Rapid Release Of Ca2 Research Articles

Ca(2+)-induced Ca2+ release amplifies the Ca2+ response elicited by inositol trisphosphate in macrophages.

We have studied the rise in intracellular calcium concentration ([Ca2+]i) elicited in macrophages stimulated by platelet-activating factor (PAF) by using fura-2 measurements in individual cells. The [Ca2+]i increase begins with a massive and rapid release of Ca2+ from intracellular stores. We have examined the mechanism of this Ca2+ release, which has been generally assumed to be triggered by inositol trisphosphate (IP3). First, we confirmed that IP3 plays an important role in the initiation of the PAF-induced [Ca2+]i rise. The arguments are 1) an increase in IP3 concentration is observed after PAF stimulation; 2) injection of IP3 mimics the response to PAF; and 3) after introduction of heparin in the cell with a patch-clamp electrode, the PAF response is abolished. Second, we investigated the possibility of an involvement of Ca(2+)-induced Ca2+ release (CICR) in the development of the Ca2+ response. Ionomycin was found to elicit a massive Ca2+ response that was inhibited by ruthenium red or octanol and potentiated by caffeine. The PAF response was also inhibited by ruthenium red or octanol and potentiated by caffeine, suggesting that CICR plays a physiological role in these cells. Because our results indicate that in this preparation IP3 production is not sensitive to [Ca2+]i, CICR appears as a primary mechanism of positive feedback in the Ca2+ response. Taken together, the results suggest that the response to PAF involves an IP3-induced [Ca2+]i rise followed by CICR.

Endothelin mediates Ca influx and release in porcine coronary smooth muscle cells

Endothelin (ET)-induced changes in intracellular free Ca (Cai) in freshly dispersed coronary artery smooth muscle cells were determined using fura-2 microfluorometry to differentiate the action of ET on Ca influx vs. release from internal stores. Comparison was made with caffeine (CAF)-induced Ca release from the sarcoplasmic reticulum (SR) to determine whether ET acts on the same Ca store. In physiological external solution, ET (5 x 10(-8) M) induced a rapid (within 90 s), transient (less than 2.5-min duration) 70% increase in Cai above baseline (n = 20). Pretreatment with diltiazem (10(-4) M; n = 10) did not change the amplitude or shape of the ET-induced Cai transient. In Ca-free solution, ET elicited a Cai response similar in duration but smaller (P less than 0.05) in peak magnitude (31% increase; n = 7). CAF (5 x 10(-3) M) also elicited a rapid (less than 60 s), transient 82% increase in Cai (n = 7). In the continued presence of CAF, ET caused no change in Cai. In contrast, ET elicited a transient 69% increase in Cai (n = 8), and in the continued presence of ET, CAF caused a 24% increase in Cai. Ryanodine (5 x 10(-5) M) suppressed the subsequent ET-induced Cai transient. These data on porcine cells suggest ET induces a rapid release of Ca from a CAF- and ryanodine-sensitive store and causes rapid influx of Ca, which is different from bovine smooth muscle cells. The return of Cai to baseline values in the continued presence of ET suggests the ET-sensitive store is depleted and increased Ca efflux matches Ca influx.

Purification and reconstitution of the ryanodine- and caffeine-sensitive Ca2+ release channel complex from muscle sarcoplasmic reticulum.

Studies in our laboratory are directed towards obtaining a better understanding of the process of excitation-contraction coupling in muscle, primarily using purified membrane and protein preparations. In excitable tissues, release of Ca2+ ions can be triggered by a change in surface membrane potential (Ebashi, 1976), or it can occur via a chain of voltage-independent steps that involve agonist-induced formation of inositol 1,4,5-trisphosphate (IP3) and its subsequent binding and activation of an intracellular membrane receptor/channel complex, the IP3 receptor (Van Breemen and Saida, 1989). The voltage-dependent mechanism, commonly referred to as excitation -contraction (E-C) coupling, has been most extensively studied in skeletal and cardiac muscle. In striated muscle, rapid release of Ca2+ ions from the intracellular membrane compartment, sarcoplasmic reticulum (SR), is triggered by a surface membrane action potential that is thought to be communicated to the SR at specialized areas where the SR comes in close contact with tubular infoldings of the surface membrane (T-tubule). Spanning the gap between the two membrane systems are protein bridges (Peachey and Armstrong, 1983) which have been variously termed “feet” (Franzini-Armstrong, 1970), “bridges” (Somlyo, 1979), “pillars” (Eisenberg and Eisenberg, 1982), or “spanning” proteins (Caswell and Brandt, 1989), and are now believed to be identical with the ryanodine receptor or SR Ca2+ release channel complex (Fleischer and Inui, 1989; Lai and Meissner, 1989).

Inhibition of Rapid Ca Release from Isolated Cardiac Sarcoplasmic Reticulum Cisternae

Inhibition of Rapid Ca Release from Isolated Cardiac Sarcoplasmic Reticulum Cisternae

The muscle ryanodine receptor and its intrinsic Ca2+ channel activity

In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca2+ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca2+ release is mediated by a high-conductance, ligand-gated Ca2+ channel. Using the Ca2+ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides of Mr approximately 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca2+ channel currents with conductance and gating kinetics similar to those of native Ca2+ release channels. Electron microscopy revealed structural similarity with the protein bridges ("feet") that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca2+ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures.

Rapid kinetic analysis of the calcium-release channels of skeletal muscle sarcoplasmic reticulum: the effect of inhibitors

During excitation of skeletal muscle fibers, Ca ions stored in the cisternal compartments of the sarcoplasmic reticulum (SR) are released to the cytosol within milliseconds. In this study, the kinetics of the fast release of Ca were analyzed by means of a newly developed rapid filtration apparatus. Isolated SR vesicles of cisternal origin were preloaded with 1 mM 45CaCl2, and Ca efflux was studied (between 20 and 1000 ms) after dilution into media of various composition. The effect of extravesicular Ca on the gating of the Ca-release channels and its susceptibility to the influence of drugs were thoroughly investigated. In the presence of 1 mM MgCl2 and 3 mM ATP, highest rates of Ca release were observed at a free Ca concentration between 1 and 50 microM. In the lower micromolar Ca range, compounds such as neomycin and FLA 365 inhibited the release monophasically and with an IC50 of 0.37 and 3.4 microM, respectively. At Ca concentrations between 10 and 50 microM, the inhibitors could not block Ca release effectively. Close analysis of the dose-response curves revealed a biphasic pattern, indicative of the presence of two substrates of the Ca-release channel, displaying high- and low-affinity binding sites for the inhibitors. Interestingly, neomycin (or ruthenium red) and FLA 365 at low concentrations acted synergistically and blocked release completely. The results indicate the existence of various open substates of the Ca channels that can be distinguished pharmacologically. Effective blockade of rapid Ca release requires inhibition of all substates coexisting under a given condition.

Sarcoballs: direct access to sarcoplasmic reticulum Ca2+-channels in skinned frog muscle fibers

In skeletal muscle, twitch contraction is caused by the rapid release of Ca2+ from the sarcoplasmic reticulum (SR) (Endo, M. 1977. Physiol. Rev. 57:71-108) via Ca2+ conducting channels in the SR membrane (Smith, J. S., R. Coronado, and G. Meissner, 1985. Nature (Lond.). 316:446-449; Suarez-Isla, B. A., G. Orozco, P. F. Heller, and J. P. Froehlich. 1986. Proc. Natl. Acad. Sci. USA. 83:7741-7745). To facilitate study of these and other intracellular channels, we have developed a method which allows direct patch-clamp recording of currents through SR channels in native membrane. The Ca2+-release channel studied using this method exhibits two predominant conductance levels (80-100 pS and 120-160 pS), conducts Ca2+ preferentially over K+ (PCa/Pk = 6.5), is highly voltage sensitive, blocked on one side by ruthenium red (1 microM), and displays enhanced activity in the presence of caffeine (5 mM). Studied in skinned fibers, this channel appears fundamentally similar to homologous channels from isolated rabbit SR incorporated into bilayers, with some distinct differences.

Inhibition of rapid Ca-release from isolated skeletal and cardiac sarcoplasmic reticulum (SR) membranes

Rapid Ca-release from the cisternal compartments isolated from skeletal and cardiac muscle SR was characterized by the use of inhibitors. Ruthenium red (RR) completely blocked (IC 50 = 90 nM) the Ca-channels of skeletal SR. Its effect on the rapid Ca-release from cardiac SR was marginal but became optimal (IC 50 = 200 nM) in the presence of FLA 365 ([2,6-dichloro-4-dimethyl-aminophenyl] isopropylamine) which by itself had no measurable effect. The antibiotic neomycin mimicked the properties of RR. The strong synergistic effect of RR or neomycin and FLA 365 indicates that either cardiac cisternae contain two distinct isoforms of Ca-release channel, or that different drugs are needed to effectively block the same channel.

A role for Ca 2+ in mediating hormone-induced biphasic pepsinogen secretion from the chief cell determined by luminescent and fluorescent probes and X-ray microprobe

In isolated chief cells from the guinea pig, cholecystokinin (10 nM) and a high concentration of ionomycin each caused a biphasic pattern of pepsinogen secretion. The initial fast response to cholecystokinin was not dependent on medium Ca 2+ and was mimicked by low concentration of ionomycin (100 nM). Inositol 1,4,5-trisphosphate caused a similar fast release from permeabilized cells. The slow component of release was dependent on medium Ca 2+, however, and was mimicked by the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA) (100 nM) or the diacylglycerol analogue 1-oleoyl-2-acetylglycerol (OAG) (100 μM). Ionomycin (100 nM) and TPA (and/or OAG), when applied together, reproduced the biphasic pattern of pepsinogen secretion, suggesting that the signalling pathways utilized by both types of agonist contribute to the response evoked by cholecystokinin-hormone stimulation. Both fura-2 and aequorin were used to monitor changes of intracellular Ca 2+. Three pathways were found to contribute to the Ca 2+ transient. A rapid release of Ca 2+ from intracellular store(s), a rapid Ca 2+ entry from the extracellular space, and a more sustained Ca 2+ entry from the extracellular space. Cholecystokinin induced a rapid increase in cytoplasmid Ca 2+ ([Ca 2+] i) as estimated with fura-2 and aequorin. This rise was reduced but not abolished upon removal of extracellular Ca 2+, suggesting that both Ca 2+ entry from the extracellular space and Ca 2+ mobilization from the intracellular store(s) contribute to the initial, fast component of the Ca 2+ transient. A second, more sustained component of the Ca 2+ transient induced by cholecystokinin was abolished by lanthanum. TPA and OAG induced a biphasic Ca 2+ transient that could be detected only with aequorin. The late, sustained component of this response was again abolished by lanthanum as well as by removal of extracellular Ca 2+. It appears that the late component of the Ca 2+ transient is dependent on Ca 2+ influx from the extracellular space and is too localized to be detected by fura-2. Prestimulation of cells with TPA or OAG preventes the aequorin transient caused by cholecystokinin and vice versa, suggesting that TPA, OAG and cholecystokinin activate the same pathways of Ca 2+ entry into the cytosol from the intracellular store(s) or the extracellular space. The stimulation-sensitive Ca 2+ pool was examined with electron probe X-ray microanalysis. It appears to be restricted to an area enriched in secretory granules or peripheral endoplasmic reticulum just beneath the apical plasma membrane and in close association with the microtubular-microfilamentous system. Ionomycin stimulation decreased the Ca level in secretory granules and reciprocally increased the cytoplasmic level of Ca. These changes were accompanied by entry Na + and Cl - from the extracellular space and by K + efflux from the cell. Our data suggest that the initial fast component of pepsinogen secretion is associated with a high, fast and more global rise of cytoplasmic Ca 2+, and that the sustained component is associated with a late, prolonged and more localized elevation of cytoplasmic Ca 2+ that is dependent on influx of Ca 2+ from the extracellular space.

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca 2+ uptake in permeabilized hepatocytes and Ca 2+ release by inositol trisphosphate

The relationships between Ca 2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca 2+ uptake. A progressive increase in the stimulation of Ca 2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20–40 μM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca 2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca 2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca 2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca 2+.

GTP mobilization of Ca2+ from the endoplasmic reticulum of islets. Comparison with myo-inositol 1,4,5-trisphosphate

The effect of the guanine nucleotide GTP on Ca2+ release from the endoplasmic reticulum of digitonin-permeabilized islets was investigated. maximal and half-maximal Ca2+ release were observed at 5 microM- and 2.5 microM-GTP respectively. GTP caused a rapid release of Ca2+ from the endoplasmic reticulum, which was complete within 1 min. GTP-induced Ca2+ release was structurally specific and required the hydrolysis of GTP. The combination of maximal concentrations of GTP (10 microM) and myo-inositol 1,4,5-trisphosphate (IP3) (10 microM) resulted in an additive effect on Ca2+ release from the endoplasmic reticulum. GDP (100 microM), which inhibits GTP-induced Ca2+ release, did not affect IP3-induced Ca2+ release. Furthermore, GTP-induced Ca2+ release was not independent on submicromolar free Ca2+ concentrations, unlike IP3-induced Ca2+ release. These observations suggest that mechanistically GTP-induced Ca2+ release is different from IP3-induced Ca2+ release from the endoplasmic reticulum.

Inositol 1,4,5-trisphosphate induced mobilization of Ca2+ from rat brain synaptosomes

Inositol 1,4,5-trisphosphate (IP3) was found to release Ca2+ from presynaptic nerve endings (synaptosomes) made permeable with saponin. ATP-dependent Ca2+ uptake was carried out until equilibrium was reached. Addition of IP3 produced a rapid release of Ca2+, which was complete within 60 sec, followed by Ca2+ reaccumulation to the original level in 5-7 min. Cholinergic receptor stimulation with muscarine also produced a similar Ca2+ release from synaptic endoplasmic reticulum. Ca2+ release by IP3 was not detectable in the absence of the mitochondrial inhibitors oligomycin or sodium azide. Reaccumulation of Ca2+ was prevented by the presence of vanadate, a potent inhibitor of Ca2+/Mg2+ ATPase. Half maximal and near complete release of Ca2+ took place at 0.4 microM and 3 microM IP3 concentrations, respectively. These studies demonstrate for the first time IP3 mobilization of Ca2+ from endoplasmic reticulum within synaptic plasma membranes.

Effects of sulfhydryl reagents and other inhibitors on Ca2+ transport and inositol trisphosphate-induced Ca2+ release from human platelet membranes.

The effects of modifiers of Ca2+ uptake and release in sarcoplasmic reticulum were studied in human platelet membranes. AgNO3,p-chloromercuribenzoate (pClHgBzO), N-ethylmaleimide (MalNEt), quercetin, vanadate, A23187, and caffeine all had the same effects on Ca2+ uptake in platelet membranes as had been observed for sarcoplasmic reticulum. These results strengthen our earlier conclusion that the Ca2+-pump proteins from internal human platelet membranes and muscle sarcoplasmic reticulum are very similar in functional properties. The sulfhydryl reagents Ag+ and pClHgBzO elicited rapid release of Ca2+ from platelet membranes in the presence of ATP, whereas MalNEt induced slow release. Quercetin also caused slow release of Ca2+ from platelet membranes in the presence of ATP. The effects of all three sulfhydryl reagents could be reversed by dithiothreitol, and Ag+-induced release was also reversed by ruthenium red. These effects are similar to those observed in sarcoplasmic reticulum, but in contrast caffeine did not induce Ca2+ release. In the absence of ATP, passively loaded platelet membranes did not release Ca2+ when exposed to sulfhydryl reagents. However, AgCl and pClHgBzO inhibited inositol trisphosphate (InsP3)-induced Ca2+ release from platelet membranes and this effect was reversed by dithiothreitol. Ruthenium red also inhibited InsP3-induced release, but ATP was found not to be required for InsP3-mediated release. LiCl enhanced Ca2+ release from platelet membranes. These results demonstrate that the InsP3-gated Ca2+ release channel is a separate entity from the Ca2+-pump and that essential protein sulfhydryls are involved in the release process.

Inositol 1,4,5-trisphosphate induces calcium release from a non-mitochondrial pool in amoebae of Dictyostelium

Evidence is presented for two distinct Ca 2+ pools in amoebae of Dictyostelium discoideum. One pool, presumably mitochondrial, was sensitive to the mitochondrial inhibitors oligomycin and dinitrophenol and showed an affinity for Ca 2+ in the μM concentration range. The other Ca 2+ pool, which was insensitive to these inhibitors, was of lower capacity but had higher affinity (in the nM range). Inositol 1,4,5-trisphosphate (5 μM) added to saponin-permeabilized amoebae induced a rapid release of Ca 2+ from the latter pool but had no effect on the presumed mitochondrial pool. Controls using addition of inositol 1,4-bisphosphate (the hydrolytic product of IP 3) induced no such Ca 2+ release. The results provide strong support for the involvement of IP 3 in signal transmission during chemotaxis of D. discoideum.

Establishment of a differentiated mesodermal line from P19 EC cells expressing functional PDGF and EGF receptors

Aggregation of pluripotent P19 embryonal carcinoma (EC) cells in the presence of DMSO induces differentiation to various mesodermal cell types, including spontaneously contracting muscle [9]. We have established clonal cell lines from these cultures and characterized one (MES-1) in particular for its response to growth factors. In contrast to the undifferentiated stem cells, but as a number of myoblast and muscle cell lines, MES-1 cells respond to both carbachol and bradykinin by the rapid release of Ca 2+ from intracellular stores. In addition, MES-1 express receptors for and respond mitogenically to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Isolated membranes from these cells retain the capacity to bind both ligands; addition of EGF to membranes induces endogenous phosphorylation of several proteins, including the EGF receptor itself and a 38 kD protein, while addition of PDGF specifically induces phosphorylation of the PDGF receptor. By contrast, other derivatives of P19, isolated from retinoic acid (RA)-treated aggregates and resembling neuroectodermal or endodermal cell types [10] respond only to EGF; PDGF neither binds nor induces phosphorylation and a mitogenic response in these cells. During differentiation from EC cells therefore MES-1 cells developed a combination of growth factor receptor characteristics typical of somatic mesodermal cells and indicate that such receptors on EC-derived mesodermal cells are also functional.

On the role of thiol groups in the inhibition of liver microsomal ca 2+ sequestration by toxic agents

ATP-dependent Ca 2+ sequestration by rat liver microsomes was assayed using three different methods, and characterized with regard to the effect of various inhibitors. When glucose and hexokinase were added in combination to deplete ATP in the incubation, Ca 2+ uptake was followed by rapid release of Ca 2+ from the microsomes. Ca 2+ sequestration was inhibited by reagents that cause alkylation (e.g. p-chloromercuribenzoate) or oxidation (e.g. diamide) of protein sulfhydryl groups. Moreover, pretreatment of the microsomes with cystamine, which causes formation of mixed disulfides with protein thiols, also resulted in the inhibition of Ca 2+ sequestration. It is concluded that microsomal Ca 2+ sequestration is critically dependent on protsin sulfhydryl groups, and that modification of protein thiols may be an important mechanism for the inhibition of microsomal Ca 12+ sequestration by a variety of toxic agents.

Inositol 1,4,5-trisphosphate-induced release of sequestered Ca2+ from highly purified human platelet intracellular membranes.

Evidence has accumulated in support of a role for intracellularly generated inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in raising cytosol [Ca2+] when various hormones, neurotransmitters, growth factors and other stimulants act on cell surfaces. The increase in [Ca2+] that follows stimulant-receptor interaction is accompanied by rapid hydrolysis of phosphoinositides. One product, Ins(1,4,5)P3, arising from the breakdown of phosphatidylinositol 4,5-bisphosphate was shown to promote the release of Ca2+ from non-mitochondrial stores in a variety of cells. Although platelet intracellular membranes have been implicated in the control of cytosol [Ca2+] and we previously characterized a Ca2+-sequestering mechanism associated with them, we have as yet no knowledge of how this Ca2+ store is mobilized after a stimulus-receptor interaction at the platelet surface. Using free-flow electrophoresis, we isolated and purified human platelet intracellular membranes. They show high enrichment and exclusive localization of the endoplasmic-reticulum marker NADH:cytochrome c reductase, and they sequester Ca2+ by an ATP-dependent process, reaching steady-state values in 10-12 min. Saturation with Ca2+ occurs at around 10-30 microM external Ca2+. When Ins(1,4,5)P3 is added to the 45Ca-loaded vesicles, a rapid release of Ca2+ occurs (approx. 35% in 15-30s). The magnitude of the release depends upon external [Ca2+], being maximum in the range 0.3-0.8 microM and low at external [Ca2+] greater than 1 microM. After release there is a rapid re-uptake of Ca2+, with restoration of the former steady-state values within 1 min. Half-maximal release occurs at approx. 0.25 microM-Ins(1,4,5)P3. This release and re-uptake pattern is not observed with ionophore A23187 or arachidonic acid, both of which liberate Ca2+ irreversibly. Inositol 1,4-bisphosphate was ineffective in releasing Ca2+ from these intracellular membranes. The results support the role of Ins(1,4,5)P3 as a specific intracellular mediator, transducing the action of excitatory agonists acting on the platelet surface into metabolic, mechanochemical and other functional events, known to occur during platelet activation.

Effects of platelet-derived growth factor on Ca 2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca 2+ was examined in BALB/c-3T3 cells. PDGF induced: 1. 1. A decrease in cell 45Ca 2+ content. 2. 2. An apparent increased rate of efflux of preloaded 45Ca 2+. 3. 3. A decrease in residual intracellular 45Ca 2+ remaining after rapid efflux. 4. 4. When added after the rapid phase of efflux of 45Ca 2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca 2+. All of the observed changes in 45Ca 2+ induced by PDGF are consistent with a rapid release of Ca 2+ from an intracellular Ca 2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca 2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca 2+ stores and CTC fluorescence intensity were identical with the effects of the Ca 2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca 2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca 2+ stores. Release of Ca 2+ from intracellular sequestration sites may be a mechanism by which PDGF Stimulates Cell growth.

Inositol 1,4,5-trisphosphate may be a signal for f-Met-Leu-Phe-induced intracellular Ca mobilisation in human leucocytes (HL-60 cells)

Pemeabilised, dimethyl sulphoxide-differentiated HL-60 human myelomonocytic leukemia cells accumulate 45Ca in an ATP-dependent manner. The 45Ca is taken up by a pool thought to be a component of the endoplasmic reticulum. Inositol trisphosphate induced a rapid release of Ca from this pool, suggesting that this molecule which is formed in these cells in response to f-Met-Leu-Phe may play a role in agonist-induced Ca metabolism.

The effect of lipid intermediates on Ca 2+ and Na + permeability and (Na + + K +)-ATPase of cardiac sarcolemma. A possible role in myocardial ischemia

The effect of fatty acid and acylcarnitine on Ca 2+ and Na + transporting enzymes and carriers was studied in sealed cardiac sarcolemma vesicles of mixed polarity. Palmitoylcarnitine markedly reduced the Na + gradient-induced Ca 2 uptake. Half-maximal reduction was obtained at 15 μM of the carnitine derivative. In a same concentration range palmitoylcarnitine caused a rapid release of accumulated Ca 2+ when added to Ca 2+-filled vesicles, which suggests that palmitoylcarnitine increases the permeability of the sarcolemma vesicles to Ca 2+. A rapid release of Ca 2+ was also observed if Ca 2+ was taken up by action of the Ca 2+ pump. The (Ca 2+ + Mg 2+)-ATPase, which most likely drives this active Ca 2+ uptake, was 90% increased by 50 μM palmitoylcarnitine and evidence was presented that the acylcarnitine effect again was linked to an alteration of Ca 2+ permeability of the vesicles. At the same concentration acylcarnitine was not able to unmask the latent protein kinase, so that probably the sarcolemma ATP permeability was not affected. Palmitoylcarnitine at 25 μM did not affect the ouabain-sensitive (Na + + K +)-ATPase in native sarcolemma vesicles, however, it inhibited markedly if the enzyme was measured in SDS-treated vesicles. The effect of increased free fatty acid concentration on some of the sarcolemma transporting properties was tested by adding oleate-albumin complexes with different molar ratios to the sarcolemma vesicles. In contrast to molar ratios 1 and 5, the ratio of 7 was able to induce a rapid Ca 2+ release and to inhibit (Na + + K +)-ATPase in either native or SDS-treated vesicles markedly. 22Na release from 22Na-preloaded sarcolemma vesicles was shown to be stimulated by either palmitoylcarnitine (50 μM) or olcate-albumin complex (with a molar ratio of 7). The possible significance of the observed effects of lipid intermediates on ion permeability and (Na + + K +)-ATPase activity in isolated sarcolemma vesicles for the derangement of cardiac cell function in ischemia is discussed.