Comparative aspects and temperature dependence of [3H]1,4-dihydropyridine Ca2+ channel antagonist and activator binding to neuronal and muscle membranes

Binding of [3H]nitrendipine, [3H]nimodipine, and (+)[3H]PN 200-110 to microsomal preparations of guinea pig smooth and cardiac muscle and brain synaptosomes revealed high affinity interaction with KD values in the sequence, (+)PN 200-110 greater than nitrendipine greater than nimodipine. Bmax values for a particular tissue were independent of the 1,4-dihydropyridine employed in radioligand binding at 25 degrees C. The temperature dependence of [3H]nitrendipine binding in cardiac and smooth muscle microsomal preparations and brain synaptosomes was measured from 0 degrees to 37 degrees C and for skeletal muscle preparations from 0 degrees to 30 degrees C. Bmax values increased with temperature for cardiac membranes, but did not vary in other tissues. van't Hoff plots were nonlinear in all tissues, enthalpy and entropy changes becoming increasingly negative with increasing temperature. Competition binding of the activator-antagonist enantiomeric 1,4-dihydropyridine pairs of Bay k 8644 and PN 202-791 for [3H]nitrendipine in smooth muscle did not reveal significant thermodynamic differences between activator and antagonist molecules.     

Specific high affinity binding of the calcium channel antagonist, [3H]nitrendipine, to rat liver microsomes

Some key properties of the binding of [3H]nitrendipine, an analogue of the 1,4-dihydropyridine, nifedipine, to a plasma membrane enriched microsomal fraction from the rat liver are described. Specific binding was saturable, linear with protein concentration, and reversible. The apparent equilibrium dissociation constant, KD, was 4.20 +/- 0.22 nM and the maximum density of binding, Bmax, was 3.02 +/- 0.17 pmol/mg of protein determined from Scatchard analysis of binding at 10 degrees C. Inhibition of binding was specific for dihydropyridines with competitive inhibition being noted with nifedipine and 4-chloronifedipine, as well as BAY K-8644, a calcium channel agonist. A biphasic displacement curve was recorded for methoxy verapamil (D-600), and a triphasic competition curve with lanthanum (La3+), and diltiazem demonstrated competitive kinetics. The high affinity binding site for nitrendipine in the liver, although having some similar properties to those sites described in skeletal muscle, would appear to be distinctive with respect to its unique sensitivity to D-600 and diltiazem. We speculate that this binding site may represent a Ca2+ channel responsible for regulating Ca2+ influx and hepatic glycogenolysis.     

Inhibition of [3H]nitrendipine binding by phospholipase A2

Phospholipase A2 from several sources inhibited [3H]nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC50 values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A2 was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A2 enzymatic activity, shifted the bee venom phospholipase A2 dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A2 (10 ng/ml) for 15 min caused a 2-fold increase in the Kd without changing the Bmax compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the Kd but significantly decreased the Bmax to 71% the value for untreated membranes. [3H]Nitrendipine, preincubated with bee venom phospholipase A2, was recovered and found to be fully active, indicating that the phospholipase A2 did not modify the ligand. It is concluded that phospholipase A2 acts on the membrane at or near the [3H]nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site.     

3H]nitrendipine receptors in skeletal muscle

The richest source of receptors for the organic calcium channel blocker [3H]nitrendipine in muscle is the transverse tubule membrane. The tubular membrane preparation binds [3H]nitrendipine with a high affinity and has a very high number of [3H]nitrendipine binding sites. For example, for the transverse tubule membrane preparation from rabbit muscle, the dissociation constant of the nitrendipine-receptor complex is 1.8 +/- 0.3 nM and the maximum binding capacity Bmax = 50 +/- 6 pmol/mg of protein. Similar results have been found with a membrane preparation from frog muscle. The dissociation constant found at equilibrium is near that determined from the ratio of rate constants for association (kappa 1) and dissociation (kappa-1). Binding of [3H] nitrendipine is pH-dependent and reveals the presence of an essential ionizable group with a pK of 5.4 on the nitrendipine receptor. The binding is destroyed by proteases showing that the receptor is a protein. Three different classes of Ca2+ channel blockers inhibit [3H]nitrendipine to its specific site. (i) The dihydropyridine analogs of nitrendipine which are competitive inhibitors of [3H]nitrendipine. These molecules form tight complexes with the nitrendipine receptor with dissociation constants between 1.4 and 4.0 nM. (ii) Other antiarrhythmic molecules like verapamil, amiodarone, bepridil, and F13004 which are noncompetitive inhibitors of [3H]nitrendipine binding with dissociation constants between 0.2 and 1 microM. (iii) Divalent cations like Ni2+, Co2+, Mn2+, or Ca2+ which are noncompetitive inhibitors of [3H]nitrendipine binding with the following rank order of potency: Ni+ (K0.5 = 1.8 mM) greater than Co2+ (K0.5 = 2.7 mM) greater than Mn2+ (K0.5 = 4.8 mM) greater than Ca2+ (K0.5 = 65 mM).     

Differentiation of receptor sites for [3H]nitrendipine in chick hearts and physiological relation to the slow Ca2+ channel and to excitation-contraction coupling

The properties of interaction of the Ca2+ channel antagonist [3H]nitrendipine have been investigated in chick hearts at various stages of in ovo and post-natal development and in cultured cells. The dissociation constant of the [3H]nitrendipine-receptor complex is between 0.4 nM and 0.5 nM for intact ventricle and cultured cells. [3H]Nitrendipine binding is antagonized by nitrendipine analogs. The order of efficacy of the different dihydropyridine molecules is nitrendipine greater than nimodipine greater than nifedipine greater than nisoldipine with Kd values ranging from 0.5 to 4 nM. Inhibition of [3H]nitrendipine binding by other antiarrhythmic molecules like amiodarone, F13004 and bepridil was observed. Half-maximum inhibitions (K0.5) were found for verapamil and D600 at concentrations between 0.23 and 0.26 microM. The potency of organic Ca2+ blockers to depress by 50% the maximum amplitude of spontaneous beating of heart cells is closely related to K0.5 values obtained from [3H]nitrendipine binding experiments. Electrophysiological results indicate that the slow channel is insensitive to nitrendipine at the younger stage of development (3-day-old) whereas, in adult like cells, nitrendipine (50 nM) abolished both slow action potential due to the slow Ca2+ channel and contraction. The maximum binding capacity for [3H]nitrendipine is found to increase during development of the embryonic heart from 40 fmol/mg protein at day 3 to 100 fmol/mg protein at day 14, to stay relatively stable until day 18. Then the number of sites increases rapidly to reach a second plateau at 210 fmol/mg protein on day 4 after hatching. Treatment with 6-hydroxydopamine results in 35% increase in [3H]nitrendipine binding, whereas reserpine treatment is without effect. Developmental properties of nitrendipine-sensitive Ca2+ channels have been compared with those of tetrodotoxin-sensitive Na+ channels and muscarinic receptors. These results indicate that nitrendipine receptors exist at the early stage of development (3-day-old-hearts) but that they do not correspond to functional slow Ca2+ channels, that in ovo development corresponds both to an increase of the number of [3H]nitrendipine receptors and to the transformation of silent Ca2+ channels into functional Ca2+ channels, and that there is a regulation of the level nitrendipine-sensitive Ca2+ channels by innervation.     

Calcium channel antagonist binding and pharmacology in rat uterine smooth muscle

The calcium channel antagonists altered Ca-dependence of high K+-contractions of the estrogen dominant rat myometrium with the following pA2 values: PN-200-110, 10.63; nitrendipine, 9.56; nifedipine, 9.41; D-600, 9.05; and diltiazem, 7.57. Specific binding of 3H-nitrendipine occurred to the isolated plasma membrane vesicles with Kd of 0.1 to 0.3 nM and was inhibited by PN-200-110, nitrendipine, nifedipine and D-600, and slightly activated by diltiazem. The binding studies and the contractility studies were in excellent agreement for the three dihydropyridines, but not for D-600 and diltiazem.     

Characterization of alpha-adrenoceptor subtypes by [3H]prazosin and [3H]rauwolscine binding to canine venous smooth muscle membranes

Postsynaptic alpha-adrenoceptor subtypes were studied using [3H]prazosin and [3H]rauwolscine binding to plasmalemma-enriched microsomal fractions isolated from dog saphenous veins and mesenteric veins. Both radioligands showed saturable binding consistent with the presence of a single homogeneous binding site in each case, based on Scatchard analysis. The Kd values of [3H]prazosin and [3H]rauwolscine, calculated from kinetic studies were similar to those from equilibrium binding data in both venous muscle membranes. The microsomal membranes of dog saphenous vein and mesenteric vein contained about a fourfold higher density of the high affinity [3H]rauwolscine binding sites than those for [3H]prazosin binding. In competition studies, IC50 values for displacement of rauwolscine or prazosin suggested that the sites of interaction for the antagonists prazosin and rauwolscine were independent. Phenylephrine, a functionally selective alpha-adrenoceptor agonist, competed with a similar IC50 value for the specific binding sites of [3H]prazosin and [3H]rauwolscine; but B-HT 920, a functionally selective alpha 2-adrenoceptor agonist, competed for [3H]rauwolscine and [3H]prazosin binding with distinctly different IC50 values. Our data show the existence of two populations of alpha-adrenoceptor antagonist binding sites in the plasma membranes of dog saphenous vein and mesenteric vein, and raise the question whether agonist selectively depends on different affinities or on differential efficacies at one or two sites.     

The distribution of [3H]kainate binding sites in primate hippocampus is similar to the distribution of both Ca2+-sensitive and Ca2+-insensitive [3H]kainate binding sites in rat hippocampus

The distribution of [³H]kainate binding sites was determined by quantitative autoradiography in three vertebrate species: rat, monkey, and human. These animals displayed a similar pattern of binding site density in the hippocampus. Highest levels were found within the stratum lucidum and moderate levels in the inner portion of the dentate gyrus molecular layer. Although the distribution is similar, there is a lower density of binding sites in the stratum lucidum of primates than in rodents. Experiments using rat brain synaptic plasma membrane fractions indicated that inclusion of Ca²⁺ ions results in a selective reduction in binding at the high affinity sites. The Ca²⁺-inhibited and Ca²⁺-insensitive binding sites in the rat hippocampus exhibited a similar distribution. Together, these results suggest that in a variety of mammalian species kainate receptors exhibit similar regional distributions, and that the high anf loe affinity kainate binding sites also exhibit similar regional distributions.Special Issue dedicated to Prof. Eduardo De Robertis.     

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.     

Decreased [3H]nitrendipine binding in the brainstem of deoxycorticosterone-NaCl hypertensive rats

Binding studies with the 1,4-dihydropyridine calcium channel antagonist [3H]nitrendipine [( 3H]NTD) were performed in uninephrectomized, deoxycorticosterone (DOCA)-NaCl hypertensive rats and vehicle treated normotensive control littermates. After 6 weeks of treatment, hypertensive (199 mmHg, systolic arterial pressure) DOCA rats showed significantly increased heart, left ventricle, and kidney weight in contrast to normotensive (135 mmHg) controls. [3H]NTD binding in the brainstem was significantly reduced (51 +/- 5 fmol/mg protein) in DOCA-NaCl rats, as compared to controls (116 +/- 24 fmol/mg protein). However, no significant differences were found in the [3H]NTD dissociation constants for DOCA-NaCl (0.43 +/- 0.03 nM) or control rats (0.62 +/- 0.06 nM). Cerebral cortical and left ventricular tissue showed no significant alterations in receptor binding density or affinity. Specific [3H]NTD binding was not significantly altered in other selected brain regions or the atria. These data suggest that alterations in the dihydropyridine binding sites associated with calcium channels in the brainstem may be involved in the etiology of DOCA-NaCl-induced hypertension.     

Diltiazem enhancement of [3H]nitrendipine binding to calcium channel associated drug receptor sites in rat brain synaptosomes

The binding of the dihydropyridine calcium channel antagonist [³H]nitrendipine to whole rat brain synaptosomes was studied. Binding was specific, saturable, and of high affinity (K_d = 170 pM). The calcium channel antagonist diltiazem enhanced [³H]nitrendipine binding in synaptosomes in concentrations of 1 and 10 μM. Equilibrium saturation analysis demonstrated that this effect was mediated by a decrease in the dissociation constant, due to a 3-fold reduction in the rate of ligand-receptor complex dissociation. It is concluded that diltiazem allosterically modulates the calcium channel drug receptor labeled by [³H]nitrendipine in this preparation.     

Ca2+ microdomains in smooth muscle

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store. A single Ca(2+) channel can create a microdomain of several micromolar near (approximately 200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca(2+)] and the rapid rates of decline target Ca(2+) signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca(2+) by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca(2+). In this review, the generation of microdomains arising from Ca(2+) influx across the plasma membrane and the release of the ion from the SR Ca(2+) store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

Ca2(+)-sensitive K+ channel in aortic smooth muscle of rats

We measured K+ channel activity in inside-out patches of cell membrane from aortic vascular smooth muscle cultured (Passages 1-3) from Wistar, Wistar-Kyoto, and spontaneously hypertensive rats (SHR). With [Ca2+]i between 25 and 100 nm and 150 mm K+ on both sides of the membrane, the conductance of this channel was 55 +/- 7 pS (slope of current-voltage curve through 0 mV) and the current was outwardly rectified. There was no difference in single-channel conductance among the three rat strains. Increasing negative holding voltages or increasing [Ca2+]i, increased the probability of this type channel being open (Npo; P less than 0.01); SHR had a larger NPo (P less than 0.01). Compared with cells from Wistar and Wistar-Kyoto, cells from SHR also had the longest mean open time. The increased NPo and mean open time we observed in this K+ channel of cells from SHR could contribute, at least in part, to the increased membrane K+ permeability, reported previously.     

Ca2+ channel ligand activities in uterine smooth muscle: influence of hormonal status

The effects of estrogen and progesterone domination, achieved by administering estrogen (E) and estrogen plus progesterone (E + P), on rat uterine reactivity to Ca2+ and to Ca2+ channel ligands (antagonist and activator) were compared. The inhibitory activities of nifedipine, diltiazem, and D 600 against K+ depolarization-induced responses were not significantly different between E- and E + P-dominated states in longitudinal or circular muscle preparations. Tonic responses were significantly more sensitive than phasic responses, but the rank orders of activity for a series of 14 antagonists were identical, suggesting the existence of a common structure-activity relationship which paralleled that seen previously in other smooth muscles. E + P-dominated uteri were slightly more sensitive to Ca2+ responses in K+ depolarizing media, but pA2 values for nifedipine, diltiazem, and D 600 inhibition were not significantly different in tissues from animals in either hormone-dominated state. Binding of [3H]nitrendipine did not differ between hormonal states. Responses to Bay K 8644 were larger in E + P-dominated uteri but the binding density was twofold greater in the E-dominated uterus. This study suggests that pathways of Ca2+ mobilization through potential-dependent Ca2+ channels in rat uterus are not significantly altered between E- and E + P-dominated environments.