Jesús Hernández

Functional and biochemical evidence for diazepam as a cyclic nucleotide phosphodiesterase type 4 inhibitor

1 The responses of the electrically‐driven right ventricle strip of the guinea‐pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors. 2 Diazepam, at concentrations ranging from 1 μM to 100 μM, was devoid of effect on the contractile force in this preparation. 3 Conversely, diazepam (5 μM–100 μM) produced a consistent positive inotropic response in the presence of a concentration (1 μM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram. 4 This effect of diazepam was not γ‐aminobutyric acid (GABA)‐dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 μM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and β‐carboline‐3‐carboxylic acid methyl ester (βCCMe), or by the inverse agonists, β‐carboline‐3‐carboxylic acid N‐methylamide (βCCMa) and methyl 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM, 0.1 μM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 μM), did not influence the effect of diazepam. 5 Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea‐pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non‐competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4. 6 Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea‐pig eosinophils, where only type 4 PDE is present. 7 The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally‐related benzodiazepines, that also displaced [3H]‐rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site. 8 The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors.

Diazepam increases the hypothalamic‐pituitary‐adrenocortical (HPA) axis activity by a cyclic AMP‐dependent mechanism

Previous studies in this laboratory have shown that diazepam behaves as a phosphodiesterase 4 (PDE 4) inhibitor. It has been reported that PDE‐4 inhibitors activate the hypothalamic‐pituitary‐adrenocortical (HPA) axis in the rat. In the present study we have examined whether activation of the cyclic AMP‐dependent protein kinase (PKA) is involved in the effect of diazepam on basal HPA axis activity. Acute systemic administration of diazepam (10 mg kg−1 i.p.) was found to increase the basal HPA axis activity, increasing the plasma concentrations of corticotrophin (ACTH) and corticosterone 30 min post injection. Diazepam also elevated cyclic AMP content of the hypothalamus. Pretreatment of the animals with dexamethasone (1 mg kg−1 s.c.) for 3 days completely abolished the effect of diazepam on HPA axis activity. The antagonists of central and peripheral benzodiazepine receptors, flumazenil (10 mg kg−1 i.p.) and PK 11195 (5 mg kg−1 i.p.) did not affect the diazepam induced increase of HPA axis activity nor did they have an effect per se. The increase in ACTH and corticosterone levels was significantly reduced by the cyclic AMP‐dependent protein kinase (PKA) inhibitor, H‐89, given either subcutaneously (5 mg kg−1 s.c.) or intracerebroventricularly (i.c.v.; 28 μg in 10 μl). The results indicate that diazepam can stimulate basal HPA axis activity in the rat by a cyclic AMP‐dependent PKA mediated pathway.

Diazepam potentiates the positive inotropic effect of isoprenaline in rat ventricle strips: role of cyclic AMP

The responses of the electrically driven right ventricle strip of the rat heart to isoprenaline and other cyclic AMP-related inotropic agents were recorded in the absence and in the presence of diazepam. Isoprenaline, in concentrations ranging from 10 nM to 1 microM, significantly increased, in a concentration-dependent manner, the contractile force in this preparation. Diazepam (10 microM) produced a leftward shift in the isoprenaline concentration-response curve and significantly reduced its EC50. Higher concentrations of diazepam (100 microM) produced no further shift, but reduced the maximum of the concentration-response curve of isoprenaline. Forskolin (0.5-10 microM), which directly stimulates adenyl cyclase, also produced a concentration-dependent increase in cardiac contractility. Diazepam (10 microM) displaced to the left the concentration-response curve for forskolin and reduced its EC50. The cyclic AMP analogous dibutyryl cyclic AMP (0.1-1 mM) produced concentration-dependent positive inotropic effects which were not significantly modified in the presence of diazepam (10 microM). Diazepam (10 microM) significantly enhanced the cyclic AMP production induced by isoprenaline (0.1 microM) and forskolin (10 microM) by about 136% and 35% respectively. These results indicate that diazepam potentiates the positive inotropic effect induced by beta-adrenoceptor agonists, probably by increasing cyclic AMP production induced by these agents.

Phosphodiesterase PDE3 blunts the positive inotropic and cyclic AMP enhancing effects of CGP12177 but not of noradrenaline in rat ventricle

The cardiostimulant effects of CGP12177, mediated through a β1‐adrenoceptor site with low affinity for (−)‐propranolol, are potentiated by the nonselective PDE inhibitor IBMX but the role of PDE isoenzymes is unknown. We studied the effects of the PDE3‐selective inhibitor cilostamide (300 nM) and PDE4‐selective inhibitor rolipram (1 μM) on the positive inotropic and cyclic AMP‐enhancing effects of CGP12177 and noradrenaline in right ventricular strips of rat. CGP12177 (under (−)‐propranolol 200 nM) only increased contractile force in the presence of either cilostamide or rolipram with −logEC50M 6.7 (Emax=23% over basal) and 7.1 (Emax=50%) respectively. The combination of cilostamide and rolipram caused CGP12177 to enhance contractile force with −logEC50M=7.7 and Emax=178%. The positive inotropic effects of noradrenaline (−logEC50M=6.9) were potentiated by rolipram (−logEC50M=7.4) but not by cilostamide (−logEC50M=7.0). In the presence of rolipram and (−)‐propranolol, noradrenaline (2 μM) and CGP12177 (10 μM) produced matching inotropic effects but failed to increase cyclic AMP levels. 20 μM (−)‐noradrenaline increased cyclic AMP levels, a response further enhanced by rolipram. Both PDE3 and PDE4 of rat ventricle appear to hydrolyse cyclic AMP generated through the low‐affinity β1‐adrenoceptor site, thereby preventing inotropic responses of CGP12177. When (−)‐noradrenaline interacts with the β1‐adrenoceptor, the generated cyclic AMP is hydrolysed only by PDE4, thereby reducing cardiostimulation.

Interaction between halothane and morphine on isolated heart muscle

The present study describes the effects of halothane on morphine activity in the isolated left atria of the rat. Concentration-response curves were obtained for the negative inotropic effects of morphine on electrically stimulated left atria. Morphine significantly decreased the contractile force, with an inhibitory concentration 16 (IC16) of 3.130.698 +/- 22.5 X 10(-9) M. The opiate agonist was more potent in reserpinized rats, causing a consistent negative inotropic action over a wide range (10(-8)-10(-4) M) or morphine concentrations. The IC16 of morphine was significantly (P less than 0.001) decreased in the presence of 1.5% v/v halothane. The administration of L-naloxone (3 X 10(-7)-10(-6) M) but not D-naloxone (10(-6) M) antagonized the inhibitory effects of morphine in the presence of halothane. These results demonstrate that halothane increases the potency of morphine on the isolated left atria and suggest that this effect is mediated by opioid receptors.

Diazepam enhances inotropic responses to dopamine in rat ventricular myocardium.

Diazepam inhibits phosphodiesterase type 4 and enhances the effect of some 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent positive inotropic drugs. We sought to determine whether diazepam and the selective phosphodiesterase type 4 inhibitor rolipram enhances the contractile response and cAMP levels induced by dopamine in rat myocardium. Dopamine (3–100 &mgr;M) produced concentration-dependent positive inotropic effects (−log EC50 = 5.21 ± 0.2, n = 5), which were augmented in the presence of 10 &mgr;M diazepam (-log EC50 = 5.40 ± 0.08, n = 6, P < 0.05) or 1 &mgr;M rolipram (-log EC50 = 5.41 ± 0.1, n = 6, P < 0.05). The effect of diazepam was not mimicked by 100 &mgr;M &ggr;-aminobutyric acid nor it was antagonized by a 5 &mgr;M concentration of the blockers of central and peripheral type benzodiazepine receptors, flumazenil and PK 11195. cAMP levels (pmol/g) produced by dopamine (744.4 ± 111.8, n = 5) in this tissue were enhanced by the presence of diazepam (1073 ± 97.7, n = 6, P < 0.05) or rolipram (1034.0 ± 245.2, n = 5, P < 0.05). Therefore, diazepam, like rolipram, augments the inotropic and biochemical effects of dopamine in rat myocardium. This effect is not mediated by benzodiazepine receptors but is probably the consequence of the phosphodiesterase type 4 inhibitory activity of diazepam.

Theophylline antagonizes the effect of diazepam on ventricular automaticity

The effect of diazepam on ventricular automaticity induced by a local injury was investigated in the isolated right ventricle of the rat in the presence of the diazepam central antagonist, RO 15-1788, and of the adenosine antagonist, theophylline. Theophylline but not RO 15-1788 antagonized the inhibitory effect induced by diazepam on ventricular automaticity. The inhibitory effect of adenosine but not that of 2-chloroadenosine was potentiated in the presence of diazepam. The results suggest that the inhibitory effect of diazepam on ventricular automaticity results from the inhibition of adenosine uptake.

The beta-agonist drug tulobuterol decreases cardiac automaticity in the rat: Comparison with isoproterenol

The effect of the new beta-agonist drug tulobuterol, has been compared with isoproterenol in an experimental model of ectopic cardiac automaticity induced by local injury in the isolated right ventricle of the rat. Isoproterenol significantly increases ventricular automaticity even with the lowest concentrations tested (10(-9)M). Tulobuterol, at concentrations ranging from 10(-9)M to 10(-6)M do not induce any change on the automatic ventricular frequency. Nevertheless, at higher concentrations (10(-5) and 5 X 10(-5)M), tulobuterol decreases the experimentally induced ventricular automaticity. Our results suggest that the new beta-agonist drug tulobuterol does not increase ventricular automaticity but, in fact, decreases it.

Diazoxide blocks the morphine induced lengthening of action potential duration on guinea-pig papillary muscle

1. Intracellular microelectrodes were used to evaluate the possible involvement of potassium currents in the action potential prolongation induced by morphine. To this purpose we investigated the electrophysiological effect of morphine on the isolated guinea pig right ventricular papillary muscle in the presence of the potassium channel opener and inhibitor diazoxide and glibenclamide respectively. 2. Diazoxide (1 microM), which is devoid of effect on its own, blocks the lengthening of action potential duration (APD) induced by morphine (5 mM). 3. However, in the presence of glibenclamide (1 microM), morphine (5 mM) prolonged APD in approximately the same proportion as that observed when used alone. 4. These results suggest that diazoxide but not glibenclamide sensitive potassium channels could mediate the APD prolongation induced by morphine.

Electrophysiological effects of opioid receptor selective agonists on guinea-pig papillary muscle.

The cardiac electrophysiological effects of DAMGO, DPDPE and U-50,488H which are selective opioid agonists for mu, delta and kappa receptors, respectively, were studied in the isolated guinea-pig right ventricular papillary muscles. Neither DAMGO (5.10(-6)-5.10(-5)M) nor DPDPE (5.10(-6)-5.10(-5)M) produced any significant effect on the action potential characteristics. However, U.-50,488H (10(-5),5.10(-5) M) caused a concentration-dependent reduction in the maximum rate of depolarization of phase 0 (Vmax) and in the action potential duration measured at 50% repolarization, APD50 and 90% repolarization, APD90. At 5.10(-5) M it also produced a decrease of action potential amplitude (APA). These results suggest that the reported electrophysiological effects of U-50,488H on the cardiac muscle action potential, may be, at least in part, explained by a direct cardiac action.