High affinity binding of a potassium channel agonist to intact rat insulinoma cells.

The specific binding of a novel tritiated K+ channel opener, [3H]BAY X 9228, has been characterized in a rat insulinoma (RINm5F) cell line. The KD was 2.1 nM and Bmax 50 fmol/mg total protein as determined by saturation analysis. The high affinity binding to intact cells was inhibited by pinacidil and by a series of BAY X 9228 analogs with an activity sequence correlating well with that for producing glyburide-reversible relaxation of partially depolarized rat aorta. This represents the first report of the specific binding of a K+ channel opener to cultured cells.

Effect of lipoxygenase inhibitors on Ca2(+)-induced constriction of the rabbit ear artery.

1. The effects of nafazatrom, nordihydroguaiaretic acid (NDGA) and quercetin on Ca2(+)-induced vasoconstriction were studied in isolated rabbit ear arteries. 2. The arteries were perfused with Ca2(+)-free and high K+ (75 mM) Krebs bicarbonate buffer. Constriction of the artery was induced by addition of Ca2+ (1.5 mM) to the perfusion fluid. 3. Indomethacin (1 microM) did not alter the response to Ca2+. 4. Nafazatrom (2 or 5 microM) produced a concentration-dependent inhibition of the constrictor response to Ca2+ ranging from 4 to 23% after 1 hr of perfusion and 26 to 62% after 3 hr. 5. Similar effects were obtained with NDGA and quercetin (0.5 and 1 microM). 6. The inhibitory effects of nafazatrom and quercetin were antagonized by Ca2+ (2.5 mM) or Bay K 8644 (1 microM), a calcium channel activator. 7. Ca2(+)-induced contractions recovered within 30 min after discontinuation of perfusion with quercetin, whereas nafazatrom and NDGA had longer durations of action. 8. These results suggest that inhibitors of lipoxygenase antagonized Ca2(+)-induced vasoconstriction and that products of lipoxygenase metabolism may facilitate Ca2+ entry into vascular smooth muscle cells.

Pharmacological basis for the use of nimodipine in central nervous system disorders.

Nimodipine, a Ca2+ antagonist with cerebrovasodilatory and anti-ischemic effects, binds to rat, guinea pig, and human brain membranes with high affinity (less than 1 nM). Only at higher concentrations has nimodipine been reported to block the release of some neurotransmitters and hormones from neuronal tissue. Nimodipine has no consistent effect on brain oxygen consumption or cortical ATP or phosphocreatine levels, although the ischemia-induced fall of brain ATP levels in gerbils or the lowering of intracellular brain pH in rabbits with focal cerebral ischemia were antagonized by the drug. In rats and baboons with middle cerebral artery occlusion, nimodipine was found to reduce neurological deficits without an increase in intracranial pressure or brain edema. Electrophysiological studies with nimodipine suggested a direct neuronal action. In rabbit dorsal root ganglion cells, concentrations as low as 20 nM were reported to block inward Ca2+ currents. Recent studies have suggested that nimodipine may also improve memory in brain-damaged or old rats, restore sensorimotor function and abnormal walking patterns of old rats, and accelerate acquisition of associative learning in aging rabbits. Blockade of age-related changes in Ca2+ fluxes in rat hippocampal neurones by nimodipine in vitro pointed to neuronal plasma membrane as the site of nimodipine action. The therapeutic usefulness of nimodipine appears not to be limited to cerebral ischemia, but may include dementia, age-related degenerative diseases, epilepsy, and ethanol intoxication.

Pharmacological basis for use of calcium antagonists in hypertension.

Many different classification systems for Ca2+ antagonists were proposed. They are mostly based on structural aspects or profiles of biological activity. 1,4-dihydropyridines, with Ca2+ channel antagonistic activity, including nifedipine and nitrendipine, are highly effective as antihypertensive agents. Although Ca2+ antagonists have multiple sites of antihypertensive action, their main mechanism of action is inhibition of Ca2+ entry into the vascular smooth muscle cells. Ca2+ channel antagonists bind to specific receptors at Ca2+ channels and stabilize the channels in a mode unavailable for opening. Their effect is enhanced by depolarization of the cell membrane. Currently used pharmacological methods for detection of Ca2+ antagonistic action of drugs include: (1) inhibition of 45Ca2(+)-uptake; (2) displacement of [3H]nitrendipine from isolated membranes, and (3) inhibition of Ca2+ current in single cells or channels. Ca2+ antagonists were reported to prevent hypertension-induced vascular changes and other vascular pathology, probably related to Ca2+ overload. Vascular lesions in Dahl salt-sensitive hypertensive rats and in spontaneously hypertensive rats were prevented by chronic administration of nifedipine or nitrendipine. Hemodynamic effects of Ca2+ antagonists are characterized by reduction in total peripheral vascular resistance, increase in cardiac output, reduction in systemic left ventricular end-diastolic, pulmonary arterial and capillary wedge pressures. Ca2+ antagonists differ in potency, duration of action and their therapeutic ratios. DHPs enhance sympathetic tone and have little or no negative dromotropic action. They are, therefore, safer in combination with beta-adrenoceptor antagonists than either verapamil or diltiazem. In comparison with other Ca2+ antagonists nitrendipine is highly potent as a vasodilator. As a negative inotropic agent, it is, however, less potent than either verapamil or nifedipine. Nitrendipine has, therefore, a better therapeutic ratio than some of the other well-known Ca2+ antagonists. Unlike older vasodilators, e.g. hydralazine and minoxidil, Ca2+ antagonists have diuretic properties which are primarily due to inhibition of tubular reabsorption of salt and water. Under certain experimental conditions, e.g. infusion of angiotensin II, DHPs can increase GFR. Nitrendipine has also renal cytoprotective activity. It protected rats from aminoglycoside-induced nephrotoxicity and antagonized proliferative glomerular changes in nephritic rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterization of a fraction from brain that inhibits 1,4-[3H]dihydropyridine binding and L-type calcium channel current.

Bovine brain was subjected to acid extraction and several purification steps. A fraction from brain that eluted from C18 reverse-phase columns at 30-35% acetonitrile inhibited [3H]nitrendipine binding to cardiac membranes. Further purification of this fraction on a sizing column in the presence of 40% acetonitrile yielded a low molecular mass fraction (less than 1 kDa) that produced a time- and voltage-dependent inhibition of L-type (but not T-type) Ca2+-channel current in GH3 cells. The results suggest that this fraction contains an endogenous substance that binds directly to slowly-inactivating Ca2+ channels and thereby inhibits current flow.

Effects of BAY K 8644 on the responses of rabbit ear artery to electrical stimulation.

BAY K 8644 at 6.25 nM to 1 microM enhanced, in a concentration-dependent manner, both phases of the vasoconstrictor response of the isolated perfused rabbit ear artery to electrical stimulation. At 1 microM, BAY K 8644 enhanced the constrictor response by more than 250%. To study possible involvement of neurotransmitter release in the enhancement of the vasoconstrictor response by BAY K 8644, rabbit ear arteries were preincubated with [3H]norepinephrine and stimulated either electrically (for 1 or 5 min) or by 60 mM K+. BAY K 8644 (1 microM) had no effect on tritium release caused by 1-min periods of electrical stimulation. However, tritium release caused by 5-min periods of electrical stimulation or by 60 mM K+ was enhanced in the presence of BAY K 8644. It was concluded that BAY K 8644 enhances vasoconstrictor effects of electrical stimulation of rabbit ear artery by primarily a direct agonist action on Ca2+ channels in vascular smooth muscle cells. Following sustained depolarization, however, the drug may also enhance the release of neurotransmitter from sympathetic nerve endings. Under certain conditions, this release may contribute to the overall action of BAY K 8644.

Pharmacology of nimodipine. A review.

The major pharmacological findings with nimodipine reviewed in this chapter are summarized in TABLE 3. On the basis of these findings, the following conclusions appear to be justified: 1. Nimodipine is a 1,4-dihydropyridine with Ca2+ channel antagonist properties. It is more lipophilic than nifedipine and its distribution volume in the brain of rats is higher than that of nifedipine. 2. Nimodipine dilates cerebral vessels at considerably lower concentrations than required for dilatation of peripheral blood vessels. It can, therefore, improve cerebral blood flow at doses that do not reduce systemic arterial pressure. 3. Nimodipine inhibits 45Ca uptake into vascular smooth muscle and neuronal cells. 4. Nimodipine antagonized postischemic cerebral hypoperfusion in cats and prolonged life of stroke-prone spontaneously hypertensive (SH) rats at doses that have little if any effect on arterial blood pressure. 5. Nimodipine reduced neurological deficits in dogs and monkeys with global cerebral ischemia. In focal ischemia (MCA occlusion) nimodipine reduced infarct size and neurological deficits and normalized intracellular brain pH. 6. In addition to its cerebral vasodilator effect, nimodipine appears to have a direct neuronal action. The suggested evidence for the neuronal site of action of nimodipine includes: a. Presence of nimodipine binding sites in brain. b. Blockade by nimodipine of Ca2+ channels in single nerve cells and in endocrine cells under conditions of sustained depolarization. c. Interactions with centrally acting drugs. d. Effects on release of various neurotransmitters from neuronal tissue or endocrine cells. e. Demonstration of anticonvulsant action of nimodipine. f. Blockade of behavioral effects of Ca2+ channel agonists by calcium channel antagonists.

Ca2+ antagonists and inhibitory effects of dopamine on isolated rabbit ear artery.

Rabbit ear arteries were isolated and perfused with Krebs-bicarbonate buffer. Brief periods of supramaximal nerve stimulation caused reproducible constriction of the arteries, which was inhibited by dopamine (EC50 approximately equal to 0.1 microM). Verapamil or flunarizine (at 1 or 5 microM) antagonized this inhibitory effect of dopamine. The maximal effect of verapamil, 5 microM was reached after 8 min and that of flunarizine, 5 microM after 40 min of perfusion with the drug. The antagonism of dopamine by verapamil, even at 5 microM, was incomplete (approximately equal to 80%), whereas flunarizine, 5 microM completely antagonized the effect of dopamine. Nitrendipine, nimodipine, or nisoldipine, at either 1 or 5 microM, or solvent (ethanol, 0.03%) had no significant effect on dopamine-induced inhibition of vasoconstriction even after perfusion for 40 min. Antagonism of dopamine at central synapses may conceivably explain reported Parkinsonian side effects of flunarizine. Our results suggest a mechanism for the tendency of flunarizine to cause Parkinsonism and an explanation why 1,4-dihydropyridines are not likely to have this side effect.

Pharmacological basis for use of calcium channel antagonists in hypertension.

The major mechanism of antihypertensive action of Ca2+ channel antagonists is the inhibition of Ca2+ entry into the vascular smooth muscle cell. Ca2+ channels are stabilized by these drugs in a mode unavailable for opening. The inhibition of Ca2+ entry leads to vasodilatation and lowering of arterial pressure. The advantages of Ca2+ channel antagonists in hypertension include improvement of blood supply to vital organs, diuretic activity, coronary vasodilatation, reduction in heart size and vascular cytoprotective effect.

Comparative pharmacology of 1,4-dihydropyridines and other Ca2+ channel ligands.

Experimental and clinical pharmacology of Ca2+ channel antagonists is reviewed. Advantages and disadvantages of three major classes of Ca2+ antagonists, 1,4-dihydropyridines (DHPs), benzothiazepines, and phenylalkylamines, are discussed. Reduction of peripheral vascular resistance, freedom from adverse effects on lipid metabolism, mild diuretic activity without an increase in plasma aldosterone, and absence of negative inotropic action at therapeutic dose levels are considered the major advantages of DHPs in the treatment of hypertensive patients.

BAY K 8644-induced enhancement of 45Ca uptake by rabbit aortic rings.

BAY K 8644, a Ca2+ channel activator, enhances uptake of 45Ca by rabbit aortic rings. This effect depends on the concentration of K+ in the medium: at 20 mM K+ the effect of BAY K 8644 was more pronounced than at 5 mM, whereas at 80 mM, no significant enhancement of 45Ca uptake by BAY K 8644 was found. In the medium containing 5 mM K+, BAY K 8644 was effective in experiments involving 10 or 30 (but not 3) min exposure of aortic rings to 45Ca. The dose-response curve for BAY K 8644 was established in 5 mM K+-containing medium and for 30 min exposure to 45Ca. BAY K 8644 was effective at 0.01 microM and higher concentrations. In the presence of norepinephrine (0.1 or 10 microM), BAY K 8644 had no greater effect on 45Ca uptake than in control 5 mM K+ medium. Our observation that the presence of norepinephrine in 5 mM K+ did not enhance BAY K 8644-induced 45Ca uptake suggests that activation of alpha-adrenergic receptor does not depolarize aortic membranes to the same extent as an increase in K+ concentration to 20 mM or that BAY K 8644 does not enhance Ca2+ entry through receptor-operated channels.

Plasma membrane calcium flux, protein kinase C activation and smooth muscle contraction.

Isolated perfused rabbit ear arteries contract when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of the calcium-activated, phospholipid-dependent protein kinase or C-kinase. Under conditions where the calcium concentration in the perfusate is 1.5 mM and the potassium concentration is 4.8 mM, there is a latent period of 70 +/- 19 min (mean +/- S.E.M., n = 10) between TPA addition and the onset of the contractile response. Once initiated, the contractile response is progressive and sustained. When perfusion conditions are altered in such a way as to modify calcium flux across the plasma membrane (i.e., raising the extracellular calcium concentration to 2.5 mM Ca++, raising the extracellular potassium concentration to 10 mM, and/or preincubating the tissues in media containing 100 nM Bay K 8644, a potent calcium channel agonist), the latency period between TPA addition and initiation of the contractile response is significantly reduced (2.5 mM Ca++, 37 +/- 7 min; 10 mM K+ and 2.5 mM Ca++, 11 +/- 3 min; 100 nM Bay K 8644 and 1.5 mM Ca++, 20 +/- 7 min; 100 nM Bay K 8644 and 2.5 mM Ca2+, 8.5 +/- 1.7 min; 10 mM K+ and 100 nM Bay K 8644, 11 +/- 5 min). Likewise, the combination of 2.5 mM calcium, 100 nM Bay K 8644, and 3.3 microM ouabain results in a contractile response 4.5 +/- 2.0 min after TPA addition (means +/- S.E.M., n = 4). Control tissues (absence of TPA addition) run simultaneously show no contractile responses to the various Ca++ flux regulators even after 90 min of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative effects of nitrendipine and verapamil on neuroeffector transmission in the rabbit ear artery.

The purpose of the present study was to evaluate and compare the effects of the calcium antagonists nitrendipine and verapamil on nerve-evoked constrictions and on norepinephrine (NE) release in the isolated, perfused rabbit ear artery. The response of the ear artery to nerve stimulation consists of an early phasic constriction (P response) and a late, slow-developing tonic constriction (T response). Both calcium antagonists were much more potent in causing inhibition of the T than P responses. For nitrendipine the EC50 (6.6 +/- 2.3 X 10(-8)M) for inhibition of T response was 450 times lower than the EC50 for inhibition of P response. Verapamil exhibited less selectivity for T response: the EC50 for inhibition of T response was 17 times lower than the EC50 for inhibition of the P response. Nitrendipine (3.3 X 10(7) to 3.3 X 10(-5) M) did not alter the nerve-evoked release of [3H]NE from arteries preincubated with [3H]NE. Verapamil at 3.3 X 10(-6) and 3.3 X 10(-5)M enhanced both spontaneous and nerve-evoked [3H]NE release.

TPA-induced contraction of isolated rabbit vascular smooth muscle.

Myosin light chain phosphorylation may not regulate the sustained phase of vascular smooth muscle contraction. Another, unidentified, calcium-dependent pathway may be involved in this process. TPA, an activator of C-kinase, at concentrations of 10 to 333 nM induces a calcium-dependent contraction of vascular smooth muscle which develops slowly but progressively to reach values of 50-300 mm Hg. Arteries exposed to the ionophore A23187, in a calcium-free medium, display a uniform series of contractile responses when exposed to 1.5 mM Ca2+ for 2 min once every 10 min. Exposure to 100 nM TPA as well as ionophore leads to a progressive enhancement of these calcium-induced, contractile responses. Arteries stimulated by brief (10 sec), repetitive (every 3 min) electrical pulses, respond with a series of comparable phase 1 responses. Prior exposure of vessels to 10 nM TPA, causes a progressive increase in the magnitude of these responses to repetitive electrical stimulation. Addition of 25 microM forskolin, an activator of adenylate cyclase, to TPA-treated, partially-contracted muscle leads to the immediate inhibition of the TPA-induced contraction. These data suggest that the activation of C-kinase plays a significant role in regulating vascular smooth muscle contraction.

Some recent pharmacological findings with nitrendipine.

The available evidence indicates that nitrendipine and other dihydropyridines with a similar pharmacological action exert their therapeutic effects by inhibiting Ca2+ channels. In our recent experiments, nitrendipine was shown to block K+-stimulated 45Ca2+ uptake and K+-induced contractions of isolated rabbit aortic rings. Its IC50 were 4.7 and 8.9 nM for inhibition of Ca2+ uptake and of contractions, respectively. There was no statistically significant difference between the two values. At higher concentrations, nitrendipine also reduced norepinephrine-induced 45Ca2+ uptake and norepinephrine-induced contractions of rabbit aortic strips. The norepinephrine-induced contractions were only slightly (21%) reduced by nitrendipine at 10 microM. Nitrendipine at 10 nM and higher concentrations inhibited K+- or angiotensin-II-(AII) induced release of aldosterone from isolated bovine adrenal glomerulosa cells. The drug was more potent and more effective in inhibiting K+- than AII-induced aldosterone release. Dantrolene, 25 microM, enhanced the inhibitory activity of nitrendipine on AII-stimulated aldosterone release. Acute renal failure produced by either glycerol or gentamicin in rats was antagonized by nitrendipine at oral doses of 15-25 mg/kg/day. Our studies confirmed previously reported observations that the usefulness of nitrendipine in the treatment of hypertension may be determined not only by its vasodilator action. We demonstrated that nitrendipine has a direct inhibitory effect on the release of aldosterone from adrenal glomerular cells. In addition to a previously described diuretic action, nitrendipine was shown to have renal cytoprotective activity.

Antagonism of Ca2+-induced constriction of isolated rabbit ear artery by nitrendipine in high K+- and/or norepinephrine-containing medium.

Nitrendipine is a Ca2+ channel antagonist with vasodilator and antihypertensive properties. In the present experiments, the isolated rabbit ear artery was perfused with Ca2+-free Krebs-bicarbonate solution. Constriction of the artery was induced by addition of Ca2+ to perfusion fluid in the presence of KCl (75 mM, high K+), norepinephrine (NE) at various concentrations, or high K+ and NE. Nitrendipine produced concentration-dependent inhibition of the constrictor response to Ca2+ in high K+ medium. NE, added to high K+ medium, reversed the inhibitory effect of nitrendipine. In NE-containing medium, nitrendipine also inhibited the constrictor effect of Ca2+. Nitrendipine-induced inhibition was inversely proportional to the concentration of NE in the medium. The inhibitory effect of nitrendipine in the medium containing high K+ and NE was significantly less pronounced than in normal K+- and NE-containing medium. The experiments demonstrate relative resistance of "receptor-operated" channels to nitrendipine and suggest that under depolarizing conditions these channels may operate more effectively.