"Calcium antagonists": a class of drugs with a bright future. Part I. Cellular calcium homeostasis and calcium as a coupling messenger

The aim of this series of minireviews is to present material from multidisciplinary sources to facilitate the understanding of the pharmacology and the ample clinical potential of a class of drugs that were originally designated as "calcium antagonists" and more recently have been referred to as "calcium entry blockers", "calcium slow channel blockers" or "calcium modulators". In this first report our attention will be focussed on the pivotal role of Ca++ as a messenger linking stimuli of extracellular origin to the intracellular environment. Eucaryotic cells have a number of powerful means to control their cytosolic Ca++ concentration. Firstly, in a cell at rest the cellular membrane is relatively impermeable to passive Ca++ movements. This property of the plasmalemma prevents the high free Ca++ concentration (approximately 1 mM) of the extracellular compartment from invading the cytosol (approximately 0.1 microM). However, extracellular Ca++ can reach the cytosol through the Na+/Ca++ exchange mechanism and the plasmalemma possesses special Ca++ channels the conductance of which is controlled by gates that are opened by critical changes in cellular polarization (voltage-operated channels: VOC) or by receptor activation (receptor-operated channel: ROC). The Ca++ entering via VOC or ROC can subsequently trigger the liberation of Ca++ from the sarcoplasmic reticulum or from calcium stores located in the inner side of the plasmalemma. The intracellular message generated by external stimuli is transferred to the response mechanism by several cytosolic proteins that require Ca++ as activator. Finally, the termination of the response is the result of a reduction in the cytosolic Ca++ concentration that is accomplished by the Na+/Ca++ exchange mechanism or by energy-dependent pumps which extrude Ca++ from the cell or store it in subcellular organelles. Therefore, any of the numerous steps of the excitation-response coupling which employ Ca++ as a messenger or as a protein activator can be the site of action of a pharmacological agent. In the follow-up minireview, some methods to determine the basic pharmacological profile of compounds interfering with cellular Ca++-dependent functions will be described.     

Hepatic insulin effects independent of changes in Ca++ metabolism

The possibility of Ca++ acting as second messenger for insulin in rat liver was investigated using the net stimulation of 14C-glucose incorporation into glycogen by isolated hepatocytes as an index of insulin action. An insulin effect could be partially sustained in the virtual absence of Ca++ and Mg++ and a maximal insulin effect could be observed in the presence of either Ca++ or Mg++, suggesting that extracellular Ca++ is not required for insulin action. Inhibiting the activity of calmodulin, an intracellular mediator of Ca++ action, with trifluoperazine had little effect on insulin action. The efflux of 45Ca from prelabeled hepatocytes was not altered by the presence of insulin arguing against insulin-induced changes in Ca++ fluxes. Collectively, these results do not support the role of Ca++ as second messenger for insulin action in liver.     

The blocking effect of magnesium on the secretion of adrenal catecholamines induced by the omission of sodium from the extracellular medium.

The blocking action of Mg++ on catecholamine release induced by the substitution of extracellular Na+ by an osmotic equivalent amount of sucrose was studied in isolated, perfused bovine adrenal glands. Perfusing glands with 10 mM Mg++ produced at 51.1% inhibition on catecholamine release evoked by Na+ omission. Increasing the concentration of Mg++ to 20 mM this inhibitory effect was enhanced to 90.3%. D-600 (0.3 mM) promoted a marked blockade of acetylcholine-induced release of catechol hormones that was partially and significantly reverted increasing the concentration of Ca++ in the perfusion medium. D-600 (0.3 mM) failed to inhibit the catecholamine-releasing effect of Na+ deprivation. In adrenal glands previously perfused with D-600 (0.3 mM) and then exposed to a Locke solution containing D-600 (0.3 mM) + Mg++ (10 or 20 mM) the inhibition of the secretory responses evoked by the omission of Na+ was of the same magnitude as that obtained when the glands were perfused with Mg++ (10 or 20 mM) in the absence of D-600. These results are compatible with the view that the blocking effect of Mg++ may involve an intracellular site of action and that the access of Mg++ into the chromaffin cell may not be mediated through the Ca++ channels.     

细胞膜毒素对线粒体上钙通道的抑制

本文探讨膜毒素对鼠肝线粒体Ca~(++)传递和Ca~(++)结合亲和力的影响。当膜毒素的浓度为7.14毫微克分子/毫克线粒体蛋白时,处理过的线粒体传递Ca~(++)能力下降至原来一半左右。本实验做Ca~(++)结合膜毒素处理线粒体的Scatchard图呈直线(K_d=48.2μM,结合Ca~(++)数目N=341毫微克分子/毫克线粒体蛋白)。就是说,膜毒素抑制线粒体高亲和力Ca~(++)结合部位,而不影响低亲和力Ca~(++)结合部位。我们认为膜毒素作用位点在于线粒体高亲和力Ca~(++)结合部位。     

Transduction persists in rod photoreceptors after depletion of intracellular calcium

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.     

Mitogen-stimulated glucose transport in thymocytes. Possible role of Ca++ and antagonism by adenosine 3'':5''-monophosphate

The plant lectin, concanavalin A (Con-A), and the ionophore, A-23187 (specific for divalent cations), stimulated glucose transport in rat thymocytes. Con-A stimulation developed more slowly and was somewhat less extensive than that of stimulation developed more slowly and was somewhat less extensive than that of A-23187. Both responses showed saturation dose dependencies. The two responses were poorly additive, suggesting that A-23187 may saturate regulatory processes shared by the two stimulatory mechanisms. Doses of methylisobutylxanthine (MIX) and prostaglandin E2 which raised adenosine 3':5'-monophosphate (cAMP) levels in these cells also antagonized the Con-A stimulation of glucose transport but did not inhibit basal glucose transport or the A-23187 stimulation. Dibutyryl-cAMP and 8-bromo-cAMP also natagonized Con-A stimulation without inhibiting basal glucose transport. MIX antagonized high Con-A doses about as strongly as it did low Con-A doses, suggesting that MIX did not compete in the Con-A binding step or other process saturable by Con-A. [3H-A1Con-A binding was not affected by MIX. The stimulatory effects of Con-A and A-23187 were reduced by reduction of Ca++ in the medium. Both Con-A and A-23187 enhanced 45Ca++ influx and cellular Ca++ content. The A-23187 dose, which was saturating for glucose transport stimulation, enhanced Ca++ influx and cellular Ca++ content more than did the Con-A dose which was saturating for glucose transport stimulation. The dose fo MIX which specifically antagonized Con-A stimulation of glucose transport proved also to reduce Ca++ influx and cellular Ca++ in the presence of Con-A but not in the presence of A-23187. Thus, glucose transport correlates rather well with cellular Ca++. These results are compatible with the view that Ca++ in a cellular compartment can promote glucose transport, the Con-A's enhancement of Ca++ entry contributes to its stimulation of glucose transport, and the MIX antagonized Con-A action at least partly by reducing Ca++ entry. The action of MIX is apparently mediated by cAMP.     

Properties of tetraethylammonium ion-resistant K+ channels in the photoreceptor membrane of the giant barnacle

After the offset of illumination, barnacle photoreceptors undergo a large hyperpolarization that lasts seconds or minutes. We studied the mechanisms that generate this afterpotential by recording afterpotentials intracellularly from the medial photoreceptors of the giant barnacle Balanus nubilus. The afterpotential has two components with different time-courses: (a) an earlier component due to an increase in conductance to K+ that is not blocked by extracellular tetraethylammonium ion (TEA+) or 3-aminopyridine (3-AP) and (b) a later component that is sensitive to cardiac glycosides and that requires extracellular K+, suggesting that it is due to an electrogenic Na+ pump. The K+ conductance component increases in amplitude with increasing CA++ concentration and is inhibited by extracellular Co++; the Co++ inhibition can be overcome by increasing the Ca++ concentration. Thus, the K+ conductance component is Ca++ dependent. An afterpotential similar to that evoked by a brief flash of light is generated by depolarization with current in the dark and by eliciting Ca++ action potentials in the presence of TEA+ in the soma, axon, or terminal regions of the photoreceptor. The action potential undershoot is generated by an increase in conductance to K+ that is resistant to TEA+ and 3-AP and inhibited by Co++. The similarity in time-course and pharmacology of the hyperpolarization afterpotentials elicited by (a) a brief flash of light, (b) depolarization with current, and (c) an action potential indicates that Ca++-dependent K+ channels throughout the photoreceptor membrane are responsible for all three hyperpolarizing events.     

Effect of in vivo and in vitro application of glucagon, insulin and epinephrine on Ca++-transport properties of liver mitochondria.

In vivo administration of glucagon, insulin or epinephrine, respectively, gives rise to an increase of Ca++-retention time as well as of the Ca++-uptake rate in subsequently isolated rat liver mitochondria. Whereas the changes of Ca++-transport properties after pretreatment with glucagon or epinephrine occur already 6--15 min after their administration, the effect of insulin is observed not earlier than 30 min after its application. Under diabetic and starving conditions the Ca++-retention time of isolated liver mitochondria is prolonged, whereas no alteration of the uptake rate occurs. Since alloxan as well as streptozotocin induced qualitatively similar changes, a specific action of alloxan on liver mitochondria can be ruled out. Application of insulin 60--90 min prior to decapitation normalizes the changes of mitochondrial Ca++-transport observed under chronic alloxan diabetic conditions. Cycloheximide abolishes the prolongation of Ca++-retention in mitochondria from alloxan diabetic rats, but has no influence on the changes induced by glucagon pretreatment.     

Ca++ and Na+ channels involved in neuronal cell death. Protection by flunarizine.

Flunarizine, a class IV Ca++ antagonist non-selective for slow Ca++ channels, has been shown to be beneficial in the prophylactic treatment of migraine, the treatment of vertigo, and as add-on treatment in therapy-resistant forms of epilepsy. Flunarizine protects the brain against functional and/or structural neuronal damage in various animal models of cerebral ischemia. In addition to its cerebrovascular effect, flunarizine has also direct neuroprotective actions. New data have emerged on flunarizine with regard to Ca++ and Na+ channels in neuronal cells. There are several possible mechanisms involved in the mode of action of flunarizine. Flunarizine may block Ca++ and Na+ channels, both of which may flux Ca++ as well as Na+. A decrease in Ca++ influx may prevent further release of glutamate, and activation of NMDA receptor gated Ca++ channels at physiological pH. A decrease in Na+ influx may prevent cytotoxicity secondary to a large gain in intracellular Ca++, by reverse operation of the Na+/Ca++ exchanger. This mechanism may be important when the glycolytic rate is increased with concomitant acidosis, and phospholipids are broken down as occurs typically during ischemia. Given the complexity of biochemical events leading to cell death, blocking exclusively one channel subtype is not likely to yield sufficient protection. Hence, it may be useful to develop anti-ischemic compounds which act on a series of pathways involved in Ca++ overload, rather than selectively block one such channel.     

Activation of the fast calcium input in the denervated smooth muscle of the cat nictitating membrane

The excitation and contraction features of innervated and sympathetically denervated smooth muscle strips from cat's nictitating membrane have been studied by single sucrose gap arrangement. Increasing of smooth muscle cells sensitivity to drugs were accompanied by elevation of membrane response and the ability to generation of action potentials. Action potentials have been induced by agonists or high potassium concentration in external solution and spontaneously. In innervated muscle action potentials have been evoked as a result of depolarization by high potassium concentration of TEA blockade of potassium conductance. Induced and spontaneously generated action potentials were blocked by organic and inorganic antagonists of potential dependent Ca++ channels. In Ca-free solution action potentials were absent but might be supported by Ba++. Decrease of Na+ had no effect on smooth muscle excitability. It is supposed that activation of potential depended Ca++ channels in smooth muscle cells with pharmaco-mechanical coupling are under influence of sympathetic nerves.     

Presynaptic uptake blockade hypothesis for LSD action at the lateral inhibitory synapse in Limulus

We investigated the action of LSD at the putative indoleaminergic lateral inhibitory synapse in the lateral eye of Limulus polyphemus. We recorded extracellular and intracellular voltage responses from eccentric cells while producing inhibition either by light or by antidromic stimulation of the optic nerve in the presence of LSD, serotonin (5-HT), chlorimipramine, or a bathing medium whose high Mg++ and low Ca++ concentrations partially or completely blocked synaptic transmission. We found (a) light-evoked and antidromically stimulated lateral inhibition is enhanced during superfusion of low (1-5 microM) concentrations of LSD and suppressed by higher (5-20 microM) concentrations; (b) these actions of LSD are markedly reduced by bathing the retina in a medium high in Mg++ and low in Ca++; (c) very low concentrations of chlorimipramine, a putative uptake blocker of serotonin, appear to mimic actions of LSD both on eccentric cell firing rate and on lateral inhibition; (d) superfused 5-HT depresses lateral inhibition at all superthreshold concentrations (0.1-25 microM). These results suggest that LSD's action may require an intact inhibitory transmitter release and postsynaptic response mechanism, whereas serotonin exerts a direct postsynaptic effect. We propose that LSD blocks presynaptic uptake of transmitter at the lateral inhibitory synapse. The concentration dependence of LSD's action can be accounted for as follows: low concentrations partially restrict transmitter reuptake, thereby prolonging the lifetime of the transmitter in the synaptic cleft and thus increasing the magnitude and duration of postsynaptic inhibition. Higher concentrations cause more presynaptic uptake sites to be blocked; this causes accumulation of transmitter in the synaptic cleft, which causes a functional blockade of the synapse because of postsynaptic desensitization. As an alternative, we propose a hypothesis based on LSD action at presynaptic autoreceptors. Similar hypotheses can account for many aspects of LSD's action in mammalian brain.     

Exposure to 4-aminopyridine prevents depressant effects of opiates on sensory-evoked dorsal-horn network responses in spinal cord cultures

The depressant effects of morphine (0.1-1 microM) on sensory-evoked dorsal-horn network responses in explants of mouse spinal cord with attached dorsal root ganglia (DRGs) were rapidly restored after addition of 4-aminopyridine (4-AP; 0.1 mM) and major components of these cord responses were stably maintained in the presence of the opiate. Moreover, prior exposure of cord-DRG explants to 0.1 mM 4-AP prevented the depressant effects of 0.1 microM morphine on DRG-evoked dorsal-horn responses, and the effects of 1-10 microM morphine were at least partly antagonized. Increased Ca++ levels (5 microM) attenuated the depression of dorsal horn responses by 1-10 micro M morphine and these effects of Ca++ were greatly enhanced in the presence of 4-AP--in some cultures, concentrations of morphine as high as 100 micro M were strongly antagonized during test periods up to 2 hours. Receptor assays showed that 0.1 mM 4-AP +/- 5 mM Ca++ had no effect on stereospecific opiate binding, indicating that the antagonist actions of these agents in our cultures do not occur at the level of the opiate receptor. The relevance of our in vitro studies of 4-AP antagonism of opiate-depressant effects on sensory-evoked dorsal-horn network responses for analyses of problems in opiate analgesia has been strengthened by a recent report demonstrating that 4-AP does, in fact, reverse morphine analgesia in rats, as determined by tail flick tests.     

Potentiation of postjunctional cholinergic sensitivity of rat diaphragm muscle by high-energy-phosphate adenine nucleotides.

The cholinergic sensitivity of rat diaphragm muscle, me-sured as the magnitude of depolarization responses to repetitive, iontophoretic pulses of acetylcholine (ACh) onto neuromuscular endplates, is increased by addition of ATP to the perfusion medium. Depolarization responses begin to increase within the first min after addition of 10 mM ATP and plateau at 60% above control levels (mean value) after 4 to 6 min. Neither the magnitude nor the time course of the potentiations corresponds to changes in resting potential or membrane resistance. Other nucleotides are equally or less effective at the same concentration: ATP=ADP greater than UTP greater than AMP=GTP (=no added nucleotide control) The duration of the individual ACh responses does not increase during continuous exposure to the active nucleotides for up to 15 min except when the muscle is pretreated with eserine. Mild enzymatic predigestion of the muscle with collagenase and then protease, increasing the availability of the postjunctional membrane to bath-applied drugs, decreases the variability and increases the magnitude of the potentiation to a given dose of ATP. The dose-response curve for ATP is then more than half-maximal at 1 mM and the ranking of the other nucleotides relative to ATP is the same as without predigestion. There is an optimum Ca++ concentration for the potentiation between zero and 2 mM: potentiation is enhanced in Ca++ -free medium, partially blocked in twice-normal Ca++ medium, and totally blocked in Ca++ -free medium 10 min after a 5 min exposure to 2.5 mM EGTA. The similar Ca++ dependence of ACh receptor activation in the absence of added nucleotide suggests that ATP directly facilitates receptor activation by ACh. This facilitory action could be one of the physiological roles for the ATP released from stimulated phrenic nerve.     

Attractant-induced changes and oscillations of the extracellular Ca++ concentration in suspensions of differentiating Dictyostelium cells

We used a Ca++-sensitive electrode to measure changes in extracellular Ca++ concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation. The cells maintained an external level of 3-8 microM Ca++ until the beginning of aggregation and then started to take up Ca++. The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca++ by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2 microM folic acid, respectively. The apparent rate of uptake amounted to 2 X 10(7) Ca++ per cell per min. Following uptake, Ca++ was released by the cells with a rate of 5 X 10(6) ions per cell per min. Specificity studies indicated that the induced uptake of Ca++ was mediated by cell surface receptors. The amount of accumulated Ca++ remained constant as long as a constant stimulus was provided. No apparent adaptation occurred. The cyclic AMP-induced uptake of Ca++ increased during differentiation and was dependent on the external Ca++ concentration. Saturation was found above 10 microM external Ca++. The time course and magnitude of the attractant-induced uptake of external Ca++ agree with a role of Ca++ during contraction. During development the extracellular Ca++ level oscillated with a period of 6-11 min. The change of the extracellular Ca++ concentration during one cycle would correspond to a 30-fold change of the cellular free Ca++ concentration.     

Effectivity of murein preparations in the phage inhibition test--effect of Ca++ions and EDTA]

The inactivating efficacy for the typing phages 54, 83A, and 187 of the peptidoglycan of Staphylococcus aureus is decreased by increasing the Ca++-content of the medium; the irreversible inhibition of phage 187 becomes reversible. The percentage of its inhibition is proportional to the Ca++-content of the medium. Minute amounts of phage 187 bound irreversibly under Ca++-deprivation can be loosened by addition of Ca++-ions. Addition of EDTA up to the equivalence concentration of Ca++ present in the medium has no influence. Addition of EDTA up to the tenfold equivalence causes a significant increase of the inactivating efficacy of the peptidoglycan.     

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity.     

Enhancement of insulin release and islet cell calcium content by an acyl-amino-alkyl benzoic acid derivative, HB 699

HB 699, a hypoglycaemic agent which lacks the structural requirements regarded as essential for the insulin releasing action of the sulfonamide group, was studied in isolated rat islets and compared with tolbutamide. In the presence of 8.3 mM glucose for long exposure (24 h) both substances induced an increase of insulin release without altering the islet insulin content. After preloading of the islets to isotopic equilibrium with 45Ca++ (24 h), HB 699 and tolbutamide induced a significant increase in total exchangeable calcium content parallelled by an increase in insulin release. Both effects could be suppressed by verapamil, a blocker of voltage-sensitive Ca++ channels. These results indicate that HB 699, like tolbutamide, stimulates insulin release by increasing Ca++ uptake by the B cells.     

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.     

Action of insulin and cell calcium: effect of ionophore A23187.

We have measured the effects of the carboxylic Ca++ ionophore A23187 on muscle tension, resting potential and 3-O-methylglucose efflux. The ionophore produces an increase in tension that is dependent on external Ca++ concentration since (a) the contracture was blocked by removing external Ca++ and (b) its size was increased by raising outside Ca++. Neither resting potential nor resting and insulin-stimulated sugar efflux were modified by the ionophore. These data imply that the action of insulin is not mediated by increasing cytoplasmic [Ca++]. Additional support for this conclusion was obtained by testing the effects of caffeine on sugar efflux. This agent, which releases Ca++ from the reticulum, did not increase resting sugar efflux and inhibited the insulin-stimulated efflux. Incubation in solutions containing butyrated derivatives of cyclic AMP or cyclic GMP plus theophylline did not modify the effects of insulin on sugar efflux. Evidence suggesting that our experimental conditions increased the cytoplasmic cyclic AMP activity was obtained.     

Mitogen and co-mitogen stimulation of lymphocytes inhibited by three Ca++ antagonists

The Ca++ requirement for in vitro lymphocyte stimulation by lectins is well known and can be demonstrated by the use of Ca++ chelators. In this study, three Ca++ antagonists were examined for their effects on lymphocyte proliferation. [3H]-thymidine incorporation was employed to measure DNA synthesis in several systems. Stimulation and proliferation were achieved by the addition of one of the following: the mitogenic lectin concanavalin A (ConA); the combination of two co-mitogens, the calcium ionophore A23187 and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), neither of which is mitogenic alone; or the non-mitogenic lectin, wheat germ agglutinin (WGA) with TPA. These mitogenic systems were tested for their sensitivity to the Ca++ channel blockers verapamil and nicardipine and the intracellular Ca++ antagonist TMB-8. We found that the ConA and WGA plus TPA treated cells were inhibited approximately 50% by 10 microM verapamil, nicardipine or TMB-8. The stimulation caused by A23187 and TPA was only inhibited by TMB-8 and nicardipine. The inhibitory effects caused by the Ca++ antagonists could not be reversed by the addition of exogenous Ca++ (0.1-1.5 mM), but were reversed by repeated washings in antagonist free media. Using TMB-8 we saw an apparent intracellular Ca++ dependence throughout the G1 phase. Previous studies using Ca++ chelators or Ca++ antagonists suggested an endpoint at about halfway through this period.