Possible role of Ca ions in the resting metabolism of frog sartorius muscle during potassium depolarization

Oxygen consumption and Ca exchangeability at different levels of potassium depolarization were studied in frog sartorius muscle. It was found that the changes in oxygen consumption parallel the changes in Ca exchangeability. Procaine (10^?3 M) and CaCl₂ (2.10?2 M) suppressed both extra oxygen consumption and Ca exchangeability at low values of depolarization. At higher values of depolarization procaine and CaCl₂ differed in their action. Procaine favored inhibition of these processes, CaCl₂ caused their activation. The effects of these compounds was not a result of a change in the membrane potential, since their effect on potassium depolarization was found to be small. Relations between oxygen consumption and Ca exchangeability similar to those observed at potassium depolarization seem to exist under conditions where caffeine was applied. It is proposed that the extra oxygen consumption caused by potassium depolarization or on application of caffeine and unaccompanied by mechanical changes is related to the release of Ca from its bound form. Oxygen consumption in isotonic sucrose solution was also studied, but some different data from the above were obtained.     

The role of free calcium ion in calcium release in skinned muscle fibers

Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.     

Inositol-1,4,5-trisphosphate releases calcium from skinned cultured smooth muscle cells

We examined the effects of inositol-1,4,5-trisphosphate on 45Ca uptake and 45Ca efflux in the saponin skinned primary cultured rat aortic smooth muscle cells. 10 microM inositol-1,4,5-trisphosphate induced a rapid (half time less than 10 sec) and large quantity of Ca release in both 45Ca uptake and 45Ca efflux in the skinned cells preloaded with 1 microM free Ca. Dose response curves showed that 100 microM inositol-1,4,5-trisphosphate produced a maximal Ca release of 97.3% of the MgATP dependent 45Ca uptake or 289 mumoles/liter cells, which was much greater than the maximal caffeine induced Ca release and would be sufficient to produce maximal tension.     

The effect of caffeine on calcium efflux and calcium translocation in skeletal and visceral muscle.

1. KCl-induced depolarization resulted in a large stimulation of the 45Ca efflux from both cockroach skeletal muscle and rat ileal smooth muscle. 2. Caffeine (10 mM) induced a large stimulation of 45Ca efflux from skeletal muscle, but a fall in the efflux from ileal muscle, especially if the efflux was previously stimulated by KCl depolarization. 3. Caffeine inhibited calcium uptake by skeletal muscle mitochondria and sarcoplasmic reticulum, was without effect on ileal muscle mitochondria, but significantly increased caclium binding by ileal muscle membrane vesicular preparations. 4. The induction of contractures and stimulation of 45Ca efflux in skeletal muscle by caffeine are clearly related to inhibition of intracellular calcium binding by the sarcoplasmic reticulum and mitochondria. 5. The relaxation of ileal muscle by caffeine and the inhibition of fibre calcium efflux correlate well with caffeine enhancement of intracellular calcium binding. These experiments suggest that the membrane vesicular compartment may be the main agency centrally involved in fibre calcium regulation in this muscle during the contraction-relaxation cycle.     

An antihypertensive extract of erythrocytes: effects on 45Ca uptake and efflux

The present report describes some aspects of the effects of a recently described antihypertensive extract of erythrocytes (AHF) on calcium uptake and efflux in rat aortae. AHF was found to be present in the erythrocytes of both spontaneously hypertensive rats and normotensive rats. Furthermore, AHF obtained from erythrocytes of SH rats was shown to be equally effective in suppressing lanthanum-resistant calcium uptake in aortae from hypertensive and normotensive rats. AHF treatment prior to incubation of aortae with 45Ca caused an apparent increase in the total 45Ca uptake. The analysis of calcium washout curves obtained for tissue in calcium-free or lanthanum-containing media indicated that AHF had no significant effect on the rate of calcium loss from the slow component of efflux, though this compartment tended to be reduced in size. This indicated that the increase in the 45Ca content of AHF-exposed aortae prior to rinsing was confined to the rapid component of efflux. The loss of calcium from the rapidly exchanging compartment was enhanced in either of the efflux media used. The results suggest that a principal action of AHF involves an increase in the lability and exchangeability of calcium stores. In addition to its effects in resting tissue, AHF abolished the increase in lanthanum-resistant calcium uptake induced in rat aortae by the addition of high K+ or norepinephrine to the incubation media. In a second part of the study, the effect of AHF on blood pressure and in vitro calcium uptake were compared with that of phosphatidylethanolamine (PEA), the probable identity of another endogenous antihypertensive (renin preinhibitor) compound earlier shown to share important functional similarities with AHF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnesium effects on activation of skinned fibers from striated muscle

The intracellular Ca movements that control contraction and relaxation of striated muscle are regulated by the membrane potential and influenced by Mg2+. In skinned fibers, the internal composition can be manipulated directly by Ca movements estimated from isometric force transients, net changes in sarcoplasmic reticulum (SR) Ca, and 45Ca flux between fiber and bath. Stimulated Ca release, unlike unstimulated 45Ca efflux at low external [Ca2+], is highly [Mg2+]-sensitive at 20 C. Force and tracer measurements indicate three major sites of Mg2+-Ca2+ interaction in situ: Mg2+ can stimulate the SR active Ca transport system, inhibit a Ca2+-dependent Ca efflux pathway of SR, and shift the force-[Ca2+] relation, presumably by reducing Ca2+ binding to myofilament regulatory sites. These mechanisms constrain the resting Ca flux and are adaptive during relaxation. However, analysis of CI-stimulated 45Ca release and reaccumulation suggests that the depolarization process may inhibit Mg2+-dependent Ca influx, the membrane potential controlling both efflux and influx; recent studies on voltage-clamped cut fibers support this hypothesis. The Ca2+ and Mg2+ dependence of caffeine-stimulated 45Ca efflux suggests that Mg2+ inhibition of the Ca2+-dependent efflux pathway is small during rapid Ca2+ efflux. Therefore, both Mg2+ mechanisms, which minimize net release, may be reversed during normal activation.     

Properties of chloride-stimulated 45Ca flux in skinned muscle fibers

Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

Presence of Na+/Ca2+ exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells.

The presence of a Na+/Ca2+ exchanger in bovine adrenal chromaffin cells was demonstrated by measuring the efflux of 45Ca2+ which had been preloaded into cells by a brief depolarization. The efflux of 45Ca2+ was dependent on extracellular Na+ (Na+o); 45Ca2+ efflux was significantly decreased by replacing Na+o with N-methylglucamine (NMG), or Li+. Replacement of Na+o by NMG increased the resting intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated chromaffin cells. This could be reversed by adding Na+, suggesting that Na+/Ca2+ exchanger activity was involved in maintaining [Ca2+]i at its resting level. The initial rate of Na(+)-dependent [Ca2+]i recovery after Ca2+ loading by depolarization was dependent on the level of [Ca2+]i. There was an apparent linear relationship between the activity of the Na+/Ca2+ exchanger and [Ca2+]i both in the presence and absence of Na+o. When cells were treated with other stimuli, including 10 microM DMPP or 40 mM caffeine, the ability of the stimulated cells to decrease [Ca2+]i was significantly reduced upon replacing Na+o with NMG. Our data show that the Na+/Ca2+ exchanger is one of the major pathways for regulating [Ca2+]i in chromaffin cells in both resting and stimulated states.     

Excitation of skinned muscle fibers by imposed ion gradients. IV. Effects of stretch and perchlorate ion

Depolarizing ion gradients stimulate 45Ca release in skeletal muscle fibers skinned by microdissection. Several lines of indirect evidence suggest that sealed transverse (T) tubules rather than sarcoplasmic reticulum (SR) are the locus of such stimulatory depolarization. Two implications of this hypothesis were tested. (a) A requirement for signal transmission was evaluated from the stimulation of 45Ca efflux in fibers that had been highly stretched, an intervention that can impair the electrical stimulation of intact fibers. Length was increased over approximately 95-115 s, after loading with 45Ca and rinsing at normal length; prestimulus 45Ca loss due to stretch itself was very small. In the first study, stimulation of 45Ca release by KCl replacement of K propionate was inhibited completely in fibers stretched to twice slack length, compared with fibers at 1.05-1.1 times slack length. Identical protocols did not alter 45Ca release stimulated by caffeine or Mg2+ reduction, implying that SR Ca release per se was fully functional and inhibition was selective for a preceding step in ionic stimulation. In a second study, stimulation by choline Cl replacement of K methanesulfonate, at constant [K+] [Cl-] product, was inhibited strongly; total 45Ca release decreased 69%, and stimulation above control loss decreased 78%, in segments stretched to twice the length at which sarcomere spacing had been 2.2 micron, compared with paired controls from the same fibers kept at 2.3 micron. (b) Perchlorate potentiation of T tubule activation was evaluated in fibers stimulated at constant [K+] [Cl-] at normal length (2.3 micron); this anion shifts the voltage dependence of intramembrane charge movement and contractile activation in intact fibers. Perchlorate (8 mM) potentiated both submaximal stimulation of Ca2+-dependent 45Ca release by partial choline Cl replacement of K methanesulfonate and the small Ca2+-insensitive 45Ca efflux component stimulated by nearly full replacement in the presence of 5 mM EGTA. These results provide independent support for the hypothesis that the T tubules are the locus of stimulation by depolarizing ion gradients, with junctional transmission of this signal causing SR 45Ca release.     

Intracellular Ca release in skinned smooth muscle

The release of internal Ca from saponin-treated skinned smooth muscle of guinea pig taenia caecum was studied. The amount of Ca released was estimated by the area under the contraction curve during treatment with 25 mM caffeine in the presence of 0.1 mM EGTA. The magnitude of the caffeine response in skinned muscle, after loading with 10(-6) M Ca for 3 min, was similar to that in the depolarized muscle in the presence of EGTA before treatment with saponin. This suggests that Ca in the skinned muscle was in a physiological range after loading. The release of Ca from the storage site could be facilitated by Ca itself when the skinned muscle was exposed to Ca above 3 x 10(-6) M. An increase in environmental MG concentration suppressed the Ca-induced Ca release mechanism. Sudden replacement of propionate with Cl in the bathing solution made it possible to release Ca from the storage site. This "depolarization"-induced Ca release occurred only immediately after the application of Cl; thereafter, the Ca release mechanism seemed to be inactivated by the prolonged presence of Cl. These results suggest that two mechanisms of Ca release operate in smooth muscle: (a) release induced by Ca itself, and (b) release by "depolarization".     

Kinetic studies of calcium release from sarcoplasmic reticulum in vitro

Release of Ca2+ from a heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum was triggered by several different methods: (a) increasing extravesicular [Ca2+] [( CaO2+] from about 0.1 microM to 10 microM), (b), adding caffeine, (c) adding quercetin, and (d) substituting a solution containing equimolar choline+ for K+-containing solution (depolarization-induced Ca2+ release). The maximal rate of Ca2+ release triggered by caffeine or quercetin in the presence of 12.5 microM [CaO2+] (21-25 nmol of Ca2+/mg/s) is similar to that of the depolarization-induced Ca2+ release (19 nmol of Ca2+/mg/s), as determined by stopped flow spectrometry of changes in [CaO2+] with arsenazo III. The release is transient and all of the released Ca2+ is reaccumulated. The rates of Ca2+ release triggered by caffeine, quercetin, or membrane depolarization sharply decrease at high [CaO2+], suggesting a negative feedback effect of the released Ca2+. Inhibition of the release pathway allows the sarcoplasmic reticulum to reaccumulate Ca2+. The rate of Ca2+ release triggered by caffeine or quercetin, but not that triggered by membrane depolarization, is also reduced upon decreasing [CaO2+] to the submicromolar range. Passive efflux of intravesicular Ca2+ in solutions containing lower [CaO2+] in the absence of Mg.ATP is attenuated at about the same time (congruent to 1 min) regardless of the amounts of Ca2+ released, indicating that the opened Ca2+ channels close spontaneously. These results suggest that kinetically identical channels are responsible for Ca2+ release independent of the methods of triggering and this in vitro release is consistent with the physiological mechanism both in terms of the rapidity and the reversibility of Ca2+ release.     

Effects of 1,25-dihydroxyvitamin D3 on bone resorption and 45Ca2+ efflux in bone cells

1,25-dihydroxyvitamin D3[1,25(OH)2D3] effects on bone resorption in organ culture and on 45Ca2+ efflux rates in bone cells were measured in presence of a calcium channel inhibitor, diltiazem. Though, diltiazem reduced the 45Ca release from mice calvaria it did not act at the same Ca compartment as 1,25(OH)2D3 to alter Ca2+ flux parameters. It therefore seems difficult to hypothesize a simple relationship between bone resorption and Ca2+ movements in bone cells.     

Peeled mammalian skeletal muscle fibers. Possible stimulation of Ca2+ release via a transverse tubule-sarcoplasmic reticulum mechanism

Single muscle fibers from rabbit soleus and adductor magnus and from semitendinosus muscles were peeled to remove the sarcolemma and then stimulated to release Ca2+ by (a) caffeine application or (b) ionic depolarization accomplished via substitution of choline chloride for potassium propionate at constant [K+] X [Cl-] in the bathing solution. Each stimulus, ionic or caffeine, elicited an isometric tension transient that appeared to be due to Ca2+ released from the sarcoplasmic reticulum (SR). The peak magnitude of the ionic (Cl- -induced) tension transient increased with increasing Cl- concentration. The application of ouabain to fibers after peeling had no effect on either type of tension transient. However, soaking the fibers in a ouabain solution before peeling blocked the Cl- -induced but not the caffeine-induced tension transient, which suggests that ouabain's site of action is extracellular, perhaps inside transverse tubules (TTs). Treating the peeled fibers with saponin, which should disrupt TTs to a greater extent than SR membrane, greatly reduced or eliminated the Cl- -induced tension transient without significantly altering the caffeine-induced tension transient. These results suggest that the Cl- -induced tension transient is elicited via stimulation of sealed, polarized TTs rather than via ionic depolarization of the SR.     

Ca2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent.     

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Bromo-eudistomin D, a novel inducer of calcium release from fragmented sarcoplasmic reticulum that causes contractions of skinned muscle fibers

Bromo-eudistomin D induced a contraction of the chemically skinned fibers from skeletal muscle at concentrations of 10 microM or more. This contractile response to bromo-eudistomin D was completely blocked by 10 mM procaine. The extravascular Ca2+ concentrations of the heavy fractions of the fragmented sarcoplasmic reticulum (HSR) were measured directly by a Ca2+ electrode to examine the effect of bromo-eudistomin D on the sarcoplasmic reticulum. After the HSR was loaded with Ca2+ by the ATP-dependent Ca2+ pump, the addition of 10 microM bromo-eudistomin D caused Ca2+ release that was followed by spontaneous Ca2+ reuptake. In the presence of 2 microM ruthenium red or 4 mM MgCl2, no Ca2+ release was induced by 20 microM bromo-eudistomin D. The rate of 45Ca2+ efflux from HSR, which had been passively preloaded with 45Ca2+, was accelerated 7 times by 10 microM bromo-eudistomin D. The concentration of bromo-eudistomin D for half-maximum effect on the apparent efflux rate was 1.5 microM, while that of caffeine was 0.6 mM. The bromo-eudistomin D-evoked efflux of 45Ca2+ was abolished by 2 microM ruthenium red or 0.5 mM MgCl2. Bromo-eudistomin D was found to be 400 times more potent than caffeine in its Ca2+-releasing action but was similar in its action in other respects. These results indicate that bromo-eudistomin D may induce Ca2+ release from the sarcoplasmic reticulum through physiologically relevant Ca2+ channels.     

Ca release induced by cyclic adenosine diphosphoribose (cADPr) in sea urchin egg homogenates: mechanisms of release and heterogeneity of the Ca compartments

A rapid superfusion system measuring the amounts, kinetics, and Ca dependencies of released 45Ca, was used to examine the effects of ryanodine (RY), caffeine (CF), and cyclic ADP ribose (cADPr) on sea urchin egg homogenates. The RY-sensitive compartment had more than twice the Ca release capacity of the CF-sensitive or cADPr-sensitive compartment. cADPr-stimulated 45Ca release required calcium with half-maximal activation at approximately 0.2 to 0.6 microM [Ca2+]. K(1/2) for cADPr activation was approximately 100 nM, and in spite of the Ca requirement for cADPr-stimulated release, the cADPr affinity was not affected by [Ca2+]. Peak 45Ca release rate with cADPr (3 microM) was greater than with CF (20 mM), yet the release amounts were similar and both were [Ca2+]-dependent. When activated with CF and cADPr simultaneously, 45Ca release was large and, no longer [Ca2+]-dependent. Mg competitively inhibited the Ca activation site(s), yet did not inhibit the activation with CF-plus-cADPr. Pre-release of 45Ca by cADPr with low (approximately 0.1 microM) [Ca2+] right-shifted the [Ca2+] dependence of the remaining cADPr-response. These data suggest that (a) only a portion of RY-sensitive compartments empty when stimulated with cADPr or CF, (b) Ca and cADPr act on non-interacting sites, and (c) cADPr-sensitive compartments represent a heterogeneous population with different [Ca2+] dependencies.     

Depolarization of rat basophilic leukemia cells inhibits calcium uptake and exocytosis

We have investigated the unusual observation that depolarization of rat basophilic leukemia cells in high potassium not only fails to induce secretion, but also inhibits the secretion induced when receptors for IgE are aggregated by antigen. Antigen-stimulated 45Ca uptake and the rise in cytoplasmic free ionized calcium measured with the fluorescent indicator quin2 were both inhibited in depolarized cells. 45Ca efflux, on the other hand, was unaffected, which confirms that IgE receptor activation was not impaired in high potassium. Unlike the large increase in total cell calcium seen when cells in normal saline solution were stimulated with antigen, there was a decrease in total cell calcium when depolarized cells were stimulated. This is consistent with our finding that 45Ca uptake was inhibited while 45Ca efflux was unaffected. Inhibition of 45Ca uptake and secretion closely paralleled the decrease in membrane potential, and could be overcome by increasing the extracellular calcium concentration. We conclude that changes in the electrochemical gradient for calcium are important in determining calcium influx and the magnitude of antigen-stimulated secretion from rat basophilic leukemia cells, while the release of calcium from intracellular stores is unaffected.