PTH release stimulated by high extracellular potassium is associated with a decrease in cytosolic calcium in bovine parathyroid cells

We employed the calcium (Ca⁺⁺)-sensitive, intracellular dye QUIN-2 to examine the role of cytosolic Ca⁺⁺ in the stimulation of PTH release by high extracellular potassium (K⁺) concentrations. Addition of 55 mM KCl to cells incubated with 115 mM NaCl and 5 mM KCl lowered cytosolic Ca⁺⁺ at either low (0.5 mM) extracellular Ca⁺⁺ (from 194±14 to 159±9 nM, p<.01, N=6) or high (1.5 mM) extracellular calcium (from 465±38 to 293±20 nM, p<.01, N=10). This reduction in cytosolic Ca⁺⁺ was due to high K⁺$\text{per}\text{se}$ and not to changes in tonicity since addition of 55 mM NaCl was without effect while a similar decrease in cytosolic Ca⁺⁺ occurred when cells were resuspended in 60 mM NaCl and 60 mM KCl. PTH release was significantly (p<.01) greater at 0.5 and 1.5 mM Ca⁺⁺ in QUIN-2-loaded cells incubated with 60 mM NaCl and 60 mM KCl than in those exposed to 115 mM NaCl and 5 mM KCl. In contrast to most secretory cells, therefore, stimulation of PTH release by high K⁺ is associated with a decrease rather than an increase in cytosolic Ca⁺⁺.     

Effects of PTH and Ca2+ on renal adenyl cyclase

The effects of calcium ion on the adenylate cyclase system was studied in isolated, renal basal-lateral plasma membranes of the rat. Bovine parathyroid hormone (bPTH) and a guanyl triphosphate analogue, Gpp(NH)p were used to stimulate cyclase activity. Under conditions of maximal stimulation, calcium ions inhibited cyclic adenosine monophosphate (cAMP) formation, the formation rate falling exponentially with the calcium concentration. Fifty percent inhibition of either bPTH- or Gpp(NH)p-stimulated activity was given by approximately 50 μM Ca⁺⁺. Also the Hill coefficient for the inhibition was close to unity in both cases. The concentration of bPTH giving half-maximal stimulation of cAMP formation (1.8 × 10^?8 M) was unchanged by the presence of calcium. These data suggest that calcium acts at some point other than the initial hormone-receptor interaction, presumably decreasing the catalytic efficiency of the enzymic moiety of the membrane complex.     

Theoretical description of the calcium channel in rat spinal ganglion neurons

The surface charge density (σ′₀) and the binding constant of Ca⁺⁺ with charged groups on the outer surface of the membrane (K_Ca) were calculated from experimentally determined values of the shift of the current-voltage characteristic curves of calcium currents in the membrane of rat spinal ganglion neurons: σ′₀ = 0.15 ± 0.05 e/nm² and K_Ca = 70 ± 10 liters/mole. Using a three-barrier model the energy profile of the calcium channel of the membrane of these neurons was calculated for Ca⁺⁺, Ba⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and verapamil. The calcium current was shown to be determined mainly by the depth of the potential hole corresponding to the outer binding site of the calcium channel. It is concluded from the results that the outer binding site of the calcium channel contains only one carboxyl group.     

Vitellogenesis in the cockroach Nauphoeta cinerea: Separation of two classes of ovarian binding sites and calcium effects on binding and uptake

Two classes of vitellogenin binding sites with K_d-values of 7.3 nM and 290 nM were observed in follicle-membrane preparations of the cockroach Nauphoeta cinerea using a membrane-binding assay at pH 8. Separation of follicle cells and basal laminae from oocyte membranes prior to binding studies showed that the fraction consisting of follicle cells and basal laminae (FC/BL) contained high-affinity binding sites for vitellogenin (K_d=16.6 nM), whereas loweraffinity binding sites (K_d=200 nM) were found in the oocyte membrane fraction. The concentration of Ca²⁺ had a distinct effect on vitellogenin binding and uptake: maximal binding to the oocyte membrane fraction was observed at 0.3 mM Ca²⁺ and to the FC/BL fraction at 10 mM, whereas uptake of vitellogenin by oocytes in vitro was highest at 4 mM Ca²⁺. The calcium ionophore A23187 decreased vitellogenin uptake. This effect of A23187 could be counteracted by the calcium chelator Quin2. A hypothetical model for the uptake of vitellogenin into follicles of Nauphoeta cinerea is suggested.     

Ca++ fluxes in resealed synaptic plasma membrane vesicles

The effect of the monovalent cations Na⁺, Li⁺, and K⁺ on Ca⁺⁺ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K₂/succinate and ⁴⁵CaCl₂, rapidly released approximately 60% of the intravesicular Ca⁺⁺ when exposed to NaCl or to the Ca⁺⁺ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca⁺⁺. The EC₅₀ for Na⁺ activation of Ca⁺⁺ efflux from the vesicles was approximately 6.6mM. exposure of the Ca⁺⁺-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca⁺⁺ from the vesicles, but the Na⁺-induced efflux could still be detected above this K⁺ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid ⁴⁵Ca uptake with an estimated EC₅₀ for Ca⁺⁺ of 7–10 μM. This Ca⁺⁺ uptake was blocked by dissipation of the Na⁺ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na⁺Ca⁺⁺ exchange process and of a K⁺ - sensitive Ca⁺⁺ flux across the membranes.     

Susceptibility Test of Two Ca2+-ATPase Conformers to Denaturants and Polyols to Outline Their Structural Difference

To determine the effect of denaturants [guanidine hydrochloride (GdnHCl) and urea] and polyols [with various molecular masses (62.1–600)] on calcium binding at the two hypothesized conformers (A and B forms) of the chemically equivalent sarcoplasmic reticulum Ca²⁺-ATPase, which bind two calcium ions in different manners, we examined the effect of these reagents on the calcium dependence of ATP-supported phosphorylation of the ATPase molecules and of their calcium-activated, acetyl phosphatate hydrolytic activity. (1) GdnHCl (~0.05 M) and urea (~0.5 M) increased the apparent calcium affinity (K _0.5) of 2–6 μM of noncooperative binding [Hill coefficient (n _H) ~ 1] of the A form to 10–40 μM. (2) The employed polyols transformed the binding of the A form into cooperative binding (n _H ~ 2), accompanying the approach of its K _0.5 value to that (K _0.5 = 0.04–0.2 μM) of the cooperative binding (n _H ~ 2) of the B form; the transition concentration (0.025–2 M) of the polyols, above which such transformation occurs, was in inverse relation to their molecular mass. (3) The binding of the B form was resistant to these denaturants and polyols. Based on these data, a structural model of the two forms, calcium-binding domains of which are loosely and compactly folded, is presented.     

Identification of Voltage Operated Calcium Channels by Binding Studies: Differentiation of Subclasses of Calcium Antagonist Drugs with 3H-Nimodipine Radioligand Binding

Abstract

³H-Nimodipine (³H -NIM) is a high affinity radioligand suitable to study Ca²⁺ -channels in a variety of tissues. The binding is saturable, reversible, and stereospecific in purified bovine heart and partially purified guinea-pig brain membranes. In the latter a Bmax of 600fmol/mg protein, dissociation constants (K_D) of 0.4-0.8nM and a Hill slope of 1.0 are found. At 37^C the optimal pH in 50mM TRIS-HCl buffer is 7.1-7.4. The calcium channel is a metalloprotein, and the divalent cation which is essential for the binding of ³ H -NIM can be removed by EDTA (EC₅₀ 20μM); the nimodipine binding site of the channel may then be reconstituted by divalent cations with Mn²⁺ > Ca²⁺>Mg²⁺>Sr²⁺. Ca²⁺-antagonist drugs can be divided into three main classes based on their interaction with the ³H -NIM binding site: Class I has one site law of mass action-displacement isotherms with ³H -NIM, Class II exhibits complex biphasic inhibition profiles and Class III drugs increase the affinity of 1,4 dihydropyridines for the Ca²⁺ -channel. Diltiazem is a Class III Ca²⁺ -antagonist. Our in vitro studies lead us to conclude that the Ca²⁺ -channel contains multiple regulatory sites at which drugs can act.     

R 24571: a new powerful inhibitor of red blood cell Ca++-transport ATPase and of calmodulin-regulated functions

Compound R 24571 (1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4-dichlorobenzyloxy)phenethyl]imidazoliniumchloride) is found to be a powerful inhibitor of red blood cell Ca⁺⁺-ATPase as well as Ca⁺⁺ transport into inside-out red blood cell vesicles with an IC₅₀-value of 0.5 and 2 μM, respectively. The inhibitory action of R 24571 is more specific on the calmodulin-dependent fraction of Ca⁺⁺-transport ATPase as compared to the basal Ca⁺⁺-transport ATPase (determined in the absence of calmodulin) and can be antagonized by increasing concentrations of calmodulin in an apparently competitive manner. With respect to other ATPases the action of R 24571 is relatively specific for red blood cell Ca⁺⁺-transport ATPase. Mg⁺⁺-ATPase requires a 40 times higher concentration for halfmaximal inhibition (IC₅₀ = 20 μM) whereas (Na⁺ + K⁺)-transport ATPase is only slightly affected in the investigated concentration range (≤20 μM).     

Calcium binding to the subunit c ofE. coli ATP-synthase and possible functional implications in energy coupling

The 8-kDa subunit c of theE. coli F₀ ATP-synthase proton channel was tested for Ca⁺⁺ binding activity using a⁴⁵Ca⁺⁺ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds⁴⁵Ca⁺⁺ strongly with Ca⁺⁺ binding properties very similar to those of the 8-kDa CF₀ subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca⁺⁺ binding capacity, shown by loss of Ca⁺⁺ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca⁺⁺ binding, shown by Ca⁺⁺ binding being retained in twoE. coli mutants, Asp61Asn and Asp61Gly. The Ca⁺⁺ binding is pH dependent in both theE. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca⁺⁺ binding affinity to its F₀ binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca⁺⁺ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca⁺⁺ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80–100 nM was obtained using an EGTA-Ca⁺⁺ buffer system to control free Ca⁺⁺ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca⁺⁺ bound to the periplasimic side of theE. coli F₀ proton channel can block H⁺ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca⁺⁺ binding site is on the lumen side of the thylakoid, where Ca⁺⁺ binding can modulate acid-base jump ATP formation. The Ca⁺⁺ binding to the F₀ and CF₀ complexes is consistent with a pH-dependent gating mechanism for control of H⁺ ion flux across the opening of the H⁺ channel.This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia.     

Properties of an ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase in brain synaptosome membrane vesicles from the bertha armyworm,Mamestra configurata Wlk

Biochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase had single, high-affinity binding sites for ATP (K_m = 14 and 116 μM, respectively), Ca²⁺_free (K_m = 0.13 nM and 0.072 nM, respectively), and Mg²⁺ (K_m = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K⁺ and were insensitive to ouabain, an inhibitor of (Na⁺ + K⁺)-ATPase. The results indicate that the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca²⁺ transport in the brain of M. configurata. Although moth brain (Ca²⁺ + Mg²⁺)-ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one-half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high-affinity site at nanomolar Ca²⁺_free and a low-affinity site at micromolar Ca²⁺_free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two-stage, concentration-dependent process in which a first stage, low-affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high-affinity pump becomes activated.     

Decavanadate displaces inositol 1,4,5-trisphosphate (IP3) from its receptor and inhibits IP3 induced Ca release in permeabilized pancreatic acinar cells

Inositol 1,4,5-trisphosphate (IP₃) induced Ca²⁺ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca²⁺ release induced with 0.18 μM IP₃ is half maximally inhibited with approximately 5 μM decavanadate. Complete inhibition is achieved with around 20 μM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP₃, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca²⁺ uptake into intracellular stores, does not influence IP₃ induced Ca²⁺ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP₃ to the same cellular preparations was investigated. We found that binding of IP₃ to a high affinity receptor site (K_d approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 μM decavanadate, IP₃ binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP₃ with the same affinity (K_d approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP₃ was displaced from these subcellular membranes with similar kinetics by unlabelled IP₃ and decavanadate. The data suggest that the inhibitory action of decavanadate on IP₃ induced Ca²⁺ release is a consequence of its effect on binding of IP₃ to its receptor.     

Reversible shift between two states of Ca2+-ATPase in human erythrocytes mediated by Ca2+ and a membrane-bound activator

The (Ca²⁺ + Mg²⁺)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) from human erythrocytes occurred in two different states, A-state and B-state, depending on the membrane preparation.The A-state showed low maximum activity (V) and the Ca²⁺ activation was characterized by a Hill coefficient, n_H, of about 1 and a Michaelis constant, K_Ca, about 30 μM.The B-state showed high V, a n_H above 1, which indicates positive cooper-activity of Ca²⁺ activation, and a K_Ca of about 1 μM.With varying ATP concentrations, both the A-state and the B-state showed negative cooperativity and slightly different values of K_m.The B-state was shifted to the A-state when the membranes were exposed to low Ca²⁺ concentrations. The shift reached 50% at approx. 0.5 μM Ca²⁺. At the low Ca²⁺ concentrations an activator was released from the membranes.The A-state was shifted to the B-state when the membranes were exposed to Ca²⁺ in the presence of the activator. The shift reached 50% at about 30 μM Ca²⁺. The recovery of high V was time dependent and lasted several minutes. Increasing concentrations of Ca²⁺ and activator accelerated the recovery.It is suggested that the A-state and the B-state correspond to enzyme free of activator and enzyme associated with activator, respectively. Furthermore, the two states may represent a resting and an active state, respectively, of the calcium pump.     

Effects of thiol modification and Ca++ on agonist-specific state transitions of nicotinic acetylcholine receptor from Torpedo californica electroplax.

The agonist binding affinity of nicotinic acetylcholine receptor (nAChR) from $\text{Torpedo}$$\text{californica}$ electroplax, as inferred from ability of agonist to inhibit specific curaremimetic neurotoxin binding to nAChR, is sensitive to the duration of exposure to agonist. The concentration of carbachol necessary to prevent one-half of toxin binding over a 30 min incubation with nAChR (K₃₀) is 10 μM when toxin and carbachol are simultaneously added to membrane-bound nAChR, and 3 μM when nAChR are pretreated with carbachol for 30 min prior to the addition of toxin. These alterations in agonist affinity may be mimicked by modification of nAChR thiol groups. Affinity of nAChR for carbachol is decreased following treatment with dithiothreitol (DTT). Dithio-bis-nitrobenzoic acid treatment of DTT-reduced membranes yields K₃₀ values of 5 μM for carbachol, while N-ethylmaleimide treatment of DTT-reduced nAChR produces nAChR with reduced affinity for carbachol, reflected in K₃₀ values of about 400 μM. In the absence of Ca⁺⁺, K₃₀ values for carbachol binding to native and DTT-reduced nAChR are diminished 3–6 fold. These affinity alterations are not observed with d-tubocurarine (antagonist) binding to nAChR. Thus, Ca⁺⁺ and the oxidation state of nAChR thiols appear to affect the affinity of nAChR for agonists (but not antagonists), and may therefore be related to agonist-mediated events in receptor activation and/or desensitization.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of ¹²⁵I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca²⁺, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (K_d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca²⁺-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca²⁺ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca²⁺ atoms. The concentration of the active calmodulin ·Ca²⁺ species required for half-maximal activation of adenylate cyclase is very similar to the K_d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca²⁺-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

Intracellular calcium requirements for β1 integrin activation

Human polymorphonuclear leukocytes (PMNs) express β1 integrins that mediate adhesion to extracellular matrix proteins following stimulation with agonists that induce an increase in intracellular calcium. The purpose of these studies was to determine the contribution made by alterations in intracellular calcium ([Ca⁺⁺]_i) to inside-out activation of β1 integrins using dimethyl sulfoxide (DMSO)-differentiated granulocytic HL60 cells as a model of human PMNs. Activation of β1 integrins was determined by measuring the expression of an activation-dependent epitope on the β1 subunit that is recognized by monoclonal antibody (mAb) 15/7. Exposure of granulocytic HL60 cells to calcium ionophore ionomycin (800 nM) alone did not increase the binding of mAb 15/7 to the cell surface, nor did it increase β1 integrin-mediated adhesion of the cells to fibronectin. Similarly, exposure of the cells to the direct protein kinase C (PKC) activator, dioctanoylglycerol (di-C₈) at 100 μM, neither increased binding of mAb 15/7 to these cells nor adhesion to fibronectin. Simultaneous addition of di-C₈ and ionomycin, however, caused a significant increase in the expression of the 15/7 epitope and cell adhesion, suggesting synergy between elevating [Ca⁺⁺]_i and stimulating PKC in β1 integrin activation. Chelation of [Ca⁺⁺]_i with Quin-2 and EGTA reduced both basal (unstimulated) expression of the 15/7 epitope and basal adhesion of granulocytic HL60 cells to fibronectin. In addition, chelation of [Ca⁺⁺]_i caused a significant decrease in 15/7 binding and adhesion stimulated by low (1 ng/ml) concentrations of phorbol myristate acetate (PMA). The inhibitory effect of [Ca⁺⁺]_i chelation on β1 integrin activation was reversed by repleting [Ca⁺⁺]_i with ionomycin in a Ca⁺⁺-containing buffer, or by the addition of higher concentrations of PMA (10 ng/ml). These data suggest a role for [Ca⁺⁺]_i in inside-out activation of β1 integrins, probably through a synergistic effect with PKC activation. J. Cell. Physiol. 175:193–202, 1998. © 1998 Wiley-Liss, Inc.     

Isolation, identification and synthesis of an endogenous arachidonic amide that inhibits calcium channel antagonist 1,4-dihydropyridine binding

This study was part of a broad search for endogenous regulators of L-type calcium channels. The screening for active fractions was done by measuring inhibition [³H]1,4-dihydropyridine (DHP) binding to rat cardiac and cortex membranes. An inhibitory fraction, termed lyophilized brain hexane-extractable inhibitor (LBHI), was isolated from hexane extracts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. ¹³C nuclear magnetic resonance (NMR), ¹H coherence spectroscopy (COSY) NMR and fast atom bombardment (FAB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hydroxyethylamide. Synthesis of this substance and subsequent high performance liquid chromatography (HPLC) and NMR analysis confirmed this structure. Synthetic LBHI (SLBHI) inhibited [³H]DHP binding to rat cortex membranes with an IC₅₀ value of 15 μM and a Hill coefficient of 2. Saturation analysis in the presence of SLBHI showed a change in K_D (equilibrium dissociation constant), but not maximal binding capacity (B_max). SLBHI produced an increased dissociation rate, which, along with the Hill slope of > 1, suggested a non-competitive interacton with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the DHP calcium antagonist binding site.     

Binding of calcium and zinc to the acetylcholine receptor purified from Torpedo Californica

Binding of [⁶⁵Zn⁺⁺] and [⁴⁵Ca⁺⁺] to the acetylcholine (ACh)-receptor, purified from the $\text{Torpedo}$ electric organ, was studied by equilibrium dialysis. Whereas [⁶⁵Zn⁺⁺] bound to 56 nmoles of sites per mg protein with a dissociation constant of 2.5 × 10⁻⁶M, no binding of [⁴⁵Ca⁺⁺] at concentrations up to 10⁻³M could be detected with this method. However, the binding of [acetyl-³H]choline to the receptor was blocked equally by very high Zn⁺⁺ or Ca⁺⁺ concentrations, and the K_i for this low affinity binding was 7 × 10⁻³M. The high affinity binding of [⁶⁵Zn⁺⁺] to the receptor was blocked best by Cd⁺⁺ then Co⁺⁺ and Mn⁺⁺, but least by Mg⁺⁺ and Ca⁺⁺. When the purified ACh-receptor itself was analyzed for the presence of cations by atomic absorption, it was discovered that 4.7% of its weight was due to bound Ca⁺⁺ that could not be removed even by extensive dialysis. When Ca⁺⁺-free solutions (containing 1 mM EDTA) were used during purification, 0.6% of the molecular weight of the receptor was still due to bound Ca⁺⁺. This was equivalent to 15 moles of Ca⁺⁺ for each mole of ACh bound at saturation. It is suggested that the source of this Ca⁺⁺ is endogenous, and that it is tightly bound to the ACh-receptor molecule.     

Binding of 45Ca2+ to particulate fractions of coleoptile tissue

Summary Using recently developed techniques, we have investigated the binding of ⁴⁵Ca²⁺ to membrane preparations from corn (Zea mays L) and oat (Avena sativa L) coleoptile tissue. Scatchard plot analysis reveals at least two Ca²⁺-binding sites in each tissue, a high affinity binding site (K _m=7.7×10^-7 M, n=6.9×10^-10 mol·0.5 g f.w.^-1 in corn, K _m=4.93×10^-6 M, n=2.29×10^-9 mol·0.5 g f.w.^-1 in Avena) and a low affinity binding site (K _m=9.01×10^-5 M, n=5.4×10^-8 mol·0.5 g f.w.^-1 in corn; K _m=1.03×10^-4 M, n=3.40×10^-8 mol·0.5 g f.w.^-1 in Avena). There is also some evidence of a third, lower affinity binding site in each tissue, especially corn.More detailed studies with corn coleoptile homogenates show that they contain a potent dialyzable inhibitor of Ca²⁺ binding. Monovalent cations were observed to be ineffective as inhibitors of Ca²⁺ binding in corn. However, of six divalent cations tested, all were capable of strong inhibition of Ca²⁺-binding and there appeared to be a relationship between size of the atomic radius of the ion and potency as an inhibitor of calcium binding.Abbreviations CSM corn suspensiom medium - EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide - GEE glycine ethyl ester     

Calcium alters the properties of [3H]diazepam binding sites in rat brain membranes

• 1.1. [³H]diazepam ([³H]DZ) was used as a ligand to study the effects of Ca²⁺ on benzodiazepine binding to rat brain membranes.
• 2.2. [³H]DZ bound at a single class of binding sites showing K_D and B_max values of 5.4 nM and 852 fmol/mg protein respectively. These values are consistent with previous reports.
• 3.3. Amongst the various divalent cations tested Mg²⁺, Fe²⁺, Cd²⁺, and Sr²⁺ had no significant effect on [³H]DZ binding. Mn²⁺, Ba²⁺, Co²⁺, Ni²⁺ and La²⁺ enhanced radioligand binding, whereas Ca²⁺, Zn²⁺ and Pb²⁺ inhibited [³H]DZ binding to brain freeze-thawed membranes.
• 4.4. The inhibition of [³H]DZ binding by Ca²⁺ was concentration-dependent. 50% inhibition occurred at a Ca²⁺ concentration of 5.6mM. The Hill coefficient for the inhibition was 1.03, displaying noncoperativity. The effect of Ca²⁺ on [³H]DZ binding could be prevented by La³⁺ but was not reversed by EGTA.
• 5.5. A kinetic analysis of Ca²⁺ inhibition of [³H]DZ binding indicates that Ca²⁺ inhibited competitively. Analysis of binding isotherms indicates that both K_D and B_max were altered at the [³H]DZ binding sites. The marked increase in K_D value in the presence of Ca²⁺ (5 mM) can be explained by a drastic increase in the dissociation rate constant.
• 6.6. It was suggested that Ca²⁺ may induce a conformational change in the diazepam binding sites on rat brain membranes. The unchanged Hill coefficient in the presence or absence of Ca²⁺ indicates that a single population of binding sites was labeled by [³H]DZ.
• 7.7. The calmodulin antagonists, trifluoperazine and W-7 were weak inhibitors of [³h]dz binding.

     

Interaction of the Calcium Channel Activating 3H-Bay K 8644 and Inhibiting 3H-Verapamil with Specific Receptor Sites on Cultured Beating Myocardial Cells

Abstract

Binding properties of the calcium channel activating dihydropyridine (DHP), ³H-BAY K 8644, and the inhibiting ³H-verapamil were demonstrated in monolayer cultures of beating cardiac cells. ³H-BAY K 8644 specific binding was dependent on the presence of extracellular calcium, the affinity was modulated by Ca²⁺, but Hill coefficients remained unaffected. BAY K 8644 stimulated myocardial contractility in resting and beating myocytes. In contrast to ß-adrenoceptor agonists, however, cellular levels of cyclic AMP and cyclic GMP in cultured myocytes remained unchanged by the compound. Dihydropyridine derivatives of both the calcium channel activating BAY K 8644 as well as the Ca²⁺ entry blocking DHPs of the nifedipine or nimodipine type yielded very low affinity to other receptors measured in brain and heart membranes. ³H-BAY K 8644 binding sites proved to be highly specific for various potently displacing DHP derivatives and discriminated between optical isomers (stereoselectivity) with inhibition constants (K_i) in the nanomolar range. The heterogeneous shapes of the competition curves also imply interactions of these compounds with different (sub-)sites of the DHP receptor that represents one locus of interaction in regulating transmembranal Ca²⁺ currents. The other specific site of action for the potent diphenylalkylamines, clearly different to the DHP receptor was characterized with ³H-verapamil. The equilibrium dissociation constant, K_d in cultured myocytes ranged between 16-25 nM, and binding capacity, B_max amounted to about 1.85 pmol/mg of protein. The different mode of competitions indicates the involvement of more than one ³H-verapamil binding site. The interrelation of the structurally heterogeneous channel modulators with the differently radiolabelled receptor (sub-)sites located in or near by the calcium channel may represent new approaches in investigating the nature of action of these potent compounds.