Binding Ca2+ to intracellular or to extracellular sites of dihydropyridine receptor of rabbit skeletal muscle discriminates between in vitro binding of Ca2+-channel agonist and antagonist

Transverse tubule membrane vesicles contain dihydropyridine receptor of rabbit skeletal muscle in an insideout orientation. Digitonin-solubilized, purified dihydropyridine receptor is embedded in digitonin vesicles in an outside-out orientation. Ca2+ selectively stimulates binding of the Ca2+-channel antagonist [3H]PN200-110 to dihydropyridine receptor in the outside-out but not the inside-out orientation. The dissociation constant for binding Ca2+ to the extracellular Ca2+-specific binding site of dihydropyridine receptor is 2-3 microM. The data demonstrate that binding Ca2+ to the extracellular high-affinity Ca2+-binding site is required for binding dihydropyridines to dihydropyridine receptor. This binding is inhibited, however, by 1-10 mM concentrations of any divalent cation tested (Ba2+, Mn2+, Mg2+). Also, Ca2+ selectively stimulates binding of the Ca2+-channel agonist [3H]BayK8644 to dihydropyridine receptor in the inside-out orientation. The titration of this Ca2+ dependence indicates that the dissociation constant for binding Ca2+ to the intracellular Ca2+-specific binding site of dihydropyridine receptor is in the millimolar range. Thus, binding Ca2+-channel agonist or antagonist to dihydropyridine receptor is modulated by binding Ca2+ to different sites of the receptor. Measurements of dissociation rate constants for binding [3H]PN200-110 to dihydropyridine receptor in the presence of diltiazem, verapamil and/or Ca2+ indicate that Ca2+, like diltiazem or verapamil, is an allosteric effector of this receptor.     

Specific binding of tritium-labeled inositol 1,4,5-trisphosphate to human platelet membranes: ionic and GTP regulation.

Specific, saturable and reversible binding of tritium-labeled inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) to human platelet membranes is demonstrated. The Ins(1,4,5)P3-binding sites are abundant and display high selectivity for Ins(1,4,5)P3. Other inositol phosphates exhibit much lower affinity for this site. The specific [3H]Ins(1,4,5)P3 binding was found to be modulated by pH, monovalent and divalent cations, and GTP. A sharp increase in binding occurs at slightly alkaline pH. The monovalent cations, Na+, K+ and Li+ almost double the binding at 30 mM. Mg2+ inhibits the specific [3H]Ins(1,4,5)P3 binding. At low concentrations of Ca2+, the binding is inhibited, but at concentrations higher than 5 mM the binding is potentiated and increases by almost 5-fold at 100 mM. Similar pattern of the effects is also observed for Mn2+ and Sr2+. The specific [3H]Ins(1,4,5)P3 binding is specifically inhibited by GTP. Other nucleotides also inhibit the binding but at higher concentrations. From saturation binding studies, Ca2+ potentiation seems to be due to the conversion of the receptor from the low-affinity state to the high-affinity one. In the absence of Ca2+, the Scatchard plot is nonlinear and concave, and statistically can be fitted best with two equilibrium dissociation constants (Kd values), 0.19 +/- 0.11 and 13.2 +/- 18.1 nM, respectively, for high- and low-affinity binding sites. However, in the presence of 100 mM CaCl2, the Scatchard plot reveals only the high-affinity binding sites with a Kd value of 0.32 +/- 0.15 nM. The specific Ins(1,4,5)P3 receptor in human platelets could therefore exist in multiple conformational states to regulate the intracellular Ca2+ concentration.     

Characterization of the Ca2+ coordination site regulating binding of Ca2+ channel inhibitors d-cis-diltiazem, (+/-)bepridil and (-)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes

Ca2+ inhibits (-)[3H]desmethoxyverapamil, d-cis-[3H]diltiazem and (+/-)[3H]bepridil binding to skeletal muscle transverse-tubule membranes with a half-maximum inhibition constant, K0.5 = 5 +/- 1 microM. This value is close to that of the high affinity Ca2+ binding site which controls the ionic selectivity of the Ca2+ channel found in electrophysiological experiments suggesting that the Ca2+ coordination site which regulates the ionic selectivity is also the one which alters binding of the Ca2+ channel inhibitors investigated here. Ca2+ and (-)D888 bind to distinct sites. Occupation of the Ca2+ coordination site decreases the affinity of (-)D888 for its receptor by a factor of 5. Other divalent cations have the same type of inhibition behavior with the rank order of potency Ca2+ (K0.5 = 5 microM) greater than Sr2+ (K0.5 = 25 microM) greater than Ba2+ (K0.5 = 50 microM) greater than Mg2+ (K0.5 = 170 microM).     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

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).     

A novel 1,4-dihydropyridine-binding site on mitochondrial membranes from guinea-pig heart, liver and kidney.

The 1,4-dihydropyridine (+/-)-[3H]nitrendipine reversibly binds to mitochondrial preparations from guinea-pig heart with a dissociation constant (Kd) of 593 +/- 77 nM and a maximum density of binding sites (Bmax.) of 1.75 +/- 0.27 nmol/mg of protein. This low-affinity high-capacity 1,4-dihydropyridine-binding site does not discriminate between the enantiomers of nitrendipine and is also found in mitochondrial membranes from guinea-pig liver (Kd 586 +/- 91 nM; Bmax. 0.36 +/- 0.04 nmol/mg of protein) and kidney (Kd 657 +/- 149 nM; Bmax. 0.56 +/- 0.12 nmol/mg of protein). Phenylalkylamines (e.g. verapamil) inhibit ( +/- )-[3H]nitrendipine binding with micromolar inhibition constants, but the benzothiazepine D-cis-diltiazem, a potent Ca2+-channel blocker, is without effect. The binding is heat-stable, shows a V-shaped pH-dependence with a minimum around pH 7.0, and is strongly dependent on ionic strength in the incubation medium. The cations La3+ greater than Cd2+ much greater than Co2+ greater than Ca2+ much greater than Ba2+ greater than Mg2+ greater than Li+ greater than Na+ and the anions NO3- greater than C1- greater than or equal to F- stimulate the binding, whereas PO4(3-) greater than SO4(2-) slightly inhibit it. The low-affinity ( +/- )-[3H]nitrendipine-binding site located on the mitochondrial inner membrane is biochemically and pharmacologically different from the 1,4-dihydropyridine-receptor domain of the L-type Ca2+ channel. Furthermore, it is not identical with any of the low-affinity 1,4-dihydropyridine-binding sites described so far.     

Subcellular distribution and isolation of the Ca2+ antagonist receptor associated with the voltage regulated Ca2+ channel from rabbit heart muscle

The Ca²⁺ antagonist binding sites associated with the voltage dependent calcium channel in rabbit myocardium were found to distribute with the sarcolemmal Na^– + K⁺ ATPase and adenylate cyclase activities during subcellular fractionation on sucrose-density gradients. The equilibrium dissociation constants (K_D) for the binding of [³H]nitrendipine and [³H]verapamil were 0.31 ± 0.04 nM and 4.1 ± 0.5 nM respectively, and displayed an average density of 0.55 ± 0.05 pmol/mg and 0.4 ± 0.03 pmol/mg protein respectively for the most enriched membrane fraction. The Ca²⁺2 antagonist binding sites were solubilized from the membranes with the detergent 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and specific binding sites for [³H]PN200-110, [³H]verapamil and [³H]diltiazem were isolated on a wheat-germ lectin column. The binding sites for [³H]PN200-110 were enriched about 2500 fold as compared with the original homogenate and displayed a density of 28.5 ± 8 pmole/mg protein in the isolated fraction. Sodium dodecyl sulfate gel electrophoresis of the isolated drug binding proteins indicated enrichment of proteins of Mr 170000, 140000, 130000, 100 000 and 53000. The isolated receptor contained an intrinsic kinase activity that phosphorylated glycoproteins of Mr 170 000 and 53000. Exogenously added cAMP-kinase stimulated phosphorylation of the 170000, 100000, 53 000 and 28000 Mr glycoproteins in the receptor fraction. The results of this study indicate that the binding sites for [³H]nitrendipine, [³H]PN200-110, [³H]verapamil and [³H]diltiazem residue on glycoprotein(s) which are of sarcolemmal origin, and co-purify together on wheat germ lectin columns. The polypeptide composition of the Ca²⁺ antagonist binding sites from cardiac muscle appears to be very similar to that of the dihydropyridine receptor in skeletal muscle.Abbreviations CHAPS 3-[-(3-cholamidopropyl) dimethylammonio]-propanesulphonate - SDS sodium dodecyl sulphate Scholar of the Ontario Heart and Stroke Foundation.     

Interaction of alkali metal ions with Ca2+-binding center of Ca2+- ATPase of the sarcoplasmic reticulum in skeletal muscles

Sodium ion interaction with sarcoplasmic reticulum (SR) membranes leads to considerable alterations of the [23Na]NMR lineshape. Na+ binding to SR in the presence of Ca2+ and H+ is well described by a model which postulates a competitive ion binding to high and low affinity sites of Ca2+-ATPase. The dissociation constant, Kd, for high and low affinity sites is 5 and 10 mM, respectively, for Na+ and (3-5).10(-8) and 1.5.10(-3) M, respectively, for Ca2+. The pK value for high and low affinity sites is 7.3 and 6.1, respectively. Other alkaline metal ions compete with Na+ for the low affinity sites of Ca2+-ATPase; their affinities decrease in the following order: Na+ = K+ greater than Rb+ greater than Cs greater than Li+. Some of the Na+ binding sites (approximately 10%) do not interact with Ca2+.     

Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.     

The purified Ca2+ antagonist receptor from skeletal muscle: subunit structure, photoaffinity labeling and endogenous protein kinase activity

Tritiated analogues of the Ca2+ channel blockers such as [3H] PN200-110, [3H] verapamil and [3H] diltiazem have been used to identify and isolate Ca2+ antagonist receptors. The Ca2+ antagonist binding sites were solubilized from skeletal muscle transverse tubules with the detergent CHAPS and purified by wheat germ lectin column chromatography and sucrose density gradient centrifugation. The isolated proteins retained their ability to bind the various classes of Ca2+ channel blockers. Polypeptides of 170, 150, 108, 56, and 32 kDa were found to be present in the purified receptor fraction when analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weight of the 170 kDa polypeptide changed to 145 kDa in the presence of reducing agents, as where the apparent molecular weight of the 150, 108, 56 and 32 kDa peptides remained unchanged. An endogenous protein-kinase present in the original membranes, co-purified with the receptor and stimulated the phosphorylation of the 150 and 56 kDa polypeptides in the isolated fraction.     

Specific binding of the calcium antagonist [3H]verapamil to membrane fractions from plants

Specific binding of the Ca2+ channel blocker [3H] verapamil to a membrane fraction from plants has been characterized. Binding to zucchini membranes was saturable and reversible. The apparent equilibrium dissociation constant is KD = 102 nM and the maximum number of binding sites is Bmax = 60 pmol/mg of protein. The KD determined from the association and dissociation rate constants is 130 nM. [3H]Verapamil binding to zucchini membranes could not be inhibited by the Ca2+ antagonists nifedipine and diltiazem. However, [3H]verapamil could be displaced by diltiazem but not by nifedipine from corn membranes. Sucrose density fractionation of zucchini membrane preparations revealed that [3H]verapamil binding sites are located primarily at the plasma membrane.     

Comparison of the Effects of Ions and GTP on Substance P Binding to Membrane-Bound and Solubilized Specific Sites

Mg2+ increased but Na+ and GTP decrease [3H]substance P (SP) binding to rat cerebral cortical membranes and to 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized membrane fraction. To determine the binding parameters that are modified by the cations and GTP, inhibition experiments of [3H]SP binding by unlabeled SP were performed in both of the preparations. Nonlinear least-squares regression analysis of data in the membrane fraction indicated that optimal fitting of the inhibition curves in the presence of 10 mM MgCl2 was attained with a two-site model, corresponding to a "high-affinity (H)" and a "low-affinity (L)" state. By omitting MgCl2, or by addition of NaCl and GTP, the [3H]SP specific binding was decreased, the H state disappeared, and the L state and a new "super-low affinity (SL)" state observed. The SP/[3H]SP inhibition curves in the cerebral cortical membranes by in vivo treatment with pertussis toxin (islet-activating protein) were similar to that in the presence of GTP in control membranes. The effects of MgCl2, NaCl, and GTP were greater in the CHAPS-solubilized fraction than in the membrane fraction. In contrast to the membrane fraction, the inhibition curves of [3H]SP binding by unlabeled SP in the presence of MgCl2 in the CHAPS-solubilized fraction were best fitted to a one-site model. The KD value was relatively close to that of the low-affinity state in the membrane fraction. Even with the addition of NaCl or GTP, or by reducing MgCl2 concentration to 1 mM, although the inhibition curves consistently fit the one-site model, the KD values changed only slightly.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Human red-blood-cell Ca2+-antagonist binding sites. Evidence for an unusual receptor coupled to the nucleoside transporter

The human red blood cell ghost Ca2+-antagonist binding sites were characterized with (+/-)-[3H]nimodipine. The labelled 1,4-dihydropyridine bound in a non-cooperative, reversible manner with a Kd of 52 nM at 25 degrees C to 9.65 pmol sites/mg ghost protein. The stereochemistry of the binding domain was evaluated with the optically pure enantiomers of chiral 1,4-dihydropyridines. In contrast to the 1,4-dihydropyridine-selective receptors on Ca2+ channels in electrically excitable tissues, the (+) enantiomer of nimodipine and the (-) enantiomer of the benzoxadiazol 1,4-dihydropyridine (PN 200-110) were bound with higher affinity than the respective optical antipodes. The human red blood cell ghost [3H]nimodipine-labelled sites also interacted with the inorganic Ca2+-antagonist La3+ (increase in the number of binding sites), and were allosterically regulated by the optical enantiomers of the phenylalkylamine-type Ca2+-antagonists (e.g. verapamil, desmethoxyverapamil, methoxyverapamil). The benzothiazepines d- or l-cis-diltiazem were without effect. Nucleosides (adenosine approximately equal to inosine greater than cytidine) were inhibitory at the nimodipine-labelled site, as were the nucleoside uptake inhibitors dipyridamole, hexobendine, dilazep, nitrobenzylthioinosine and nitrobenzylthioguanosine. The binding sites have essential sulfhydryl groups, show trypsin sensitivity, but are relatively heat stable. When nitrobenzylthioinosine was employed as a covalent probe to inactivate the red blood cell ghost nucleoside carrier, [3H]nimodipine binding was irreversibly lost. (+)-Nimodipine greater than (-)-nimodipine inhibited [14C]adenosine transport into human red blood cells. A good correlation between IC50 values for inhibition of [3H]nimodipine binding and IC50 values for inhibition of [14C]adenosine uptake was found for 18 compounds. Sheep red blood cells (which lack the nucleoside transporter) had no detectable [3H]nimodipine binding sites. It is concluded that the Ca2+-antagonist receptor sites of the human erythrocyte are coupled to the nucleoside transporter.     

Modulation of GABA Receptor Binding by Ca+2

In frozen-thawed repeatedly washed rat cortical synaptic membranes, Ca2+ (1-5 mM) decreased the binding of [3H]muscimol whereas it increased the binding of [3H]gamma-aminobutyric acid (GABA). However, the binding of [3H]GABA was decreased by the same extent as the binding of [3H]muscimol when the membranes were incubated with baclofen (a selective ligand for the GABAB binding site) and Ca2+. Scatchard analysis of [3H]muscimol binding revealed that Ca2+ reduced the density of GABA binding sites without affecting the dissociation constant. Ca2+ was more potent than Ba2+, Mg2+ was ineffective, and the Ca2+ antagonist La3+ stimulated [3H]muscimol binding. The inhibition of [3H]muscimol binding by Ca2+ was not influenced by calmodulin (50 micrograms/ml), trifluoperazine (10(-5) M), verapamil (10(-6) M), quinacrine (10(-4) M), cordycepin (0.1 mM), leupeptin (20 microM), or soybean trypsin inhibitor (0.1 mg/ml). Moreover, the effect of Ca2+ was additive to that of GABA-modulin. These results indicate that Ca2+ decreases the number of GABAA binding sites while unveiling GABAB binding sites.     

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mn2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ greater than Co2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ approximately equal to control without cations. Addition of Na2+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 +/- 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 +/- 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, Bmax and affinity were reduced. The addition of 100 microM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K-1). The equilibrium dissociation constant (KD) calculated with these rate constants (K-1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.     

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.     

(-)-[3H]Desmethoxyverapamil, a novel Ca2+ channel probe. Binding characteristics and target size analysis of its receptor in skeletal muscle

(-)-[3H]Desmethoxyverapamil (2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan- 7-aza-9-(3-methoxyphenyl)-nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (-)-[3H]desmethoxyverapamil co-purified with t-tubule membrane markers in an established subcellular fractionation procedure. (-)-[3H]Desmethoxyverapamil bound to partially purified t-tubule membranes with a KD of 2.2 +/- 0.1 nM and a Bmax of 18 +/- 4 pmol/mg membrane protein at 25 degrees C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non-competitive fashion by the benzothiazepine Ca2+ antagonist d-cis-diltiazem and the 1,4-dihydropyridine Ca2+ antagonist (+)-PN 200-110. Target size analysis of the (-)-[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 +/- 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d-cis-[3H]diltiazem, was 130.5 +/- 4 kDa (p less than 0.01) and of the 1,4-dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (-)-[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine-selective receptor site of the t-tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites.     

Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils.

We have undertaken a detailed study of the mechanisms of maintenance of intracellular Ca2+ homeostasis in human polymorphonuclear neutrophils (PMN) and its implications for phagocytosis and IgG Fc receptor (FcR) signaling. When PMN were incubated in Ca(2+)-free medium, cytoplasmic calcium concentration ([Ca2+]i) was markedly depressed and intracellular stores were depleted of calcium. [Ca2+]i in these depleted cells increased within 1 min when PMN were placed in medium containing Ca2+ and then decreased to a level close to the normal basal [Ca2+]i, replenishing the intracellular Ca2+ pools. LaCl3 prevented entry of Ca2+ into Ca(2+)-depleted PMN, but the calcium channel blockers nifedipine, diltiazem, and verapamil did not. Nifedipine and diltiazem but not verapamil inhibited the movement of Ca2+ from cytosol to intracellular stores. Nifedipine and diltiazem inhibited the normal increase in [Ca2+]i from aggregated IgG binding to FcR and also prevented formyl-methionyl-leucyl-phenyl-alanine (fMLP)-induced [Ca2+]i rise. Verapamil had no effect on either an fMLP- or IgG-mediated increase in [Ca2+]i. Consistent with this, nifedipine and diltiazem inhibited fMLP-stimulated phagocytosis (which is dependent on an increase in [Ca2+]i) when PMN had repleted intracellular stores. In contrast, LaCl3 inhibited fMLP-stimulated ingestion only in PMN which had intracellular store depleted. None of these compounds had any effect on phorbol dibutyrate-stimulated ingestion (which is independent of a [Ca2+]i rise). In summary, these data show that Ca2+ is in rapid equilibrium between intracellular and extracellular compartments in PMN. Exchange of cytoplasmic Ca2+ with the extracellular space is inhibited by LaCl3, while exchange of Ca2+ between the cytosol and intracellular stores is inhibited by the dihydropyridine nifedipine and the benzothiazepine diltiazem. These data suggest that these drugs, which are known to regulate some plasma membrane Ca2+ channels in excitable cells, can also regulate Ca2+ release from intracellular stores in PMN and that this regulation may have significant effects on PMN function.     

Comparative Effects of Chronic Exposure to Ethanol and Calcium Channel Antagonists on Calcium Channel Antagonist Receptors in Cultured Neural (PC12) Cells

Treatment with 200 mM ethanol for 6 days increased binding of the Ca2+ channel antagonist, (+)-[3H]PN 200-110, to intact PC12 cells in culture. Enhancement of binding by ethanol was due to an increase in binding site number without appreciable change in binding affinity. Long-term exposure to Ca2+ channel antagonist drugs (nifedipine, verapamil, or diltiazem), which, like ethanol, acutely inhibit Ca2+ flux, failed to alter (+)-[3H]PN 200-110 binding to PC12 membranes. Cotreatment of ethanol-containing cultures with the Ca2+ channel agonist, Bay K 8644, did not attenuate the response to ethanol; instead, chronic exposure to Bay K 8644 alone increased (+)-[3H]PN 200-110 binding. These results suggest that chronic exposure to ethanol increases Ca2+ channel antagonist receptor density in living neural cells, but that acute inhibition of Ca2+ flux by ethanol is unlikely to trigger this response.