Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.     

Different sensitivity to trypsin of the human platelet plasma and intracellular membrane Ca2+ pumps

The Ca2+ pumps associated with human platelet plasma and intracellular membranes have been further characterized by their sensitivity to trypsin. (a) Tryptic degradation of the Ca2+-ATPases has been followed by immunoblotting. It resulted in fragmentation into peptides of 80, 55, 35, and 24 kDa for both enzymes. Subcomplete hydrolysis obtained with a ratio of trypsin/membrane protein of 0.05-0.1 for the two Ca2+ pumps resulted in the total disappearance of the 100-, 80-, and 35-kDa fragments. However, maximum degradation was reached within 1 min for the intracellular enzyme but needed 5 min of incubation for the plasma membrane enzyme. (b) This effect of trypsin has been correlated with its effect on both the Ca2+-ATPase activities. The plasma membrane enzyme showed a maximum inhibition of 50-60% which was obtained using a trypsin/protein ratio of 0.1 and 5 min of incubation. A much higher trypsin sensitivity was observed for the intracellular enzyme because the maximum inhibition reached 80% after only 1 min of incubation. (c) Finally, the two Ca2+ transport systems studied showed different trypsin reactivities; the Ca2+ uptake by the plasma membrane vesicles was inhibited by 20-25%, and this maximum inhibition was observed after 5 min of incubation with trypsin. In contrast, the Ca2+ transport associated with the intracellular membrane vesicles was difficult to detect after trypsin treatment. Taken together, the results show that the two Ca2+ pumps can be distinguished by their trypsin sensitivity.     

Inhibition of inorganic pyrophosphatase of animal mitochondria by calcium

Calcium ion is an uncompetitive inhibitor of the inorganic pyrophosphatases of bovine heart and rat liver mitochondria with respect to substrate MgPPi at pH 8.5 and a non-competitive inhibitor of the former enzyme at pH 7.2. The concentration of Ca2+ required to decrease the maximal velocities for both enzymes at pH 8.5, 0.4 mM Mg2+ was about 10 microM. The inhibition results from the binding of two Ca2+ ions to both free enzymes and their complexes with the substrate. The results suggest that Ca2+ regulates pyrophosphatase activity and hence PPi level in mammalian mitochondria.     

Factors controlling the intracellular concentration of calcium and the spontaneous activity of the ureter

The role of the electrogenic Na(+)-Ca(2+)-exchange mechanism in regulating the spike activity of the ureter was studied. The ureter cells were shown to be capable of generating action potentials (AP) in sodium-free Krebs solution. The time during which the spikes are generated is in exponential dependence on the concentration of calcium ions in the medium, [Ca2+]o within 2.5 to 15 mmol/l. Simultaneously with the generation of the spikes, accumulation of calcium in the muscles is observed, proportional to the increase of [Ca2+]o. The addition of as little as 20 mmol/l Na+ or Li+ ions into the solution restores the prolonged electrical activity of the ureter. Under these conditions, the decrease of intracellular Ca2+ within 5 min was more than two times larger as compared with that in sodium-free medium. Upon substituting Ba2+ ions for Ca2+ ions in Krebs solution AP are generated within an interval which was the longer the higher the Ba2+ concentration in the medium. Li+ ions can replace Na+ ions in maintaining AP and in extruding calcium from the cell. It is supposed that the generation of the stable spike activity of the ureter depends on the functioning of Na(+)-Ca(2+)-exchange mechanism.     

Elucidation of conservative elements of calmodulin-dependent enzymes with the use of monoclonal antibodies

Monoclonal antibodies against human erythrocyte membrane Ca2+-ATPase were obtained. The binding of monoclonal antibodies to the enzyme resulted in a decrease in the enzyme sensitivity to calmodulin (CaM). The effects of monoclonal antibodies on other CaM-dependent enzymes, namely, on the phosphodiesterase of cAMP, phosphorylase kinase, and Ca2+-CaM-dependent protein kinase II (PK II), were studied. It was found that all four enzymes contain a common antigenic site. However, the inhibitory effect of antibodies was observed only with respect to Ca2+-ATPase and PK II. The kinetics of the binding of monoclonal antibodies and their inhibitory action were investigated. It was shown that the antigenic site is confined to the calmodulin-binding portion of Ca2+-ATPase and PK II.     

Pancreatic enzyme activity in early phases of acute experimental pancreatitis in rats

Experiments were performed on laboratory rats with acute pancreatitis caused by local freezing the pancreas with chlorethyl. An active action of enzymes alpha-amilase, lipase, phospholipase A2, was revealed. During the first hours, an increase in action of all three enzymes, particularly that of phospholipase A2, was found. It was established that the lipid spectrum of pancreas had changed. It shows that cell membranes were destroyed. Experiments revealed an activating role of Ca2+ ions for all the enzymes and a correcting action of chlorpromasine.     

Cellular mechanisms of burn-related changes in contractility and its prevention by mesenteric lymph ligation

Major burn injury results in impairment of left ventricular (LV) contractile function. There is strong evidence to support the involvement of gut-derived factor(s) transported in mesenteric lymph in the development of burn-related contractile dysfunction; i.e., mesenteric lymph duct ligation (LDL) prevents burn-related contractile depression. However, the cellular mechanisms for altered myocardial contractility of postburn hearts are largely unknown, and the cellular basis for the salutary effects of LDL on cardiac function have not been investigated. We examined contractility, Ca(2+) transients, and L-type Ca(2+) currents (I(Ca)) in LV myocytes isolated from four groups of rats: 1) sham burn, 2) sham burn with LDL (sham + LDL), 3) burn ( approximately 40% of total body surface area burn), and 4) burn with LDL (burn + LDL). Myocytes isolated from hearts at 24 h postburn had a depressed contractility ( approximately 20%) at baseline and blunted responsiveness to elevation of bath Ca(2+). Myocyte contractility was comparable in sham + LDL and sham burn hearts. LDL completely prevented burn-related changes in myocyte contractility. Mechanistically, the decrease in contractility in myocytes from postburn hearts occurred with a decrease in the amplitude of Ca(2+) transients ( approximately 20%) without changes in resting Ca(2+) or Ca(2+) content of the sarcoplasmic reticulum. On the other hand, I(Ca) density was decreased ( approximately 30%) in myocytes from postburn hearts, with unaltered voltage-dependent properties. Thus burn-related myocardial contractile dysfunction is linked with depressed myocyte contractility associated with a decrease in I(Ca) density. These findings also provide strong evidence that mesenteric lymph is involved in the onset of burn-related cardiomyocyte dysfunction.     

Changes in guinea pig gallbladder smooth muscle Ca2+ homeostasis by acute acalculous cholecystitis

Impaired smooth muscle contractility is a hallmark of acute acalculous cholecystitis. Although free cytosolic Ca2+ ([Ca2+]i) is a critical step in smooth muscle contraction, possible alterations in Ca2+ homeostasis by cholecystitis have not been elucidated. Our aim was to elucidate changes in the Ca2+ signaling pathways induced by this gallbladder dysfunction. [Ca2+]i was determined by epifluorescence microscopy in fura 2-loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. F-actin content was quantified by confocal microscopy. Ca2+ responses to the inositol trisphosphate (InsP3) mobilizing agonist CCK and to caffeine, an activator of the ryanodine receptors, were impaired in cholecystitic cells. This impairment was not the result of a decrease in the size of the releasable pool. Inflammation also inhibited Ca2+ influx through L-type Ca2+ channels and capacitative Ca2+ entry induced by depletion of intracellular Ca2+ pools. In addition, the pharmacological phenotype of these channels was altered in cholecystitic cells. Inflammation impaired contractility further than Ca2+ signal attenuation, which could be related to the decrease in F-actin that was detected in cholecystitic smooth muscle cells. These findings indicate that cholecystitis decreases both Ca2+ release and Ca2+ influx in gallbladder smooth muscle, but a loss in the sensitivity of the contractile machinery to Ca2+ may also be responsible for the impairment in gallbladder contractility.     

Osmoregulatory reactions of frog erythrocytes under conditions of activation and blockade of Ca2+-channels

The kinetics of cell osmoregulatory reactions under conditions of activation and blockade of Ca2+-channels was studied on a model of frog polyfunctional nuclear erythrocyte. Both activation and blockade of Ca2+-channels has been established to promote swelling of nuclei and an increase of the nuclear-cytoplasmic ratios under conditions of hypotonic exposure. The osmoregulatory cell reactions after activation of Ca2+-channels are expressed as a decrease of the cell volume. The blockator of Ca2+-channels verapamil produces an alternated increase and decrease of the erythrocyte volume with time intervals of 30 and 60 s. The clearly expressed functional activity of the nuclear membrane in response to the hypotonic action under conditions of activation and blockade of Ca2+-channels indicates participation of Ca2+ ions in mechanisms of the nuclear-cytoplasmic transfer.     

Influence of oxidative processes in mitochondria on contractility of the frog Rana temporaria heart muscle. Effects of cadmium

The inotropic Cd2+ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd2+ at concentrations of 1, 10, and 20 microM is established to decrease dosedependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Ca2(+)-channels of the L-type (Ca 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd2+ at concentrations of 15 and 25 microM produces swelling of non-energized and energized mitochondria in isotonic (with KNO2 and NH4NO3) and hypoosmotic (with 25 mM CH3COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 microM Cd2+ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd2+ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP-uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd2+ is due not only to its direct blocking action on Ca2(+)-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K+ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

Effect of detergents,trypsin, and bivalent metal ions on interfacial activation and functioning of phospholipase D

The effects of detergents, trypsin, and bivalent metal ions on production of phosphatidic and lysophosphatidic acids by the action of phospholipase D (PLD) on lecithin and lysolecithin were studied. It was found that these reaction products and dodecyl sulfate ions activate PLD, whereas other anionic detergents are less effective. A protective effect of the functioning enzyme against its hydrolytic inactivation by trypsin was found. Bivalent metal ions can be arranged in the following sequence by their ability to activate PLD in the hydrolysis of lecithin and lysolecithin: Ca²⁺ > Sr²⁺ > Ba²⁺ > Mg²⁺. These results are considered in relation to a proposed mechanism of activation and functioning of PLD with the participation of clusters of phosphatidates and lysophosphatidates. Such Me²⁺-induced formation of rafts or microdomains from the products of hydrolysis of phospholipids can rationalize not only PLD activation and self-regulation, but also the action of this mechanism on other components and properties of biomembranes. PLD and other lipolytic enzymes can be classified as lateral vector enzymes.     

Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.     

Letter: Mechanism of K+-dependent swelling of Ehrlich ascites carcinoma cells]

Limiting values of cell volume measured under the conditions of K+-dependent swelling of EAC cells decrease when the medium is hypertonized, the extent of the decrease depending more on the type of the agent applied than on the concentration gradient. According to the action efficiency the agents studied can be placed in the following sequence: Ca2+, succrose Na+,K+. It is concluded that Ca ions strengthen the cell membrane, while K+ ions produce a contrary effect.     

Effect of the irradiation conditions on the radiation inactivation of subtilisin-72

A comparative study was made of gamma-inactivation of subtilisin-72 solutions in 5 X 10(-3) M acetate buffer and 0.1 M NaCl in the presence and absence of Ca2+ ions. It was shown that the acetate buffer had a protective action, and the influence of Ca2+ ions depended on the ionic strength of the solution. In general, Ca2+ ions exerted a stabilizing effect irrespective of the subtilisin concentration in the acetate buffer, but this effect competed with the destabilizing influence of the ionic strength increased by Ca2+ ions.     

The role of calcium in the response of cardiac muscle to stretch

This review focuses on the complex interactions between two major regulators of cardiac function; Ca2+ and stretch. Initial consideration is given to the effect of stretch on myocardial contractility and details the rapid and slow increases in contractility. These are shown to be related to two diverse changes in Ca2+ handling (enhanced myofilament Ca2+ sensitivity and increased intracellular Ca2+ transient, respectively). Interaction between stretch and Ca2+ is also demonstrated with respect to the rhythm of cardiac contraction. Stretch has been shown to alter action potential configuration, generate stretch-activated arrhythmias, and increase the rate of beating of the sino-atrial node. A variety of Ca(2+)-dependent mechanisms including attenuation of Ca2+ extrusion via Na+/Ca2+ exchange, Ca2+ entry through stretch-activated channels (SACs) and mobilisation of intracellular Ca2+ stores have been proposed to account for the effect of stretch on rhythm. Finally, the interaction between stretch and Ca2+ in the secretion of natriuretic peptides and onset of hypertrophy is discussed. Evidence is presented that Ca2+ (entering through L-type Ca2+ channels or SACs, or released from sarcoplasmic reticular stores) influences secretion of both atrial and B-type natriuretic peptide; there is data to support both positive and negative modulation by Ca2+. Ca2+ also appears to be important in the pathway that leads to expression of precursors of hypertrophic protein synthesis. In conclusion, two of the major regulators of cardiac muscle function, Ca2+ and stretch, interact to produce effects on the heart; in general these effects appear to be additive.     

DNA structural transitions induced by divalent metal ions in aqueous solutions

Using methods of IR spectroscopy, light scattering, gel-electrophoresis DNA structural transitions are studied under the action of Cu2+, Zn2+, Mn2+, Ca2+ and Mg2+ ions in aqueous solution. Cu2+, Zn2+, Mn2+ and Ca2+ ions bind both to DNA phosphate groups and bases while Mg2+ ions-only to phosphate groups of DNA. Upon interaction with divalent metal ions studied (except for Mg2+ ions) DNA undergoes structural transition into a compact form. DNA compaction is characterized by a drastic decrease in the volume occupied by DNA molecules with reversible formation of DNA dense particles of well-defined finite size and ordered morphology. The DNA secondary structure in condensed particles corresponds to the B-form family. The mechanism of DNA compaction under Mt2+ ion action is not dominated by electrostatics. The effectiveness of the divalent metal ions studied to induce DNA compaction correlates with the affinity of these ions for DNA nucleic bases: Cu2+>Zn2+>Mn2+>Ca2+>Mg2+. Mt2+ ion interaction with DNA bases (or Mt2+ chelation with a base and an oxygen of a phosphate group) may be responsible for DNA compaction. Mt2+ ion interaction with DNA bases can destabilize DNA causing bends and reducing its persistent length that will facilitate DNA compaction.     

Calcium-dioxolene complexes: rate constants of pyrocatechol oxidation in the presence of Ca2+

The effect of calcium ions on the rate of pyrocatechol autoxidation at pH 9.0 has been studied by mathematical modeling. The effect of Ca2+ on the oxygen absorption rate has been studied, and a kinetic model has been suggested, which takes different stages of interaction of pyrocatechol and its radical form with oxygen into account. It has been shown that the prooxidant action of Ca2+ is related to an abrupt increase (approximately by three orders of magnitude) in the rate constant of comproportionation (reaction of chain branching and formation of o-semiquinonates) and a marked decrease (by two orders of magnitude, from 1.4 10(7) to 0.6 10(5) M(-1)s(-1)) in the rate constant of disproportionation of o-semiquinones. The system can be used as a model for studying the prooxidant action of calcium ions.     

Modulation of an Intracellular Calmodulin-Stimulated Ca2+-Pumping ATPase in Cauliflower by Trypsin (The Use of Calcium Green-5N to Measure Ca2+ Transport in Membrane Vesicles)

The effect of controlled trypsin digestion of a calmodulin-stimulated Ca2+-ATPase in low-density intracellular membranes from cauliflower (Brassica oleracea L.) inflorescences was investigated. Ca2+ uptake into vesicles was measured either continuously with the fluorescent Ca2+ indicator Calcium Green-5N or with a radio-active filter technique. Trypsin treatment of vesicles resulted in a 3-fold activation of Ca2+ uptake and loss of calmodulin sensitivity. Immunoblotting experiments with an antiserum raised against the Ca2+-ATPase showed that the trypsin activation was accompanied by a decrease in the amount of intact Ca2+-ATPase (111 kD) and by successive appearances of polypeptides of 102 and 99 to 84 kD. 125I-Calmodulin overlays showed that only the intact Ca2+-ATPase bound calmodulin. Removal of the calmodulin-binding domain (about 9 kD) was not enough to obtain full activation. Trypsin proteolysis resulted in a Ca2+ concentration necessary for half-maximal activity of 0.5 [mu]M, whereas a value of about 2 [mu]M was obtained with untreated membranes in the presence of calmodulin. Without trypsin treatment or calmodulin the activity was not saturated even at 57 [mu]M free Ca2+. The data suggest that trypsin digestion and calmodulin activate the cauliflower Ca2+-ATPase by at least partly different mechanisms.     

A novel specific binding site for anxiolytic homophthalazines in the rat brain.

Radioligand binding studies were performed in order to elucidate the mechanism of action of anxiolytic-neuroleptic homophthalazines. Rat striatal membrane preparations were found to bind 3H-EGIS 6775 [3H-GYKI-52 322, 3H-(1-(4-aminophenyl)-4-methyl-7,8-dimethoxy-5H-homophthalazine)] in a specific and displaceable manner. Several other brain regions tested were devoid of similar binding activity. Saturation analysis revealed that binding affinity was in the 10(-8)-10(-7) M range. Binding was enhanced by Mg2+ ions and, to a smaller extent by Ca2+ ions. The binding principle was sensitive to heat or trypsin treatment. This specific binding site appears, according to competition studies, different from the receptors whose presence in the rat striatum has been reported earlier.     

cAMP, calmodulin-dependent stimulation and stability to proteolysis of Ca 2+ transport in the heart sarcoplasmic reticulum

The calmodulin content in cardiomyocyte cytosol of hypoxic myocardium is increased compared to normal level. This is unaccompanied by differences in the stimulating effect of calmodulin on Ca2+ transport in sarcoplasmic reticulum (SR) of ischemic heart. The decrease of the endogenous cAMP-dependent protein kinase activity in ischemia is associated with the lowered resistance to trypsinolysis of Ca2+ transport in SR (trypsin/microsomal protein ratio is 1:10) with simultaneous Ca-ATPase activation. In the presence of exogenous protein kinase and cAMP the protective effect of phosphorylation on Ca2+ transport in SR vesicles of hypoxic cardiomyocytes treated with trypsin for 10 min reaches the same level as in intact heart.