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Long chain fatty acids bind to carnitine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca(2+) dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10μM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX Red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20 μM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca(2+) leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca(2+) leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongkrekic acid and NAC significantly reduced spontaneous Ca(2+) wave occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca(2+) handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cardiac ryanodine receptors (RYR2s) infrequently exhibit coupled gating that is manifested by synchronous opening and closing. To better characterize this phenomenon, we investigated the regulation of coupled RYR2 channels by luminal Ca(2+) focusing on effects that are likely mediated by the true luminal activation mechanism. By reconstituting an ion channel into a planar lipid bilayer and using substantially lower concentration of luminal Ba(2+) (8mM, the virtual absence of Ca(2+)) and luminal Ca(2+) (8mM), we show that response of coupled RYR2 channels to caffeine at a diastolic cytosolic Ca(2+) (90nM) was affected by luminal Ca(2+) in a similar manner as for the single RYR2 channel except the gating behavior. Whereas, the single RYR2 channel responded to luminal Ca(2+) by prolongation in open and closed times, coupled RYR2 channels seemed to be resistant in this respect. In summary, we conclude that the class of Ca(2+) sites located on the luminal face of coupled RYR2 channels that is responsible for the channel potentiation by luminal Ca(2+) is functional and not structurally hindered by the channel coupling. Thus, the idea about non-functional luminal Ca(2+) sites as a source of the apparent gating resistance of coupled RYR2 channels to luminal Ca(2+) appears to be ruled out., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The Ca(2+) binding sites of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) have been identified as two high-affinity sites orientated towards the cytoplasm, two sites of low affinity facing the lumen, and a transient occluded species that is isolated from both membrane surfaces. Binding and release studies, using (45)Ca(2+), have invoked models with sequential binding and release from high- and low-affinity sites in a channel-like structure. We have characterised turnover conditions in isolated SR vesicles with oxalate in a Ca(2+)-limited state, [Ca(2)](lim), where both high- and low-affinity sites are vacant in the absence of chelators (Biochim. Biophys. Acta 1418 (1999) 48-60). Thapsigargin (TG), a high-affinity specific inhibitor of the Ca(2+)-ATPase, released a fraction of total Ca(2+) at [Ca(2+)](lim) that accumulated during active transport. Maximal Ca(2+) release was at 2:1 TG/ATPase. Ionophore, A23187, and Triton X-100 released the rest of Ca(2+) resistant to TG. The amount of Ca(2+) released depended on the incubation time at [Ca(2+)](lim), being 3.0 nmol/mg at 20 s and 0.42 nmol/mg at 1000 s. Rate constants for release declined from 0. 13 to 0.03 s(-1). The rapidly released early fraction declined with time and k=0.13 min(-1). Release was not due to reversal of the pump cycle since ADP had no effect; neither was release impaired with substrates acetyl phosphate or GTP. A phase of reuptake of Ca(2+) followed release, being greater with shorter delay (up to 200 s) following active transport. Reuptake was minimal with GTP, with delays more than 300 s, and was abolished by vanadate and at higher [TG], >5 microM. Ruthenium red had no effect on efflux, indicating that ryanodine-sensitive efflux channels in terminal cisternal membranes are not involved in the Ca(2+) release mechanism. It is concluded that the Ca(2+) released by TG is from the occluded Ca(2+) fraction. The Ca(2+) occlusion sites appear to be independent of both high-affinity cytoplasmic and low-affinity lumenal sites, supporting a multisite 'in line' sequential binding mechanism for Ca(2+) transport.

The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.

A reproducible in situ crystallization of the Ca2+-ATPase in isolated sarcoplasmic reticulum (SR) membranes was studied. The addition of various nucleotides to the washing buffer allowed the formation of tubular crystals, which is induced by vanadate. SR membranes washed with nucleotide-free buffer could not form tubular crystals upon subsequent incubation with vanadate.

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.

The actions of two organic mercurial compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and p-chloromercuribenzoic acid (p-CMB) on the calcium release channel (ryanodine receptor) from rabbit skeletal muscle were determined by single channel recordings with the purified calcium release channel, radioligand binding to sarcoplasmic reticulum vesicles (HSR) and calcium release from HSR. p-CMB or 4-CMPS (20-100 microM) increased the mean open probability (Po) of the calcium channel at subactivating (20 nM), maximally activating (20-100 microM and inhibitory (1-4 mM) Ca2+ concentrations, with no effect on unitary conductance. This activation was partly reversed by 2 mM DTT. Both compounds affected the channels only from the cytosolic side, but not from the trans side. 100 microM 4-CMPS caused a transient increase in Po, followed by a low activity state within 1 min. At inhibitory Ca2+ concentrations Po was increased to values observed with maximally activating Ca2+ or lower, inhibitory Ca2+ concentrations. The p-CMB/4-CMPS modified channels were ryanodine sensitive and blocked by ruthenium red. [3H]Ryanodine binding was increased up to four-fold with 3-15 microM 4-CMPS/p-CMB (Hill coefficient 1.7-2.0) at 4 microM Ca2+ and reduced at high concentrations (50-200 microM). The increase in [3H]ryanodine binding by 10 microM 4-CMPS was completely inhibited by 2 mM DTT. 4-CMPS significantly increased the affinity for the high affinity calcium activation sites and decreased the affinity of low affinity calcium inhibitory sites of specific [3H]ryanodine binding. 4-CMPS increased the affinity of the ryanodine receptor for high affinity ryanodine binding without a change in receptor density. 4-CMPS induced a rapid, concentration-dependent, biphasic calcium release from passively calcium-loaded HSR vesicles at subactivating Ca2+ concentrations (20 nM), which was partly inhibited by 4 mM DTT and completely blocked by 20 microM ruthenium red. It is suggested that the 4-CMPS-induced modulation of essential sulfhydryls involved in the gating of the calcium release channel results in a modulation of the apparent calcium affinity of the activating high affinity and inhibitory low affinity calcium binding sites of the calcium release channel.

Several effects of the neuroleptic agent haloperidol on the sarcoplasmic reticulum (SR) Ca-dependent adenosine triphosphatase (Ca-ATPase) and Ca transport are described. Haloperidol inhibits the Ca-ATPase activity in the presence of calcimycin. The effect depends on the conditions of preexposure of the membranes to the drug: the inhibition increases with the preincubation time; Ca and Mg protect the enzyme against the effect of the drug. The inhibitory effect of haloperidol decreases upon increasing [Ca2+], at constant [Mg], and disappears at 20 mM [Mg] for any [Ca2+], and at 0.5 mM [Ca2+] for any [Mg2+]. Haloperidol also inhibits phosphorylation of the enzyme by Pi, and ATP-dependent Ca2+ uptake, in both cases with apparent Ki = 0.10-0.15 mM, and increases the rate of Ca efflux from preloaded vesicles in this concentration range. The results suggest that haloperidol interacts with the catalytic site, interfering with the effect of the divalent catalytic cation, but not at other steps of the enzymatic cycle, where Mg2+ and Ca2+ are also activators. They are consistent with a reaction model where haloperidol interacts with the E2 conformers of the enzyme, with lower affinity for the phosphoenzyme than for the dephospho species. The inhibition of Ca uptake by SR vesicles is ascribed to an increased Ca2+ permeability rather than to the inhibition of the Ca-ATPase, which requires higher concentrations of the drug.

Quinolidomicin A1, a 60-membered macrolide purified from an actinomycete Micromonospora sp. markedly induced 45Ca2+ release from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR), but induced only slightly from the light fraction of sarcoplasmic reticulum (LSR), showing a lack of the ionophoretic activity even at a high concentration (300 microM). This was also confirmed by measuring the 45Ca2+ transport activity of quinolidomicin A1 across an organic solvent barrier. Quinolidomicin A1 (3-300 microM) increased 45Ca2+ release from HSR with an EC50 value of approx. 20 microM. The potency of quinolidomicin A1 was approx. 100-fold higher than that of caffeine. The bell-shaped profile of Ca2+ dependence for quinolidomicin A1 was different from that for caffeine. Blockers of Ca2+ release channels such as Mg2+ (10 mM), procaine (10 mM) and ruthenium red (10 microM) partially blocked quinolidomicin A1 (30 microM)-induced 45Ca2+ release from HSR. At 0 degrees C, quinolidomicin A1-induced 45Ca2+ release was ascertained not to be due to the inhibition of Ca2+ ATPase by the ATPase assay. Quinolidomicin A1 potentiated [3H]ryanodine binding to HSR with a decrease in KD but without a change in Bmax. These results suggest that quinolidomicin A1-induced Ca2+ release from HSR is consisted of two components, which are both sensitive and insensitive to blockers of Ca2+ release channels, and that the former component is associated with the ryanodine receptor.

Differential cardiac contractile depression by volatile anesthetics is well documented, and evidence points to differing actions on the myocardial sarcoplasmic reticulum (SR). Since the Ca(2+)-release channel (CaRC) of the SR binds ryanodine with high-affinity when opened by micromolar Ca2+ concentrations, ryanodine binding to cardiac SR membrane vesicles was employed as an assay of anesthetic modulation of CaRC activity. Canine ventricle was homogenized, centrifuged preparatively and then differentially on a sucrose gradient. A fraction enriched with CaRCs was defined by: the presence of a approximately 450 kDa protein consistent with CaRC; approximately 3-fold enhancement of vesicular 45Ca2+ uptake by ruthenium red; Ca(2+)-activated [3H]ryanodine binding. Specific binding of 10 nM ryanodine was activated by > 0.5 microM Ca2+ and was maximal at approximately 6 pmol/mg protein in > or = 20 microM Ca2+. Halothane (1.5%), but not isoflurane, shifted the Ca(2+)-dependence of ryanodine binding to lower [Ca2+]. With submaximal activation by 5 microM Ca2+, 1.5% and 0.75% halothane enhanced binding of 10-80 microM ryanodine, while 2.5% isoflurane and 3.5% enflurane did not. A plot of bound/free vs. bound ryanodine suggests that halothane causes a dose-dependent increase in ryanodine binding to a high-affinity site, while isoflurane has no such action. In intact myocardium, this effect will decrease Ca2+ retention in the SR so that less Ca2+ will be available to activate contractions, consistent with halothane's depressant action.

Comparison of the effects of fluoride (NaF, 1-10 mM) on the catalytic and ion transport functions of the Ca(2+)-ATPase in sarcoplasmic reticulum (SR) vesicles isolated from rabbit cardiac and fast-twitch skeletal muscles revealed similarities as well as striking tissue-specific differences depending on the experimental conditions employed. Short preincubation (3 min at 37 degrees C) of cardiac or fast muscle SR with fluoride in the absence of Ca2+ and ATP prior to initiating enzyme turnover by simultaneous addition of Ca2+ and ATP to the assay medium resulted in a strong inhibitory effect of fluoride on ATP-energized (oxalate-facilitated) Ca2+ uptake and Ca(2+)-ATPase activity. On the other hand, when turnover was initiated by the addition of ATP to SR preincubated with fluoride in the presence of Ca2+ but in the absence of ATP, fluoride caused concentration-dependent stimulation of active Ca2+ uptake by fast muscle SR with no appreciable change in Ca(2+)-dependent phosphoenzyme (EP) formation (from ATP) or Ca(2+)-ATPase activity but inhibition of active Ca2+ uptake by cardiac SR with concomitant inhibition of EP formation and Ca(2+)-ATPase activity. Exposure of cardiac or fast muscle SR to fluoride in the presence of both Ca2+ and ATP resulted in concentration-dependent stimulatory effect of fluoride on Ca2+ uptake with no change in EP formation or Ca(2+)-ATPase activity, this effect diminished substantially at saturating oxalate concentration in the assay. Assessment of the effects of deferoxamine (1 mM) and exogenous aluminum (10 microM) did not indicate a requirement for aluminum in the inhibitory or stimulatory effect of fluoride. These results suggest that (a) the Ca2+ and ATP-deprived (E1/E2) but not the Ca2+ plus ATP-liganded (CaE1ATP) conformation of the SR Ca(2+)-ATPase is susceptible to inhibition by fluoride in both cardiac and fast muscle; (b) the Ca(2+)-bound conformation (CaE1) of the SR Ca(2+)-ATPase is susceptible to inhibition in cardiac muscle but is refractory to fluoride in fast muscle; and (c) the stimulatory effect of fluoride is largely secondary to its ability to mimic the action of oxalate in intravesicular Ca2+ trapping when the fluoride-resistant enzyme is turning over normally. Fluoride inhibited phosphorylation of the Ca(2+)-free enzyme by Pi in cardiac and fast muscle SR indicating that fluoride sensitivity of the phosphorylation site of the SR Ca(2+)-ATPase is similar in cardiac and fast muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of the organophosphorus insecticide parathion (O,O-diethyl O-(p-nitrophenyl)phosphorothioate) on the physical state of synthetic and native membranes was investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and by its anionic propionic acid derivative (DPH-PA), probing the outer regions of the bilayer. Parathion disorders the gel phase of liposomes reconstituted with dimyristoylphosphatidylcholine (DMPC), broadening the transition profile and shifting the temperature midpoint of the phase transition, as detected by both probes. The insecticide strongly orders the fluid phase either in the hydrophobic core or in the outer regions of the membrane, as evaluated by DPH and DPH-PA, respectively. These ordering effects of parathion were further confirmed in fluid models of egg-yolk phosphatidylcholine. Parathion increases to some extent the ordering promoted by cholesterol in DMPC bilayers, but high cholesterol concentrations (> or = 30 mol%) prevent parathion interaction. The results in native membranes correlate reasonably with those obtained in models of synthetic lipids. Thus, parathion does not exert detectable effects in cholesterol-rich membranes, namely, erythrocytes, but moderate ordering effects of parathion are detected by both probes in brain microsomes, i.e., membranes with a lower content of cholesterol. Again, in agreement with the models of synthetic lipids, pronounced ordering effects of parathion are detected in cholesterol-poor membranes, e.g., sarcoplasmic reticulum and mitochondria.

Nitrendipine and valinomycin act synergistically to stimulate ATP-dependent Ca2+ accumulation by rat skeletal muscle sarcoplasmic reticulum vesicles 3-fold. The stimulation is not caused by activation of the Ca(2+)-ATPase or by inhibition of the sarcoplasmic reticulum Ca2+ channel, but is due to an increased efficiency of transport by Ca(2+)-loaded vesicles. At low Ca2+ concentrations, nitrendipine+valinomycin inhibits Ca2+ uptake by increasing the Ca2+ KM but does not effect equilibrium Ca2+ binding to the Ca(2+)-ATPase (Kd = 0.75 microM). In the presence of 50 mM phosphate, nitrendipine+valinomycin increases the steady-state coupling ratio (Ca2+ accumulated per ATP hydrolyzed) from 0.6 to 1.9 by decreasing the rate of ATP hydrolysis by 72%, while reducing the Ca2+ accumulation rate by only 13%. The rates of both passive and Ca(2+)-ATPase-mediated Ca2+ release are reduced by nitrendipine+valinomycin. The data indicate that nitrendipine and valinomycin act directly on the Ca(2+)-ATPase to decrease the ATP hydrolysis rate, increase the Ca2+ KM, decrease Ca2+ efflux, and increase the Ca2+/ATP coupling ratio of Ca(2+)-loaded vesicles.

The effects of hydroxytamoxifen (OHTAM) on lipid organization of pure phospholipid liposomes, native sarcoplasmic reticulum (SR) membranes and liposomes of SR lipids were evaluated by intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py(3)Py) and by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its derivative 3-[p-(6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (DPH-PA). OHTAM promotes alterations in the thermotropic profiles of DMPC, DPPC and DSPC. As detected by Py(3)Py and DPH-PA, OHTAM induces an ordering effect in the fluid phase and a fluidizing effect in the temperature range of the cooperative phase transition. In the gel phase, no significant effects are noticed, except for DSPC bilayers, where Py(3)Py and DPH-PA detect a disordering effect. In the hydrophobic region of the above membrane systems probed by DPH, OHTAM induces only a slight fluidizing effect in the range of the phase transition and a small ordering effect in the fluid phase. As detected by all probes, the drug broadens the transition profile of DMPC and shifts the main transition temperature to lower values. However, these effects, and so those observed for the fluid phase, decrease as the fatty acyl chain length increases. Moreover, the drug removes the pre-transitions of DPPC and DSPC bilayers, as probed by Py(3)Py. In fluid SR native membranes and liposomes of SR lipids, OHTAM induces a moderate ordering effect in the outer regions of the lipid bilayer, as monitored by Py(3)Py and by DPH-PA, DPH failing to detect any apparent effect, as observed for the fluid phase of pure phospholipids. Apparently, OHTAM distributes preferentially in the outer region of the lipid bilayer, without significant effect in the bulk lipid organization of the bilayer interior. The changes of OHTAM in the bilayer dynamic properties and the different location across the bilayer thickness relative to its drug promoter (Custódio et al. (1993) Biochim. Biophys. Acta 1150, 123-129) may be involved in the cytostatic activity of tamoxifen.

Competition between Ca2+ binding and Pi phosphorylation showed that the affinity of Ca(2+)-ATPase for Pi was not changed by 20% DMSO. Thus, the enhancement of Pi phosphorylation in the presence of DMSO should not be attributed to a solvent effect on the affinity for Pi, but rather on the phosphorylation reaction itself.

We have studied the effect of Ruthenium red on the sarcoplasmic reticulum Ca(2+)-ATPase. Ruthenium red does not modify the Ca2+ pumping activity of the enzyme, despite its interaction with cationic binding sites on sarcoplasmic reticulum vesicles. Two pools of binding sites were distinguished. One pool (10 nmol/mg) is dependent upon the presence of micromolar Ca2+ and may therefore represent the high-affinity Ca2+ transport sites of the Ca(2+)-ATPase. However, Ruthenium red only slightly competes with Ca2+ on these sites. The other pool (15-17 nmol/mg) is characterized as low-affinity cation binding sites of sarcoplasmic reticulum, distinct from the Mg2+ site involved in the ATP binding to the Ca(2+)-ATPase. The interaction of Ruthenium red with these low-affinity cation binding sites, which may be located either on the Ca(2+)-ATPase or on surrounding lipids, decreases tryptophan fluorescence level of the protein. As much as 25% of the tryptophan fluorescence of the Ca(2+)-ATPase is quenched by Ruthenium red (with a dissociation constant of 100 nM), tryptophan residues located near the bilayer being preferentially affected.

(Z)-5-Methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, AU-1421, interacted at 0 degree C with the K(+)-sensitive phosphoenzymes of three transport ATPases, Ca(2+)-, H+/K(+)- and Na+/K(+)-ATPase. In the case of Ca(2+)-ATPase, AU-1421 at about 80 microM stimulated 6-fold the rate of splitting of the phosphoenzyme, on which K+ simply functions as an accelerator from one side of the membrane. Probably AU-1421 also simply interacts with the K(+)-binding site of the phosphoenzyme that is easily accessible from the aqueous phase. In the cases of H(+)/K(+)- and Na(+)/K(+)-ATPases, AU-1421 stabilized the phosphoenzymes which accept K+ as the translocating ion. The rate constants of dephosphorylation for H(+)/K(+)-ATPase and Na(+)/K(+)-ATPase were decreased to half by AU-1421 at about 5 and 10 microM, respectively. Presumably after binding of AU-1421 to a K(+)-recognition site of the phosphoenzyme, local motion of the peptide region near the binding site that serves to move the bound ion into the ion-transport pathway (occlusion center) might be inhibited. Thus AU-1421 may be able to distinguish two modes of K+ action on the K(+)-sensitive phosphoenzymes.

The photochemical release of Ca2+ from caged-Ca2+ in the absence of ATP, and the release of ATP from caged-ATP in the presence of Ca2+ induce characteristic difference FTIR spectra on rabbit sarcoplasmic reticulum that are related to the formation of Ca2-E1 and E approximately P intermediates of the Ca(2+)-ATPase, respectively. Dicyclohexylcarbodiimide (10 nmol/mg protein) abolished both the Ca(2+)-and ATP-induced difference FTIR spectra parallel with inhibition of ATPase activity. Cyclopiazonic acid (50 nmol/mg protein) inhibited the Ca(2+)-induced difference spectrum measured in the absence of ATP, but had no significant effect on the ATP-induced difference spectrum measured in the presence of 1 mM Ca2+. The dog kidney Na+,K(+)-ATPase did not give significant difference spectrum after photolysis of caged-ATP in Ca(2+)-free media containing 90 mM Na+ and 10 mM K+, with or without ouabain. We propose that both the Ca2+ and the ATP-induced difference FTIR spectra of the Ca(2+)-ATPase reflect the occupancy of the high-affinity Ca2+ transport site of the enzyme.

While studying the effects of membrane phosphorylation on active Ca2+ transport in cardiac sarcoplasmic reticulum (SR) we used NaF (a conventional phosphatase inhibitor) in the Ca2+ transport assay medium to suppress protein dephosphorylation by endogenous phosphatases. Unexpectedly, depending on the experimental conditions employed, NaF was found to cause a strong inhibitory or stimulatory effect on ATP-dependent, oxalate-facilitated Ca2+ uptake (Ca2+ pump) activity of SR. Investigation of this phenomenon using canine cardiac SR revealed the following. Exposure of SR to NaF in the absence of Ca2+ or ATP in the Ca2+ transport assay medium (prior to initiating Ca2+ transport by the addition of Ca2+ or ATP) promoted a striking concentration-dependent inhibitory effect of NaF (50% and 90% inhibition with approx. 4 and 10 mM NaF, respectively) on Ca2+ uptake by SR; the magnitude of inhibition did not differ appreciably with varying oxalate concentrations. In contrast, exposure of SR to NaF in the presence of both Ca2+ and ATP resulted in a concentration-dependent stimulatory effect of NaF (half-maximal stimulation at approx. 2.5 mM NaF with 2.5 mM oxalate in assay) on Ca2+ uptake; the magnitude of stimulation decreased with increasing oxalate concentration (greater than 2-fold at 1 mM oxalate, 10% at 5 mM oxalate). The inhibitory effect prevailed when SR was exposed to NaF in the presence of Ca2+ alone (without ATP) or ATP alone (without Ca2+). Both the inhibitory and stimulatory effects of NaF were specific to fluoride ion, as NaCl (1-10 mM) showed no effect on Ca2+ uptake by SR under identical assay conditions. A persistently less active state of the Ca2+ pump (evidenced by decreased Ca2+ transport rates) resulted upon pretreatment of SR with NaF in the absence of Ca2+ or ATP; presence of Ca2+ and ATP during pretreatment prevented this transition. The inhibitory action of NaF on the Ca2+ pump was accompanied by a two-fold increase in K0.5 for Ca2+ and decrements in Hill coefficient (nH) and Ca(2+)-stimulated ATP hydrolysis, as well as steady-state level of Ca(2+)-induced phosphoenzyme. The stimulatory effect of NaF, on the other hand, was associated with an increase in the ratio of Ca2+ transported/ATP hydrolysed with only minor changes, if any, in the above parameters. These findings imply that the divergent effects of fluoride are dependent on specific conformational states of the Ca(2+)-ATPase which evolve during the catalytic and ion transport cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Light-induced Ca2+ release from the Ca2+ complex of Nitr-5 altered the FTIR spectra of sarcoplasmic reticulum vesicles and purified Ca(2+)-ATPase preparations. The principal changes seen in difference spectra obtained after and before illumination in the presence of Nitr-5.Ca2+ consisted of an increase in absorbance at 1663 and 1676 cm-1 and a decrease in absorbance at 1653 cm-1. The light-induced changes in FTIR spectra were prevented by vanadate or EGTA, indicating that they were associated with the formation of Ca2E1 enzyme intermediate. Other light-induced changes in the FTIR spectra at 1600-1250 cm-1 were not clearly related to the sarcoplasmic reticulum, and were attributed to photolysis of Nitr-5. The difference absorbance bands are narrow, suggesting that they originate from changes in side chain vibrations, although some changes in secondary structures may also contribute.

Illumination of sarcoplasmic reticulum vesicles by ultraviolet light in the presence of 1 mM vanadate causes photocleavage of the Ca(2+)-ATPase into two fragments (Vegh et al. (1990) Biochim. Biophys. Acta 1023, 168-183). In the absence of Ca2+ the photocleavage occurs in the N-terminal half of the molecule near the phosphate acceptor Asp-351. In the presence of 2 mM Ca2+ the photocleavage shifts to the C-terminal half of the ATPase, near the FITC binding site (Lys-515). About half of the Ca(2+)-ATPase was cleaved rapidly, accompanied by nearly complete, irreversible loss of ATPase activity when illuminated in the presence of 2 mM CaCl2; further cleavage of the enzyme was slow and affected primarily the C-terminal fragment produced in the presence of Ca2+. Solubilization of the Ca(2+)-ATPase with C12E8 did not affect the site of photocleavage in either conformation. The vanadate-induced Ca(2+)-ATPase crystals were disrupted during photocleavage, while the binding of anti-ATPase antibodies directed against the phosphorylation site (PR-8) and against the FITC binding region (PR-11) was enhanced. The bovine kidney Na+,K(+)-ATPase was insensitive to photocleavage under conditions where about half the Ca(2+)-ATPase was fragmented. The slight cleavage of the pig gastric H+,K(+)-ATPase after prolonged illumination produced fragments that are distinct from the fragments of the Ca(2+)-ATPase.

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.

Thyroid hormones inhibit Ca2+ accumulation and ATPase activity of isolated sarcoplasmic reticulum vesicles. Half-maximal inhibition was obtained by about 2.5 microM. The ATP hydrolysis activity of the purified (Ca2+ + Mg2+)-ATPase or of the SR vesicles, in the presence of the Ca2+ ionophore A23187, is not inhibited by T3 or T4. Modification of T3 or T4 in the ring portion, but not in the amino portion, of the molecules results in T4 and T3 analogues which are unable to inhibit Ca2+ accumulation. T3 and T4 have no significant effect on various partial reactions of the transport cycle such as: the binding of ATP and Ca2+, or ADP-ATP exchange and E-P formation from ATP, but they inhibit the E-P formation from inorganic phosphate (Pi) and ATP-Pi exchange. The inhibition of both Ca2+ accumulation and ATPase activity by T3 or T4 is increased in the presence of Pi. Binding sites for [125I]T3 and for [125I]T4 in SR proteins were demonstrated using either equilibrium dialysis or gel overlay techniques. The results suggest that the thyroid hormones inhibit the ATP-dependent Ca2+ accumulation, probably by inhibiting the transport of anions which act as the Ca2+ precipitating anion.

The sarcoplasmic reticulum (SR) of skeletal muscle contains a 53 kDa glycoprotein of unknown function, as well as the (Ca(2+)-Mg2+)-ATPase. It has been suggested that the glycoprotein couples the hydrolysis of ATP by the ATPase to the transport of calcium. It has been shown that if SR vesicles are solubilized in cholate in media containing low K+ concentrations followed by reconstitution, then vesicles are formed containing the glycoprotein and with ATP hydrolysis coupled to Ca2+ accumulation, as shown by a large stimulation of ATPase activity by addition of A23187. In contrast, if SR vesicles are solubilized in media containing a high concentration of K+, then the vesicles that are produced following reconstitution lack the glycoprotein and show low stimulation by A23187 (Leonards, K.S. and Kutchai, H. (1985) Biochemistry 24, 4876-4884). We show that the effect of K+ on reconstitution does not follow from any changes in the amount of glycoprotein but rather from an effect of K+ on the detergent properties of cholate. In low K+ media, the cmc of cholate is high, cholate is a relatively poor detergent and incomplete solubilization results in 'reconstitution' of vesicles with the correct orientation of ATPase molecules. In high K+ media, the cmc of cholate is reduced and more complete solubilization of the SR leads to a true reconstitution with the formation of vesicles with a random orientation of ATPase molecules. The experiments provide no evidence for an effect of the glycoprotein on the (Ca(2+)-Mg2+)-ATPase.

The extra uptake of Ca2+ by vesicles of sarcoplasmic reticulum (SR) observed in the presence of Pi, attributable to transport of Pi by the Pi-transporter, has been studied. It has been shown that the Pi transporter is stimulated by ATP. Single channel conductance measurements have shown that the Cl- channel in the SR membrane is impermeable to Pi. It is suggested that the transporter could be an ion antiporter system. Studies of uptake as a function of pH and Mg2+ concentration suggest that transport of MgHPO4 and H2PO-4 are faster than transport of HPO2-4. For oxalate and pyrophosphate, Mg2+ binding inhibits transport. It is suggested that protonation of lysine residue(s) at the anion binding site increase the rate of transport.

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.

The sarcoplasmic reticulum (SR) of skeletal muscle contains a Pi transporter which transports Pi into the lumen of the SR, increasing the level of accumulation of Ca2+ by SR by forming insoluble salts with Ca2+. Phosphonocarboxylic acids inhibit the transport of Pi by the transporter, phosphonoformic acid itself being transported into the SR increasing the level of accumulation of Ca2+. Phenylphosphonic acid also inhibits Pi transport, distinguishing the Pi transporter of SR from the Na+/Pi transporter of brush-border membranes. Oxalate transport is also inhibited by the phosphono-carboxylic acids, consistent with the suggestion that oxalate and phosphate are carried on the same transporter. The effects of maleate are, however, not inhibited, suggesting a separate carrier for the dicarboxylic acids. Acetic anhydride and phenylglyoxal inhibit the transporter, Pi providing protection against the effects of acetic anhydride, suggesting the presence of a lysine residue at the Pi binding site. ATP provides protection against the effects of acetic anhydride and phenylglyoxal, suggesting the presence of an ATP binding site on the transporter.

m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was used to cross-link the protein components of rabbit skeletal muscle sarcoplasmic reticulum. Analysis of cross-linked material by SDS-polyacrylamide gel electrophoresis showed that both the (Ca(2+)-Mg2+)-ATPase and the 53 kDa glycoprotein could be cross-linked, since the amount of protein at the locations on the gel corresponding to uncross-linked material was reduced in the presence of 1.0 mM MBS. Cross-linked products of 130 kDa, 200-260 kDa and approx. 300 kDa were identified. Probing the cross-linked products with monoclonal antibodies against ATPase, 53 kDa glycoprotein and calsequestrin revealed no cross-linked products containing the ATPase and either calsequestrin or the 53 kDa glycoprotein over the range of molecular weights examined here. Possible interactions between the ATPase and calsequestrin or the 53 kDa glycoprotein were also investigated by studying the ATPase activity for the purified ATPase and for the ATPase in sarcoplasmic reticulum vesicles made permeable to Ca2+ with A23187. Effects of Ca2+ and ATP on the two systems were indistinguishable, providing no evidence for a major modulatory role of calsequestrin or the 53 kDa glycoprotein on the ATPase.

The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.

In the presence of divalent cations, the metallochromic Ca2+ indicator arsenazo III is reduced by sulfhydryl groups to form an azo anion radical. Reduced arsenazo III is reoxidized back to its original state by oxygen. The formation of the arsenazo III azo anion radical in the presence of sarcoplasmic reticulum vesicles leads to the rapid inhibition of the (Ca2+ + Mg2+)-ATPase. These data indicate that several factors should be considered when arsenazo III is used as a Ca2+ indicator; (1) Functionally important sulfhydryl groups may be oxidized by arsenazo III; (2) the generation of free radicals by arsenazo III reduction may be toxic to the system being studied; (3) the absorbance spectrum of arsenazo III is altered when reduced by sulfhydryl groups.

The fluorescence quenching properties of a brominated derivative of androstenol 5 alpha,6 beta-dibromoandrostan-3 beta-ol have been used to study binding to phospholipid bilayers and to the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum of rabbit skeletal muscle. It is shown that androstenol is excluded from the phospholipid/protein interface of the ATPase but can bind to other (non-annular sites) on the ATPase. Binding to these sites increases in strength with decreasing chain length for the phospholipids present in the system. Binding is also stronger in the presence of phospholipids in the gel phase than in the liquid crystalline phase. Androstenol increases the ATPase activity of the ATPase reconstituted with phosphatidylcholines of chain lengths less than C18, but has no effect on activity for the ATPase reconstituted with phosphatidylcholines of chain lengths C18 or greater. The effects of cholestanols on the activity of the ATPase reconstituted with dimyristoleoylphosphatidylcholine depend on the configuration of the sterol, with 5 alpha-cholestan-3 alpha-ol having little effect but the other isomers causing a marked stimulation.

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of DDT with model and native membranes. DDT decreases the phase transition midpoint temperature (Tm) of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), and broadens the thermotropic profile of the transition. The effects of DDT are concentration dependent and are more pronounced in bilayers of short-chain lipids, e.g., DMPC. The insecticide fails to alter DPH polarization in the fluid phase of the above lipids. Similar effects were observed in binary mixtures of DMPC plus DPPC. Furthermore, DDT alters the single broad transition of the equimolar mixture of DMPC plus DSPC into a biphasic transition. The lower temperature component has a midpoint at 25 degrees C, i.e., a value close to the Tm of DMPC. DDT inhibits to some extent the cholesterol-induced ordering in DMPC bilayers and high cholesterol concentrations (greater than or equal to 30 mol%) do not prevent insecticide interaction, conversely to the effect observed for lindane (Antunes-Madeira, M.C. and Madeira, V.M.C. (1989) Biochim. Biophys. Acta 982, 161-166). Apparently, the bilayer order is not disturbed by DDT in fluid native membranes of mitochondria and sarcoplasmic reticulum, but moderate disordering effects are noticed in membranes enriched in cholesterol, namely, brain microsomes and erythrocytes.

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.

The effect of caesium on the cation transport system in sarcoplasmic reticulum vesicles has been analysed kinetically through Tris+ influx. The Tris+ influx was measured by following the change in K+ diffusion potential due to the mutual diffusion between K+ and Tris+ in the presence of valiomycin using a potential probe; 3,3'-dipropylthiadicarbocyanine iodide. The main results were as follows. (1) Tris+ influx increased when membrane potential became inside-negative. This suggests that Tris+ permeates through the channel which has a voltage-dependent gate. (2) Cs+ reacted with the cation transport system only from the outside of the vesicle and inhibited Tris+ influx. The inhibition follows a single-site titration curve with a voltage-dependent dissociation constant of 18 mM at -60 mV. The inhibition can be explained by assuming that Cs+ binds to a site located about 45% of the way through the membrane from the outside of the vesicle in the open state of the channel. These results are in good agreement with those reported by Coronado and Miller (Coronado, R. and Miller, C. (1979) Nature 288, 495-497), which were gained electrically by using sarcoplasmic reticulum vesicles incorporated into an artificial planar phospholipid bilayer.

Sarcoplasmic reticulum vesicles were prepared from rabbit skeletal muscle. It could be demonstrated that the anion binding sites on this membrane can be studied by 35Cl-NMR spectroscopy. Titration of sarcoplasmic reticulum vesicles with the sulfate exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) revealed specific binding of this compound to the sarcoplasmic reticulum membrane. A new inhibitor, pyridoxalphosphate-6-azophenyl-2'-sulfonic acid (PPAPS) was introduced and proved to displace chloride equally well from its binding sites. Two binding sites could be distinguished by titration with inorganic phosphate in the presence and absence of the inhibitors. Because of the insensitivity of 35Cl-NMR spectroscopy these anion binding sites have to be located on a protein being present in considerable amount in the sarcoplasmic reticulum membrane.

Passive Ca2+ permeability of sarcoplasmic reticulum vesicles has been studied after maximal loading with Ca2+ (150-200 nmol/mg protein) in the presence of Ca2+, MgATP and an ATP generating system of limited capacity. Outflow of accumulated Ca2+ in the non-energized state of the system was studied by depletion of the medium of one of the substrates, either MgATP (by complete consumption) or Ca2+ (by complexation with EGTA). It was found that Ca2+ outflow under these conditions is relatively slow and independent of the medium concentration of Ca2+ (5 X 10(-9)-5 X 10(-5) M) or MgATP (0.7-730 microM). Outflow curves were steep at the beginning of the outflow phase (30-60 nmol/min per mg protein), and outflow proceeded at a much lower rate below 100 nmol Ca2+/mg protein. Outflow could be completely inhibited by La3+. The Ca2+ release curves are not compatible with simple diffusion, and cannot be accounted for by Ca2+ binding inside the vesicles. Neither are our observations consistent with permeation mediated via the Ca2+ translocation sites involved in active transport. We suggest that non-energized Ca2+ outflow may proceed by a process of ion-exchange through negatively charged, water-filled channels in the membrane, the properties of which are altered by a high intravesicular concentration of Ca2+.

Calcium accumulation by two fractions of sarcoplasmic reticulum presumably derived from longitudinal tubules (light vesicles) and terminal cisternae (heavy vesicles) was examined radiochemically in the presence of various free Mg2+ concentrations. Both fractions of sarcoplasmic reticulum exhibited a Mg2+-dependent increase in phosphate-supported calcium uptake velocity, though half-maximal velocity in heavy vesicles occurred at a much higher free Mg2+ concentration than that in light vesicles (i.e., approx. 0.90 mM vs. approx. 0.02 mM Mg2+). Calcium uptake velocity in light vesicles correlated with Ca2+-dependent ATPase activity, suggesting that Mg2+ stimulated the calcium pump. Calcium uptake velocity in heavy vesicles did not correlate with Ca2+-dependent ATPase activity, although a Mg2+-dependent increase in calcium influx was observed. Thus, Mg2+ may increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles. Analyses of calcium sequestration (in the absence of phosphate) showed a similar trend in that elevation of Mg2+ from 0.07 to 5 mM stimulated calcium sequestration in heavy vesicles much more than in light vesicles. This difference between the two fractions of sarcoplasmic reticulum was not explained by phosphoenzyme (EP) level or distribution. Analyses of calcium uptake, Ca2+-dependent ATPase activity, and unidirectional calcium flux in the presence of approx. 0.4 mM Mg2+ suggested that ruthenium red (0.5 microM) can also increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles, with no effect in light vesicles. These functional differences between light and heavy vesicles suggest that calcium transport in terminal cisternae is regulated differently from that in longitudinal tubules.

The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly.

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium X calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca2+ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium X calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca2+ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium X calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca2+ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent 45Ca2+-40Ca2+ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium X calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca2+ release and 45Ca2+-40Ca2+ exchange.

Effects of Na+ and K+ on Ca-2+ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg-2+ and ATP (2mM) and low Ca-2+ (0.44muM) concentrations. Under these conditions, Na+ and K+ inhibit Ca-2+ uptake, ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca-2+ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca-2+ pump of the sarcoplasmic reticulum operates below its maximal capacity.

Sarcoplasmic reticulum vesicles are used here as model membrane system to question the hypothesis of enhancement of permeability of cations by anesthetics, particularly that of Ca2+ and of Mg2+. The effects of dibucaine (up to 800 microM), tetracaine (up to 2 mM), lidocaine (up to 10 mM) and procaine (up to 10 mM) on the permeability of these membranes to Ca2+ and Mg2+ have been measured. We have used an experimental approach based on the light scattering method (Kometani, T. and Kasai, M. (1978) J. Membrane Biol. 41, 295-308). It has been found that all the local anesthetics cited above markedly increase the permeability of sarcoplasmic reticulum vesicles to Mg2+ and, in the concentration range tested herein, only dibucaine and tetracaine increase the permeability to Ca2+. The kinetic analysis of the time dependence of the light-scattering data after the osmotic shock shows that, in the absence of local anesthetics, the Mg2+ influx can be described as proceeding through a unique type of channel. However, Ca2+ influx appears to involve two channel of different kinetic properties. Because the relative fraction of both types of Ca2+ channel is similar to the average ratio between light and heavy vesicles in unfractionated sarcoplasmic reticulum, we suggest that each type of channel can be preferentially located in one of these fractions. The determined rate constants for Ca2+ permeability through both types of channel are 0.77 +/- 0.08 min-1 (fast channels) and 0.025 +/- 0.005 min-1 (slow channels) and that for Mg2+ is 0.08 +/- 0.02 min-1. These results agree with data obtained by other groups using different experimental approaches. Dibucaine and tetracaine significantly alter the rate of Mg2+ and Ca2+ influx through the slow channels. In addition, these two local anesthetics also produce the effect that the Mg2+ influx cannot be described with only one exponential process, thus suggesting a differential effect on vesicles of different density. The increase of Ca2+ and Mg2+ permeability by dibucaine and by tetracaine is found at concentrations of these drugs that do not produce a noticeable inhibition of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles.

The uptake and release of Ca2+ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca2+ indicator. Incorporation of the Ca2+ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca2+ by several orders of magnitude. Therefore in addition to participating in active Ca2+ transport, the (Mg2+ + Ca2+)-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca2+ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca2+ release from the sarcoplasmic reticulum during activation of muscle.

2H2O (99.8%) Ringer's solution greatly reduces the twitch and tetanus of frog sartorius muscle and, as specially shown here, slows the onset features of the mechanical output of the twitch by: (a) increasing the time (LR) from stimulus to start of latency relaxation; (b) slowing the development of the latency relaxation, and (c) greatly decreasing the rate of onset of tension development. These changes reflect effects of 2H2O on excitation-contraction coupling and they represent the critical direct effects of 2H2O on muscle since it does not depress either the action potential or the intrinsic myofibrillar contractility. The increase in LR is attributed to slowed inward electrical propagation in the T-tubule. But the critical effect of 2H2O on frog muscle is to greatly depress mobilization of activator Ca2+. The depression of the Ca2+ mobilization and of its effects on the activation of contraction evidently result from (a) a lowered rate of release of Ca2+ from the sar coplasmic reticulum, as indicated by the slowed development of the latency relaxation, (b) a decreased amount of Ca2+ released in a twitch, and (c) a reduced speed of diffusion of the Ca2+ to the contractile filaments. The depressed mobilization of Ca2+ is apparently the essential cause of 2H2O's general depression of twitch and tetanus output.

Calcium transport into sarcoplasmic reticulum fragments isolated from dog cardiac and mixed skeletal muscle (quadriceps) and from mixed fast (tibialis), pure fast (caudofemoralis) and pure slow (soleus) skeletal muscles from the cat was studied. Cyclic AMP-dependent protein kinase and phosphorylase b kinase stimulated the rate of calcium transport although some variability was observed. A specific protein kinase inhibitor prevented the effect of protein kinase but not of phosphorylase b kinase. The addition of cyclic AMP to the sarcoplasmic reticulum preparations in the absence of protein kinase had only a slight stimulatory effect despite the presence of endogenous protein kinase. Cyclic AMP-dependent protein kinase catalyzed the phosphorylation of several components present in the sarcoplasmic reticulum fragments; a 19000 to 21 000 dalton peak was phosphorylated with high specific activity in sarcoplasmic reticulum preparations isolated from heart and from slow skeletal muscle, but not from fast skeletal muscle. Phosphorylase b kinase phosphorylated a peak of molecular weight 95000 in all of the preparations. Cyclic AMP-dependent protein kinase-stimulated phosphorylation was optimum at pH 6.8; phosphorylase b kinase phosphorylation had a biphasic curve in cardiac and slow skeletal muscle with optima at pH 6.8 and 8.0. The addition of exogenous phosphorylase b kinase or protein kinase increased the endogenous level of phosphorylation 25-100%. All sarcoplasmic reticulum preparations contained varying amounts of adenylate cyclase, phosphorylase b and a (b:a = 30.1), "debrancher" enzyme and glycogen (0.3 mg/mg protein), as well as varying amounts of protein kinase and phosphorylase b kinase which were responsible for a significant endogenous phosphorylation. Thus, the two phosphorylating enzymes stimulated calcium uptake in the sarcoplasmic reticulum of a variety of muscles possessing different physiologic characteristics and different responses to drugs. In addition, the phosphorylation catalyzed by these enzymes occurred at two different protein moieties which make physiologic interpretation of the role of phosphorylation difficult. While the role phosphorylation in these mechanisms is complex, the presence of a glycogenolytic enzyme system may be an important link in this phenomenon. The sarcoplasmic reticulum represents a new substrate for phosphorylase b kinase.

The effect of a synthetic neutral ligand on the Ca2+ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca2+ -ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca2+ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca2+ -ionophore complex across the hydrophobic core of the membrane. In mitochondria, the neutral ionophore promotes the active transport of Ca2+ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.

Vesicular fragments of sarcoplasmic reticulum isolated from rabbit skeletal muscle were actively loaded with Ca2+ in the presence of ATP and an ATP-regenerating system using Arsenazo III as metallochromic indicator to monitor Ca2+ movements across the membrane. Once the Ca2+ release is triggered by the presence of tetraphenylboron in the reaction medium, the addition of verapamil or diltiazem gives rise to a net Ca2+ entry inside the vesicles. Preincubation in the presence of verapamil does not abolish the tetraphenylboron-induced Ca2+ release, the verapamil-induced Ca2+ accumulation being still observed. There appears to be a high-affinity site for verapamil titrated in the micromolar concentration range, whereas diltiazem demonstrates more complex behavior when its concentration is raised. This study suggests the existence of a Ca2+ pathway (putative channels) which is blocked by the drugs tested allowing Ca2+ accumulation inside the vesicles owing to the Ca2+-dependent ATPase activity.

The permeability of sarcoplasmic reticulum vesicles to sulfate ions was inhibited by diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (H2DIDS), which is a potent inhibitor of anion permeability in red blood cell membrane. The amount of H2DIDS bound to the vesicles was determined by using [3H]-H2DIDS. Apparent half inhibition of sulfate permeation was observed on the binding of 2.5 mumol/g protein. SDS-polyacrylamide gel electrophoresis on the vesicles treated with [3H]H2DIDS showed that about 10% of the total bound H2DIDS corresponds to a 100 000-dalton protein, but the remaining 90% to non-protein components. The content of the H2DIDS-binding protein was about 0.5 mumol/g protein. These results suggest that the H2DIDS-binding protein is different from the calcium pump protein and is possibly an anion transport system similar to band 3 in red blood cell membrane.

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.

The Ca2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca2+ and surface membrane-bound Ca2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca2+ in the absence of ATP, 10-12 h are necessary for measurable amount of Ca2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca2+ from 'loaded' vesicles only after this period of incubation. A fraction of Ca2+ of 50-60 nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg2+ and K+ in the medium and is readily released by ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca2+ (30-40 nmol/mg protein) is rapidly released X-537A. The results indicate that most of the Ca2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.