A non-specific Ca2+ (or Mg2+)-stimulated ATPase in rat heart sarcoplasmic reticulum

ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Increasing concentrations of Mg²⁺ produced an apparent inhibition of the Ca²⁺-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca²⁺ with a high affinity (K_0.5 Ca²⁺ = 0.41 M) suggesting the presence of a high affinity Ca²⁺-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations and failed to stimulate membrane phosphorylation and Ca²⁺ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48180 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca²⁺-pumping ATPase previously characterized in dog heart and other species.Abbreviations cAMP adenosine 3,5-monophosphate - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PLB phospholamban - SR sarcoplasmic reticulum - TFP trifluoperazine     

Biochemical characterization of the Ca2+ release channel of skeletal and cardiac sarcoplasmic reticulum

Summary Rapid mixing-vesicle ion flux and planar lipid bilayer-single channel measurements have shown that a high-conductance, ligand-gated Ca²⁺ release channel is present in heavy, junctional-derived membrane fractions of skeletal and cardiac muscle sarcoplasmic reticulum. Using the release channel-specific probe, ryanodine, a 30S protein complex composed of polypeptides of M_r 400 000 has been isolated from cardiac and skeletal muscle. Reconstitution of the complex into planar lipid bilayers has revealed a Ca²⁺ conductance with properties characteristic of the native Ca²⁺ release channel.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Evolutionary temperature adaptation of fish sarcoplasmic reticulum

Summary Sarcoplasmic reticulum has been isolated from the white muscle of 15 species of teleost fish adapted to diverse thermal environments. Evidence has been obtained that the Ca²⁺-dependent ATPase of fish sarcoplasmic reticulum has undergone evolutionary modification for function at different temperatures. Compared with tropical fish, cold adapted species have higher rates of Ca²⁺ transport and Ca²⁺-ATPase activities at low temperatures. Most species have linear Arrhenius plots over the temperature range 0–30°C. Activation enthalpies (H ) of the ATPase ranged from 53–190 kJ mol^–1 and were positively correlated with environment temperature. Activation entropy (S ) varied from negative values in cold adapted species to positive values in tropical fish.In contrast to the Ca²⁺-ATPase, the basal ATPase of fish sarcoplasmic reticulum showed no relationship between either ATPase activity or thermodynamic activation parameters and environmental temperature.Only the Ca²⁺-dependent ATPase is coupled to Ca²⁺ transport. The percentage of total ATPase activity which is Ca²⁺ activated is higher at low temperatures in cold than in warm adapted species. For example, ratios of Ca²⁺-dependent/total ATPase at 2°C varied from 80–98% in Arctic, Antarctic and North Sea species to only 2–50% in various tropical fish. Above 20°C, similar ratios in the range 80–98% were obtained for all species. The nature of the basal ATPase and mechanisms of temperature adaptation of fish sarcoplasmic reticulum are discussed.Abbreviations ET environmental temperature - EGTA ethylene glycol-bis (-aminolethyl ether)-N, N-tetraacetic acid - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - SR sarcoplasmic reticulum     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Characterization of Melittin Effects in Synaptosomes

The effects of melittin at increasing concentrations on: [³H]GABA release from mouse brain synaptosomes; on the radioactivity released from [³H]arachidonic acid labeled synaptosomal membranes; on synaptosomes ultrastructure and on the leakage of the cytoplasmic marker, lactate-dehydrogenase (LDH) was investigated. Melittin 0.3, 1, 3, 7, and 10 M progressively increases [³H]GABA release, but the efficacy of melittin is decreased when the amount of tissue exposed to a constant concentration of the toxin increases. The release of [³H]GABA induced by melittin below 3 M is Ca²⁺ dependent, but not that induced by the higher concentrations. The Ca²⁺ dependent fraction of the [³H]GABA released by 0.3 M melittin is selectively inhibited by 10 M quinacrine and 1 M nordihydroguaiaretic acid (NDGA) and facilitated by 3 M indomethacin, whereas the Ca²⁺ independent fraction of the [³H]GABA released by melittin is not. In the presence of Ca²⁺, melittin 0.3, 1 and 10 M progressively increases [³H]arachidonic acid release over control release, but the effectiveness of melittin is also decreased as the amount of tissue increases. No apparent changes in synaptosomes ultrastructure are observed in 0.3 M treated synaptosomes, but a noticeable disorganization is produced in 10 M melittin-treated synaptosomes, independently on the presence of external Ca²⁺. LDH activity only increases over control activity in the supernatant solutions of 10 M melittin treated synaptosomes, also in a Ca²⁺ independent manner. Our interpretation of these results is that the Ca²⁺-dependent, pharmacologic sensitive component of melittin-induced release of [³H]GABA, unmasked when 0.3 M melittin was used, involves the activation of a Ca²⁺-dependent type of membrane PLA₂. The Ca²⁺-independent release of [³H]GABA is in contrast, highly probable to be due to the membrane perturbation produced by complex melittin/lipid interactions.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

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 ofM _r 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.     

Use of mitochondrial inhibitors to differentiate kinetic properties of the ATP-dependent Ca2+ uptake system in synaptic membranes

Synaptosomal membranes accumulate 3–6 times more Ca²⁺ in the presence of ATP (50–1000 M) than basal Ca²⁺ accumulation (-ATP). The location of this Ca²⁺ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca²⁺ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 g/ml), sodium azide (100 M) and dinitrophenol (100 M) differentiate mitochondrial from nonmitochondrial Ca²⁺ accumulation under conditions that are [Ca²⁺]- and ATP-dependent. In the presence of low concentrations of ATP (<150 M) and Ca _free ²⁺ (2.5 or 6.8 M), Ca²⁺ accumulation occurs as one process in both lysed synaptosomal membranes and purified synaptic plasma membranes in the presence and/or absence of MI. When ATP levels are increased (>200 M), the Ca²⁺ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca _free ²⁺ =2.5 M. When Ca _free ²⁺ is increased to 6.8 M, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca²⁺ accumulating mechanisms in synaptosomal membranes depend on both [Ca²⁺] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca²⁺ uptake may be a viable mechanism for the regulation of synaptosomal Ca²⁺ levels.     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Ca2+-antagonists inhibit the N-methyltransferase‐dependent synthesis of phosphatidylcholine in the heart

Evidence indicates that, in addition to the Ltype Ca²⁺ channel blockade, Ca²⁺antagonists target other functions including the Ca²⁺pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca²⁺pumps by verapamil and diltiazem could be due to druginduced depression of phosphatidylethanolamine (PE) Nmethylation which modulates these Ca²⁺transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of SadenosylLmethionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10^–6–10^–3 M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10^–3 M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10^–5 M) was associated with a depression of the V_max value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE Nmethylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [³H-methyl]methionine following the treatment of animals with verapamil, reduced the synthesis of PC by Nmethyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca²⁺pump and although diltiazem also inhibited the SR Ca²⁺pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca²⁺-release. These results suggest that verapamil and diltiazem alter the cardiac PE Nmethyltransferase system. This action is apparently additional to the drugs' effect on Ltype Ca²⁺ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca²⁺pumps and altered cardiac function.     

Critical sulfhydryls regulate calcium release from sarcoplasmic reticulum

Rapid Ca²⁺ release from the sarcoplasmic reticulum (SR) can be triggered by either binding of heavy metals to a sulfhydryl (SH) group or by catalyzing the oxidation of endogenous groups to a disulfide. Ca²⁺ release has been monitored directly using isolated vesicle preparations or indirectly by monitoring phasic contractions in a skinned fiber preparation. SH oxidation triggered by addition of Cu²⁺ /mercaptans, phthalocyanine dyes, reactive disulfides, and various anthraquinones appears to involve a direct interaction with the Ca²⁺ release protein from the SR. A model is presented in which reversible oxidation and reduction of endogenous SH groups results in the opening and closing of the Ca²⁺ release channel from the SR.Abbreviations SR sarcoplasmic reticulum - SH sulfhydryl - T-tubule transverse tubule - 2,2-DTDP 2,2-dithiodipyridine - 4,4-DTDP 4,4-dithiodipyridine - DTT dithiothreitol     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca²⁺ (EC₅₀=2.4 M). The stable GTP analogue GTPS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca²⁺ concentrations. At optimal Ca²⁺ (10 M) the effect of GTPS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A₂. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca²⁺ dependent effects, (ii) phospholipase C was not activated in the absence of Ca²⁺ and insulin secretion due to the phorbol ester TPA displayed a different Ca²⁺ dependency, (iii) arachidonic acid did not elicit Ca²⁺ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca²⁺ or TPA is attenuated by the inhibitory guanine nucleotide GDPS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.Abbreviations InsP₃ inositol trisphosphate - Ptd-InsP₂ phosphatidylinositol 4,5-bisphosphate - GTPS guanosine 5-(3-O-thio)triphosphate - GDPS guanosine 5-(2-O-thio)diphosphate - Gpp(NH)p guanyl-5-yl imidodiphosphate - TPA 12-O-tetradecanoylphorbol-13-acetate - OAG 1-oleoyl-2-acetylglycerol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid - DAG diacylglycerol - [Ca²⁺]_i cytosolic free Ca²⁺ concentration     

Phosphorylation of cardiac junctional and free sarcoplasmic reticulum by PKCα, PKCβ, PKA and the Ca2+/calmodulin-dependent protein kinase

Phosphorylation of cardiac junctional and free sarcoplasmic reticulum (SR) by protein kinase C (PKC) isoforms and was investigated. Both SR and PKC were isolated from canine heart. Junctional and free SR vesicles were prepared by calcium-phosphate-loading. The substrate specificities of PKC and PKC were found to be similar in both SR fractions. A high molecular weight junctionally-associated protein was phosphorylated by PKA, PKC and an endogenous Ca²⁺/calmodulin-dependent protein kinase activity: the highest levels of phosphate incorporation being catalysed by the latter kinase. In addition to this high molecular weight junctionally-associated protein, PKC induced phosphorylation of 45, 96 kDa and several proteins of greater than 200 kDa in junctional SR. A protein of 96 kDa was phosphorylated by both isoforms in junctional and free SR. The major substrate for PKA, PKC, PKC and the Ca²⁺/calmodulin-dependent protein kinase, in both junctional and free SR, was phospholamban. Although the phosphorylation of phospholamban by PKC was activated by Ca²⁺, a component of this activity appeared to be independent of Ca²⁺. PKC-mediated phosphorylation of phospholamban was fully activated by 1 M Ca²⁺ whereas the Ca²⁺/calmodulin dependent kinase required concentrations in excess of 5 M Ca²⁺. In the in vitro system employed in these studies, the concentrations of either PKC or the catalytic subunit of PKA required to phosphorylate phospholamban were found to be similar. In addition, in the presence of a 15 kDa sarcolemmal-associated protein, which becomes phosphorylated upon activation of PKC in vivo, phosphorylation of phospholamban by PKC was unaffected. These results demonstrate that, although substrates for both subtypes are found in both junctional and free SR, PKC and PKC do not show differences in selectivity towards these substrates.Abbreviations Ca²⁺ free calcium - CaM kinase Ca²⁺/calmodulin-dependent protein kinase - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(b-aminoethylether)-N,N,N,N-tetraacetic acid - FSR free sarcoplasmic reticulum - JSR junctional sarcoplasmic reticulum - PKC protein kinase C - PS phosphatidylserine - SDS sodium dodecyl sulfate - SAG 1-stearoyl-2-arachidonylglycerol - TPCK L-1-tosylamido-2-phenylethyl chloromethyl ketone - Tris/HCI tris(hydroxymethyl)aminomethane hydrochloride This work was supported by a grant (to S.K.) from the Heart and Stroke Foundation of B.C. and Yukon. The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Recipient of a Studentship form the Heart and Stroke Foundation of Canada.     

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney α-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification

Summary NADH inhibition of bovine kidney -ketoglutarate dehydrogenase complex was compared at 10 m free Ca²⁺ or in the absence of Ca²⁺ (i.e., < 1.0 nM free Ca²⁺). In the presence of Ca^2–, NADH inhibition was appreciably decreased for a wide range of NADH : NAD⁺ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 m free Ca²⁺ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca²⁺ on the S_0.5 for -ketoglutarate which was decreased by Ca²⁺ with a half-maximal effect at a similar concentration. The effect of Ca²⁺ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the -ketoglutarate dehydrogenase complex is required to elicit the effect of Ca²⁺ on NADH inhibition.At a fixed -ketoglutarate concentration (50 m), removal of Ca² reduced the activity of the -ketoglutarate dehydrogenase complex by 8,5-fold (due to an increase in S_0.5 for -ketoglutarate) and, in the presence of different NADH : NAD⁺ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxP_i) or a combination of the phosphate potential and NADH :NAD⁺ ratio are also described. The possibility that the level of intramitochondrial free Ca²⁺ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.     

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd²⁺⁺⁺ influx via the Mn²⁺ porter down membrane potential () was fast due to involvement of energy generated by two proton pumps—the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd²⁺ influx is suggested to be due to a series of toxic events elicited by Cd²⁺ accumulation down generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient ( _H ⁺) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of _H ⁺-dependent ATP synthesis, (iii) acceleration of the initial Cd²⁺ influx down generated the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd²⁺ of _H ⁺-dependent ATP synthesis and of pH gradient (pH)-linked [¹⁴C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of _H ⁺ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd²⁺ accumulation was prevented due to activity of the plasmid-coded Cd²⁺ efflux system. Consequently, _H ⁺-producing and -requiring processes were not affected by Cd²⁺.     

Decreased expression of phospholamban is not associated with lower β-adrenergic activation in rat atria

The aim of the study was to find out whether low phospholamban level in atria as compared with ventricles is associated with differences in sarcoplasmic reticular Ca²⁺-uptake and contractile performance. Relationship between phospholamban and -adrenergic stimulation in rat left atria and papillary muscles were examined by means of contractile measurements, sarcoplasmic reticular oxalate-supported Ca²⁺-uptake, and Western blotting of phosphorylated phospholamban. Phosphoprotein determination after -adrenergic stimulation demonstrated that the levels of Ser¹⁶ and Thr¹⁷ phosphorylated phospholamban in atria remained at about one-third of that in ventricles. However, comparison of sarcoplasmic reticular Ca²⁺-uptake in control and isoproterenol perfused preparations demonstrated that the effect of -adrenergic stimulation on sarcoplasmic reticular Ca²⁺-uptake was stronger in atrial preparations. Moreover, atria responded to isoproterenol with much larger increases in developed tension, contractility and relaxation rates than papillary muscles. Thus, despite lower level of phospholamban, the -adrenergic activation of sarcoplasmic reticular Ca²⁺-uptake and contractile indices are higher in atria.