Effects of diltiazem or verapamil on calcium uptake and release from chicken skeletal muscle sarcoplasmic reticulum

The purpose of this study was to determine the effects of 2 Ca2+ channel blockers, verapamil and diltiazem, on calcium loading (active Ca2+ uptake) and the following Ca2+ release induced by silver ion (Ag+) and Ca2+ from the membrane of heavy sarcoplasmic reticulum (SR) of chicken skeletal muscle. A fluorescent probe technique was employed to determine the calcium movement through the SR. Pretreatment of the medium with diltiazem and verapamil resulted in a significant decrease in the active Ca2+ uptake, with IC50 of about 290 micromol/L for verapamil and 260 micromol/L for diltiazem. Inhibition of Ca2+ uptake was not due to the development of a substantial drug-dependent leak of Ca2+ from the SR. It might, in part, have been mediated by a direct inhibitory effect of these drugs on the Ca2+ ATPase activity of the SR Ca2+ pump. We confirmed that Ca2+ channel blockers, administered after SR Ca2+ loading and before induction of Ca2+ release, caused a dose-dependent inhibition of both Ca2+- and Ag+-induced Ca2+ release rate. Moreover, if Ca2+ channel blockers were administered prior to SR Ca2+ loading, in spite of Ca2+ uptake inhibition the same reduction in Ca2+- and Ag+-induced Ca2+ release rate was seen. We showed that the inhibition of Ag+-induced Ca2+ release by L-channel blockers is more sensitive than Ca2+-induced Ca2+ release inhibition, so the IC50 for Ag+- and Ca2+-induced Ca2+ release was about 100 and 310 micromol/L for verapamil and 79 and 330 micromol/L for diltiazem, respectively. Our results support the evidence that Ca2+ channel blockers affect muscle microsome of chicken skeletal muscle by 2 independent mechanisms: first, reduction of Ca2+ uptake rate and Ca2+-ATPase activity inhibition, and second, inhibition of both Ag+- and Ca2+-induced Ca2+ release by Ca2+ release channels. These findings confirm the direct effect of Ca2+ channel blockers on calcium release channels. Our results suggest that even if the SR is incompletely preloaded with Ca2+ because of inhibition of Ca2+ uptake by verapamil and diltiazem, no impairment in Ca2+ release occurs.     

Calcium release from vesicles of heavy sarcoplasmic reticulum of rabbit skeletal muscles

The release of Ca2+ from vesicles of heavy sarcoplasmic reticulum after its accumulation due to hydrolysis of ATP, GTP, CTP, UTP or ITP has been studied using Antipyrylazo III, a metal-chromic Ca-indicator. All the studied substrates of the Ca-pump provide Ca2+ accumulation inside the heavy sarcoplasmic reticulum vesicles, the spontaneous Ca2+ outflux rate being different for different nucleoside triphosphates. It is only ATP that provides Ca-(caffeine)-induced Ca2+ release, however AMP, ADP, beta, gamma-methylene-ATP induce Ca2+ ejection in the presence of nonadenylic nucleotides. The ruthenium red (10(-7M) inhibits the induced ejection of Ca2+ from vesicles of the heavy sarcoplasmic reticulum, but does not prevent the spontaneous release of Ca2+ in the same concentrations. A conclusion is drawn that besides Ca-channels sensitive to Ca2+ and caffeine in the presence of ATP (or to AMP, ADP, beta, gamma-methylene-ATP in the presence of nonadenylic nucleotides) and possessing high sensitivity to the ruthenium red there is another pathway for Ca2+ in the heavy reticulum membranes along which its spontaneous release occurs after the substrate exhaustion. It is supposed that this release is provided by the presence of the Ca-ATPase protein.     

Oxidation of Analogs of l-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine by Monoamine Oxidases A and B and the Inhibition of Monoamine Oxidases by the Oxidation Products

Intraterminal free Ca2+ concentration modulates the subsequent release of neurotransmitters. Depolarization of synaptosomes with 29 mM K+ augments cytosolic free Ca2+ concentration, which is triphasic, the peak times being at 10, 60, and 180 s. We examined the characteristics of each elevation of cytosolic free Ca2+ concentration in rat brain synaptosomes which had been preincubated for 3 min with a Ca2+-channel blocker, such as La3+, diltiazem, nifedipine, or verapamil, and under conditions of hypoxia or acidosis. The concentration of free Ca2+ in the quin-2-loaded rat brain synaptosomes was detected fluorometrically. All these elevations were suppressed in the presence of 200 microM EGTA or 100 microM La3+. At the first phase, the elevation of cytosolic free Ca2+ concentration with high K+ stimuli was significantly inhibited by La3+ (20 microM) or by acidosis (pH 6.7). On the other hand, diltiazem, which is a more potent blocker of the release of Ca2+ from the mitochondria, inhibited the increasing cytosolic free Ca2+ concentration at the third phase in a concentration-dependent manner. Hypoxia also showed inhibition at the third phase. These results suggest that the augmentation of high K+-evoked cytosolic free Ca2+ concentration may be due to the influx of extracellular Ca2+. The increase in cytosolic free Ca2+ concentration at the third phase is no doubt linked to the mitochondrial function.     

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.     

Effects of anions, pH and magnesium on calcium accumulation and release by sarcoplasmic reticulum vesicles

In the absence of oxalate, Ca2+ accumulation by isolated sarcoplasmic reticulum vesicles may show a transient behavior in which the vesicles accumulate during the first 2 min of incubation as much as twice the amount of Ca2+ which is retained after 5-7 min, when Ca2+ accumulation approaches a steady state. Before Ca2+ release begins, the Ca2+ accumulation can reach 200-250 nmol/mg protein. The spontaneous release of the "extra" Ca2+ initially accumulated appears to be triggered by the attainment of a sufficiently high concentration of free Ca2+ inside the vesicles. The amplitude of the transient phase of Ca2+ accumulation reaches a high value near pH 6.0 and is increased by free Mg2+. At optimal concentrations of H+ and Mg2+, the amount of Ca2+ accumulated during the transient is augmented by various anions, in the order maleate > or = propionate > or = succinate > chloride > sulfate > acetylglycine. The divalent anions have their maximum effects at 20-40 mM and the monovalent anions, at 40-200 mM. At 200 mM, all of the carboxylic anions tested significantly reduce the amount of Ca2+ retained in the steady state.     

Release of growth hormone from purified somatotrophs: effects of the calcium channel antagonists diltiazem and nifedipine on release induced by growth hormone-releasing factor

We examined the effect of the voltage-sensitive Ca2+ channel antagonists, diltiazem and nifedipine, on basal and stimulated growth hormone (GH) release from purified somatotrophs. Our aim was to ascertain whether an influx of Ca2+ from the extracellular to the intracellular compartment is essential for augmented release. Basal release was decreased in a concentration-dependent manner by both diltiazem and nifedipine, while cAMP accumulation was unaffected. The release of GH induced by 29 mM K+ was blocked by diltiazem and nifedipine, at 10(-7) and 10(-8) M, respectively. Again cAMP was unaffected. The release of GH induced by growth hormone-releasing factor was significantly reduced by 10(-4) M diltiazem and completely blocked by nifedipine at a concentration of 10(-6) M or greater. Where the antagonists were effective, the growth hormone-releasing factor induced increase in cAMP accumulation was augmented. We conclude that an influx of Ca2+ from the extracellular compartment is essential for stimulated GH release.     

Mechanism of passive Ca2+ permeability of vesicular sarcolemmal preparations from rat hearts

Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).     

Effects of phospholipase A2 inhibitors on ruthenium red-induced Ca2+ release from mitochondria

The pharmacologic agents verapamil, nifedipine, diltiazem, prenylamine, N-oleoylethanolamine, R 24571, trifluoperazine, dibucaine, and quinacrine are examined as potential inhibitors of rat liver mitochondrial phospholipase A2 acting on endogenous phospholipid. Their potency as inhibitors of the enzyme is compared to their activities as inhibitors of phospholipase A2-dependent swelling and ruthenium red-induced Ca2+ release in intact mitochondria. For verapamil, diltiazem, trifluoperazine, dibucaine, and quinacrine, there is complete agreement between the relative potencies as inhibitors of phospholipase A2 and the two other processes. Nifedipine and prenylamine, which are weak inhibitors of phospholipase A2, produce a permeable inner membrane, provided that the mitochondrial have accumulated Ca2+. R 24571, which strongly inhibits the enzyme, disrupts mitochondria by a Ca2+-independent mechanism. N-Oleoylethanolamine, which is an effective inhibitor of swelling, does not inhibit phospholipase A2 or ruthenium red-induced Ca2+ release. The results support a proposed scheme wherein ruthenium red-induced Ca2+ release is viewed as reverse activity of the Ca2+-uptake uniporter occurring subsequent to decline in the proton motive force. The latter effect is proposed to arise from a specific phospholipase A2-dependent increase in inner-membrane H+ conductance of mitochondrial subpopulations. It is further shown that mitochondrial membranes display cyclic oscillations in free fatty acid content which are not dependent on the presence of Ca2+ or on the capacity to generate acylcoenzyme A.     

Passive Ca2+ permeability of vesicular sarcolemmal preparations from myocardium

Vesicular preparations of sarcolemma isolated from rat myocardium possessed high ATPase (4.32 +/0 0.57 micromole/min per mg), adenylate cyclase (121 +/- 11 pmole/min per mg) and creatine kinase (1.74 +/- 0.35 micromole/min per mg) activities and a Na-Ca exchange activity specific for sodium. The ATPase activity was inhibited by digitoxigenin by 50-70% and was not changed by ouabain, EGTA, ionophore A23187 and oligomycin, thus showing the absence of mitochondrial and sarcoplasmic reticulum contaminations in the sarcolemmal preparations. The preparations consisted mostly of closed inside-out vesicles. The preparation was used to study the mechanism of Ca2+ penetration across the sarcolemmal membrane. For this purpose the vesicles were load with 45Ca2+, which relatively slowly diffused from the medium into the vesicles, and which was bound to the binding sites inside the vesicles (n = 20.5 +/- 4.6 nmoles per mg of protein, Kd approximately equal to 1.8 +/- 0.21 mM). The transmembrane movement of Ca2+ was demonstrated by the following findings: 1) the ionophore A23187 only insignificantly increased the total vesicular Ca2+ content, but strongly accelerated Ca2+ efflux from the vesicles along its concentration gradient; 2) gramicidin and osmotic shock caused a similar acceleration of Ca2+ efflux. Ca2+ efflux from these vesicles along Ca2+ concentration gradient was studied under conditions, when the extravesicular Ca2+ content was lowered due to its binding to EGTA and by dilution. The gradient of Ca2+ concentration was from 2.0 mM inside to approximately 0.1 micro M outside. The rate of 45Ca2+ efflux depended hyperbolically on the intravesicular Ca2+ efflux from the vesicles was inhibited by Mn2+, Co2+ and verapamil when they acted from the inside of the vesicles. An increase in ionophore A23187 concentration increased the efflux of Ca2+ hyperbolically and enhanced only the maximal rate of the efflux. It is concluded that the passive permeability of Ca2+ across the sarcolemmal membrane along its concentration gradient is controlled by Ca2+ binding to the membrane.     

Histamine stimulated synaptosomal Ca2+ uptake through activation of calcium channels

Histamine stimulated Ca2+ uptake in synaptosomes was completely inhibited by the slow Ca2+ channel antagonists verapamil, cinnarizine and flunarizine, and slightly inhibited by nifedipine and diltiazem. Ca2+ uptake in synaptosomes depolarized or predepolarized with varying K+ concentrations was increased by histamine, in both conditions, until 30mM K+. At higher K+ concentrations histamine was not able to alter K+ effects in either conditions. 30mM K+ stimulated uptake of Ca2+ in the absence or presence of histamine was not inhibited by verapamil and diltiazem. However nifedipine slightly inhibited K+ and K+ +histamine effects. 3-Isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP potentiated (10%) the uptake of Ca2+ in synaptosomes induced by histamine. Dibutyryl cyclicAMP alone however decreased the basal Ca2+ uptake in a concentration-dependent manner. Verapamil, but not diltiazem, antagonized the effects elicited by 3-isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP in the presence of histamine. The data suggest that the increase in synaptosomal Ca2+ uptake induced by histamine is mediated by the activation of the voltage sensitive calcium channels, and possibly a cyclicAMP-dependent protein kinase phosphorylation can modulate the opening of Ca2+ channels.     

Changes in the intensity of the fluorescence of potential-sensitive fluorescent probes in the active transport of Ca2+ in the fragmented sarcoplasmic reticulum

The dependence of fluorescence intensity changes of potential-sensitive fluorescent probes 3,3'-dipropyl-2,2'-thyodicarbocianine and 1-anilino-8-naphtalenesulphonae on the ATP concentration during Ca2+ transport in fragmented SR of the rabbit skeletal muscle has been studied. An increase in the accumulation of Ca2+ in the SR vesicles caused by ATP is accompanied by an increase in the fluorescence intensity of the potential-sensitive probes. These fluorescence changes are related neither to ATP or Ca2+ effect but are coupled with cation accumulation inside the vesicles since they are not observed in the presence of either EGTA or triton X-100 or in the absence of Mg2+. The results obtained prove the membrane potential generation in SR in the course of ATP-dependent Ca2+ transport.     

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.     

A model for the uptake and release of Ca2+ by sarcoplasmic reticulum.

On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase.     

Ca2+ release triggered by NAADP in hepatocyte microsomes

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.     

Interaction of different nucleotides with Ca-release channels from heavy sarcoplasmic reticulum

Calcium accumulation and release from the heavy fraction of sarcoplasmic reticulum vesicles have been studied in the presence of different nucleotides with the use of the Ca-sensitive dye antipyrylazo III for monitoring of the free Ca2+ concentration. The calcium- and caffeine-induced Ca2+ release is observed only with ATP but not with any of nonadenine nucleotides used as substrates for the Ca-pump. Adenine nucleotides provide for a rapid ruthenium red sensitive Ca2+ release from the vesicles, when nonadenine nucleotides are used as energy sources for Ca2+ uptake. A comparison of the nucleotides interaction with Ca-channels and Ca-ATPase supports the hypothesis that Ca-ATPase is involved in the operation of Ca-channels.     

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.     

Charge changes in sarcoplasmic reticulum and Ca2+-ATPase induced by calcium binding and release: a study using lipophilic ions

Changes in the charge of sarcoplasmic reticulum (SR) vesicles are studied using lipophilic ions, which are adsorbed by the membrane phase. Upon addition of MgATP, phenyldicarbaundecaborane (PCB-) and tetraphenylboron (TPB-) are taken up by the SR vesicles, while tetraphenylphosphonium (TPP+) is released into the water phase. The PCB- uptake occurs as well under conditions when SR membrane is shunted by high Cl- concentration. MgATP induces minor additional binding of PCB- in the presence of oxalate and it is followed by release of the lipophilic anion from the vesicles. EGTA partly reverses the ATP effect, and calcium ionophore A23187 plus EGTA reverses it completely. Vesicles that were preliminarily loaded by Ca2+ demonstrated higher passive and lower ATP-dependent PCB- binding. Activation of isolated Ca2+-ATPase in the presence of 0.1 mM EGTA results in PCB- release into the medium and additional TPP+ binding to the enzyme. We suggest that the redistribution of the lipophilic ions between the water phase and SR membrane reflects charge changes in Ca2+-binding sites inside both SR vesicles and Ca2+-ATPase molecules in the course of Ca2+ translocation.     

Nifedipine, verapamil and diltiazem block shock-wave-induced rises in cytosolic calcium in MDCK cells

Nifedipine and verapamil have been shown previously to protect against renal function alterations induced by shock wave lithotripsy (SWL) in humans and rats; however, the mechanism is unclear. This study was aimed to examine whether these drugs could protect cultured kidney cells following shock wave exposure (SWE). The effect of nifedipine, verapamil and diltiazem on Madin Darby canine kidney (MDCK) cells following SWE was examined by determining the release of glutamate oxalactate transferase (GOT) and lactate dehydrogenase (LDH) in cell suspensions; and also cytosolic Ca2+ concentration ([Ca2+]i). Immediately after SWE, there was a transient release of GOT and LDH (16% and 4 fold, respectively). In contrast, [Ca2+]i measured within 1-6 hr after SWE gradually increased by 15-156%. The Ca2+ entry blockers (1 or 10 microM) failed to inhibit the enzyme release; however, they abolished the progressive rises in [Ca2+]i. The Ca2+ entry blockers may protect the cells from damage of SWE via maintaining a low resting [Ca2+]i.     

ATP-dependent transport of Ca2+ in myocardium sarcolemma vesicles and its activation by phorbol esters

Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.     

Clinical potency of calcium channel blockers in asthma may be related to their inhibition of receptor-mediated phasic responses in vitro.

The calcium channel blockers (CCB) have been clinically effective in exercise-induced asthma. The completeness of protection with the CCB might be related specifically to inhibition of Ca2+ influx or release. To examine this hypothesis, the rank order of potency of inhibition of the CCB, nicardipine, diltiazem and verapamil on the steady-state and kinetic parameters of the phasic and tonic responses to the muscarinic receptor agonist carbachol (10 microM) and KCl (40 mM) in the intact isolated guinea-pig trachea was determined. The Ca2+ channel agonist Bay K 8644 was also examined for its effects on intracellular Ca2+. Nicardipine abolished the KCl response at both 0.1 microM and 1 microM concentrations. The amplitude of the KCl response was inhibited equally by 1 microM diltiazem (61% inhibition) and 1 microM verapamil (68% inhibition). The rate constant of onset of the KCl response was similarly inhibited 60% by diltiazem and 66% by verapamil. Nicardipine abolished the carbachol phasic response at the 1 microM concentration. The amplitude of the phasic response was inhibited equally by 0.1 microM nicardipine (61.3% inhibition), 1 microM diltiazem (64.5% inhibition) and 1 microM verapamil (71% inhibition). The rate constant of decay of the phasic response was inhibited equally by 0.1 microM nicardipine (43% inhibition) and 1 microM diltiazem (29% inhibition). The rate constant of onset of the phasic response was unaffected by nicardipine, diltiazem and verapamil. Only 1 microM nicardipine inhibited the amplitude and rate constant of onset of the tonic response. The only effect of Bay K 8644 (1 microM) was to increase the phasic response amplitude. The CCB demonstrate a similar order of potency for inhibition of the phasic responses and clinical efficacy of the CCB in exercise-induced asthma (nicardipine > verapamil > diltiazem).(ABSTRACT TRUNCATED AT 250 WORDS)