Antioxidant drug mechanisms: transition metal-binding and vasodilation

In our work evaluating the antioxidant properties of a number of cardiovascular drugs, we have emphasized the importance of lipophilicity as a property contributing to antioxidant potency. Thus, the dihydropyridine calcium channel blockers and propranolol, one of the most lipophilic beta-blockers, were found to exhibit the greatest potency in membrane and cellular models. Both beta-blockers and calcium channel blockers are classified as antihypertensive agents. We found that the specific chemical moieties of various drugs may participate in the antioxidant mechanism of action. While reviewing relevant work from the past literature, it became apparent that some of the chemical moieties of antihypertensive and vasodilator drugs may bind transition metals. Thus, this present review focuses on common properties of transition metal-interaction that are shared, to a greater or lesser degree, by a number of vasoactive drugs and chemical agents. Although this observation has been pursued by other investigators in the past, we submit that the potential relevance to the newer pharmacological agents needs to be explored further. In addition, new information regarding the role of transition metals and free radicals involving vascular cells focuses greater importance on transition metal-interaction as a potential mechanism in vasodilation. This review does not intend to be inclusive of all chemical structures capable of binding transition metals; only those that are clinically relevant will be considered in some detail. Potential mechanisms of metal-chelating actions leading to vasodilation are also discussed.     

The effects of 4-hydroxy-2,3-trans-nonenal on beta-adrenoceptors of rat lung membranes

Lung membranes are susceptible to oxygen radicals, formed during inflammation, redox cycling of toxic agents, exposition to ozon etc. Oxygen radicals may modify the beta-adrenergic response. However, at the same time beta-adrenoceptors of the lung are frequently addressed in therapy. We embarked upon this problem by studying the effects of the aldehyde 4-hydroxy-2,3-transnonenal (HNE), one of the major products of lipid peroxidation, on the density of beta-adrenoceptors of rat lung membranes. It is shown, that the physiological important sulfhydryl blocking agent HNE inactivates the beta-adrenoceptors in a time- and concentration dependent (0.5-2.5 mM) way, indicated by a decrease in (-)-[3H]dihydroalprenolol (DHA) binding to lung membranes. Moreover, it is shown that combined treatment of HNE with (-)-isoproterenol (0.5 microM) or 1-alprenolol (0.5-10 nM) does not influence the extent of inactivation of beta-adrenoceptors by HNE. This is in contrast with previous studies, conducted with other, synthetic, sulfhydryl blocking agents, such as N-ethylmaleimide (NEM), suggesting that an other mechanism of inactivation is involved upon HNE treatment.     

The inhibitory actions of acebutolol and propranolol on the contractile response to 5-hydroxytryptamine in various isolated vascular smooth muscles

Pretreatment with acebutolol or propranolol at high concentrations had an inhibitory effect on the contractile response to 5-hydroxytryptamine (5-HT) in most vascular smooth muscles such as rabbit aorta and basilar, mesenteric, renal, femoral arteries and cat coronary artery. The inhibitory actions of both agents were generally greater than on the responses to excess Ca2+ and potassium. In rabbit renal arteries, acebutolol had no effect on the response to 5-HT but inhibited the responses to excess Ca2+ and potassium. Propranolol had a marked inhibitory effect on the response to 5-HT. In all preparations used, the contractions induced by norepinephrine (NE) and histamine showed a much greater resistance to the effect of acebutolol and propranolol than the contractions induced by 5-HT, Ca2+ and potassium. Nifedipine had no inhibitory effect on the response to 5-HT in most of the preparations. Nifedipine inhibited the response to 5-HT only in the basilar arteries. The inhibitory actions of propranolol on the response to 5-HT was greater than that of acebutolol. The inhibitory action of acebutolol and propranolol on the response to 5-HT may be related to mechanisms other than the beta-adrenoceptor blocking action of the drugs. The possible mechanisms of inhibitory action of both beta-adrenoceptor antagonists on 5-HT are discussed.     

Antioxidant activity of β-blockers: An effect mediated by scavenging reactive oxygen and nitrogen species?

The therapeutic effects of beta-blockers are normally explained by their capacity to block the beta-adrenoceptors, however, some of the beneficial cardiovascular effects shown by this group of compounds have already been associated with the antioxidant properties that some of them seem to possess. The beta-blockers atenolol, labetalol, metoprolol, pindolol, propranolol, sotalol, timolol, and carvedilol were tested for their putative scavenging activity for ROS (O(2)(-), H(2)O(2), HO(.), HOCl, and ROO(.)) and RNS ((.)NO and ONOO(-)). Some of the studied compounds are effective ROS and/or RNS scavengers, these effects being possibly useful in preventing oxidative damage verified in hypertension as well as in other cardiovascular diseases that frequently emerge in association with oxidative stress.     

Actions of cimetidine and ranitidine at some cholinergic sites: implications in toxicology and anesthesia

Cimetidine and ranitidine are specific and potent H2-receptor antagonists widely used in the effective therapy of peptic ulcer disease. The drugs also possess other pharmacological properties unrelated to H2-receptor antagonism. More recently large experimental doses of cimetidine or ranitidine were found to have anticholinesterase, ganglion blocking and neuromuscular blocking activities. Actions of the drugs at such cholinergic sites may account for some of their clinically documented adverse effects. The toxicological implications of these findings including the potential for drug interactions to occur, especially during some anesthetic procedures, are discussed.     

Propranolol modifies platelet serotonergic mechanisms in rats.

Though the mechanisms for the vascular actions of vasodilatory beta-blockers are mostly determined, some of their interactions with monoaminergic systems are not elucidated. Because there are evidences supporting a possible involvement of serotonin (5-HT) in the actions of beta-blockers, we studied the effect of propranolol on peripheral serotonergic mechanisms in normotensive and Goldblatt two-kidney - one clip (2K1C) hypertensive rats. In both groups of animals propranolol decreased systolic blood pressure, significantly increased whole blood serotonin concentration and at the same time it decreased platelet serotonin level. The uptake of the amine by platelets from hypertensive animals was lower than that of normotensive animals and it was decreased by propranolol only in the latter. In both groups propranolol inhibited potentiation of ADP-induced platelet aggregation by serotonin. In conclusion, this study provides evidence that propranolol modifies platelet serotonergic mechanisms in normotensive and renal hypertensive rats.     

Lipophilic beta-blockers inhibit monocyte and endothelial cell-mediated modification of low density lipoproteins.

The effects of propranolol, pindolol and metoprolol on the modification of low density lipoprotein (LDL) by U937 monocyte-like cells, endothelial cells and copper ions were studied by determination of the lipid peroxidation product content and measurement of the relative electrophoretic mobility of the particle. Propranolol and pindolol inhibited LDL oxidation by U937 cells in a dose-dependent manner from 10 to 100 microM, whereas metoprolol had no effect. In the case of LDL modification by endothelial cells, all the three beta-blockers were efficient within the same range of concentrations, and the order of potency was propranolol greater than pindolol greater than metoprolol. In vitro oxidation of LDL in the presence of copper ions was also inhibited by propranolol; pindolol and metoprolol had no significant protective effect in this system. These results concerning the inhibitory action of beta-blockers were confirmed by testing the degradation of modified LDL by J774 macrophages. Although the concentrations of the drugs utilized in this study are relatively high, in long-term treatment beta-blockers might accumulate in target tissues, and the protective effect of propranolol against LDL oxidation might be involved in its inhibitory action on atherosclerosis previously reported in animal models.     

Effect of lyso-phosphatidylserine on rat hypothalamic cAMP, in vivo

Lyso-Phosphatidylserine, like its precursor phosphatidylserine, increases the concentration of cAMP in the rat hypothalamus. The effect is dose-dependent and reaches its maximum 10–20 minutes from the injection. Antiadrenergic drugs, such as reserpine, α-methyl-p-tyrosine (α-MpT), haloperidol and chlorpromazine, but not phentolamine or propranolol, prevent the accumulation of hypothalamic cAMP induced by lysophosphati dylserine. Serotoninergic, histaminergic, cholinergic and GABAergic drugs do not modify the lyso-phosphatidylserine effect. The obtained results suggest a primary involvement of the dopaminergic system in the mediation of the pharmacological effect of lyso-phosphatidylserine.     

Comparison of propranolol,metoprolol, and acebutolol on insulin-induced hypoglycaemia.

Metoprolol and acebutolol, two supposedly cardio-selective beta-adrenergic recptor blocking agents, were tested in healthy volunteers against propranolol, a non-selective drug, for their effect on blood glucose levels during insulin-induced hypoglycaemia. There was not significant difference between propranolol and metoprolol, which both potentiated the initial hypoglycaemic action of the insulin and delayed the return to normoglycaemia. Acebutolol, even though potentiating the initial hypoglycaemia, did not possess a significant delaying effect. A similar trial should be undertaken in diabetics to determine with certainty the safety of such drugs in diabetes mellitus.     

Recent studies of the actions of cholinomimetic drugs on adrenergic nerves and their implications for the cholinergic link hypothesis

This review analyzes the results of recent studies of the actions of cholinomimetic drugs on adrenergic nerve terminals and their implications for the cholinergic link hypothesis. Thus far, evidence suggests that the only possible action of endogenous acetylcholine (ACh) present near noradrenaline (NA) stores is an inhibition of the release of NA from the adrenergic nerve terminals and that NA is released only when the action of acetylcholinesterase is inhibited. Nicotinic agents have been shown to act on adrenergic nerve terminal membranes, a finding that casts doubt on the proposed intraneuronal cholinergic sites for the action of endogenous ACh. Evidence also indicates that the mode of adrenergic neurone blocking action of bretylium and guanethidine is independent of the proposed cholinergic process in NA release. Current findings do not support the proposal that nicotinic agents in higher concentrations interfere with adrenergic neurotransmission. It is therefore concluded that nicotinic agents, in causing the release of NA from adrenergic nerve terminals, are merely exhibiting a pharmacological action and not mimicking the physiological function of ACh, as proposed by the cholinergic link hypothesis.     

Coexistence of beta 1- and beta 2-adrenoceptors in the melanophore of the goby Tridentiger obscurus

The effects of beta-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the beta-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these beta-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both alpha- and beta-adrenoceptors, and that the activation of the beta-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two beta 1-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of beta 1-receptors. By contrast, the pigment-dispersing effects of two beta 2-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the beta-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest co-existence of beta 1- and beta 2-adrenoceptors in the melanophores. The relative numbers of beta 1- and beta 2-adrenoreceptors as a percentage of the total population of beta-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the beta-agonists on the melanophores in the presence of butoxamine or metoprolol.     

Effect of gamma-aminobutyric acid on corticosterone secretion: involvement of the noradrenergic system.

The administration of gamma-aminobutyric acid (GABA) in the brain right lateral ventricle reduces serum corticosterone levels, and induces significant variations of hypothalamus biogenic amines in conscious male rats. After pretreatment with either alpha 1-adrenergic (prazosin) or alpha 2-adrenergic (yohimbine) blocking agents, the inhibitory effect of GABA on ACTH secretion was prevented. However, we observed that pretreatment with a beta-adrenergic blocking agent (propranolol), did not preventing the inhibitory effect of GABA on serum corticosterone levels. These results indicate that GABA has an inhibitory effect on ACTH secretion mediated by the activation of alpha 1 and alpha 2-adrenergic receptors.     

Sympathetic control on salivary secretion by the parotid gland in rabbit. II. Effects upon the gland submitted to a weak parasympathetic stimulation (author's transl)]

Superior cervical ganglion stimulation significantly increases both, flow and amylase activity of saliva. Stimulation provokes two markedly different periods of flow: during the first half, flow is very high and differs significantly from the flow of the second half which closely resembles the one previous to stimulation. Alpha-adrenergic blocking agents, when administered intravenously, abolish the hypersecretion induced by sympathetic stimulation; beta-adrenergic blocking agents do not. These facts strengthen the hypothesis that alpha-adrenoceptors are most important in fluid secretion. The infusion of epinephrine acts similarly on cervical ganglion stimulation, but it differs because of its more diffuse effects and deeper cardiovascular alterations. Isoproterenol, after a long latency period, slightly increases salivary flow, which seemingly indicates that beta-adrenoceptors are involved in the fluid secretory processes although in a lesser degree than alpha-adrenoceptors.     

Studies on rat sympathetic neurons developing in cell culture. III. Cholinergic transmission.

Principal neurons were dissociated from the superior cervical ganglia of newborn rats and grown in culture with several types of non-neuronal cells. As described in the second paper of this series, the neurons in such mixed cultures formed two types of excitatory synapses with each other, electrical and chemical. Evidence is presented here that transmission at the chemical synapses was cholinergic. Four nicotinic ganglionic blocking agents (curare, hexamethonium, tetraethylammonium, and mecamylamine) strongly attenuated or eliminated the excitatory postsynaptic potentials (e.p.s.p.'s) at moderate concentrations; atropine at relatively high concentrations also blocked transmission. Iontophoretic application of acetylcholine (ACh) to the surface of the neurons gave rise to depolarizations that could be made to resemble the e.p.s.p.'s in size and time course; the ACh potentials and the e.p.s.p.'s were then similarly affected by nicotinic blocking agents. The sensitivity to ACh was often distributed nonuniformly on the neuronal surface; it was common to find small, sharply localized regions of high sensitivity. Catecholamines (norepinephrine, epinephrine, and dopamine) had only inhibitory actions; in a few experiments adrenergic blocking agents (phenoxybenzamine, propranolol) were found to have no effect on the e.p.s.p.'s. These observations leave no doubt that the neurons released ACh and had ganglionic, nicotinic ACh receptors on their surfaces. The significance of the fact that a high proportion of the sympathetic neurons in mixed cultures formed cholinergic synapses is discussed.     

Drug Withdrawal Syndromes: A Literature Review

Drug withdrawal syndromes reportedly have been caused by numerous pharmacological agents, but only a few drugs have been adequately studied in this regard. Criteria for evaluating drug withdrawal syndromes have been proposed. Sedative-hypnotic agents, opiates, corticosteroids, clonidine, tricyclic antidepressant medications and beta-adrenergic blocking agents meet the criteria for such syndromes. Gradual tapering of the dose of these drugs is recommended when therapy must be discontinued. Whether or not other drugs cause rebound reactions is questionable, but caution should be used when discontinuing drugs for which numerous reports of withdrawal syndromes exist.     

Interaction of alpha adrenergic antagonists with calmodulin

Several alpha-adrenergic antagonists inhibited the activation of calmodulin-stimulated phosphodiesterase at concentrations that had little or no effect on basal phosphodiesterase activity. The most potent of these compounds were phenoxybenzamine and dibenamine (IC50 values of about 1 microM); the amino acid ergot alkaloids ergocryptine, ergocristine, ergotamine and their dihydrogenated derivatives were less potent calmodulin-inhibitors (IC50 values of 35-80 microM). The amino ergot alkaloids ergonovine and methysergide were essentially devoid of inhibitory activity. A variety of other alpha 1-antagonists (phentolamine, tolazoline and prazosin), an alpha 2-antagonist (yohimbine), alpha-agonists (norepinephrine, phenylephrine and clonidine), beta-adrenergic antagonists (propranolol and practolol) and the beta-adrenergic agonist methoxyphenamine displayed little or no anti-calmodulin activity (IC50 values greater than 300 microM). Similarly, the alkylating agents chlorambucil and mechlorethamine also failed to inhibit calmodulin activity. Phenoxybenzamine and dibenamine inhibited calmodulin activity irreversibly, whereas the inhibition caused by other alpha adrenergic blocking agents was reversible. Phenoxybenzamine inhibited calmodulin activity by binding directly to it. This binding was calcium-dependent and irreversible. The irreversible binding and inhibition of calmodulin activity by phenoxybenzamine (or dibenamine) may serve as a useful tool for studying the sites at which drugs bind to calmodulin and may also be useful for studying the distribution and turnover of calmodulin.     

Cholinergic and adrenergic effects on diffusional water flux in the toadfish, Opsanus beta

Intraperitoneal injections of adrenaline resulted in increased tritiated water efflux rate in the toadfish, Opsanus beta. Adrenaline-stimulated water flux was inhibited by the beta-adrenergic blocker, propranolol, but not by the alpha-adrenergic blocker, phentolamine. Propranolol on its own had no effect but phentolamine significantly stimulated water flux; this action was attributed to a beta-mimetic effect of the drug. The cholinergic neurotransmitter acetylcholine, had no effect while the parasympathico-mimetic carbachol, significantly stimulated water flux. Arguments were advanced to explain the similarity in the effects of the adrenergic and cholinergic drugs although they are both known to produce opposing vascular haemodynamic effects in fish gills. Adrenaline substantially stimulated tritiated water flux in the toadfish, Opsanus beta. The adrenaline-stimulated water flux exhibited a linear dose-response curve up to an adrenaline dosage of 750 micrograms kg-1; wt. At higher doses there was apparently a desensitization of the beta-adrenergic receptor sites. The adrenaline effect was inhibited by the beta-blocker propranolol, but not by the alpha-blocker, phentolamine. This suggests that the adrenaline-stimulated water flux was due predominantly to beta-receptor site stimulation. Stimulation of water flux by phentolamine on its own could be due to the stimulation of endogenous catecholamine release by the drug. We have proposed that the beta-stimulated water efflux could be due to an increase in surface area of the branchial epithelium, a decrease in water to blood diffusion distance, a direct metabolic effect or any combination of these effects by adrenaline. Carbachol caused an increase in tritiated water efflux. The carbachol-stimulated water flux was inhibited by atropine thus suggesting that the drug acts via muscarinic receptor sites. We have suggested that the action of the drug on hydraulic water conductivity, water to blood diffusion distance, hydrostatic pressure or a direct effect on membrane diffusion coefficient.     

Muscarinic cholinergic inhibition of beta-adrenergic stimulation of phospholamban phosphorylation and Ca2+ transport in guinea pig ventricles

The effects of muscarinic cholinergic stimulation on beta-adrenergic induced increases in phospholamban phosphorylation and Ca2+ transport were studied in intact myocardium. Isolated guinea pig ventricles were perfused via the coronary arteries with 32Pi, after which membrane vesicles were isolated from individual hearts. Isoproterenol produced reversible increases in 32P incorporation into phospholamban. Associated with the increases in 32P incorporation were increases in the initial rate of phosphate-facilitated Ca2+ uptake measured in aliquots of the same membrane vesicles isolated from the perfused hearts. The increases in 32P incorporation and calcium transport were significantly attenuated by the simultaneous administration of acetylcholine. Acetylcholine also attenuated increases in phospholamban phosphorylation and Ca2+ uptake produced by the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin. The contractile effects of all agents which increased cAMP levels (increased contractility and a reduction in the t1/2 of relaxation) were also attenuated by acetylcholine. The inhibitory effects of acetylcholine were associated with attenuation of the increases in cAMP levels produced by isoproterenol and isobutylmethylxanthine but not by forskolin. Acetylcholine also increased the rate of reversal of the functional and biochemical effects of isoproterenol by propranolol without affecting cAMP levels. These results suggest that cholinergic agonists inhibit the functional effects of beta-adrenergic stimulation in part by inhibition of phospholamban phosphorylation. This inhibition may be mediated by two potential mechanisms: inhibition of beta-adrenergic activation of adenylate cyclase and stimulation of dephosphorylation.     

The influence of adrenoceptor activity on cell proliferation in colonic crypt ipithelium and in colonic adenocarcinomata.

The effects of chemical sympathectomy and of the injection of amines or amine-receptor blocking drugs on cell proliferation in colonic crypts and in dimethylhydrazine-induced colonic carcinomata is examined in rats using a stathmokinetic technique. In animals which had been chemically sympathectomized by injection of 6-hydroxydopamine cell proliferation essentially ceased in the colonic crypts but continued at a normal rate in the tumours. Stimulation of alpha-adrenoceptors by metaraminol, a drug with properties similar to noradrenaline, caused acceleration of cell proliferation in colonic crypts but not in tumours. Conversely, blockade of alpha-adrenoceptors by phentolamine inhibited cell proliferation in crypts but not in tumours. Injection of adrenaline, predominantly a beta-adrenergic agonist, inhibited cell proliferation in the tumours but not in colonic crypts whereas blockade of beta-adrenoceptors by propranolol accelerated cell proliferation in tumours but not in colonic crypts. It is postulated that cell proliferation in the crypts of Lieberkühn in rat colon resembles that in rat jejunum in being controlled by the autonomic nervous system. However, tumour cell proliferation does not appear to be subject to such regulation.     

Pharmacological characterization of postsynaptic potentials evoked in the bimodal pacemaker neuron of Helix pomatia L

Stimulation of various peripheral nerve trunks evokes very similar compound postsynaptic potentials (PSP) composed of one or more excitatory postsynaptic potentials (EPSP) followed by fast and slow inhibitory postsynaptic potentials (IPSP) on the identified RPal neuron of Helix pomatia L. Evoked EPSPs were reduced or blocked by nicotine, atropine and d-tubocurarine. The two components of IPSP were different in their pharmacological sensitivity. Slow IPSP was partly or totally eliminated by ergometrine and chlorpromazine and was reduced by atropine, nicotine as well as by propranolol. Fast IPSP was reduced only in the presence of ergometrine and could not be blocked by either of the applied drugs. Participation of cholinergic transmission seems to be essential in the evoked EPSP but its partial involvement in the slow IPSP can also be supposed. A dopaminergic mechanism may take part in the generation of both components of IPSP but the receptors responsible for the slow IPSP were sensitive to other catecholamine antagonists as well, referring to a more complex origin, or to the involvement of an unknown transmitter. Comparison of PSPs evoked by stimulation of different nerves shows that presynaptic areas belonging to various peripheral sources are overlapped on the RPal neuron, and they probably act by similar transmitter substances.