Inhibition of glucose-induced insulin secretion by calcium channel blocking drugs in-vitro but not in-vivo in the rat

The organic calcium channel blocking drugs darodipine, nicardipine, diltiazem and verapamil inhibited glucose-induced (8.3 mmol L-1) insulin secretion by rat isolated islets in a concentration-dependent manner. The IC50 values (mumol L-1) for nicardipine, diltiazem and verapamil were 0.0025, 1.94 and 3.6, respectively. There were no significant differences between the potencies of any one drug when compared at two different glucose concentrations (27.8 and 8.3 mmol L-1). Isolated islets were also responsive to the calcium channel activating drugs BAY K 8644 and CGP 28392, which enhanced glucose-induced insulin secretion and prevented the inhibitory effect of verapamil. BAY K 8644 was more potent than CGP 28392. In the anaesthetized rat, neither verapamil (1 mg kg-1 i.v. 10 min before glucose) nor nicardipine (0.2 mg kg-1 i.v.) modified the glucose or insulin response to intravenous glucose whilst producing marked cardiovascular effects. The plasma concentrations of nicardipine (3.9 X 10(-8) M at 10 min post injection) were similar to those producing effects in-vitro whereas the plasma concentrations of verapamil (5 X 10(-7) M) were lower. It thus appears that the islet B cell calcium channel is less sensitive to calcium channel blocking drugs in-vivo than in-vitro. Moreover, in-vivo, the cardiovascular system is more sensitive to these drugs than are the islet B cells, although the potencies of the calcium channel blocking drug in isolated islets are similar to those reported for cardiac muscle and vascular smooth muscle in-vitro.     

Cardiovascular effects of LAS 30538, a new vascular selective Ca(2+)-channel blocker.

A new compound, 1-[2-(2,6-dimethylphenoxy)ethyl]-alpha,alpha-bis-(p-fluorphenyl)-4 -piperidine methanol (LAS 30538), was found to have potent vasodilator effects. Its vasorelaxant activity was demonstrated in rat perfused hindlimbs contracted with 80 mM K+, having an IC50 value of 40 nM. In conscious spontaneously hypertensive rats, LAS 30538 administered orally, caused dose-dependent sustained falls in systolic blood pressure with an ED30 value of 11 mg kg-1. In pithed rats, LAS 30538, strongly inhibited vasoconstriction induced by the alpha 2-adrenoceptor agonist B-HT 933 and the calcium agonist compound Bay K8644 with ED50 values of 4 mg kg-1 p.o. and 1.3 mg kg-1 i.v., respectively. Results from electrophysiological studies carried out using guinea-pig papillary muscles partially depolarized by 22 mM K+ are consistent with LAS 30538 acting as a Ca(2+)-channel blocker. When compared with verapamil, in guinea-pig and rabbit isolated heart preparations, LAS 30538 caused less cardiodepression and bradycardia. The results suggest that LAS 30538 may have some advantages over other Ca(2+)-channel blockers such as verapamil in causing less myocardial depression for a given level of vasodilatation.     

Effects of calcium channel blockers and hydralazine on epinephrine-induced hyperglycemia in vivo

Effects of calcium channel blockers from structurally different classes and hydralazine on epinephrine-induced hyperglycemia were studied in vivo. Nifedipine (0.05-0.20 mg/kg, i.p.) and nicardipine (0.40-0.80 mg/kg, i.p.) markedly potentiated the epinephrine-induced hyperglycemia in a dose-dependent manner. In contrast to these dihydropyridine calcium channel blockers, verapamil and diltiazem did not significantly affect the epinephrine-induced hyperglycemia at doses of 0.10-1.0 mg/kg, i.p. At higher doses (10 mg/kg, i.p.), significant potentiation of epinephrine-induced hyperglycemia was observed by these non-dihydropyridine calcium channel blockers. Hydralazine also markedly increased the epinephrine-induced hyperglycemia. These calcium channel blockers and hydralazine had no significant effect on the basal plasma glucose levels at any dose used here. As judged from the rates of glucose disappearance (K values), dihydropyridines significantly impaired the glucose tolerance in much lower doses than those of non-dihydropyridines and hydralazine. Furthermore, epinephrine-induced impairment of glucose tolerance was markedly potentiated by these calcium channel blockers and hydralazine at doses which potentiated the epinephrine-induced hyperglycemia. These results suggest that, at least in part, the potentiation of epinephrine-induced hyperglycemia by dihydropyridines, non-dihydropyridines and hydralazine is related to the inhibition of peripheral glucose utilization produced by insulin.     

Analgesic effects of several calcium channel blockers in mice

The possibility that calcium channel blockers might produce antinociception and increase morphine analgesia was examined using the acetic acid writhing test in mice. Subcutaneous injections of diltiazem, verapamil, nicardipine, flunarizine and cinnarizine produced a dose-dependent antinociception. This activity of diltiazem was stereospecific; d-cis-diltiazem was more potent than 1-cis-diltiazem. All the calcium channel blockers studied increased morphine analgesia and displaced to the left the morphine dose-response curve. This effect of diltiazem was also stereospecific. These results suggest that calcium channel blockers can induce analgesia and increase morphine analgesia, possibly through a decrease in cellular calcium availability.     

Midazolam alpha-hydroxylation by human liver microsomes in vitro: inhibition by calcium channel blockers, itraconazole and ketoconazole

The inhibitory effects of five calcium channel blockers (diltiazem, isradipine, mibefradil, nifedipine and verapamil) and three azole antifungal agents (itraconazole, hydroxyitraconazole and ketoconazole) on the alpha-hydroxylation of midazolam, a probe drug for CYP3A4-mediated interactions in humans, were studied in vitro using human liver microsomes. IC50 and Ki values were determined for each inhibitor. The kinetics of the formation of alpha-hydroxymidazolam were best described by simple Michaelis-Menten kinetics. The estimated values of Vmax and Km were 696 pmol min.-(1) mg(-1) and 7.46 micromol l(-1), respectively. All the compounds studied inhibited midazolam alpha-hydroxylation activity in a concentration-dependent manner, but there were marked differences in their relative inhibitory potency. Ketoconazole was the most potent inhibitor of midazolam alpha-hydroxylation (IC50 0.12 micromol l (-1)), being 10 times more potent than itraconazole (IC50 1.2 micromol l(-1)). The inhibitory effect of hydroxyitraconazole (IC50 2.3 micromol l (-1) was almost as large as that of itraconazole. Among the calcium channel blockers, mibefradil was the most potent inhibitor of the alpha-hydroxylation of midazolam, with an IC50 value (1.6 micromol l (-1)) similar to that of itraconazole. The other calcium channel blockers were much weaker inhibitors than mibefradil: verapamil exhibited a modest inhibitory effect with an IC50 of 23 micromol l(-1), while isradipine, nifedipine and diltiazem, with IC50 values ranging from 57 to >100 micromol l (-1), were weak inhibitors. This rank order of potency against the alpha-hydroxylation Qf midazolam was verified by the Ki values. With the exception of diltiazem, these in vitro results conform with the observed interaction potential of these agents with midazolam and many other CYP3A4 substrates in vivo in man.     

Effects of calcium channel blockers on gastric emptying and acid secretion of the rat in vivo.

Experiments were designed to evaluate the effects of three calcium channel blockers (verapamil, diltiazem and cinnarizine) on gastric emptying and secretion in the rat. Pretreatment with the calcium blockers delayed gastric emptying of phenol red in a dose-dependent manner. Verapamil was the most effective of the agents tested. Verapamil and diltiazem inhibited gastric acid secretion in the pylorus-ligated rat without affecting pepsin output. Cinnarizine was ineffective in this model. When the perfused lumen of the anaesthetized rat was used, verapamil was found to inhibit responses to carbachol or histamine more than those to pentagastrin. Further, we found a greater sensitivity to verapamil for basal compared with vagal-stimulated (2-deoxy-D-glucose) acid secretion. Neither diltiazem nor cinnarizine modified gastric acid secretion in this experimental model. These findings are discussed in relation to the role of extracellular calcium in gastric motility and secretion, and the existence of a regional and functional selectivity for calcium blockers is proposed.     

Comparative effects of LN 5330 and diazoxide on insulin release and 86Rb+ permeability in perifused rat islets of Langerhans

The benzothiadiazine derivative LN 5330 (chloro-7 trifluoromethyl-6 benzothiadiazine-1,2,4-dioxide-1,1) has been shown to inhibit insulin secretion and calcium uptake. The present study was carried out to investigate the effects of LN 5330 on insulin release and 86Rb- efflux from perifused rat pancreatic islets; a comparison was made with the structural analogue diazoxide. In the presence of 8.3 mM glucose, LN 5330 (100 microM) accelerated 86Rb+ efflux while reducing insulin output from the islets. LN 5330 induced a dose-dependent acceleration of 86Rb+ efflux and appeared to be a more potent activator of 86Rb+ efflux than diazoxide. The stimulatory effect of LN 5330 on 86Rb+ efflux persisted in the absence of extracellular calcium. In the absence of glucose, 86Rb+ permeability also increased, LN 5330 being again significantly more efficient than diazoxide at an equimolar concentration. These data indicate that the benzothiadiazine derivative LN 5330 inhibits insulin secretion by increasing the potassium permeability of the plasma membrane. It is suggested that, like diazoxide, this drug could activate the ATP-sensitive K+ channel.     

Effect of verapamil and diltiazem on isolated gastro-oesophageal sphincter of the rat

The effect of verapamil and diltiazem on the contraction induced by agonists on the rat lower oesophageal sphincter in-vitro has been studied. Both calcium entry blockers inhibited the contractile response to acetylcholine, carbachol and KCl. The potency of the inhibitory action was diltiazem greater than verapamil. The results give substance to the use of calcium entry blockers in the treatment of oesophageal spasm.     

Verapamil, diltiazem and nifedipine inhibit vascular responses to noradrenaline, acetylcholine and 5-hydroxytryptamine in human saphenous vein

Verapamil (0.02-2 microM), diltiazem (0.22-8.14 microM) and nifedipine (0.29-8.96 microM) produced concentration-dependent relaxation of human isolated saphenous vein. Based on the EC50 values of the calcium entry blockers, verapamil (relative potency = 1) was 7 and 5 times as potent as nifedipine and diltiazem, respectively, in producing relaxation of the human saphenous vein. In addition, verapamil, diltiazem and nifedipine inhibited the vascular responses (i.e. contractions) produced by noradrenaline (0.03-36 microM), acetylcholine (0.05-55 microM) and 5-hydroxytryptamine (0.02-25 microM) and to KCl (10-100 mM). It was concluded that verapamil, diltiazem and nifedipine relaxed the human isolated saphenous vein, verapamil being more potent than diltiazem or nifedipine, and modified the vascular response to vasoconstrictor agents, e.g. noradrenaline, acetylcholine, 5-hydroxytryptamine and KCl. Thus verapamil, diltiazem and nifedipine may inhibit calcium influx through both potential and receptor-operated calcium ion channels.     

Comparative cardiovascular actions of clentiazem, diltiazem, verapamil, nifedipine, and nimodipine in isolated rabbit tissues.

Clentiazem is an 8-chloro-substituted derivative of diltiazem. We compared the relative potency of clentiazem with that of diltiazem, verapamil, nifedipine, and nimodipine in isolated rabbit right atria and vascular smooth muscle removed from various arterial beds. In experiments with isolated right atria, calcium channel blockers were added cumulatively to study relative cardiodepressive potencies (as compared with vascular effects) with the following results: verapamil greater than or equal to diltiazem greater than clentiazem greater than or equal to nimodipine much greater than nifedipine. The aorta, pulmonary, renal, mesenteric, coronary, and basilar arteries were removed, cut helically in strips, and mounted in isolated tissue baths to measure isometric force. Vessels were contracted with either 40 mM KCl (opening voltage-operated calcium channels) or 1 x 10(-5) M norepinephrine (NE, opening receptor-operated calcium channels). Cumulative dose-response curves were generated for relaxation with each calcium channel blocker. All compounds were more potent at relaxing potassium-induced contractions than NE-induced contractions. In vessels precontracted with KCl, neither diltiazem, verapamil, or nifedipine showed selectivity for basilar artery as compared with the mesenteric artery. Both clentiazem and nimodipine were selective (6- and 30-fold, respectively) for basilar artery in blocking potassium-induced contractions. When NE was used to contract the arteries, clentiazem (12-fold), diltiazem (8-fold), verapamil (8-fold), and nifedipine (153-fold) were all more potent in relaxing the contraction in basilar artery than in mesenteric artery. Nimodipine failed to demonstrate selectivity for basilar artery as compared with mesenteric artery contracted with NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by calcium channel blockers of the binding of platelet-activating factor to human neutrophil granulocytes

The inhibitory action of calcium channel blockers on platelet-activating factor (PAF)-induced activation of human polymorphonuclear granulocytes (PMNL) and on the binding of [3H]PAF to neutrophils was studied. Verapamil and diltiazem inhibited PAF (10(-8)-10(-15) M)-induced degranulation and superoxide production in a dose-dependent manner, with pA2 values of 5.6 and 6.1 for verapamil and 5.9 and 6.2 for diltiazem, respectively. Both channel blockers inhibited the specific binding of [3H]PAF to PMNL in dose-dependent fashion, with Ki values of 3.9 +/- 0.6 X 10(-5) M and 3.2 +/- 0.4 X 10(-5) M for verapamil and diltiazem, respectively. Scatchard analysis of the binding data revealed that both calcium channel blockers decreased the receptor binding affinity and slightly increased the number of high-affinity PAF receptors, whereas they did not affect the binding affinity and number of low-affinity receptors. These results indicate that calcium channel blockers can inhibit neutrophil responses elicited by PAF by a mechanism other than inhibition of calcium influx and suggest that the PAF receptor may be closely associated with calcium channels.     

Effect of diltiazem on insulin secretion. I. Experiments in vitro.

Effect of diltiazem on glucose-induced insulin secretion was investigated in the rat islets of Langerhans isolated by a collagenase digestion technique. It was found that B-cells, main constituents of isolated islet preparations, had a well-preserved ultrastructural appearance immediately following isolation or after incubation with glucose or glucose and diltiazem. The islets released a large amount of insulin upon stimulation with glucose and CaCl2. Diltiazem (10(-6)-10(-4) M) produced a dose-related inhibition of glucose-induced insulin secretion and this effect was antagonized by the increase in extracellular concentration of CaCl2. The inhibitory effect of diltiazem on the insulin secretion was also counteracted by dibutyryl-3',5'-cyclic AMP or by theophylline. Among calcium-antagonists tested, nifedipine produced the most powerful inhibitory action on the insulin secretion, while the effect of verapamil was similar to or somewhat stronger than that of diltiazem. It was suggested that diltiazem may reduce the intracellular concentration of free calcium ion, thus causing an inhibitory effect on the glucose-induced insulin secretion by the isolated islets of Langerhans.     

Effects of calcium channel blockers on neuromuscular blockade induced by aminoglycoside antibiotics

The effects of several calcium channel blockers (nifedipine, verapamil and diltiazem) on rat phrenic-hemidiaphragm preparations were studied. The calcium channel blockers were used either alone or associated with two aminoglycoside antibiotics, neomycin and streptomycin. All drugs investigated produced a concentration-dependent decrease in indirectly elicited diaphragmatic contractions. The order of potency was: verapamil greater than neomycin congruent to nifedipine greater than diltiazem greater than streptomycin. Moreover, neomycin-induced neuromuscular blockade was significantly increased by nifedipine (1 and 10 microM), verapamil (1 and 10 microM) and diltiazem (10 microM), whereas the streptomycin-induced neuromuscular blockade was increased only by nifedipine (1 and 10 microM) and verapamil (10 microM).     

Effects of calcium-channel blockers on cytosolic free calcium and amylase secretion in rat pancreatic acini.

We investigated the effects of verapamil and diltiazem on cytosolic free calcium and amylase secretion in rat pancreatic acini. Verapamil and diltiazem reduced a rise in cytosolic free calcium and amylase release stimulated by the maximal concentration (10(-5) M) of carbachol in a dose-dependent manner. High concentrations (500 microM) of verapamil and diltiazem inhibited both the initial and the sustained amylase secretion stimulated by 10(-5) M carbachol. However, at low concentration (1 microM), they showed no effect on amylase secretion by 10(-5) M carbachol. These calcium-channel blockers did not affect calcium mobilization and amylase secretion stimulated by either caerulein or neuromedin C. Binding of 3H-N-methylscopolamine to pancreatic acini was inhibited by verapamil and diltiazem in a dose-dependent manner. These findings suggested that verapamil and diltiazem reduced carbachol-induced amylase secretion probably not due to their calcium-channel blocking activities but due to their non-competitive effects on the level of muscarinic receptors.     

Calcium antagonists and blood glucose in normal rabbits

The effects of the calcium antagonists verapamil and nifedipine on blood glucose levels, glucose tolerance, insulin secretion during glucose tolerance and hypoglycaemic effect of tolbutamide were studied in normal nondiabetic rabbits. Daily dosage of 40 mg/kg verapamil and 5 mg/kg nifedipine given orally up to 7 days did not affect blood glucose level, glucose tolerance, insulin secretion during glucose tolerance and hypoglycaemic activity of tolbutamide 250 mg/kg p.o.     

Effects of calcium channel blockers on formalin-induced nociception and inflammation in rats.

The possible anti-inflammatory and antinociceptive effects of nitrendipine, nicardipine, diltiazem and verapamil were examined with formalin test in the rat paw. Pretreatment with these calcium channel blockers 1 h before formalin injection diminished formalin-induced inflammatory changes and nociceptive responses. Formalin-induced nociceptive responses were inhibited 20-90% by the calcium channel blockers. Nitrendipine and nicardipine were found to be highly effective in inhibiting the inflammatory changes, whereas the effects of verapamil and diltiazem were partial. Administration of naloxone affected neither the inflammatory changes and nociceptive responses induced by formalin nor the antiinflammatory and antinociceptive effects of the calcium channel blockers. The results suggest the possible anti-inflammatory and naloxone-insensitive antinociceptive properties of calcium channel blockers.     

Comparison of negative inotropic potency, reversibility, and effects on calcium influx of six calcium channel antagonists in cultured myocardial cells.

The negative inotropic effects of calcium channel antagonists on the myocardium were used as a standard for the definition and determination of potency of this group of drugs. The effects of six calcium channel antagonists (verapamil, methoxyverapamil (D600), nifedipine, lidoflazine, perhexiline and diltiazem) were compared on cultured chick embryo ventricular cells. Drug concentrations producing 50% inhibition of contractile amplitude, derived from linearized concentration-response curves, varied from 2.8 X 10(-8)M for nifedipine to 8.3 X 10(-7)M for perhexiline. Equipotent negative inotropic concentrations of verapamil, D600, perhexiline, diltiazem and lidoflazine produced a similar inhibitory effect on 45Ca uptake into cultured cells. Nifedipine produced no significant inhibition of 45Ca uptake. The time required for recovery of contractility after cessation of drug superfusion varied in the order lidoflazine greater than perhexiline greater than D600 greater than verapamil greater than nifedipine greater than diltiazem. This relative order accords closely with the reported in vivo half-lives of these drugs. It is concluded that while some inhibition of 45Ca2+ uptake into cardiac cells can be demonstrated with five of the six calcium channel blockers studied, the relationship between the degree of inhibition of calcium influx and negative inotropic effects may not be uniform for all calcium channel antagonists.     

Effects of Calcium-Channel Blockers on Cytosolic Free Calcium and Amylase Secretion in Rat Pancreatic Acini

Abstract: We investigated the effects of verapamil and diltiazem on cytosolic free calcium and amylase secretion in rat pancreatic acini. Verapamil and diltiazem reduced a rise in cytosolic free calcium and amylase release stimulated by the maximal concentration (10^-5 M) of carbachol in a dose-dependent manner. High concentrations (500 μM) of verapamil and diltiazem inhibited both the initial and the sustained amylase secretion stimulated by 10^-5 M carbachol. However, at low concentration (1 μM), they showed no effect on amylase secretion by 10^-5 M carbachol. These calcium-channel blockers did not affect calcium mobilization and amylase secretion stimulated by either caerulein or neuromedin C. Binding of ³H-N-methylscopolamine to pancreatic acini was inhibited by verapamil and diltiazem in a dose-dependent manner. These findings suggested that verapamil and dilitiazem reduced carbachol-induced amylase secretion probably not due to their calcium-channel blocking activities but due to their non-competitive effects on the level of muscarinic receptors.     

Effects of hypoxia, elevated K+ and acidosis on the potency of verapamil, diltiazem and nifedipine in the guinea-pig isolated papillary muscle.

1 The effects of the structurally diverse calcium channel blockers verapamil, nifedipine and diltiazem were investigated on the force of contraction of guinea-pig, electrically stimulated papillary muscles in vitro. 2 Calcium channel blocking potency was assessed either as a direct negative inotropic effect or as the ability of the drugs to antagonise the positive inotropic effects of added calcium (Ca2+). By either method, the rank order of potency was found to be nifedipine greater than verapamil greater than diltiazem. 3 Various factors which mimic some of the consequences of acute ischaemia in vivo, namely low pH, hypoxia and elevated K+, in combination, but not singly, enhanced the negative inotropic potency of verapamil and to lesser extent that of diltiazem, but not that of nifedipine. 4 Whilst the various interventions, especially in combination, produced a profound negative inotropic effect themselves, this was not responsible per se for the potentiation of the negative inotropic effects of verapamil and diltiazem, since the negative inotropic effects of nifedipine and dinitrophenol were not potentiated under the 'ischaemic' conditions. 5 By use of antagonism of the positive inotropic action of exogenous Ca2+ as an alternative measure of potency, the differential influence of 'ischaemia' on calcium channel blocker potency was confirmed. The effect of verapamil was potentiated some 9 fold, that of diltiazem about 2 fold, and that of nifedipine was unchanged. 6 The differential effect of 'ischaemia' on the potencies of the calcium channel blockers was unexpected. Verapamil (but not nifedipine) is thought to bind to the calcium channel at a specific site not easily accessible from the extracellular space. 'Ischaemic' conditions may cause membranal perturbations which allow verapamil easier access to its binding site thus increasing its negative inotropic potency.     

High sensitivity of chicken's skeletal muscle sarcoplasmatic reticulum to effects of diltiazem or verapamil on calcium uptake and release

In this study, the effects of two different calcium channel blockers, diltiazem and verapamil on calcium uptake and release from the membrane of heavy sarcoplasmic reticulum (SR) of chicken skeletal muscle were investigated. A fluorescent chelate probe technique was employed to determine calcium movement through the SR. Chlortetracycline was used as a fluorescent indicator which is able to penetrate the membrane, bind to the calcium on the inner face of the membrane and show an increase in fluorescence intensity when calcium uptake occurs. Addition of tris-ATP to the microsomes caused ATP-induced calcium uptake in a concentration dependent manner with half-maximal calcium uptake around 0.126 mM. Pretreatment of the medium containing sarcoplasmic reticulum with different concentrations of diltiazem or verapamil followed by added tris-ATP resulted a significant decrease in the fluorescence intensity of chlortetracycline, showing that these calcium channel blockers can diminish ATP-induced calcium uptake in a concentration-dependent manner. The maximum fluorescence intensity of tris-ATP falls to 50% in the presence of 1.75 microM diltiazem and 25 nM verapamil. In addition, diltiazem and verapamil can significantly induce rapid calcium release from the membrane of sarcoplasmic reticulum in a concentration-dependent manner. Therefore, membrane-bound or sequestered calcium in the sarcoplasmic reticulum may be targeted by these two calcium channel blockers in chicken skeletal muscle. Chicken SR is about 1000 times more sensitive to the effects of diltiazem on Ca2+ uptake and release than rabbit SR as shown previously.