"Calcium antagonists": a class of drugs with a bright future. Part II. Determination of basic pharmacological properties

This minireview discusses some simple pharmacological tests useful in detecting biological activity (screening), characterizing mechanisms of action and predicting possible therapeutic applications for calcium antagonists in general and calcium slow channel blockers in particular. In smooth muscle preparations these agents inhibit mechanical effects evoked by K+-depolarization which selectively opens voltage-operated calcium channels (VOC) to allow extracellular Ca++ into the cytosol. In contrast, any inhibition of receptor-mediated responses by calcium antagonists appears to depend on the transduction system and the specific cellular mechanism (e.g. VOC opening consequent to partial depolarization) activated by the receptor and, evidently, on ancillary pharmacological properties of the studied compound. For instance, whereas calcium slow channel blockers antagonize contractions produced by norepinephrine and K+-depolarization in the rat isolated portal vein, they inhibit effectively only the latter response in the rabbit aorta. This apparent discrepancy may be accounted for by the different pool of Ca++ mobilized in the two tissues by norepinephrine. Agents (e.g. diphenylalkylamines, calmodulin blockers) that impair the interaction of Ca++ with intracellular proteins produce effects which are less specific than those of slow channel blockers. Currently, the pharmacological profile of calcium antagonists can be appropriately defined by studying their effects on radioligand (dihydropyridine) binding, radioactive calcium movements through biological membranes, electrophysiological parameters in cardiac and vascular smooth muscle and on various in vivo cardiovascular preparations. Together, these approaches allow a functional classification of new calcium antagonists in relation to already known compounds and some hypotheses on their potential clinical applications. Finally, desirable pharmacokinetics and pharmacological properties for novel calcium antagonists are mentioned. This point will be further explored in the forthcoming minireview which will deal with the clinical applications of calcium antagonists.     

The effects of extracellular K+ and angiotensin II on cytosolic Ca++ and steroidogenesis in adrenal glomerulosa cells

We evaluated changes in cytosolic calcium concentration (Ca++) and steroidogenesis in rat adrenal glomerulosa cells (GC) stimulated with potassium (K+) or angiotensin II (AII). Cytosolic Ca++ concentration was determined using the Ca++-sensitive, fluorescent dye QUIN 2. Raising extracellular K+ increased cytosolic Ca++ from 267 +/- 23 nM at 3.7 mM K+ to a maximum of 377 +/- 40 nM at 8.7 mM K+ (p less than 0.01, N = 23). AII also increased cytosolic Ca++ from 238 +/- 20 nM to a maximum of 427 +/- 42 nM at 10(-7) M (p less than 0.01, N = 16). In parallel studies, K+ and AII stimulated aldosterone secretion from QUIN 2-loaded GC at concentrations similar to those which raised cytosolic Ca++. QUIN 2-loaded cells were as responsive steroidogenically as unloaded cells and showed trypan blue exclusion of 98% suggesting that QUIN 2 did not compromise cellular viability. These results provide direct support for a role of cytosolic Ca++ as a second messenger during stimulation of aldosterone secretion by both K+ and AII.     

Disease causing mutations of calcium channels

Calcium ions play an important role in the electrical excitability of nerve and muscle, as well as serving as a critical second messenger for diverse cellular functions. As a result, mutations of genes encoding calcium channels may have subtle affects on channel function yet strongly perturb cellular behavior. This review discusses the effects of calcium channel mutations on channel function, the pathological consequences for cellular physiology, and possible links between altered channel function and disease. Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca(2+) ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca(2+) stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores.     

Regulation of cytosolic free calcium concentration by intrasynaptic mitochondria.

By the use of digitonin permeabilized presynaptic nerve terminals (synaptosomes), we have found that intrasynaptic mitochondria, when studied "in situ," i.e., surrounded by their cytosolic environment, are able to buffer calcium in a range of calcium concentrations close to those usually present in the cytosol of resting synaptosomes. Adenine nucleotides and polyamines, which are usually lost during isolation of mitochondria, greatly improve the calcium-sequestering activity of mitochondria in permeabilized synaptosomes. The hypothesis that the mitochondria contributes to calcium homeostasis at low resting cytosolic free calcium concentration ([Ca2+]i) in synaptosomes has been tested; it has been found that in fact this is the case. Intrasynaptic mitochondria actively accumulates calcium at [Ca2+]i around 10(-7) M, and this activity is necessary for the regulation of [Ca2+]i. When compared with other membrane-limited calcium pools, it was found that depending on external concentration the calcium pool mobilized from mitochondria is similar or even greater than the IP3- or caffeine-sensitive calcium pools. In summary, the results presented argue in favor of a more prominent role of mitochondria in regulating [Ca2+]i in presynaptic nerve terminals, a role that should be reconsidered for other cellular types in light of the present evidence.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

Characterization of the Ca2+ influx into embryonic cells after stimulation of the embryonic muscarinic receptor.

Embryonic cells transiently express an embryonic muscarinic system during morphogenesis. Stimulation of the embryonic muscarinic receptor results in biphasic intracellular Ca2+ mobilization: an initial "peak" due to Ca2+ release from intracellular stores is followed by a sustained "plateau" of enhanced cytoplasmic Ca2+ due to influx of extracellular Ca2+. In the present investigation, we characterized the Ca2+ influx by measuring the cytoplasmic free Ca2+ concentration [Ca2+]i using the Ca2+ indicator fura-2: 1. The increase of [Ca2+]i during the plateau depended linearly on the logarithm of the extracellular calcium concentration whereas the initial peak was almost independent from extracellular calcium. 2. The organic Ca2+ entry blockers verapamil, gallopamil, nifedipine, nitrendipine and the inorganic blockers Mn2+, Mg2+ and La3+ were without effect on both phases of Ca2+ mobilization. Only Ni2+ at concentrations above 1 mM was able to reduce the influx without affecting the intracellular Ca2+ release. 3. Substitution of extracellular Na+ by guanidine+, choline+ or tris+ and membrane depolarisation by increasing the extracellular K+ concentration had no effect on either phase of Ca2+ mobilization. We conclude that a non-voltage dependent, receptor-operated influx mechanism, probably a "second messenger operated Ca2+ channel", is responsible for the Ca2+ influx after stimulation of the embryonic muscarinic receptor.     

A direct mass-action mechanism explains capacitative calcium entry in Jurkat and skeletal L6 muscle cells

We examined capacitative calcium entry (CCE) in Jurkat and in L6 skeletal muscle cells. We found that extracellular Ca2+ can enter the endoplasmic reticulum (ER) of both cell types even in the presence of thapsigargin, which blocks entry into the ER from the cytosol through the CaATPase. Moreover, extracellular Ca2+ entry into the ER was evident even when intracellular flow of Ca2+ was in the direction of ER to cytosol due to the presence of caffeine. ER Ca2+ content was assessed by two separate means. First, we used the Mag-Fura fluorescent dye, which is sensitive only to the relatively high concentrations of Ca2+ found in the ER. Second, we transiently expressed an ER-targeted derivative of aequorin, which reports Ca2+ by luminescence. In both cases, the Ca2+ concentration in the ER increased in response to extracellular Ca2+ after the ER had been previously depleted despite blockade by thapsigargin. We found two differences between the Jurkat and L6 cells. L6, but not Jurkat cells, inhibited Ca2+ uptake at very high Ca2+ concentrations. Second, ryanodine receptor blockers inhibited the appearance of cytosolic Ca2+ during CCE if added before Ca2+ in both cases, but the L6 cells were much more sensitive to ryanodine. Both of these can be explained by the known difference in ryanodine receptors between these cell types. These findings imply that the origin of cytosolic Ca2+ during CCE is the ER. Furthermore, kinetic data demonstrated that Ca2+ filled the ER before the cytosol during CCE. Our results suggest a plasma membrane Ca2+ channel and an ER Ca2+ channel joined in tandem, allowing Ca2+ to flow directly from the extracellular space to the ER. This explains CCE; any decrease in ER [Ca2+] relative to extracellular [Ca2+] would provide the gradient for refilling the ER through a mass-action mechanism.     

Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production

The recently available compound quin-2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0-2 production by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin-2, the production of 0-2 was measured in conjunction with the transfer of 45Ca+2 and changes in quin-2 fluorescence upon stimulation with FNLLP. When cells were maintained in low (10 microM) extracellular calcium medium the presence of 1.5 mM quin-2 in the cytosolic space partially inhibited the rate of 0-2 production upon stimulation by FNLLP. Addition of 1 mM Ca+2 to the medium prior to stimulation rapidly restored the cell's capability to produce 0-2 upon stimulation at rates equal to control and extended the duration of stimulated 0-2 production as well. Quin-2 fluorescence measurements indicated an increase in cytosolic Ca+2 upon stimulation with FNLLP. This increase was lowest under conditions in which 0-2 production was inhibited. The addition of 1 mM Ca+2 to the medium caused by itself a rapid but transient increase in cytosolic Ca+2 as measured with quin-2 without stimulating 0-2 production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2 transfer demonstrated a buffering of cytosolic Ca+2 changes by quin-2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0-2 response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0-2 production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimulation is a concomitant but nonregulatory event only.     

Phosphorylation and the control of calcium fluxes

Cell activation, e.g. stimulus-contraction or stimulus-secretion coupling, is brought about by a 100-fold increase in cytosolic free Ca2+ concentration from 0.1 to 10 microM, upon release of Ca2+ from intrareticular or extracellular stores along the concentration gradient. A return to steady state is achieved by either Na+-Ca2+ exchange or ATP-dependent Ca2+ transport against the concentration gradient. Both processes, Ca2+ influx and Ca2+ efflux, are regulated by sophisticated covalent mechanisms. The positive inotropic effect of adrenalin is mediated by the cyclic-AMP-dependent phosphorylation of cardiac sarcolemmal proteins, among which calciductin is the major phosphate acceptor. Upon cyclic-AMP-dependent phosphorylation, the slow Ca2+ channel is activated 3.5 time above its basal low-conductance state, and retains its characteristics, competition by divalent metals, inhibition by La3+ and Ca2+ entry blockers. The adrenalin-induced abbreviation of systole is also explained in terms of the dual phosphorylation of the cardiac sarcoplasmic reticulum calcium pump activator, phospholamban, by cyclic-AMP-dependent protein kinase on the one hand and Ca2+-calmodulin-dependent phospholamban kinase on the other. Calciductin and phospholamban are closely similar acidic proteolipids. A phospholamban-like protein is also found in platelet Ca2+-accumulating vesicles, where its cyclic-AMP-dependent phosphorylation doubles the rate of Ca2+ efflux. These observations raise the possibility that calcium fluxes are regulated by phosphorylation of membrane-bound proteolipids. More generally, phosphorylation modulates K+, Na+ and Ca2+ fluxes through membranes, i.e. the general excitability properties of the cell.     

Mechanisms involved in the cellular calcium homeostasis in vascular smooth muscle: calcium pumps

The regulation of cytosolic Ca2+ homeostasis is essential for cells, and particularly for vascular smooth muscle cells. In this regulation, there is a participation of different factors and mechanisms situated at different levels in the cell, among them Ca2+ pumps play an important role. Thus, Ca2+ pump, to extrude Ca2+; Na+/Ca2+ exchanger; and different Ca2+ channels for Ca2+ entry are placed in the plasma membrane. In addition, the inner and outer surfaces of the plasmalemma possess the ability to bind Ca2+ that can be released by different agonists. The sarcoplasmic reticulum has an active role in this Ca2+ regulation; its membrane has a Ca2+ pump that facilitates luminal Ca2+ accumulation, thus reducing the cytosolic free Ca2+ concentration. This pump can be inhibited by different agents. Physiologically, its activity is regulated by the protein phospholamban; thus, when it is in its unphosphorylated state such a Ca2+ pump is inhibited. The sarcoplasmic reticulum membrane also possesses receptors for 1,4,5-inositol trisphosphate and ryanodine, which upon activation facilitates Ca2+ release from this store. The sarcoplasmic reticulum and the plasmalemma form the superficial buffer barrier that is considered as an effective barrier for Ca2+ influx. The cytosol possesses different proteins and several inorganic compounds with a Ca2+ buffering capacity. The hypothesis of capacitative Ca2+ entry into smooth muscle across the plasma membrane after intracellular store depletion and its mechanisms of inhibition and activation is also commented.     

A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering by cytoplasmic calcium binding proteins. Numerical integration of the model allows us to study the fluctuations in the cytosolic calcium concentration, the ER membrane potential, and the concentration of free calcium binding sites on a calcium binding protein. The model demonstrates the physiological features necessary for calcium oscillations and suggests that the level of calcium flux into the cytosol controls the frequency and amplitude of oscillations. The model also suggests that the level of buffering affects the frequency and amplitude of the oscillations. The model is supported by experiments indirectly measuring cytosolic calcium by calcium-induced chloride currents in Xenopus oocytes as well as cytosolic calcium oscillations observed in other preparations.     

Extracellular calcium ion depletion in frog cardiac ventricular muscle.

The extracellular free [Ca++] in frog ventricular muscle strips was monitored using single-barrel calcium ion-selective microelectrodes. During trains of repetitive stimulation, a heart rate-dependent, sustained fall (depletion) of the extracellular free [Ca++] occurs, which is most likely a consequence of net Ca++ influx into ventricular cells. The magnitude of the [Ca++]0 depletion increases for higher Ringer's solution [Ca++], and is reversibly blocked by manganese ion. Prolonged repetitive field stimulation (20-30 min) activates additional cellular Ca++ efflux, which can balance the additional Ca++ influx caused by stimulation, resulting in abolition of extratrabecular [Ca++]0 depletion in 20-30 min, and hence zero net transmembrane Ca++ flux at steady state. In the poststimulation period of quiescence, cellular Ca++ efflux persists and causes an elevation (accumulation) of the extracellular free [Ca++]. From these [Ca++]0 depletions, quantitative estimates for the net transmembrane Ca++ flux were derived using an analytical solution to the diffusion equation. In the highest Ringer's solution [Ca++] used (1 mM) the calculated net increase of the total intracellular calcium per beat was 6.5 +/- 1.4 mumol/l of intracellular space. This corresponds to an average net transmembrane Ca++ influx of 0.81 +/- 0.17 pmol/cm2/s during the 800-ms action potential. In lower bath [Ca++] the net transmembrane [Ca++] flux was proportionately reduced.     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.     

The calcium dependence of pigment translocation in freshwater shrimp red ovarian chromatophores

The roles of calcium in cell signaling consequent to chromatophorotropin action and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chromatosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes were perfused with known concentrations of free Ca++ (10(-3) to 10(-9) M) prepared in Mg(++)-EGTA-buffered physiological saline after selectively permeabilizing with 25 microM calcium ionophore A23187 or with 10(-8) M red pigment concentrating hormone (RPCH). The degree of pigment aggregation and the translocation velocity of the leading edges of the pigment mass were recorded in individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca++. Aggregation is Ca++ dependent, showing a dual extracellular and intracellular requirement. After perfusion with reduced Ca++ (10(-4) to 10(-9) M), RPCH triggers partial aggregation (approximately 65%), although the maximum translocation velocities (approximately 16.5 microns/min) and velocity profiles are unaffected. After aggregation induced at or below 10(-5) M Ca++, spontaneous pigment dispersion ensues, suggesting a Ca++ requirement for RPCH coupling to its receptor, or a concentration-dependent, Ca(++)-induced Ca(++)-release mechanism. The Ca(++)-channel blockers Mn++ (5 mM) and verapamil (50 microM) have no effect on RPCH-triggered aggregation. An intracellular Ca++ requirement for aggregation was demonstrated in chromatosomes in which the Ca++ gradient across the cell membrane was dissipated with A23187. At free [Ca++] above 10(-3) M, aggregation is complete; at 10(-4) M, aggregation is partial, followed by spontaneous dispersion; below 10(-5) M Ca++, pigments do not aggregate but disperse slightly. Aggregation velocities diminish from 11.6 +/- 1.2 microns/min at 5.5 mM Ca++ to 7.4 +/- 1.3 microns/min at 10(-4) M Ca++. Half-maximum aggregation occurs at 3.2 x 10(-5) M Ca++ and half-maximum translocation velocity at 4.8 x 10(-5) M Ca++. Pigment redispersion after 5.5 mM Ca(++)-A23187-induced aggregation is initiated by reducing extracellular Ca++: slight dispersion begins at 10(-7) M, complete dispersion being attained at 10(-9) M Ca++. Dispersion velocities increase from 0.6 +/- 0.2 to 3.1 +/- 0.5 microns/min. Half-maximum dispersion occurs at 7.6 x 10(-9) M Ca++ and half-maximum translocation velocity at 2.9 x 10(-9) M Ca++. These data reveal an extracellular and an intracellular Ca++ requirement for RPCH action, and demonstrate that the centripetal or centrifugal direction of pigment movement, the translocation velocity, and the degree of pigment aggregation or dispersion attained are calcium-dependent properties of the granule translocation apparatus.     

Dual pathways of calcium entry in spike and plateau phases of luteinizing hormone release from chicken pituitary cells: sequential activation of receptor-operated and voltage-sensitive calcium channels by gonadotropin-releasing hormone

It has previously been shown that, in pituitary gonadotrope cells, the initial rise in cytosolic Ca2+ induced by GnRH is due to a Ca2+ mobilization from intracellular stores. This raises the possibility that the initial transient spike phase of LH release might be fully or partially independent of extracellular Ca2+. We have therefore characterized the extracellular Ca2+ requirements, and the sensitivity to Ca2+ channel blockers, of the spike and plateau phases of secretion separately. In the absence of extracellular Ca2+ the spike and plateau phases were inhibited by 65 +/- 4% and 106 +/- 3%, respectively. Both phases exhibited a similar dependence on concentration of extracellular Ca2+. However, voltage-sensitive Ca2+ channel blockers D600 and nifedipine had a negligible effect on the spike phase, while inhibiting the plateau phase by approximately 50%. In contrast, ruthenium red, Gd3+ ions, and Co2+ ions inhibited both spike and plateau phases to a similar extent as removal of extracellular Ca2+. A fraction (35 +/- 4%) of spike phase release was resistant to removal of extracellular Ca2+. This fraction was abolished after calcium depletion of the cells by preincubation with EGTA in the presence of calcium ionophore A23187, indicating that it depends on intracellular Ca2+ stores. Neither absence of extracellular Ca2+, nor the presence of ruthenium red or Gd3+ prevented mobilization of 45Ca2+ from intracellular stores by GnRH. We conclude that mobilization of intracellular stored Ca2+ is insufficient by itself to account for full spike phase LH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.     

Calcium- Versus G Protein-Mediated Phosphoinositide Hydrolysis in Rat Cerebral Cortical Synaptoneurosomes

The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0-1.0 mM), basal, potassium-stimulated, and norepinephrine-stimulated formation of 3H-inositol phosphates decreased with decreasing extracellular calcium. Potassium- and norepinephrine-stimulated formation of 3H-inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N-methyl-D-glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H-inositol phosphates. Measurement of cytosolic calcium with fura-2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium-free rather than sodium-containing medium. In the absence of sodium, potassium-stimulated formation of 3H-inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H-inositol phosphates from that in the presence of 1 mM carbachol or 100 microM norepinephrine revealed that the carbachol-stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine-stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate inhibited norepinephrine- and, to a lesser extent, carbachol but not basal or aluminum fluoride-stimulated formation of 3H-inositol phosphates in sodium-free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization-induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling

The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti-alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface.     

Involvement of calcium and cyclic AMP in controlling ixodid tick salivary fluid secretion.

Catecholamine-stimulated salivary fluid secretion (in vitro) by ixodid ticks is reduced by deletion or lowering the concentration of exogenous bathing medium Ca++. The Ca++ antagonist, verapamil, reversibly inhibits dopamine-stimulated secretion. Ionophore A-23187 is unable to induce glands to secrete. Studies in which labeled and unlabeled Ca++ flux were measured indicate that catecholamines induce release of calcium from intracellular stores during secretion. Cyclic AMP/theophylline-stimulated secretion is inhibited by verapamil, and the exclusion of calcium from the support medium. It is concluded that the primary catecholamine stimulus induces cyclic AMP formation and mobilization of Ca++ (intra- and extracellular). Cyclic AMP and calcium are thought to interact to control secretion within the fluid transporting cells of types II and III alveoli.     

The effect of Na+ on Ca2+ homeostasis in unstimulated platelets

Platelets maintain a low cytosolic free Ca2+ concentration by limiting Ca2+ influx from plasma and promoting Ca2+ efflux. The present studies examine the role of the plasma membrane Na+ gradient in these processes. The Na+ gradient in intact unstimulated platelets was altered by incubating the platelets with ouabain or by replacing extracellular Na+ with N-methyl-D-glucamine or choline. Ca2+ flux across the plasma membrane and the amount of exchangeable Ca2+ in the platelet cytosol were measured by observing 45Ca2+ influx and efflux under steady-state conditions. The cytosolic free Ca2+ concentration was measured with the fluorescent probe quin2. At extracellular Na+ concentrations below 50 mM, the size of the cytosolic exchangeable Ca2+ pool increased by 48%. The size of the exchangeable Ca2+ pool sequestered in the dense tubular system increased by 356%. Ca2+ flux across the plasma membrane increased by 38%. There was, however, no change in total platelet Ca2+ and little, if any, change in the cytosolic free Ca2+ concentration. Similar effects were produced by incubating platelets with ouabain. These observations demonstrate a marked influence of the plasma membrane Na+ gradient on Ca2+ homeostasis in platelets. The nature of the changes, however, suggests that Na+/Ca2+ exchange cannot be sole basis for Ca2+ efflux from platelets.