects of RU 24969 were antagonized neither by MDL 72222 nor by ICS 205-930 (5-HT3 receptor antagonists). The present results suggest that the cardiovascular effects of RU 24969 seem to be due to the stimulation of 5-HT1-like receptors. The participation of both 5-HT1A and 5-HT1B receptors subtypes has been considered. The results suggest a centrally-mediated inhibition of sympathetic tone and increase in vagal tone in the cardiovascular effects of RU 24969.

Vascular postsynaptic effects of some 5-HT1-like receptor agonists in the pithed rat.

5-HT induced an increase in blood pressure in the pithed rat which was antagonized by LY 53857 a selective 5-HT2 receptor antagonist. It was not antagonized by spiroxatrine, MDL 72222, idazoxan or AR-C 239, respectively 5-HT1-like and 5-HT3 receptor antagonists, alpha 2- and alpha 1-adrenoceptor antagonists. 5-MeODMT also induced an increase in blood pressure which was antagonized by LY 53857 but not by the other 5-HT receptor antagonists and alpha-adrenoceptor antagonists used, suggesting a 5-HT2 component in the pressor effect of 5-MeODMT. The maximal effect of 5-MeODMT was less marked than that of 5-HT. 8-OH-DPAT, RU 24969 and TFMPP were far less effective than 5-HT and 5-MeODMT to increase blood pressure. In contrast, 5-CT induced a vasodepressor effect. It is therefore suggested that the vasoconstriction induced by 5-HT and by 5-MeODMT in pithed rats could be due mainly to the selective stimulation of postjunctional 5-HT2 receptors because selective alpha 1- and alpha 2-adrenoceptor antagonists were ineffective against the vasoconstrictor effects of 5-HT and 5-MeODMT. The relative lack of effect of 8-OH-DPAT, RU 24969 and TFMPP to increase blood pressure suggested that postjunctional 5-HT1-like receptors play only a minor role - if any - in 5-HT induced vasoconstriction in the pithed rat.

Comparison of effects of some 5-HT1 agonists on blood pressure and heart rate of normotensive anaesthetized rats.

The present experiments served to compare the effects of the 3 5-HT1 agonists, 8-OH-DPAT, 5-MeODMT and TFMPP on the blood pressure and heart rate of normotensive anaesthetized rats. All the agonists induced, after i.v. injection, a decrease in blood pressure and heart rate. The hypotensive effects of 5-MeODMT and TFMPP were preceded by an increase, suppressed by both ketanserin and methysergide. The decrease in blood pressure induced by 5-MeODMT and 8-OH-DPAT was not antagonized by ketanserin, cocaine (and methysergide for 8-OH-DPAT) but was antagonized by methysergide (for 5-MeODMT) and spiroxatrine (for both). Bradycardia was not susceptible to ketanserin and cocaine (for 5-MeODMT) or to ketanserin and methysergide (for 8-OH-DPAT) but to methysergide and spiroxatrine (for 5-MeODMT) and cocaine and spiroxatrine (for 8-OH-DPAT). These results suggested that the hypotension and bradycardia induced by 5-MeODMT and 8-OH-DPAT are due to the stimulation of '5-HT1-like' receptors and probably to the 5-HT1A subtype; the 5-MeODMT-induced hypertension being ascribed to the stimulation of 5-HT2 receptors.