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

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

Activation of the caffeine center of the sarcoplasmic reticulum at a reduced concentration of magnesium ions

The action of caffeine and Mg2+ on the efficacy of Ca2+ transport by terminal cisterns and longitudinal tubules of rabbit skeletal muscle sarcoplasmic reticulum (SR) was studied and compared. Addition of 5 to 10 mM caffeine to the incubation medium or a decrease in Mg2+ concentration from 4 to 0.1 mM led to a 3-fold diminution of the Ca/ATP ratio for the terminal cistern fraction. In longitudinal tubules, that effect was far less pronounced. The effects of caffeine and decreases in Mg2+ concentration were blocked by ruthenium red, tetracaine and dimethylsulfoxide. It is assumed that the decrease in Mg2+ concentration is accompanied by activation of the caffeine site of the SR, induced by the intravesicular caffeine-like factor.     

Blockers of the uncoupling action of caffeine on the Ca2+-transport function of sarcoplasmic reticulum membranes

The influence of caffeine on the efficiency of Ca2+ transport in the presence of oxalate by different fractions of sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle was studied. It was shown that caffeine (5 mM) decreases 4-fold the value of the Ca/ATP ratio in terminal cisterns of the SR without having any appreciable influence on the efficiency of Ca2+ transport by the light fraction of the SR. The uncoupling effect of caffeine is completely blocked by ruthenium red and by the local anesthetics, tetracaine, procaine, and benzocaine.     

Effect of caffeine and cyclosporin A on the transmembrane exchange of Ca ions in smooth muscle mitochondria

The effects of cyclosporin A and caffeine on the active and passive transport of Ca2+ in mitochondria isolated from adult rat myometrium were studied by fluorescent technique using Ca2+-sensitive probe tetracycline (TC). It was shown that 5 microM cyclosporin increases Ca2+ accumulation by the mitochondria matrix. But it fails to exhibit such effect when 20 mM caffeine was also present in the incubation medium, while the inhibitory action of caffeine on the accumulation of Ca2+ reveals nevertheless in the absence or presence of cyclosporin A. In case of the preliminary incubation of mitochondria with 10 mM caffeine before the initiation of transport process one could also observe the inhibition of kinetic parameters of the active accumulation of Ca2+ by the mitochondria. It was also shown, that caffeine stimulates passive efflux of Ca2+ from the myometrium mitochondria. Thus we conclude, that the stimulating effect of cyclosporin on Ca2+ accumulation by the myometrium mitochondria is sensitive to caffeine, while caffeine has no direct effect on Ca2+-uniporter, but it evidently disturbs the barrier function of the inner mitochondria membrane in such way, that stimulating effect of cyclosporin A cannot develop.     

Intracellular localization of the caffeine-sensitive form of Ca-dependent ATPase in the sarcoplasmic reticulum

A Ca-selective electrode was used to study the effect of caffeine on different fractions of sarcoplasmic reticulum membranes of rabbit skeletal muscles. Caffeine was found to uncouple Ca2+ transport and ATP hydrolysis in a fraction, which is enriched with fragments of terminal cisterns according to the electron microscopy data. Caffeine does not produce any effect on the light fraction containing no fragments of terminal cisterns. It is concluded that caffeine-sensitive Ca2+-dependent ATPase is localized in terminal cisterns of the sarcoplasmic reticulum.     

The effect of total ischemia on the Ca2+-transporting activity of the sarcoplasmic reticulum of the rat myocardium

Different caffeine and calcium concentrations have been studied for their influence on Ca2(+)-pumping function of sarcoplasmic reticulum in a homogenate from control and ischemic rat myocardium. Ca2(+)-transporting system of sarcoplasmic reticulum terminal cisternae membranes from the ischemic myocardium was found to be more sensitive to Ca2+ and caffeine action, inhibiting Ca2+ uptake velocity, as compared to control. This may be one of causes leading to the contractibility disorder under myocardium ischemia.     

Effect of caffeine on active Ca2+ ion transport in a homogenate of skeletal muscles and myocardium

A Ca2-selective electrode was used to study active transport of Ca2+ by sarcoplasmic reticulum fragments of rabbit skeletal muscle and myocardium homogenates. The specific Ca2+ transport activities (mumol Ca2+/min/mg tissue) are 40 = 60 and 3 = 5 units for fast and slow muscles and the myocardium, respectively. Caffeine (5 mM) exerts a powerful inhibitory influence on Ca2+ transport in skeletal muscle homogenates. For fast muscles, the degree of inhibition exceeds 50%. The rate of Ca2+ transport in the myocardium homogenate increases in the presence of creatine phosphate. The latter produces no effect on Ca2+ transport in skeletal muscle homogenates. The high sensitivity of Ca2 transport to caffeine, a specific blocker of Ca2+ transport to the terminal cisterns of the sarcoplasmic reticulum, suggests that the terminal cisterns, apart from being a reservoir for Ca2+ needed for contraction trigger, may play an essential role in muscle relaxation.     

Effect of caffeine on kinetics of accumulation and release of Ca2+ by vesicles of the sarcoplasmic reticulum of skeletal muscle

The action of caffeine was studied on the heavy sarcoplasmic reticulum fraction enriched by vesicles derived from terminal cisterns. Caffeine lowers the ATP-dependent accumulation of Ca2+ by vesicles and enhances the first rapid phase of the Ci2+ release from vesicles. The action of caffeine was transient, reversed, Ca2+-dependent. The data obtained suggest that the reduction of ATP-dependent calcium accumulation and enhancement of calcium release by caffeine are mediated by the mechanism of Ca2+-induced Ca2+ release and support the view that caffeine may regulate the equilibrium between open and closed states of Ca2+-channel by increasing the affinity of Ca2+-receptor site of the channel.     

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

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

Fractionation of fragments of skeletal muscle sarcoplasmic reticulum according to their sensitivity to caffeine

Sarcoplasmic reticulum fragments were fractionated according to the ability of caffeine to selectively block Ca2+ uptake in the population of caffeine-sensitive membranes. The membrane suspension was loaded with calcium in the presence of oxalate, Mg-ATP and caffeine, after which the Ca2+-loaded caffeine-sensitive fragments were separated by sucrose density gradient centrifugation. In Ca2+-unloaded fragments of the supernatant, the sensitivity to caffeine estimated by its ability to diminish the rate of Ca2+ uptake, Ca/ATP ratio and Ca-oxalate capacity amounted to 91-93%. The terms of protein composition, the caffeine-sensitive fragments were identified with terminal cystern membranes, while the caffeine-insensitive ones with the SR canalicular membranes. The sensitivity to caffeine may serve as a reliable criterion for estimating the relative content of terminal cystern fragments in different microsomal preparations.     

Effects of caffeine on transport, metabolism and ultrastructure of isolated rat colon

The effect of caffeine on the transport, metabolism and ultrastructure of the colon were determined. Segments of proximal colon were excised from the anesthetized rat and prepared for radioisotopic tracing of ion transport in the flux chambers or oxidative metabolism in an incubator. Other segments were fixed before or after caffeine administration for electron microscopy. The isolated rat colon actively transported both Na+ and Cl- in the absorptive direction, mucosa to serosa. Serosal addition of 10 mmol/l caffeine abolished the smaller Na+ transport but did not significantly affect the larger Cl- transport. The electrical potential difference and the short-circuit current rose accordingly. Although the oxidation of glucose was inhibited by 35%, caffeine had no significant effect on the oxidation of the fatty acid, butyric acid. Comparable metabolic responses were obtained using the isolated terminal ileum of the rat. Neither the height nor the density of the microvilli in the proximal colon were affected significantly by caffeine. It may be concluded that caffeine, unlike theophylline, effectively preserves the normal absorptive condition of the colon. Thus, caffeine may have actions other than inhibition of phosphodiesterase in the distal intestine.     

Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system

Ca2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/Mg2+-dependent 45Ca2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca2+ imaging in living cells we distinguish two ER Ca2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45Ca2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca2+ ATPase and [32P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca2+ levels. TG-R Ca2+ accumulation represents a distinct Ca2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.     

The Mechanism of the Action of Caffeine on Sarcoplasmic Reticulum

Evidence is presented that caffeine does not act on the mitochondrial Ca uptake system and that its effect cannot be attributed to the accumulation of adenosine 3',5'-phosphate. Two distinct caffeine effects are described. At high ATP concentrations caffeine decreases the coupling between ATP hydrolysis and Ca inflow. It either inhibits inflow without any inhibition of the rate of ATP hydrolysis, or it stimulates the ATPase activity without stimulating Ca inflow. These high ATP concentrations (much higher than needed for the saturation of the transport ATPase) greatly reduce the control of the turnover rate of the transport system, by accumulated Ca. At low ATP concentrations when the transport system is under maximal control by accumulated Ca, caffeine inhibits the ATPase activity without affecting the rate of Ca inflow.     

Effect of caffeine and glycerin on the Ca transport system of sarcoplasmic reticulum fragments from frog skeletal muscles

Parameters of the Ca2+-ion transport system by a fragmented sarcoplasmic reticulum isolated from phasic and tonic frog skeletal muscles were investigated under the action of caffeine or caffeine in combination with glycerol. No changes were observed in the Ca-transport system of a light fraction of the sarcoplasmic reticulum under the influence of caffeine and caffeine-glycerol combination. Caffeine reduced the value of Ca/ATP and enhanced the outflux of Ca2+-ions from membrane fragments of the caffeine-sensitive sarcoplasmic reticulum fraction of both the muscles; the combined effect of caffeine and glycerol was analogous to the action of caffeine applied alone. It is concluded that the potentiation of muscle contraction in the presence of glycerol is not due to the excess of Ca-release from the sarcoplasmic reticulum caused by this agent.     

Effects of hirsutine, an antihypertensive indole alkaloid from Uncaria rhynchophylla, on intracellular calcium in rat thoracic aorta.

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla (MIQ.) Jackson, on cytosolic Ca2+ level ([Ca2+]cyt) were studied by using fura-2-Ca2+ fluorescence in smooth muscle of the isolated rat aorta. Noradrenaline and high K+ solution produced a sustained increase in [Ca2+]cyt. Application of hirsutine after the increases in [Ca2+]cyt induced by noradrenaline and high K+ notably decreased [Ca2+]cyt, suggesting that hirsutine inhibits Ca2+ influx mainly through a voltage-dependent Ca2+ channel. Furthermore, the effect of hirsutine on intracellular Ca2+ store was studied by using contractile responses to caffeine under the Ca(2+)-free nutrient condition in the rat aorta. When hirsutine was added at 30 microM before caffeine treatment, the agent slightly but significantly reduced the caffeine-induced contraction. When added during Ca2+ loading, hirsutine definitely augmented the contractile response to caffeine. These results suggest that hirsutine inhibits Ca2+ release from the Ca2+ store and increases Ca2+ uptake into the Ca2+ store, leading to a reduction of intracellular Ca2+ level. It is concluded that hirsutine reduces intracellular Ca2+ level through its effect on the Ca2+ store as well as through its effect on the voltage-dependent Ca2+ channel.     

Elucidation of the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells: modulation by cyclic ADP-ribose and FK506

The effects of cyclic ADP-ribose (cADPR) and the immunosuppressant drug FK506 on microsomal Ca2+ release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. After a steady state of 45Ca2+ uptake into the microsomal vesicles, ryanodine or caffeine was added. Preincubation of the vesicles with cADPR (0.5 microM) shifted the dose-response curve of ryanodine- or caffeine-induced 45Ca2+ release from the vesicles to the left. Preincubation with cADPR shifted the dose-response curve of the FK506-induced 45Ca2+ release upward. Preincubation with FK506 (3 microM) shifted the dose-response curve of the ryanodine- or caffeine-induced 45Ca2+ release to the left by the same extent as that in the case of cADPR. FK506 shifted the dose-response curve of the cADPR-induced 45Ca2+ release upward. The presence of both cADPR and FK506 enhanced the ryanodine (30 microM)- or caffeine (10 mM)-induced 45Ca2+ release by the same extent as that in the case of cADPR alone or FK506 alone. These results indicate that cADPR and FK506 modulate the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the mechanism. In addition, there is a possibility that the mechanisms of modulation by cADPR and FK506 are the same.     

Endothelin mobilizes Ca2+ from a caffeine- and ryanodine-insensitive intracellular pool in rat atrial cells

Endothelin-1 is a powerful inotropic peptide for the rat atrium. Its action can develop in the absence of L-type Ca2+ channel activity provided that the external Ca2(+)-concentration has been raised to supraphysiological concentrations. Endothelin stimulates phosphatidylinositol hydrolysis in new born rat atrial cells via a mechanism that is insensitive to pertussis toxin. The diacylglycerol/protein kinase C signaling pathway cannot account for the contractile action of endothelin but its activation by phorbol esters induces a partial desensitization of phospholipase C activity. Endothelin-1 and the related peptides, endothelin-2, endothelin-3, and sarafotoxin S6b, raise intracellular Ca2+ levels in rat atrial cells. The actions of endothelin-1, endothelin-2, and sarafotoxin on [Ca2+]i are mutually exclusive, suggesting that they act at the same receptor site. The rise in [Ca2+]i induced by endothelins results both from the mobilization of intracellular stores and from Ca2+ entry through the sarcolemma via a pathway that is not voltage-dependent L-type Ca2+ channels. The Ca2+ store that is mobilized in response to endothelin retains its Ca2+ content when cells were incubated for long periods of time in a 50 nM Ca2+ solution. It is insensitive to caffeine and ryanodine. These two properties distinguish it from the sarcoplasmic reticulum. Contraction experiments in which the pacing rate has been altered to favor Ca2+ accumulation into terminal cisternae of the sarcoplasmic reticulum also suggest that the Ca2+ load of the sarcoplasmic reticulum is increased in endothelin treated rat atria.     

Identification of a ryanodine receptor in rat heart mitochondria

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.     

Concentration of components of the adenylate system in heavy and light fractions of the sarcoplasmic reticulum of skeletal muscles and sensitivity of these fractions to the effects of imidazole-containing compounds and caffeine]

Fragments of sarcoplasmic reticulum from rabbit sceletal muscles sedimented within the range from 2000 g to 8000 g (heavy fraction) and 8000 g to 40000 g (light fraction) and washed with 0.6 M KCl, were practically free of adenylatecyclase activity. Phosphodiesterase cAMP was not found in the light fraction, while its activity in the heavy fraction was 500 pmol of cAMP/min per mg of protein. Both fractions contain bound cAMP (1-2 pmol/mg of protein) and specific sites of cAMP binding, the binding constant being approximately 10(6)M-1. The number of binding sites is 60 pmol/mg of protein for the heavy and 30 pmol/mg of protein for the light fractions. The level of phosphodiesterase activity in the heavy fraction correlates with its sensitivity to imidazole, anserine and caffeine. Imidazole and anserine increase in 1.5-1.8 times the value of Ca2+/ATP in the heavy fraction and produce no effect on Ca2+ transport by the light fraction. Caffeine decreases almost twice the Ca2+/ATP value in the heavy fraction and has practically no effect on Ca2+ absorption by enzymes of the light reticulum fraction. Imidazole and anserine activate membrane-bound phosphodiesterase, while caffeine inhibits it. It is suggested that structural rearrangements of membrane-bound phosphodiesterase under the effect of caffeine, imidazole and anserine are responsible for changes in the efficiency of Ca2+ transport by fragments of the heavy reticulum fractions.     

Caffeine-induced Ca(2+) release increases AMPK-dependent glucose uptake in rodent soleus muscle

Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because alpha-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-beta (ACCbeta) Ser221 and immunoprecipitated alpha(1)-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated alpha(2)-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCbeta and alpha(1)-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 microM), the CaM-competitive inhibitor KN-93 (10 microM), or the SR Ca(2+) release blocking agent dantrolene (10 microM) all inhibited ACCbeta phosphorylation and alpha(1)-AMPK phosphorylation, suggesting that SR Ca(2+) release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca(2+)-activated CaMKK may control alpha(1)-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.