Theophile Godfraind

Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells

With the endothelium present, the maximum response of rat isolated aorta to clonidine was much lower than that to noradrenaline. Removal of endothelium enhanced the response to both adrenoceptor agonists and the clonidine‐induced maximum contraction became almost equal to that produced by noradrenaline, although it was much more sensitive to inhibition by flunarizine and nifedipine. These results indicate that clonidine and noradrenaline activate receptors present in the endothelial cells and that these receptors are highly sensitive to clonidine.

Calcium exchange in vascular smooth muscle, action of noradrenaline and lanthanum.

1. The Na, K, Ca and Mg content and the 45Ca uptake and loss were determined in rat aortae incubated in physiological solution or in solution containing LaCl3 instead of CaCl2. 2. Aortae washed in La‐solution contained less Ca and Na than controls in physiological solution, the K content was not modified and the Mg content was slightly decreased. 3. In 50 mM‐La solution the 45Ca diffusion space was intermediate between the values found for the [14C]sorbitol space and the [14C]inulin space, indicating that there was no Ca entry within the cell nor Ca binding at superficial sites. 45Ca loss from the tissue was directly related to the La concentration. 4. Noradrenaline increased the rate of uptake of 45Ca into the Ca fraction resistant to displacement by La. This increase was dose dependent, a response of 50% of the maximum being produced by 2 x 10(‐8) noradrenaline as for the contraction. In the presence of phentolamine, the dose‐effect curves for the action of noradrenaline on 45Ca uptake were displaced in a manner characteristic of competitive antagonism. The rhoA2 for phentolamine was 7‐8. 5. In physiological solution, the rate of loss of 45Ca, from the Ca fraction resistant to displacement by La, was increased by noradrenaline the ED50 was 2 x 10(‐8) M, and the effect was abolished by phentolamine. 6. In view of the similarity of phentolamine rhoA2 estimated by measuring noradrenaline sensitive 45Ca uptake or noradrenaline evoked contraction, it is likely that the activation of alpha‐adrenergic receptors is responsible for both effects.

Blockade or reversal of the contraction induced by calcium and adrenaline in depolarized arterial smooth muscle

1 Mesenteric arteries immersed in a depolarizing solution contract in the presence of calcium. These contractions are proportional to the calcium concentration and are reversible. 2 Mesenteric arteries immersed in a calcium‐free depolarizing solution contract in the presence of adrenaline. Under the experimental conditions reported here, this response develops only about one‐third of the contractile tension developed in polarizing solution (modified Krebs bicarbonate). 3 Cinnarizine and ohlorpromazine inhibit the contractile response to calcium and induce relaxation of depolarized muscle previously contracted by calcium; cinnarizine was 4 times more potent than chlorpromazine in such activity. 4 Chlorpromazine inhibits the response to adrenaline in both polarizing and calcium‐free depolarizing solutions, whereas cinnarizine inhibits the response in polarizing solution but not that in calcium‐free depolarizing solution. 5 The significance of these results is discussed.

Postnatal maturation of excitation-contraction coupling in rat ventricle in relation to the subcellular localization and surface density of 1,4-dihydropyridine and ryanodine receptors.

To better understand excitation-contraction coupling in cardiac muscle, we investigated the main Ca2+ channels involved in that process in adult and neonatal rat ventricle. Voltage-dependent (L-type) Ca2+ channels and sarcoplasmic reticulum Ca2+ release channels were labeled by means of [3H] (+)-PN200-110 and [3H]ryanodine, respectively. The number of [3H]ryanodine binding sites (per gram tissue) increased more than that of [3H] (+)-PN200-110 binding sites over the postnatal period (2.1-fold versus 1.35-fold, respectively). After equilibration of microsomal fractions in density gradient, ryanodine receptors were characterized by a heavy distribution pattern that did not change appreciably between days 1 and 30 after birth. In neonatal tissue, 1,4-dihydropyridine receptors were found mainly in low-density subfractions, together with other sarcolemmal constituents, whereas in adult tissue, they were recovered predominantly in high-density subfractions, together with ryanodine receptors. Thus, after birth, and in parallel with the development of T tubules, there was a progressive concentration of L-type Ca2+ channels in junctional structures of high equilibrium density, where they were situated close to the Ca2+ release channels of the sarcoplasmic reticulum. In adult ventricle, L-type channels were, on an average, threefold more abundant in T tubules than in external sarcolemma. In parallel mechanical studies, we found that the inhibitory action of ryanodine on systolic contraction was much more pronounced in adult than in neonatal right ventricle, and that, conversely, neonatal tissue was more sensitive that adult tissue to inhibitors of L-type channels. We conclude that, in view of the presumed mechanism of Ca2+ release from the sarcoplasmic reticulum, that is, Ca(2+)-induced Ca2+ release, the predominant localization in adult rat ventricle of the major Ca2+ entry pathway in the vicinity of the Ca2+ release pathway is of great functional significance. Furthermore, owing to the relative stoichiometry of Ca2+ entry and Ca2+ release channels in junctional structures (about 1:9), a physical link between these channels is not likely to be involved in the modulation of Ca2+ release from the sarcoplasmic reticulum in cardiac muscle.

Classification of calcium antagonists.

Drugs of several chemical families have been identified as calcium antagonists. This article examines some pharmacologic properties of these drugs to clarify their terminology and their classification and to provide a rationale for their clinical use. Studies with nifedipine show quantitatively that the therapeutic effect in angina is related to the interaction of this drug with membrane calcium channels in human coronary arteries. This gives support to a classification based on studies at the molecular, tissue and organ levels. Among calcium antagonists, calcium entry blockers are defined as agents able to block calcium inward fluxes evoked by various stimuli. They may be subdivided in 2 groups. Group I is the group of selective calcium entry blockers. Group IA consists of those agents selective for slow calcium channels in myocardium (slow channel blockers); the leading agents are verapamil, nifedipine and diltiazem. Group IB contains agents without action on slow calcium channels in myocardium but with selective action on arteries; the leading agents are cinnarizine and flunarizine. Group II is the group of nonselective calcium entry blockers. Group IIA contains agents acting at similar concentration on calcium and on fast sodium channels. Group IIB consists of agents interacting with calcium channels while having another primary site of action. Other agents modulate calcium movements by an action on sodium-calcium exchange and by an action within the cell. Their identification requires the use of cell biology. The actual clinical uses of these drugs are consistent with this pharmacologic classification.

Heterogeneity of ouabain specific binding sites and (Na+ + K+)-ATPase inhibition in microsomes from rat heart.

Cardiac glycoside binding to microsomes prepared from rat heart ventricles and enriched in (Na+ + K+)-ATPase was measured by a rapid filtration technique. The relation between ouabain binding to microsomes and (Na+ + K+)-ATPase activity has also been examined. Data were statistically analysed by means of two different non linear regression methods. The experimental results were fitted the most closely by a model describing that ouabain specific binding occurred at two classes of independent sites. High affinity sites were characterized by a dissociation constant of 0.21 +/- 0.01 microM and a low capacity (9.4 +/- 1.4 pmoles/enzymatic unit). Low affinity sites were characterized by a dissociation constant equal to 13 +/- 3 microM and a capacity equal to 87 +/- 15 pmoles/enzymatic unit. Similar results were obtained with the more lipophilic glycoside digoxin. It was also observed that dihydroouabain, a ouabain derivative with a saturated lactone ring, competes with 3H-ouabain for the binding to the two classes of sites. Binding to these two classes of sites appeared to be associated with a corresponding inhibition of (Na+ + K+)-ATPase activity.

Evidence for heterogeneity of endothelin receptor distribution in human coronary artery.

1 The receptors mediating endothelin‐evoked contraction of human coronary artery have been investigated in isolated segments of the left anterior descending coronary artery (LAD). 2 Endothelin‐1 (ET‐1) was 10 times more potent in distal than in proximal segments but the potency ratio between ET‐1 and ET‐3 (endothelin‐3) was similar and close to 100 in any segment of the artery. 3 BQ‐123, an ETA receptor antagonist, competitively antagonized the response to ET‐1 of distal segments (pA2 equal to 7.47). In the proximal segments, part of the contractile response was BQ123 sensitive, but the antagonism was non‐competitive. In both groups of segments, the response to ET‐3 could be completely blocked by BQ‐123. 4 These observations indicate that ETA receptors mediate the contractile response to ET‐1 in distal, pre‐resistant coronary arteries, but that other ET receptors are also involved in the contractile response of proximal segments.

Selective alpha 1- and alpha 2-adrenoceptor agonist-induced contractions and 45Ca fluxes in the rat isolated aorta.

1 Contractile responses produced by the α1‐adrenoceptor selective agonist, phenylephrine, and the α2‐adrenoceptor selective agonists, oxymetazoline and clonidine, have been compared to those produced by noradrenaline (non selective) in the rat aorta. 2 The relative order of potency of the agonists was noradrenaline > phenylephrine > clonidine > oxymetazoline. Noradrenaline and phenylephrine produced similar maximal responses. The maximal responses produced by oxymetazoline and clonidine were about 59% and 24% respectively of those produced by noradrenaline. 3 Concentrations of agonists producing maximal contractions exhibited different response‐time relationships. Responses to noradrenaline and phenylephrine were biphasic while responses induced by oxymetazoline and clonidine were monophasic. 4 In calcium‐free solution, contractions stimulated by oxymetazoline and clonidine were almost abolished while those stimulated by noradrenaline and phenylephrine were reduced by about 60–70%. The calcium entry blocker, cinnarizine, almost completely inhibited responses to oxymetazoline and clonidine and reduced noradrenaline‐ and phenylephrine‐stimulated responses by about 60%. 5 All the agonists stimulated the uptake of 45Ca into the La3+‐resistant Ca2+ fraction of the artery but only noradrenaline and phenylephrine stimulated the efflux of 45Ca into calcium‐free solution. The 45Ca uptake stimulated by oxymetazoline and clonidine was abolished by cinnarizine and that stimulated by noradrenaline and phenylephrine was reduced by about 85%. 6 It is concluded that clonidine and oxymetazoline stimulate contractions that are totally dependent on extracellular calcium. Noradrenaline and phenylephrine stimulate contractions that are partly dependent on extracellular calcium and partly dependent on intracellular calcium stores.

Calcium antagonists and vasodilatation.

Calcium antagonists comprise a diverse group of chemically unrelated agents that interact with voltage-operated calcium channels (L-type) and thereby inhibit smooth muscle contractility. They are used to treat several major cardiovascular disorders, including hypertension and angina pectoris; they are also studied in congestive heart failure and in atherosclerosis. The current view is that their therapeutic action is related to vasodilatation. This view is an oversimplification, as will be shown in this review. It will also be illustrated that all calcium antagonists are not identical pharmacological agents.

Selectivity scale of calcium antagonists in the human cardiovascular system based on in vitro studies

The inhibitory effect of calcium antagonists has been studied on the calcium signal and on contractile activity in the human coronary and internal mammary arteries. We observed that rhythmic vasospasms, either spontaneous or evoked by serotonin and endothelin, were highly sensitive to calcium-channel inhibitors. Functional parameters describing the inhibition have been compared to binding parameters estimated in radioligand studies on membranes prepared from human coronary artery and from human heart. Taking the present studies and observations already published together, it was possible to build up a selectivity scale for diltiazem, verapamil, nifedipine, and nisoldipine. This showed that nisoldipine was more selective for coronary artery than the other calcium antagonists so far studied. Such a selectivity observed in functional studies on intact human preparations in vitro could be predicted considering the kinetic parameters of the interaction of nisoldipine with calcium channels.