Phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum terminal cisternal membranes. Ca2+ flux and single channel studies.

We report here that the inositol 1,4,5-trisphosphate (IP3) precursor, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent molecule (1 microM) which activates the ryanodine-sensitive Ca2+ release channel from rabbit skeletal muscle terminal cisternae incorporated into a phospholipid bilayer. It also stimulates Ca2+ release from these membrane vesicles. Therefore, it may play a modulating role in excitation-contraction coupling. In the bilayer, PIP2 added on the cytoplasmic side increased the mean channel opening probability 2-12-fold in the presence and absence of physiological Mg2+ and ATP. From flux studies, PIP2-induced Ca2+ release, occurring through the ryanodine-sensitive Ca2+ release channel, displayed saturation kinetics. The rate of Ca2+ release induced by PIP2 was approximately greater than 50% slower than the rates induced by other agents (e.g. caffeine, Ca2+, ATP). PIP2, and not IP3, effectively elicited Ca2+ release from terminal cisternae. On the contrary, IP3, and not PIP2, specifically mediated Ca2+ release from dog brain cerebellum microsomes, where IP3 receptors are known to be found. The PIP2-induced Ca2+ release from muscle membranes was not dependent on medium [Ca2+] (from less than 10(-9) to approximately 10(-4) M). However, IP3 could activate the terminal cisternae Ca2+ channel in the bilayer when there was low Ca2+ (less than 10(-7) M). The data suggest that the ionic microenvironment around the Ca2+ channel may be different for observing the two phosphoinositide actions.     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscles after treatment with caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence spectra, a comparative study of caffeine- and Ca2+-induced release of Ca2+ from the terminal cisterns of rabbit fast skeletal muscle sarcoplasmic reticulum was carried out. It was shown that the caffeine-induced release of Ca2+ depends on Ca2+ and Mg2+ concentration in the medium; Mg2+ inhibit, while Ca2+ stimulate this process. The caffeine-induced transport of Ca2+ is blocked by ruthenium red, tetracaine and dimethylsulfoxide. The Ca2+ release induced by Ca2+ was shown to occur in two ways, i. e., via Mg2+-dependent (inhibited by Mg2+ and caffeine blockers) and Mg2+-independent (insensitive to caffeine inhibitors, including Mg2+) routes. It was assumed that caffeine stimulates the Mg2+-dependent, Ca2+-induced release of Ca2+. The sensitivity of Ca2+ transport to caffeine testifies to the fact that about 80% of the total Ca2+ transport activity of fast skeletal muscle homogenates belongs to terminal cisterns. The total amount of sarcoplasmic reticulum membranes in the muscle makes up to 15-20 mg of protein/g of tissue.     

Fast release of 45Ca2+ induced by inositol 1,4,5-trisphosphate and Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle: evidence for two types of Ca2+ release channels.

The kinetics of Ca2+ release induced by the second messenger D-myoinositol 1,4,5 trisphosphate (IP3), by the hydrolysis-resistant analogue D-myoinositol 1,4,5 trisphosphorothioate (IPS3), and by micromolar Ca2+ were resolved on a millisecond time scale in the junctional sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The total Ca2+ mobilized by IP3 and IPS3 varied with concentration and with time of exposure. Approximately 5% of the 45Ca2+ passively loaded into the SR was released by 2 microM IPS3 in 150 ms, 10% was released by 10 microM IPS3 in 100 ms, and 20% was released by 50 microM IPS3 in 20 ms. Released 45Ca2+ reached a limiting value of approximately 30% of the original load at a concentration of 10 microM IP3 or 25-50 microM IPS3. Ca(2+)-induced Ca2+ release (CICR) was studied by elevating the extravesicular Ca2+ while maintaining a constant 5-mM intravesicular 45Ca2+. An increase in extravesicular Ca2+ from 7 nM to 10 microM resulted in a release of 55 +/- 7% of the passively loaded 45Ca2+ in 150 ms. CICR was blocked by 5 mM Mg2+ or by 10 microM ruthenium red, but was not blocked by heparin at concentrations as high as 2.5 mg/ml. In contrast, the release produced by IPS3 was not affected by Mg2+ or ruthenium red but was totally inhibited by heparin at concentrations of 2.5 mg/ml or lower. The release produced by 10 microM Ca2+ plus 25 microM IPS3 was similar to that produced by 10 microM Ca2+ alone and suggested that IP3-sensitive channels were present in SR vesicles also containing ruthenium red-sensitive Ca2+ release channels. The junctional SR of rabbit skeletal muscle may thus have two types of intracellular Ca2+ releasing channels displaying fast activation kinetics, namely, IP3-sensitive and Ca(2+)-sensitive channels.     

A calmodulin-stimulated Ca2+ pump in rat aorta plasma membranes

An ATP-driven Ca2+-transport system has been characterized in a microsomal fraction from rat aorta. Calmodulin enhanced 2.5-fold 45Ca accumulation by EGTA-treated microsomes incubated with 10 microM Ca2+ (in the absence of oxalate) by increasing markedly the apparent affinity of the transport system for Ca2+. The ionophore A23187 induced a rapid release of the sequestered 45Ca. The vesicles that took up 45Ca were distributed like plasmalemmal marker enzymes when the microsomal fraction was subfractionated by density gradient centrifugation. In particular, these vesicles were markedly shifted towards higher equilibrium densities after addition to the microsomes of 0.2 mg digitonin/mg protein before isopycnic centrifugation. We conclude that the calmodulin-stimulated Ca2+ pump associated with the microsomal fraction is located in plasmalemmal elements.     

Heparin induces Ca2+ release from the terminal cisterns of skeletal muscle sarcoplasmic reticulum

Using a Ca2+-selective electrode and the chlorotetracycline fluorescence technique, the effects of heparin on Ca2+ transport in the sarcoplasmic reticulum (SR) of skeletal muscles in the absence of oxalate were investigated. It was shown that heparin (0.5-10 micrograms/ml) causes a rapid release of 40-50 nmol Ca2+/mg protein from the terminal cistern SR vesicles bound to 130-150 nmol/mg protein of Ca2+ in the presence of ATP. However, heparin has practically no effect on the longitudinal cistern fraction of SR. The effects of heparin can be prevented by ruthenium red. No influence of heparin is observed in the case of the Ca2+-induced release of Ca2+ from the terminal cisterns. When the Ca2+ release is induced by heparin, no Ca2+-induced release of Ca2+ takes place.     

Characteristics of GTP-mediated microsomal Ca2+ release

Guanosine triphosphate (GTP) can release Ca2+ and enhance responses to D-myo-inositol 1,4,5-trisphosphate (IP3) in crude liver microsomes in the presence of polyethylene glycol (PEG) (Dawson et al. (1986) Biochem. J. 234, 311-315). The mechanism of these responses has been further investigated. GTP gamma S which antagonizes the actions of GTP on microsomes, does not promote Ca2+ re-uptake when added after the completion of GTP-mediated Ca2+ release. However, the effects of GTP could be reversed by washing or dilution of the microsomes. Addition of PEG to the incubation medium promoted the aggregation of microsomes. Electron microscopy provided no evidence for the fusion of microsomal vesicles in the presence or absence of GTP. In the presence of PEG, GTP produced an alteration of the permeability properties of the microsomal membrane as indicated by increased leakage of an intraluminal esterase, a reduction in the mean buoyant density of the vesicles, and a decrease in the latency of mannose 6-phosphate hydrolysis. All three effects developed relatively slowly, whereas the effects of GTP on Ca2+ fluxes occurred more rapidly (complete within 15 min). A low permeability to mannose 6-phosphate was restored upon washing away the GTP. These results suggest that non-specific permeability changes may underly the effects of GTP on Ca2+ release and that, under certain conditions, GTP can reversibly modulate the permeability of a transmembrane 'pore' in microsomal membranes that can pass ions and macromolecules. The possibility that such a pore serves to link IP3-sensitive vesicles with other Ca2+-containing compartments is discussed.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

2,2,4-Trimethylpentane induces Ca2+ release from the sarcoplasmic reticulum terminal cisterns.

Using quin2, the effects of aliphatic hydrocarbons on the system of Ca(2+)-induced Ca2+ release in isolated membranes of rabbit skeletal muscle terminal cisterns have been studied. The hydrocarbons were inserted into the membranes by means of hydrocarbon-containing liposomes. 2,2,4-Trimethylpentane (isooctane) caused a rapid release of 70-75% of Ca2+ taken up by the terminal cistern vesicles during the Ca(2+)-pump operation. This effect was inhibited by the caffeine-induced Ca2+ release blockers--Mg2+, ruthenium red and tetracaine. The same was observed with a decrease in the concentration of ATP that is known to activate the terminal cistern Ca2+ channels. The effect of 2,2,4-trimethylpentane on the longitudinal cistern fractions practically devoid of Ca(2+)-channels was insignificant. Heptane, hexane and octane caused a slow release of 5-10% of the accumulated Ca2+ from the terminal cistern vesicles; no such effect was induced by decane.     

Ca2+ release triggered by NAADP in hepatocyte microsomes

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

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.     

Microsomal Ca2+ flux modulation as an indicator of heavy metal toxicity

Inositol 1,4,5-trisphosphatee (IP3), an intracellular messenger, releases Ca2+ from microsomes. Ca2+ plays a major role in regulating various cellular events like neural transmission and regulation of hormones and growth factors. Aluminum (Al), lead (Pb) and mercury (Hg) were reported to alter Ca(2+)-regulated events thereby causing neurotoxicity. Hence, an attempt was made characterize IP3 mediated Ca2+ release from rat brain microsomes under the influence of Al, Pb and Hg. Different concentrations of metals were tested over a designated time scale and their effects on IP3 mediated Ca2+ release from microsomes were monitored using Fura-2 technique. All the three metals inhibited IP3 mediated Ca2+ release, Pb being more potent. The order of potency of these three metals was Pb>Hg>Al. Except for Al, both Hg and Pb independently released Ca2+ from microsomes. Re-uptake of Ca2+ into microsomes was inhibited by all the three metals, Pb being more potent. Microsomal Ca(2+)-ATPase activity was also inhibited by all the three metals. These results suggest that neurotoxicity exerted by Al, Pb and Hg may be due to the interference of these metals with IP3 mediated calcium release and also interfering with the microsomal Ca2+ sequestration mechanism. Differential effects of heavy metal induced changes in Ca2+ flux can be used as an index of relative toxicity.     

GTP-sensitivity of the energy-dependent Ca2+ storage pool in permeabilized pancreatic acinar cells

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 3.3 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Part of this energy-dependent pool could be released by GTP in a polyethylene glycol-dependent manner. The kinetics of GTP-induced release of Ca2+ showed a biphasic pattern with an initial rapid phase followed by a sustained slower phase. In contrast, IP3-induced release of Ca2+ was completed within 30 s following addition of IP3. No reuptake of Ca2+ was observed following GTP- or IP3-induced release of Ca2+. The GTP effect was independent of IP3 and not inhibited by Ca2+, indicating that the IP3-operated Ca2+ channel is not involved in GTP-induced release of Ca2+. The size of the IP3-releasable pool was not affected by GTP, indicating that GTP, when added to permeabilized acinar cells, does not promote the coupling between IP3-insensitive and IP3-sensitive Ca2+ accumulating organelles. Thus, in permeabilized acinar cells, GTP and IP3 act on different Ca2+ sequestering pools. Interestingly, however, comparison of the size of the GTP-releasable pool with that of the IP3-releasable pool for the cell preparations used in the present study, revealed an inversed relationship, indicating that at the time of permeabilization the GTP-releasable pool can be coupled to a greater or lesser extent to the IP3-releasable pool. This suggests that, in the intact cell, a GTP-dependent mechanism may exist that controls the size of the IP3-releasable pool by coupling IP3-insensitive to IP3-sensitive organelles. Moreover, this suggests that the extent of coupling is preserved during permeabilization.     

Effects of GTP on Ca2+ movements across endoplasmic reticulum membranes

Our initial observation that GTP could, under some experimental conditions, have profound effects on Ca2+ movements across endoplasmic reticulum membranes arose from attempts to increase the sensitivity of rat liver microsomes to inositol 1,4,5 trisphosphate (IP3). Most preparations of microsomal fractions from rat liver release only a very small percentage of accumulated Ca2+ on addition of IP3. We found, rather empirically, that the addition of microM concentrations of GTP greatly enhanced the amount of Ca2+ releasable by IP3. The initial, very appealing, hypothesis was to postulate a direct effect of GTP on the IP3-sensitive Ca2+ channel. This idea is no longer tenable, as will be described below. The more likely explanation, that GTP has its effect by either fusing small microsomal vesicles together or by allowing some form of communication between adjacent membranes is considerably more complex mechanistically and also possibly has far reaching implications for the mechanisms by which cells organise and maintain their reticular structures.     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle induced by heparin. Relation between the Ca2+ release caused by Ca2+ ions and caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence, a Ca2+ release from terminal cysterns of skeletal muscle sarcoplasmic reticulum under effects of heparin, caffeine and Ca2+ has been studied. It was shown that Ca2+ release induced by heparin is insensitive to the blockers of Mg2+-dependent system of Ca2+-induced Ca2+ release, i.e., Mg2+, tetracaine and dimethylsulfoxide. Preliminary release of Ca2+ in the presence of caffeine, which activates Mg2+-dependent Ca2+ release, does not prevent the heparin-induced Ca2+ release. At the same time, after Ca2+ release caused by Ca2+ in a Mg2+-independent system, heparin cannot cause additional efflux of Ca2+. It has been shown that the heparin-induced release of Ca2+ diminishes with a decrease in a decrease in Ca2+ concentration. This effect is less pronounced in the presence of Na+ than with K+. The data obtained suggest that sarcoplasmic reticulum terminal cysterns contain two systems of Ca2+-induced release of Ca2+, i.e., a Mg2+-dependent, caffeine-sensitive and a Mg2+-independent heparin-sensitive ones. The mechanism of activation of both systems by caffeine and heparin consists, in all probability, in their increased affinity for Ca2+.     

Glucose 6-phosphate stimulation of MgATP-dependent Ca2+ uptake by rat kidney microsomes

(1) The features of MgATP-dependent Ca2+ accumulation under stimulation with glucose 6-phosphate were studied in rat kidney microsomes. (2) Ca2+ accumulated in the presence of MgATP alone does not exceed approx. 2 nmol/mg protein. (3) Glucose 6-phosphate markedly stimulates Ca2+ accumulation, up to steady-state levels approx. 15-fold higher than in its absence. (4) The hydrolysis of glucose 6-phosphate by glucose-6-phosphatase is essential for the stimulation, as shown by inhibiting the glucose 6-phosphate hydrolysis with adequate concentrations of vanadate. Inorganic phosphate is accumulated in microsomal vesicles during glucose 6-phosphate-stimulated Ca2+ uptake in equimolar amounts with respects to Ca2+. (5) Increasing concentrations of glucose 6-phosphate result in increasing stimulations of Ca2+ uptake, until a maximal Ca2(+)-loading capacity of approx. 27 nmol/mg microsomal protein is reached. It is suggested that the enlargement of the kidney microsomal Ca2+ pool induced by glucose 6-phosphate (an important metabolite in kidney) might play a role in the regulation of Ca2+ homeostasis in kidney tubular cells.     

MgATP-dependent glucose 6-phosphate-stimulated Ca2+ accumulation in liver microsomal fractions. Effects of inositol 1,4,5-trisphosphate and GTP

Ca2+ release triggered by inositol 1,4,5-trisphosphate (IP3) and/or GTP has been studied with rough and smooth microsomes isolated from rat liver. Microsomes were loaded with Ca2+ in the presence of MgATP and in the presence or in the absence of glucose 6-phosphate (glucose-6-P) which markedly stimulated the MgATP-dependent Ca2+ accumulation in rough and smooth microsomes (5- and 10-fold, respectively). Upon addition of IP3 (5 microM), rough and smooth microsomes rapidly release a part (not exceeding 20%) of the Ca2+ previously accumulated both in the absence and in the presence of glucose-6-P. Under the same experimental conditions, inositol 1,3,4,5-tetrakisphosphate was ineffective in triggering any Ca2+ release. Upon addition of GTP (10 microM) both the microsomal fractions progressively release the Ca2+ previously accumulated in the presence of glucose-6-P, when 3% polyethylene glycol was also present. In the absence of polyethylene glycol, GTP released Ca2+ from rough microsomes only, and GTP plus IP3 caused a Ca2+ release which was the sum of the Ca2+ releases caused by GTP and IP3 independently. Both IP3 and GTP, added to microsomes at the beginning of the glucose-6-P-stimulated Ca2+ uptake, reduced the Ca2+ accumulation into rough and smooth microsomes without modifying the initial rate (3 min) of Ca2+ uptake. Also in these conditions, the effects of GTP and IP3 were merely additive. These results indicate that both rough and smooth liver microsomes are responsive to IP3 and GTP with respect to Ca2+ release and that IP3 and GTP likely act independently.     

Kinetic properties of the Ca2+-accumulation system of a rat liver microsomal fraction.

1. By using Ca-EGTA buffers, the Km for Ca2+ uptake into rat liver heavy microsomes (microsomal fraction) was found to be 0.2 microM free Ca2+. 2. In the absence of oxalate, these vesicles accumulate about 20 nmol of Ca2+/mg of protein. Efflux of Ca2+ from the vesicles is much faster at pH 7.6 than at pH 6.8, but does not apparently show saturation kinetics or any stringent requirement for external ions. 3. The steady-state distribution of Ca2+ between the microsomes and the medium in the presence of ATP and the absence of oxalate is dependent on Ca2+ load. When the vesicles are loaded to 50% capacity, the external free Ca2+ concentration is 70 nM. 4. The affinity of heavy microsomes for Ca2+ is such that is seems likely that they has a dominant role in the determination of cytoplasmic free Ca2+ concentrations.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.     

Gonadotropin-releasing hormone-induced rise in cytosolic free Ca2+ levels: mobilization of cellular and extracellular Ca2+ pools and relationship to gonadotropin secretion

Addition of GnRH to pituitary gonadotrophs preloaded with Quin 2 resulted in a rapid (approximately 8 s) mobilization of an ionomycin-sensitive intracellular Ca2+ pool. A second component of Ca2+ entry via voltage dependent channels contributed about 45% of the peak cytosolic free Ca2+ concentration ([Ca2+]i). Thereafter, influx of Ca2+ via voltage-sensitive and -insensitive channels is responsible for maintenance of elevated [Ca2+]i during the second phase of GnRH action. Addition of inositol 1,4,5-trisphosphate (IP3) to permeabilized pituitary cells resulted in a Ca2+ transient, released from a nonmitochondrial pool, which maintained ambient free Ca2+ concentration around 170 nM in an ATP-dependent mechanism. Successive stimulations of the cells with IP3 produced an attenuated response. Elevation of the gonadotroph [Ca2+]i by ionomycin, to levels equivalent to that induced by GnRH, resulted in LH release amounting to only 45% of the response to the neurohormone. Activation of the voltage-dependent Ca2+ channels by the dihydropyridine Ca2+-agonist [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate (BAYK8644)] stimulated LH release, 36% of the GnRH (100 nM) response being reached by 10(-8) M of the drug, both [Ca2+]i elevation and GnRH-induced LH release were inhibited similarly (40-50%) by the dihydropyridine Ca2+-antagonist nifedipine. The results indicate that peak [Ca2+]i induced by GnRH in pituitary gonadotrophs is derived mainly from ionomycin-sensitive cellular stores most likely via IP3 formation.(ABSTRACT TRUNCATED AT 250 WORDS)