Drug-induced lysosomal storage of sulphated glycosaminoglycans.

1. Certain compounds (e.g., the immunomodulator tilorone and congeners) are able to induce lysosomal storage of sulphated glycosaminoglycans (GAG), thus, producing cytological and biochemical alterations reminiscent of the inherited mucopolysaccharidoses. The drug-induced GAG storage has been studied in cultured fibroblasts of several species and in rats, and it is likely to occur also in humans. 2. The cytological hallmarks of GAG storage are enlarged lysosomes congested with material that is intensely stained by cationic dyes. With respect to fixation techniques, one has to keep in mind that the GAGs are highly water-soluble and are leached during conventional fixation and tissue processing. Biochemically, the elevation of GAG contents in tissues and cultured fibroblasts is due to storage of dermatan sulphate, predominantly. 3. The molecular structure of the potent inducers of GAG storage is characterized by a planar tricyclic aromatic ring system that is symmetrically substituted with two side chains of 4-5 sigma bond length, each carrying a protonizable nitrogen atom. The lysosomal storage of GAG is accompanied by lysosomal accumulation of the inducing drug, with the molar ratio of drug to GAG-disaccharide unit amounting to > 1:1. The reversibility of GAG storage is rather slow. 4. The pathogenic mechanisms underlying the drug side effects are discussed and the following hypothesis is put forward: The compounds in question are lysosomotropic weak bases. They get trapped in the acidic lysosomes and accumulate highly there. Physicochemical data suggest that the drugs form complexes with the sulphated GAGs, particularly with dermatan sulphate: The positively charged nitrogen atoms of the drug side chains interact with the negative charges of sulphate and carboxy groups of the GAGs, thereby crosslinking at least two GAG helices. Moreover, the interlinking drug molecules form parallel stacks resulting from interaction of the aromatic pi-electrons of the planar ring systems. This further stabilizes the complexes. The GAGs within the complexes are thought to be resistant to the degrading lysosomal enzymes. 5. Drug-induced GAG storage has not been directly demonstrated in man. Yet, clinical reports on keratopathy and basophilic cytoplasmic inclusions in blood lymphocytes of tilorone-treated patients suggest that this drug side effect may also occur in man.

Frequency- and potential-dependency of the negative inotropic action of various dihydropyridine and non-dihydropyridine calcium antagonists.

Transmembrane voltage and beat frequency are important determinants of the action of several organic calcium antagonists. This is well-known for the cationic amphiphilic calcium antagonists. We intended to assess the functional impact of these phenomena in cardiac muscle with special regard to dihydropyridines. Therefore, concentration-response curves were constructed in isolated guinea-pig left atria for the negative inotropic effect of various compounds. The dihydropyridines nifedipine, racemic nitrendipine, nisoldipine, and felodipine, and the enantiomers of isradipine were investigated at different stimulation frequencies (1 Hz, 2.5 Hz, 4.5 Hz), and at different extracellular K+ concentrations (2.7 mM, 5.4 mM, 10.8 mM). These drugs were compared with the cationic amphiphilic compounds gallopamil, verapamil and diltiazem. The potency of some dihydropyridines, particularly nitrendipine, could be modulated to a remarkable extent, covering several orders of magnitude. The potential-dependency of the drugs depended on stimulus frequency and ranged from less than a half to two orders of magnitude. At 2.5 Hz, the rank order of extent of potential-dependency was gallopamil greater than nitrendipine greater than diltiazem greater than verapamil = (+)-isradipine greater than (-)-isradipine greater than or equal to nisoldipine greater than or equal to felodipine = nifedipine. Based on data obtained from binding studies in intact atria and from patch-clamp measurements of calcium current blockade, a mathematical model was used which describes the observed potency changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of mucopolysaccharidosis in rats by treatment with immunostimulatory acridine derivatives.

In the accompanying paper, four dibasic acridine derivatives were reported to induce lysosomal storage of sulfated glycosaminoglycans (sGAG), i.e., mucopolysaccharidosis, in cultured fibroblast (Handrock et al. Toxicol. Appl. Pharmacol. 114, 1992). The purpose of the present morphological and biochemical investigation was to examine whether two representatives of the acridine derivatives, namely 3,6-bis[2-(diethylamino)ethoxy]-acridine and the piperidino analogue, induce mucopolysaccharidosis in intact organisms. Rats were orally treated with 60-80 mg/kg up to 22 weeks. Morphological examination of liver, spleen, and blood lymphocytes yielded cytochemical evidence of mucopolysaccharidosis. Biochemically, up to a 48-fold increase of the urinary excretion of sGAG was found. In the liver and spleen of chronically treated rats, the sGAG contents were elevated by factors up to 56 and 23, respectively. Heparan sulfate and dermatan sulfate contributed most to the total increase of sGAG; chondroitin sulfate was stored to a minor degree. For one compound, the tissue concentrations were determined. It was found that the drug was accumulated in the tissues. Due to their fluorescent properties, the drugs could be detected by fluorescence microscopy to be present in high concentrations within the sGAG-storing lysosomes. On the basis of these observations and of the biochemical data it appears justified to assume a ratio of at least one drug molecule per disaccharide unit of the sGAG to be present in the lysosomes. It is proposed that this leads to the formation of sGAG-drug complexes in the lysosomes. Such complexes may be indigestible substrates for the lysosomal enzymes, thus leading to mucopolysaccharidosis. For toxicologic practice, the cytochemical examination of lymphocytes is recommended as a simple measure for early detection and monitoring of this adverse drug effect.

The polycationic compound gentamicin inhibits the calcium paradox in guinea-pig hearts.

The polycationic drug gentamicin and two calcium antagonists were studied with respect to their protecting action against the calcium paradox in perfused guinea-pig hearts. Besides the mechanograms the release of creatine kinase was recorded; in parallel experiments the Na(+)-content of the hearts was measured before and at the end of the Ca(2+)-lack period, and during re-exposure to normal [Ca2+]0. The calcium paradox was induced by perfusion, for 50 s, with Ca(2+)-free solution containing EGTA (3 x 10(-4) M). Nifedipine and verapamil in concentrations which reduced the equilibrium contractile force by 50%, only mitigated the extent of the calcium paradox, whereas gentamicin applied in a concentration also reducing the contractile amplitude by 50% was able to suppress the calcium paradox completely. The dose-response curves for nifedipine, with respect to the reduction of contractile force and contracture, were identical. In contrast, gentamicin was more effective in attenuating the contracture of the paradox than in reducing the equilibrium contractile force. The large gain of Na+ during the Ca(2+)-lack period was diminished by both nifedipine and gentamicin. The partial protection of calcium antagonists can be related to their interference with the uptake of Na+ through L-type Ca(2+)-channels during the Ca(2+)-lack period, whereas gentamicin seems to act by an additional inhibition of the Na/Ca exchange during the re-exposure to normal [Ca2+]0.

C14-aminosteroid LNF 209, an agent with positive inotropic and antimuscarinic activity.

LNF 209 is a cardioactive steroid containing at position C14 a protonized amino group. It was tested whether LNF 209 has antimuscarinic properties besides its cardiotonic effect, which was also quantified. In guinea pig left atria (3 Hz, 0.9 mM Ca2+, 5.4 mM K+), LNF 209 increased the force of contraction at concentrations > or = 1 microM, thus having a tenfold lower potency than ouabain. Correspondingly, cardiac glycoside binding sites in guinea pig cardiac membranes labeled with [3H]ouabain were occupied by LNF 209 (Ki = 1.7 microM) at concentrations tenfold higher compared with ouabain (Kd = 0.14 microM). The negative inotropic effect of the muscarinic agonist oxotremorine was antagonized by LNF 209, but not by ouabain; in the Schild plot, the data for LNF 209 could be connected by a line with a slope of unity and a pA2 = 7.5. The binding of [3H]N-methylscopolamine ([3H]NMS) to the M2-cholinoceptors in guinea pig cardiac membranes was inhibited by LNF 209 with a Ki = 0.5 microM; LNF reduced the affinity of [3H]NMS binding without affecting the number of receptor sites. In isolated segments of guinea pig ileum, LNF 209 antagonized the effect of oxotremorine with a pA2 = 7.4. It is concluded that LNF 209 is a competitive antagonist at M-cholinoceptors without preference for the cardiac M2-cholinoceptors. Whether the antimuscarinic property is of significance in species highly sensitive to digitalis remains to be established.

Perpetuation of misinterpretations due to lack of methodical insight. A critical re-evaluation of the determination of 45Ca release from intact guinea-pig atria.

1. The evaluation of still more pretentious and complicated methods is accompanied by a decline of methodical knowledge outside of the own technical field. Interpretations or extrapolations are taken as granted without critical examination of the methodical steps applied. An example is given by re-evaluating the 45Ca release from isolated cardiac tissue and the possible interpretations. 2. 45Ca release and tissue Ca content were measured in isolated guinea-pig left atria during Ca equilibrium and under conditions known to induce net Ca movements. 3. At equilibrium condition (1.8 mM Na2+0) 3 exponential phase of 45Ca release from the atria were observed. The compartments contained 61%, 29% and 10% of total 45Ca; the t1/2 were 2, 12 and 90 min, respectively. 4. The release of 45Ca from the slowly exchanging compartment (t1/2 90 min) decreased during incubation in nominal Ca-free solution, although a net loss of tissue Ca occurred. Addition of EGTA (5 x 10(-5) M) to the washout medium abolished this retardation of 45Ca release. 5. At external Na+ concentrations below 40 mM (substituted by sucrose), the 45Ca release from the slowly exchanging compartment decreased. Simultaneously, the tissue Ca content increased massively. The 45Ca release was further reduced in Na-poor, nominal Ca-free solution. Under both conditions, the presence of EGTA in the washout medium normalized the rate of 45Ca release. 6. The results suggest that the apparent decline of 45Ca release from intact atria upon reduction of the external Ca and Na concentration does not reflect a decrease of the cellular efflux rate, but is the consequence of an enhanced re-uptake of 45Ca from the extracellular space into the myocardial cells. The probability for the released 45Ca either to escape into the organ bath or to become reabsorbed depends on the specific radioactivity of 45Ca in the extracellular space during the washout phase. Thus, this experimental procedure is not suited to demonstrate a Na-Ca exchange at the cardiac sarcolemma.

A model to check the arrhythmogenic activity of antiarrhythmic drugs.

1. In order to evaluate a condition which allows to check arrhythmogenic properties of antiarrhythmic drugs first experiences with the following model are presented. 2. Papillary muscles of guinea pig hearts were exposed to subthreshold concentration of aconitine (8 x 10(-8) M) for 30 min. 3. During the last 10 min of this period a lack of oxygen and glucose was additionally imposed on the muscle. 4. After this priming period, reoxygenation with glucose containing Tyrode solution was introduced and aconitine removed from the organ bath. 5. Restoring the normal conditions, the contractile force and the membrane potential completely recovered. Also the amplitude of the action pontial regained the control value. 6. Only the action potential duration displayed a prolongation of the late repolarization phase which can be described as a hump at a potential of about -55 mV. 7. 30 Minutes after the end of the priming period the muscles were exposed to antiarrhythmic drugs. 8. Quinidine, propafenone and gallopamil (at concentrations reducing the contractile force by 50%) induced a further prolongation of the action potential duration and provoked early after-depolarizations thus acting arrhythmogenic. 9. In contrast, lidocaine and phenytoin did not prolong the action potential duration under this condition and were devoid of any arrhythmogenic activity. 10. The proposed model might offer a possibility to differentiate antiarrhythmic drugs with respect to their unwanted arrhythmogenic activity.

Changes of isolated cardiac muscle function in response to extracellular sodium reduction.

The effects of low extracellular sodium concentration [( Na+]0, 76 mmol/l) on force of contraction, transmembrane action potentials and on calcium, sodium and potassium contents were studied in guinea-pig heart muscle using sucrose or lithium as substitutes for sodium. In papillary muscle, the positive inotropic response to low [Na+]0 was accompanied by shortening in action potential duration, in atrial muscle, prolongation at 90% of repolarization was observed. After 30 min. in low [Na+]0 solution (Ca2+ 1.8 mmol/l), the net uptake of calcium in left atria was larger in sucrose- than in lithium-substituted solution, i.e. 0.45 and 0.2 mmol Ca2+/kg wet weight, respectively. The net sodium content decreased monophasically; but the potassium content did not change consistently. The uptake of lithium by the atria was at least partially compensated for by a loss in potassium and by a transient decrease in sodium content. Increase in stimulation frequency from 0.1 to 1 Hz accelerated the time course of change by a factor of 2. In low [Na+]0, post-rest adaptation of twitch tension appeared abolished in atria because of high amplitude contractions. The recovery pattern of post-rest twitch amplitude was greatly accelerated in papillary muscle. In conclusion, low [Na+]0 elevates the cellular calcium content; this extra calcium is probably located in cellular stores that are involved in the regulation of twitch amplitude.

The binding of (+)-isradipine by guinea-pig intact atria.

1. The accumulation of [3H]-(+)-isradipine (PN 200-110) was measured in quiescent guinea-pig left atria with normal (K+ 2.7 mM) or lowered (K+ 40 mM) membrane potential. 2. Under control conditions (2.7 mM K+) a high affinity binding of (+)-isradipine could not be detected. If, however, the atria were partially depolarized to about -30 mV by 40 mM K+, high affinity binding became evident displaying a dissociation constant of 4.2 x 10(-11) M and a capacity of 9.7 nmol kg-1 wet wt. 3. The depolarization-induced binding was reversible upon repolarization of the atria although isradipine was still present in the medium. This indicates that the high affinity binding sites disappear as soon as the cell membranes become polarized. 4. Isradipine belongs to the less hydrophobic dihydropyridines, but nevertheless the unsaturable binding led to an accumulation of about 84 fold. At a concentration of 2 x 10(-8) M (+)-isradipine, which reduces the contractile force by 50%, the cellular concentration will rise to more than 10(-6) M.

The dihydropyridine derivative 202-791: interpretation of the effects of the racemate considering inverse agonistic enantiomers.

1. The influence of the enantiomers and the racemate of the dihydropyridine derivative 202-791 [isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3- pyridinecarboxylate] on force of contraction and action potential was studied in guinea-pig isolated papillary muscles. The effects were investigated during regular stimulation (1 Hz) and after a period of rest (10 min). 2. The enantiomers of the dihydropyridine derivative 202-791 had opposite effects on the mechanical and bioelectrical parameters: the (+,S)-enantiomer enhanced contractility and prolonged action potential duration whereas the (-,R)-enantiomer reduced force and shortened action potential duration. Analogous to the effects during regular stimulation, the post-rest adaptation was modified adversely: in the presence of the (+,S)-enantiomer the pattern of adaptation was intensified while the (-,R)-enantiomer caused an attenuation. The term 'inverse agonism' seems more suitable than the commonly used comparison of agonist and antagonist, because each enantiomer possesses intrinsic activity, albeit in opposite directions. 3. The racemate of 202-791 acted like the (+,S)-enantiomer. In concentrations up to 1 microM, the racemate increased the force of contraction to the same extent as if the cardiodepressant (-,R)-enantiomer was not present. Only at the highest concentration (3 microM) did the counteracting effect of the (-,R)-enantiomer become evident. The racemate prolonged the action potential duration like the (+,S)-enantiomer although to a lesser extent. Moreover, the typical post-rest adaptation of contractile force and action potential duration was accentuated by the racemate as with the (+,S)-enantiomer. 4. The results demonstrate that in case of 202-791, the effects of the racemate do not reflect the opposite actions of the two enantiomers, but rather mimic that of the (+,S)-enantiomer. A prediction concerning the effects of the enantiomers which is based on findings obtained with the racemate is not possible.

Release of N-[3H]methylscopolamine from isolated guinea pig atria is controlled by diffusion and rebinding.

This study attempts to analyze the inter-relationship between the slow fading of muscarinic antagonist action observed in isolated tissue preparations upon washout and the dissociation kinetics at the receptor level. Isolated guinea pig left atria stimulated electrically at 3 Hz were equilibrated with N-[3H]methylscopolamine ([3H]NMS) and the time course of release of [3H]NMS was studied. In parallel experiments, the fading of the antimuscarinic action of NMS was monitored by repeated determinations of the negative inotropic effect of oxotremorine. In comparison, the dissociation of [3H]NMS from muscarinic receptors was recorded in a membrane suspension of guinea pig right atria. Having been equilibrated at 10(-8) M [3H]NMS, the atria released [3H]NMS extremely slowly, when transferred into a drug-free washout bath: not even half of the initially bound [3H]NMS was lost within 3 hr. With a corresponding time course, 10(-8) M oxotremorine regained its negative inotropic action. In contrast, when 10(-4) M unlabeled NMS was present in the washout bath, it took only 5 min for the [3H]NMS binding to fall by 50%. Similarly, in a membrane-suspension of guinea pig right atria, the half-life time of the [3H]NMS receptor complexes amounted to about 5 min. It is concluded that the dissociation of NMS from its receptor sites proceeds with a half-life time of 5 min in intact atria as well as in the cardiac membrane suspension.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake and displacement of [3H]-propranolol and [3H]-chlorpromazine in isolated, recirculating perfused guinea-pig lungs.

The pulmonary uptake of tritium labelled propranolol and chlorpromazine and their displacement by amphiphilic drugs has been studied in isolated guinea-pig lungs. Lungs were perfused by recirculation with 60 ml tyrode solution (carbogen gassed, 37 degrees C, 6% hydroxy-ethyl-starch) at a flow rate of 10 ml/min. In uptake experiments, a steady state was reached within 20 min with 95% of 10(-9) M propranolol and 90% of 10(-9) M chlorpromazine removed from the perfusate. Using 10(-4) M propranolol, a saturation process became evident, whereas no concentration dependency was observed for chlorpromazine uptake. Kinetic analysis revealed similar uptake rates, but different capacities for both compounds. In displacement experiments, 10(-9) M propranolol was displaced by 10(-4) M amphiphilic drugs in the order: chlorpromazine greater than propranolol greater than alprenolol greater than tetracaine. No displacement occurred by practolol, indomethacin or phenylbutazone. The results indicate that the lungs have a large binding capacity for amphiphilic drugs. These compounds can interfere with each other, according to their lipophilicity, their steric configuration and charges.

Allosteric stabilization of 3H-N-methylscopolamine binding in guinea-pig myocardium by an antidote against organophosphate intoxication.

W84 (hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)-ammonium bromide]) protects overadditively against an organophosphate-intoxication when applied in combination with atropine. Further experimental evidence led to the hypothesis that W84 exerted an allosteric effect on muscarinic acetylcholine receptors. In order to investigate the action of W84 on the receptor level, binding studies with 3H-N-methylscopolamine were performed in homogenized and intact guinea-pig myocardium. For sake of comparison three bispyridinium oximes were included, i.e. Uno3 (trimethylene-bis-[4-hydroxyiminomethyl-pyridinium] dibromide mono-2,6-dichlorobenzylether), obidoxime, and TMB4. In cardiac membrane suspensions, all compounds inhibited 3H-NMS-binding after 2 hrs of incubation concentration-dependently by reducing its affinity, whereas leaving the number of binding sites unaltered. However, with increasing concentrations W84 suppressed 3H-NMS-binding less than expected for a competitive antagonist. Kinetic studies revealed that W84 did not only slow the association of 3H-NMS, but additionally retarded its dissociation over the entire range of concentrations that inhibited 3H-NMS-binding. At lmM, W84 augmented the half life time of the 3H-NMS-receptor complexes from a control value of 4 min to more than 120 min. The stabilization of the radioligand-receptor complexes is indicative of an allosteric effect of W84. Obidoxime, TMB4 and Uno3 at low concentrations acted like competitive inhibitors of 3H-NMS-binding. From 10(-5)M onwards, Uno3 retarded 3H-NMS-dissociation concentration-dependently. It is concluded that the effect of bisquaternary compounds on 3H-NMS-association and -dissociation is mediated via binding to two separate sites, i.e. the muscarinic receptor site and an allosteric effector site, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Interpretation of [3H]ouabain binding in guinea-pig ventricular myocardium in relation to sodium pump activity.

1. The attempt was made to analyse the complex [3H]ouabain binding curves obtained in intact cardiac ventricular preparations electrically stimulated at different frequencies. The result of this analysis was used to draw conclusions from the binding curves on the frequency dependence of sodium pump activity. 2. [3H]Ouabain binding to isolated, electrically stimulated (1.5 Hz) ventricular strips of guinea-pig hearts was investigated. The positive inotropic effects were studied in separate experiments. Specific [3H]ouabain binding barely reached an equilibrium within 3 h of incubation. A binding curve was constructed using the equilibrium values of specific [3H]ouabain binding obtained at different ouabain concentrations. This binding curve revealed a concentration-proportional component at positive inotropic concentrations and a saturating component at high, toxic concentrations. At very low, inotropically ineffective ouabain concentrations, however, binding values were higher than expected from a linear relationship between ouabain concentration and binding. 3. The peculiar shape of the binding curve could be largely accounted for by a mathematical model, which takes into consideration biochemical properties and physiological regulation of the sodium pump. The model predicts a concentration-proportional pattern of binding which takes place in the non-toxic ouabain concentration range. The slope of the concentration-proportional component of the binding curve should represent a measure of sodium pump activity. 4. Investigation of binding curves at various stimulation frequencies revealed that, as predicted by the model, the slope of the concentration-proportional component of the binding curves was increased and the maximum non-toxic equilibrium binding was decreased with increasing beat frequencies. 5. Quantitative evaluation of the binding curves led to the conclusion that sodium pump activity is a linear function of stimulation frequency in guinea-pig ventricular preparations, the activity in resting preparations amounting to about 15% of the maximum activity. Comparison of the present results with former studies on sodium pump function suggests that [3H]ouabain binding reflects steady-state sodium pump activity. If the complex pattern of binding curves is taken into consideration, [3H]ouabain binding measurements may serve as a means of studying sodium pump function.

Role of calcium in the activation of smooth muscle.

Smooth muscle depends to a large extent on the extracellular calcium concentration for activation of its contractile proteins. Calcium influx via voltage-dependent or receptor-operated channels is thought to supply the cell with Ca2+ for contractile activation. If this is so, the inhibition of transmembranal calcium influx will prevent mechanical activation. A blockade of transmembranal calcium influx has been proposed as a mode of action for calcium antagonists. This suggestion seems to be supported by experiments in which tissue was incubated in 45Ca-containing solutions for 2-3 min and thereafter transferred for 40-60 min to ice-cold calcium-free or lanthanum-containing solutions. The amount of remaining 45Ca in the tissue at the end of the wash-out phase is taken as a measure of calcium influx during the labelling period. However, procedures involving rather long-lasting wash-out periods cannot yield information on Ca2+ influx, since the calcium store relevant for contractile activation adapts rapidly to changes in extracellular Ca2+ concentration.

Acrihellin, a cardioactive steroid escaping from the organ-bath.

The concentration of acrihellin rapidly declines in oxygenated Tyrode-solution, because the compound escapes from the organ-bath being enriched in droplets sprayed from the surface of the bubbled solution. As checked by radiochromatography, acrihellin remains chemically unaltered during this process. Hellebrin and hellebrigenin persist in gassed Tyrode-solution, suggesting that the 3 beta-substituent dimethylacrylic acid endows acrihellin with amphiphilic properties, thus promoting its enrichment at gas-water interphases. Measurements of the inotropic effects in guinea pig left atria performed at concentrations of acrihellin kept constant yielded a dose-response curve, which closely resembles that of the conventional cardioactive steroid ouabain.

High and concentration-proportional accumulation of [3H]-nitrendipine by intact cardiac tissue.

The binding of [3H]-nitrendipine to intact, electrically driven isolated left atria of guinea-pigs was investigated over the concentration range 10(-10) M to 3 X 10(-5) M. A high affinity binding site saturable in the nM range as found in ventricular homogenates could not be detected. Instead the accumulation of nitrendipine in intact atria was found to be proportional to the concentration from 10(-10) M to 10(-6) M; beyond 10(-6) M the binding started to become saturated. Nitrendipine was highly accumulated in atrial tissue. The cell:medium ratios amounted to about 120 in the range from 10(-10) M to 10(-6) M. The concentration-response curve for the negative inotropic action of nitrendipine yielded an ED50 of 3 X 10(-7) M, thus lying within the range of concentration-proportional accumulation. The reduction of the contractile force proceeded faster (t1/2 less than 10 min) than the uptake process t1/2 approximately 40 min) suggesting that it is the binding of nitrendipine into a superficial compartment, which interferes with the excitation-contraction coupling. The results suggest that the high concentration of nitrendipine present in hydrophobic cellular compartments such as the plasmalemma might be involved in its pharmacological action.

Calcium, cell membrane, and excitation-contraction coupling.

Skeletal muscle, heart muscle, and smooth muscle differ with respect to morphology, function, and Ca metabolism. For the actual coupling between excitation and contraction, three different sources of Ca2+ supply are conceivable: release from intracellular organelles, release from plasmalemmal binding sites, and transmembranous influx. Considerable experimental evidence favors the membranes of the sarcoplasmic reticulum as the source of coupling Ca2+ in skeletal muscle, whereas in cardiac and smooth muscles the Ca store from which coupling Ca2+ is released has to be in intimate contact with the extracellular Ca2+ concentration. In order to distinguish between the two remaining possibilities for the source of coupling Ca2+, i.e., transmembranous Ca influx and Ca release from the plasmalemma, Ca antagonist--widely accepted tools for detecting Ca influx--were used. Ca antagonists did not have any influence on Ca exchange or Ca uptake either in cardiac or in smooth muscles. These findings may be interpreted as follows: Ca antagonists do not act by inhibiting the Ca2+ influx, although they reduce the amount of coupling Ca2+. This conclusion conflicts with current ideas concerning the mode of action of Ca antagonists. We feel, however, that the experimental support for the Ca-channel hypothesis is rather vague. This leads us to favor the plasmalemma as the main source of coupling Ca2+ in cardiac and smooth muscles.