Robert P. Hof

Stereoselectivity at the calcium channel: opposite action of the enantiomers of a 1,4-dihydropyridine.

The stereoisomers of the new dihydropyri-dine derivative 202–791 [isopropyl 4-(2,1,3-benzoxadi-azol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecar-boxylate] were synthesized separately and tested on isolated rabbit aortic rings for effects on depolarization-induced contraction and depolarization-stimulated uptake of 45Ca2+. The racemic mixture enhanced contraction of rabbit aortic rings at low levels of depolarization but inhibited contraction and 45Ca2+ uptake at high levels of depolarization. The IC50 values were 2.0 × 10−7 and 1.7 × 107 M, respectively. The R enantiomer inhibited contraction and 45Ca2+ uptake with IC50 values of 3.2 × 10−8 and 4.3 × 10−8 M, respectively. This compound showed no stimulant activity. By contrast, the S enantiomer of 202–791 shifted the concentration-response curve for depolarization-induced contraction in an almost parallel fashion to the left, thus enhancing contraction. The EC50 value for this effect at a KCl concentration of 16 mM was 1.8 × 10−7 M. This compound enhanced 45Ca2+ uptake concentration dependently at all levels of depolarization tested. Thus, the stereoisomers of a dihydropyridine derivative may behave as a calcium entry blocker or a calcium entry enhancer on vascular smooth muscle, depending only on the stereochemistry. If asymmetric compounds elicit effects suggesting a dualistic action at the calcium channel, then the stereoisomers should be prepared.

PN 200-110, a new calcium antagonist: electrophysiological, inotropic, and chronotropic effects on guinea pig myocardial tissue and effects on contraction and calcium uptake of rabbit aorta

The compound isopropyl 4-(2,1,3-benzoxadiazol-4-yl) - 1,4-dihydro-5-methoxycarbonyl-2.6-dimethyl-3-pyridinecarboxylate (code name PN 200–110 [PN]) was investigated for calcium antagonistic effects in experiments in vitro. Action potentials recorded with intracellular microelectrodes in guinea pig papillary muscles were changed little by PN, 10-7 M, except for a slight shortening of the duration of the plateau phase. Slow action potentials elicited in partially depolarized papillary muscles were gradually diminished and finally blocked by this concentration of PN. Contractile force was diminished in normal and partially depolarized muscles. The rate of spontaneously beating guinea pig right atria was decreased dose dependently, and the EC25 was 4.5 × 10-10 M. The EC25 for the negative inotropic effects measured on paced guinea pig left atria was 1.5 × 10-8 M. No membrane-stabilizing effects were found. Calcium-induced contraction of rabbit aorta in depolarizing bath solution was inhibited with an apparent pA2 of 10.3. Contraction elicited by graded depolarization at a constant calcium concentration was inhibited with an EC50 of 1.4 × 10-9 M. Under resting conditions PN did not alter net uptake of 45Ca2+. KCl-stimulated uptake was inhibited with an EC50 of 3.6 × 10-9 M. Neither noradrenaline-induced contractions nor noradrenaline-stimulated net uptake of 45Ca2+ were inhibited by a concentration of PN as high as 10 -5 M. PN thus is selective on cardiac tissue with respect to negative chronotropic versus inotropic activity and on rabbit aorta with respect to potential-operated versus receptor-operated channels.

Similarities in the mechanism of action of two new vasodilator drugs: pinacidil and BRL 34915.

Summary: The present study compares the in vitro and in vivo activities of pinacidil with another new vasodilator drug, BRL 34915, claimed to act via the opening of K+channels in vascular smooth muscle. In the rabbit aorta, BRL 34915 and pinacidil caused rightward shifts of the KCl concentration–response curve and noncompetitively antagonized angiotensin II contractions, yielding an IC50 of 5 and 10 μM, respectively. In 86Rb-loaded guinea pig portal veins, both BRL 34915 and pinacidil stimulated 86Rb+ efflux over the concentration range 0.3–30 μM. At saturating concentrations, the maximum efflux elicited by pinacidil was only one-third that of BRL 34915. Spontaneous activity measured simultaneously from the same vessels was inhibited by BRL 34915 and pinacidil with an IC50 of 12 and 32 n M, respectively. Effects on mechanical activity were thus observed at drug concentrations 100-fold lower than those required to stimulate 86Rb+ efflux. In anesthetized rats, both compounds rapidly lowered blood pressure, with BRL 34915 being threefold more potent than pinacidil in this respect. Tachycardia was more pronounced after BRL 34915 than after pinacidil. Angiotensin II pressor responses were poorly antagonized by these two vasodilators in rats, in marked contrast to the potent effects of Ca2+ antagonists on these responses at equieffective hypotensive doses. We conclude the pinacidil, like BRL 34915, is a potent antihypertensive that is able to enhance the K+ permeability of vascular smooth muscle. The similarities between these two drugs suggest they have a common mechanism of action. However, the discrepancy between the concentrations of these drugs necessary to stimulate 86Rb efflux, and those at which mechanical effects are seen in the portal vein, do not rule out the possibility that other actions may contribute to their vasodilator activities.

Effects of the new calcium antagonist PN 200-110 on the myocardium and the regional peripheral circulation in anesthetized cats and dogs.

The effects of PN 200–110 (PN), isopropyl 4-(2,1,3− benzoxadiazol - 4 - yl) −1,4 - dihydro - 5 - methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylate, on the cardiovascular system were investigated. In chloralose-urethane-anesthetized cats PN decreased blood pressure (BP) and heart rate (HR) and increased cardiac output (CO) and total peripheral resistance dose dependently after intravenous doses of 1–10 μg/kg. Regional blood flow changes effected by an intravenous dose of 10 μg/kg were measured with microspheres. Flow to the heart, brain, and skeletal muscle increased selectively, and in-tramyocardial flow was redistributed in favor of the left ventricular subepicardial layer. The duration of action differed for various effects. The bradycardia was short lasting, and the cerebral vasodilatation persisted longest. It was not possible to predict the duration of action of PN on different target tissues by observing only one variable such as BP. Chloralose-urethane-anesthetized open-chest dogs appeared to be more sensitive to PN than cats. A dose of 3 μg/kg i.v. markedly increased coronary flow, lowered BP, increased CO, and tended to lower HR and to increase myocardial contractility. Myocardial oxygen consumption was lowered. PN did not alter diastolic excitation threshold or any electrocardiographic intervals in closed-chest anesthetized dogs. Absence of myocardial depression, potent vasodilator activity, and long duration of action might render PN useful for the treatment of hypertension and angina pectoris.

Contribution of donor-specific antibodies to acute allograft rejection: evidence from B cell-deficient mice.

BACKGROUND The role of T lymphocytes in acute allograft rejection is well established. The involvement of B lymphocytes in this process, however, is more controversial. A series of reports showed that mice without a functional B-cell compartment rejected allografts with the same kinetics as control animals. In rats, however, alloantibodies were found to play a decisive role in allograft rejection. To provide an explanation for the discrepant results, we readdressed the role of B cells and antibodies in mice with disrupted immunoglobulin mu chain genes. The use of cyclosporine (CsA), which strongly suppresses T cells, allowed us to focus specifically on the function of B cells. METHODS C57BL/6 mice rendered B cell deficient by targeted disruption of the immunoglobulin mu chain gene (referred to as microMT/microMT mice) and microMT/+ control mice with one functional mu chain were heterotopically transplanted with fully MHC-disparate BALB/c hearts. CsA was administered subcutaneously by Alzet osmotic pumps. Normal and immune serum specific for donor hearts was given to assess the role of antibodies in the rejection process. RESULTS Both B cell-deficient microMT/microMT and heterozygous microMT/+ mice were found to reject transplanted hearts within a similar period of time. In contrast, when T cells were partially suppressed with CsA, graft survival was significantly prolonged in microMT/microMT mice as compared with heterozygous controls. Passive transfer of donor-specific immune serum, obtained from microMT/+ animals rejecting allogeneic hearts, to CsA-treated microMT/microMT mice significantly accelerated allograft rejection as opposed to recipients treated with normal serum. CONCLUSIONS B lymphocytes and antibodies play an important role in acute allograft rejection particularly when the dominant T-cell compartment is partially suppressed.

Effect of PY 108-068, a new calcium antagonist, on general hemodynamics and regional blood flow in anesthetized cats: a comparison with nifedipine.

The hemodynamic effects of PY 108–068 (PY), a new benzoxadiazolyle dihydropyridine derivative with calcium antagonistic effects on vascular smooth muscle, were investigated in chloralose-urethane-anes-thetized open-chest cats. Regional blood flow was measured with tracer microspheres. PY lowered blood pressure and increased cardiac output similarly to nifedipine (N). Unlike the latter drug, PY decreased heart rate without showing evidence of cardiodepression. Right atrial pressure increased slightly less than with N. All these effects were dose dependent in the dose range from 1 to 43 μg/kg i.v.. PY and N increased myocardial blood flow to the same extent initially, and redistributed it in favor of the outer layer of the left ventricle. The activity of sublingually administered PY was comparable to that of a similar intravenous dose. An approximately three times larger intraduodenal dose was needed for similar effects. Our results provide preliminary in vivo evidence that it is possible to separate the negative chronotropic from the negative inotropic effects of calcium antagonists.

Stereoselectivity at the calcium channel: different profiles of hemodynamic activity of the enantiomers of the dihydropyridine derivative PN 200-110.

Summary: The enantiomers of PN 200-110 (PN), a highly potent calcium antagonist, were synthesized by a stereoselective synthesis. Cross-contamination was <0.2% for the (S)-enantiomer and 0.5% for the (R)-enantiomer. Both isomers inhibited depolarization-induced contraction of rabbit aorta, the (S)-enantiomer being much more active: The pD′2 values were 9.1 and 6.9 for the (S)- and the (R)-enantiomer, respectively. This activity of (R)-PN on potential-operated channels can be attributed to the contamination with 0.5% of (S)-PN. In anesthetized cats, the (S)-enantiomer lowered blood pressure and heart rate dose-dependently (0.3–10 μg/kg i.v.). The (R)-enantiomer had almost no effect on heart rate (HR) and blood pressure (BP) at doses up to 300 μg/kg. However, both enantiomers increased cardiac output and blood flow to the heart and the brain. The effects on regional blood flow were tissue-dependent, the (R)-enantiomer being surprisingly potent in the subendocardium of the left ventricle. The qualitative and quantitative differences between the activities of the two enantiomers suggest that the results obtained cannot merely be explained by traces of the more active enantiomer contaminating the less active enantiomer. These results, together with those found with the enantiomers of a nitro-substituted dihydropyridine, suggest that calcium channels appear to be able not only to discriminate between enantiomers but also to respond differently to them.

Cardiovascular effects of apamin and BRL 34915 in rats and rabbits

1 The cardiovascular effects of apamin, a selective blocker of certain calcium‐activated potassium channels, and BRL 34915, a vasodilator thought to act by opening of potassium channels, have been investigated in vivo in rats and rabbits. 2 In anaesthetized normotensive rats, apamin (0.05 and 0.15 mg kg−1, i.v.) potentiated angiotensin II pressor responses but did not modify baseline blood pressure or heart rate. 3 Apamin (0.15 mg kg−1, i.v.) was without cardiovascular effects in rabbits. 4 BRL 34915 (0.1 and 0.3 mg kg−1, i.v.) lowered blood pressure in rats dose‐dependently and caused reflex tachycardia. The heart rate increase was abolished by prior administration of the β‐adrenoceptor blocker bopindolol (0.1 mg kg−1, i.v.). 5 In anaesthetized rabbits, regional blood flow measurements (with radioactive tracer microspheres) showed that BRL 34915 (3 to 30 μg kg−1, i.v.) caused marked vasodilatation in the stomach, with increases in flow also to the heart and small intestine. Brain blood flow also tended to increase. Blood flow to the kidneys was reduced by BRL 34915, whereas flow to skeletal muscle was unchanged. 6 Apamin pretreatment did not modify the blood pressure lowering activity of BRL 34915 in rats. The site at which BRL 34915 acts to cause vasodilatation in vivo thus appears to be apamin‐insensitive.

PY 108-068, a new, potent, and selective inhibitor of calcium-induced contraction of rabbit aortic rings.

PY 108–068 (PY) is a new benzoxadiazolyle dihydropyridine derivative. It potently antagonizes calcium-induced contractions of rabbit aorta in depolarizing solution (PD‘2: 8.8). This antagonism is selective, since no relevant antagonistic effects against noradrenaline (NA)-, serotonin (5-HT)-, and angiotensin II (AII)-induced contractions are found at the highest concentration of PY (10-5 M). Verapamil (V), examined for comparison, was less selective with respect to its antagonism: pA2 value against calcium-induced contractions in depolarizing solution: 7.6, pA2 against serotonin-induced contractions: 6.9. In calcium-free Krebs-Henseleil solution tachyphylaxis occurred after three to four administrations of NA, 5-HT, or All. When calcium was added in the presence of one of these agonists, the slow phase of contraction, which depends on extracellular calcium, developed. This slow phase of contraction was not inhibited to a relevant extent by a 10-5 M concentration of PY. Verapamil inhibited the tonic contraction induced by serotonin (pD’2: 5.5) but not to a relevant extent the contractions induced by the two other agonists. PY, therefore, inhibits calcium-induced contractions of rabbit aorta in depolarizing solution very selectively, and shows hardly any effects on receptor-operated channels.

A smooth muscle cell line suitable for the study of voltage sensitive calcium channels

A cell line originating from the fetal rat aorta has been studied with respect to 45Ca2+ uptake. Kinetic experiments showed an initial rapid uptake followed by a slow linear phase; both the initial rate and the maximum uptake were increased in the presence of 55 mM potassium chloride. The calcium channel antagonists, darodipine (PY 108-068) and verapamil, inhibited both the basal and the potassium chloride stimulated uptake. Neither tetrodotoxin nor furosemide affected either basal or depolarisation induced 45Ca2+ uptake. Blockade of the Na+/K+ ATPase by ouabain and of the Ca2+ ATPase by vanadate caused a net increase in cellular 45Ca2+ accumulation.