Water and sodium transport: effects of calcium channel blocker and calmodulin antagonists on the apical and basolateral membranes of amphibian epithelia

Ca2+ channel blocker (sensit) and calmodulin antagonists (thioridazine, perphenazine, oxyprothepine) applied to the mucosal side of frog urinary bladder, weakened the response of epithelial cells to vasopressin. Thioridazine (2.7 X 10(-5) mol X l-1) and sensit (1.7 X 10(-4) mol X l-1) applied to the serosal side rapidly increased the permeability of the epithelia for sodium and potassium ions along the concentration gradient (from serosa to mucosa). The same concentrations of these blockers when applied to the mucosal side of frog urinary bladder selectively decreased vasopressin stimulated water permeability and did not influence ionic permeability. Both thioridazine and sensit decreased the short-circuit current across frog skin. The results show that the Ca2+ channel blocker and the calmodulin antagonists tested influenced water and ionic transport across the epithelial cell membranes, and had different effects upon the apical and the basolateral cell membranes.     

Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells.

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+ channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni(2+)-, La(3+)-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 mumol l-1 and 2.5 mumol l-1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mumol l-1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mumol l-1 and 95.29 mumol l-1, respectively. The depolarization was significantly inhibited at 50 mumol l-1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.     

Effect of calcium entry blockade on the actions of phenylephrine on the taenia of the guinea pig caecum

The interaction between phenylephrine and calcium entry blockers was studied on the taenia of the guinea-pig caecum using the double sucrose gap method. Sustained hyperpolarization, relaxation and attenuation of evoked electrical and mechanical activity were induced by non-cumulative addition of phenylephrine (0.1 to 250 mumol.1-1) for 2 to 4 min. When the alpha 1-adrenoceptor agonist was applied for a prolonged period (20 to 60 min) the initial inhibitory response gradually disappeared both at room temperature and at 32 degrees C. The renewed action potentials were accompanied by a positive afterpotential. The initial hyperpolarization and its delayed recovery in course of the phenylephrine effect were significantly reduced in calcium-free medium containing EDTA (2 mmol.1-1), after pretreatment with nifedipine (0.1 to 1 mumol.1-1), verapamil (10 to 100 mumol.1-1) or procaine (0.5 to 2 mmol.1-1). In contrast sodium nitroprusside (10 to 100 mumol.1-1) which produced biphasic changes similar to those of phenylephrine, did not affect the initial and delayed phase of phenylephrine action. Ba2+(5 mmol.1-1) could substitute for Ca2+ in the generation of action potentials but could not substitute for Ca2+ in the mechanisms responsible for the initial and delayed recovery phase of phenylephrine effects. In the presence of La3+ and Mn2+ (0.5 to 3 mmol.1-1) the phenylephrine effects were reduced. In contrast, in the presence of extracellular Ca2+, pretreatment with Mg2+ (12 mmol.1-1) or Ba2+ (5 mmol.1-1) did not affect the action of phenylephrine. It is concluded that activation of alpha 1-adrenoceptors results in the release of Ca2+ from an intracellular store, which leads to the opening od TEA-sensitive potassium channels, causing the initial phase of alpha 1-adrenoceptor action. Ca2+ is loaded into this intracellular store by entering the cell through the potential sensitive calcium channels. Although the mechanisms responsible for the delayed phase could not be clarified, its dependence on the presence of the initial phase is apparent.     

Ca(2+)-related changes in the mouse sperm capacitation state: a possible role for Ca(2+)-ATPase.

Mammalian spermatozoa require extracellular Ca2+, some of which must be internalized, to undergo complete capacitation. At a critical threshold, a rise in intracellular Ca2+ will trigger acrosomal exocytosis. We used chlortetracycline (CTC) fluorescence patterns to assess changes in the capacitation state of mouse spermatozoa after incubation under various conditions that would affect their intracellular Ca2+ concentrations. Under standard conditions with 1.80 mmol CaCl2l-1 known to support capacitation within 120 min and subsequent fertilization in vitro, a rise in the number of capacitated, acrosome-intact cells (B pattern) was observed over the first 60 min, followed by a decline. A detectable increase in capacitated, acrosome-reacted cells (AR pattern) coincided with the maximum of B pattern cells and a continued rise was observed over the following 60 min. With incubation in 3.60 mmol Ca2+l-1, the rise in AR cells began at 30 min, suggesting that this treatment accelerates capacitation. Introduction of ionophore A23187 at 15 min to cells in standard Ca2+ produced a similar but even more rapid response, with a maximum in B pattern cells and a noticeable rise in AR cells within 10 min. Thus ionophore-treated cells proceed through capacitation, but do so very quickly. However, ionophore in the presence of 90 mumol Ca2+l-1 could promote transition from the uncapacitated F pattern to the capacitated B pattern, but could not trigger acrosomal exocytosis, indicating that the latter requires high extracellular Ca2+. After preincubation in Ca(2+)-deficient medium, most cells exhibited the uncapacitated F pattern and the introduction of millimolar Ca2+ altered this distribution only slowly, over a period of 50 min. In contrast, preincubation in 90 mumol Ca2+l-1 resulted in a minority of F pattern cells and, within 10 min of millimolar Ca2+ introduction, a significant increase in AR cells was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positive inotropic effect of ryanodine on rabbit ventricular muscle: dependence on the intracellular calcium load

Two types of electrical and mechanical responses to 1 mumol/l ryanodine, depending on the intracellular calcium load, were observed in rabbit papillary muscles. In a normal calcium solution, ryanodine induced a transient decline followed by a stable increase in the developed force (by 20 +/- 5% of the pretreatment level; n = 30) and prolonged the action potential (AP). The positive ryanodine response showed an increased time-to-peak force and was completely suppressed by 2 mumol/l nifedipine, partially blocked by 50 mumol/l tetracaine (Ca2+ release blocker), but greatly potentiated by 20 mmol/l CsCl or (-) Bay R 5414 which prolonged the AP. The prolonged time-to-peak force of the positive ryanodine response was shortened by procedures raising the content of Ca2+ in the sarcoplasmic reticulum (SR). It is suggested that the initial decline in the force amplitude results from Ca2+ leakage from the SR which is further compensated for by an elevation of both the transmembrane Ca2+ entry and intracellular Ca2+ release. In calcium overloaded myocardium, 1 mumol/l ryanodine caused irreversible contracture and dramatic AP shortening, explained by a massive Ca2+ release from the overloaded SR into the cytoplasm. It is concluded that the calcium content in the SR is the main modulator of the electrical and mechanical effects of ryanodine in ventricular myocardium.     

Calcium-sensitive potassium channel inhibitors antagonize genistein- and daidzein-induced arterial relaxation in vitro

Estradiol-17beta relaxes rabbit coronary artery rings via large conductance Ca2+-activated K+-channels (K(Ca)). Genistein and daidzein are plant-derived estrogen-like compounds. The aim of the present study was to investigate whether potassium channels participate in the genistein- and daidzein-induced arterial relaxation like they do in the case of estradiol-17beta. Endothelium-denuded superior mesenteric arterial rings from non-pregnant Wistar female rats were used. At a concentration of 10 microM, estradiol-17beta, genistein and daidzein relaxed noradrenaline precontracted arterial rings, (58 +/- 4%, 45 +/- 5% and 31 +/- 3%, respectively; (n=6-8)). Genistein- and daidzein-induced relaxations were inhibited both by iberiotoxin (1-10 nM) and charybdotoxin (30 nM), the antagonists of large conductance Ca2+-activated K+-channels (K(Ca)). Estradiol-17beta-induced relaxation was reduced by iberiotoxin (30 nM). Estradiol-17beta- and daidzein-induced relaxations were also decreased by apamin (0.1-0.3 microM), an antagonist of small conductance Ca2+-activated K+-channels. The antagonists of voltage-dependent K+-channels (K(V)) (4-aminopyridine), ATP-sensitive K+-channels (K(ATP)) (glibenclamide), or inward rectifier K+-channels (KIR) (barium) had no effect on the relaxation responses of any of the compounds studied. Estrogen receptor antagonist tamoxifen did not inhibit the relaxations. In conclusion, in the noradrenaline precontracted rat mesenteric arteries, the relaxations caused by estradiol-17beta, genistein and daidzein were antagonized by large and small conductance K(Ca)-channel inhibitors, suggesting the role of these channels as one of the relaxation mechanisms.     

Effects of sulfhydryl reagents on Na+-Ca2+ exchange in rat brain microsomal membranes

Effects of six thiol reagents with different physico-chemical properties were tested on the Na+-dependent 45Ca2+ transport into the rat brain microsomal membrane vesicles. The mercurials p-chlormercuribenzoate and Mersalyl effectively inhibited 45Ca2+ uptake with IC50 values in the order of 10(-4) mol X l-1 in the medium. N-ethylmaleimide and its more lipophilic analog N-(4-(2-benzoxazolyl)phenyl)maleimide were much less effective at the same concentrations. 2,2'-dithiodipyridine markedly reduced 45Ca2+ uptake already at concentrations below 10(-4) mol X l-1, whereas 5,5'-dithiobis-2-nitrobenzoate in a concentration range 10(-6)-10(-3) mol X l-1 was a weak inhibitor. Inhibitory effects of the most potent inhibitors p-chlormercuribenzoate and 2,2'-dithiodipyridine were readily reversed by 1 mmol X l-1 dithiothreitol. The results suggest that free SH groups of membrane polypeptides are involved in the functioning of the Na+-Ca2+ exchanger in the nerve tissue cell membranes.     

Monitoring of potassium-stimulated catecholamine changes in striatal synaptosomal preparations and in corpus striatum of rats: a comparative voltammetric study.

Voltammetric techniques were used to compare the effects of K(+)-induced depolarization on catecholamine levels in in vitro synaptosomal preparations of the corpus striatum with those in the in vivo corpus striatum of anaesthetized animals. In vitro, the catechol-oxidation currents could be recorded only in dopamine-preloaded synaptosomes. In isolated synaptosomes prepared in the presence of elevated concentrations of Ca2+ (1 mmol.l-1) and Na+ (135 mmol.l-1), K(+)-induced depolarization had variable effects on catechol-oxidation current. The stimulatory effect of K(+)-induced depolarization (a short transient increase of catechol-oxidation current lasting for 30 s) could be observed after the addition of dopamine loaded synaptosomes in EGTA into the medium with elevated K+ concentration (90 mmol.l-1) and decreased concentrations of Na+ (75 mmol.l-1) and Ca2+ (0.75 mmol.l-1). These results suggest that experimental procedures and parameters of ionic composition of incubation media have to be carefully controlled, owing to an enhanced in vitro permeability of membranes of isolated synaptosomes for Ca2+ and Na+. In in vivo experiments, microinjection of KCl (3 microliters of 0.5 mol.l-1 KCl in 10 mmol.l-1 HEPES, pH 7.4) resulted in the appearance of several phases of catechol-oxidation current: the current increased (to severalfold of the control values) followed by a decrease or even total disappearance, with a gradual return to control values. Under conditions of depletion of extracellular calcium by EGTA (5 microliters of 0.5 mol.l-1 KCl + 0.25 mol.l-1 EGTA in 10 mmol.l-1 HEPES, pH 7.4) K(+)-induced depolarization confirmed the key role of calcium in the release of catecholamine transmitters as well as that in processes regulating the uptake and metabolism of these transmitters. The voltammetric techniques used in the present study may be a useful tool in extending of our knowledge about the cellular mechanisms of stimulus-response coupling in nerve cells.     

Evaluating in vitro and in vivo the interference of ascorbate and acetaminophen on glucose detection by a needle-type glucose sensor.

The aim of this work was to assess, in vitro and in vivo, the interference of ascorbate and acetaminophen on glucose measurements by a needle-type glucose sensor detecting hydrogen peroxide generated during the enzymatic oxidation of glucose, and to ascertain whether the protection against interference by the membranes used in the construction of the electrode is feasible. The oxidation of ascorbate and acetaminophen on a platinum electrode set at a 650 mV potential yielded a current representing 75 +/- 5% and 25 +/- 6% of that generated by the oxidation of an equimolar concentration of hydrogen peroxide, respectively. The bias introduced by the presence of 100 mumol l-1 ascorbate on the reading of 5 mmol l-1 glucose by the complete sensor (electrode + membranes) would be minimal (approximately 0.4 mmol l-1). By contrast, the bias introduced by 200 mumol l-1 of acetaminophen (a plasma concentration easily reached in clinical practice) was about 7 mmol l-1. The sensor was implanted subcutaneously in anaesthetized rats (n = 3). Using the current generated in the presence of a plasma acetaminophen concentration of about 200 mumol l-1 for glucose monitoring would lead to a major underestimation (approx. 6 mmol l-1) of subcutaneous glucose concentrations.     

Na(+)-Ca2+ exchange in locust striated muscles

High Na+ + Ca2+ exchange rates comparable with those reported for crayfish striated muscle, rat heart and rat brain, were observed in locust striated muscle homogenates and membrane preparations. The Na(+)-Ca2+ exchange followed the 1st order kinetics with a Km value of 18 mumol.l-1 for Ca, the pH optimum was at 8, the temperature optimum at 30 degrees C, and the exchange was inhibited in the presence of sodium in the incubation medium, with a KiNa of approx. 25 mmol.l-1. The present results suggest a high Na(+)-Ca2+ exchange in locust striated muscles which operate on the calcium electrogenesis principle.     

Role of calcium on the contraction induced by potassium and norepinephrine in the sheep rumen

The effect of calcium on the contractile responses induced by high K+ solutions and noradrenaline has been investigated Ca2+-free-solutions and two selective antagonists of calcium channels (verapamil and sodium nitroprusside) have been used. Both types of responses were inhibited by Ca2+-free-solutions. Contractions induced by high K+ solutions were inhibited by verapamil, but not by sodium nitroprusside. However, the responses to noradrenaline were specifically inhibited by sodium nitroprusside. These results suggest that in rumen circular smooth muscle of the sheep there are two types of calcium channels, a voltage-dependent Ca2+ channel and receptor-linked Ca2+ channel.     

Increased Ca++ or mg++ concentration reduces relative tight-junction permeability to Na+ in the cortical thick ascending limb of Henle's loop of rabbit kidney

We have tested whether increased Ca++ and Mg++ concentrations have an effect on transepithelial voltage (PDte) and transepithelial resistance (Rte) in isolated perfused cortical thick ascending limbs (cTAL) of rabbit kidney. The divalent cations added at 2.5, 5.0 and 10.0 mmol.l-1 to the lumen or peritubular bath perfusate led to a concentration-dependent increase in Rte. The maximal response in Rte was observed between 5 and 10 mmol.l-1. No significant change in active transepithelial potential difference (PDte) was observed. The increase in Rte still occurred when the transcellular current was reduced by Ba++ (3 mmol.l-1) added to the lumen perfusate. This suggests that the increase in Rte caused by Ca++ and Mg++ is due to a modification of the paracellular shunt pathway. In the absence of active transport, i.e. when furosemide (5.10(-5) mol.l-1) was added to the lumen perfusate. Ca++ and Mg++ reduced the transepithelial diffusion potential generated by a NaCl gradient established across the epithelium, and thus produced a reduction of the relative permeability for Na+ over Cl- (PNa+/PCl-) of the paracellular shunt pathway. This indicates that divalent cations increase Rte by reducing the sodium permeability of the tight junctions. The observed Ca++ and Mg++ induced reduction of the sodium permeability of the paracellular pathway corresponds to a decrease in net Na+ reabsorption by 5-10%. Since it has been demonstrated that peptide hormones such as parathyrin (PTH) modulate divalent cation and NaCl reabsorptions, in a second series of experiments we tested the effects of PTH (2-20 USP.l-1) and dbcAMP (10(-3) mol.l-1) on PDte and Rte of isolated perfused cTAL segments of rabbit nephron. Neither Rte nor PDte were affected by PTH or dbcAMP.     

Role of eicosanoids, inositol phosphates and extracellular Ca2+ in cell-volume regulation of rat liver

1. In isolated perfused rat liver, the time-course of volume-regulatory K+ efflux following exposure to hypoosmolar perfusate resembled the leukotriene-C4-induced K+ efflux in normotonic perfusion. Omission of Ca2+ from the perfusion fluid had no effect on volume-regulatory K+ efflux, but abolished completely the leukotriene-C4-induced K+ efflux. 2. Volume-regulatory K+ fluxes following hypoosmolar exposure (225 mOsmol l-1) and subsequent reexposure to normotonic media (305 mOsmol l-1) were not significantly affected by the cyclooxygenase inhibitors indomethacin (5 mumol l-1) or ibuprofen (50 mumol l-1), the leukotriene D4/C4-receptor antagonist 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]etha none (YL 171883, 50 microM), the lipoxygenase inhibitor nordihydroguaiaretic acid (20 microM), the phospholipase-A2 inhibitor bromophenacyl bromide (50 microM) or the thromboxane-receptor antagonist 4-[2-(benzenesulfonamido)ethyl]-phenoxyacetic acid (BM 13.177, 20 microM). Also the effects of hypoosmotic cell swelling on lactate, pyruvate and glucose balance across the liver remained largely unaffected in presence of these inhibitors. Neither exposure of perfused rat liver to hypoosmolar (225 mOsmol l-1) nor to hyperosmolar (385 mOsmol l-1) perfusion media affected hepatic prostaglandin-D2 release. 3. When livers were 3H-labeled in vivo by an intraperitoneal injection of myo-[2-3H]inositol about 16 h prior to the perfusion experiment, cell swelling due to lowering the perfusate osmolarity from 305 mOsmol l-1 to 225 mOsmol l-1 led to about a threefold stimulation of [3H]inositol release. The maximum of hypotonicity-induced [3H]inositol release preceded maximal volume-regulatory K+ efflux by about 30 s, but came after the maximum of water shift into the cells. Hypotonicity-induced [3H]inositol release was largely prevented in presence of Li+ (10 mM), but simultaneously inositol monophosphate accumulated inside the liver within 10 min and a small, but significant increase of inositol trisphosphate 1 min after onset of hypoosmolar exposure was detectable. No stimulation of [3H]inositol release was observed during cell shrinkage by switching the perfusate osmolarity from 225 mOsmol l-1 to 305 mOsmol l-1 or from 305 mOsmol l-1 to 385 mOsmol l-1. No stimulation of [3H]inositol release was observed upon swelling of preshrunken livers by lowering the osmolarity from 385 mOsmol l-1 to 305 mOsmol l-1, although the volume-regulatory K+ efflux under these conditions was almost identical to that observed after lowering the osmolarity from 305 mOsmol l-1 to 225 mOsmol l-1. 4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium export by sodium-calcium exchange in bovine chromaffin cells

Calcium efflux from bovine chromaffin cells in tissue culture has been examined after loading them with small amounts of Ca2+ by brief depolarization in media containing 20 mumol/l to 1 mmol/l Ca2+ and 45Ca2+ in trace amounts. In the presence of normal external Na+ and Ca2+ concentrations cells depolarized in media containing up to 200 mumol/l Ca2+ exported nearly 100% of their accumulated Ca2+ loads within 10 min and 20% within the first 5 s. In the absence of external Na+ and Ca2+ the proportion of a small (i.e., depolarization in 20 mumol/l calcium) Ca2+ load exported at any time point in the range to 10 min was approximately two thirds of the total efflux measured in their presence indicating that under these conditions the external Na+/Ca(2+)-dependent and Na+/Ca(2+)-independent mechanisms both contribute significantly to the export of calcium. At higher cellular loads of calcium (i.e., depolarization in 200 mumol/l to 1 mmol/l calcium) the Na+/Ca(2+)-dependent mechanism exported a progressively greater proportion of the accumulated Ca2+. Both sodium and calcium alone promoted a component of Ca2+ efflux; Ca2+ (i.e. calcium-calcium exchange) was as effective as Na+ (i.e. sodium-calcium exchange). The Km for Na+ stimulation of Ca(2+)-efflux (KNa) was approximately 65 mM. Increased external Mg2+ (from 1.2 to 10 mmol/l) increased the apparent KNa to 90 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium regulation of the short-term plasticity of the cholinoreceptors in neurons RPa3 and LPa3 of the edible snail]

Pharmacological influences, changing intracellular content of Ca2+, reversibly change the speed and depth of extinction of the input current of the Helix RPa3 and LPa3 neurones, elicited by a repeated iontophoretic application of acetylcholine to the soma. Suppression by extracellular medium, devoid of Ca2+ and by verapamyl (100-150 mumol/l) of Ca2+ input to the cell, induced by cholinoreceptors activation, reversibly weakens the extinction. Raise of intracellular Ca2+ level by blockade with ruthenium red (5-10 mumol/l) of specific Ca2+ transport by mitochondria and by mobilization with caffeine (1-4 mmol/l) of Ca2+, deposited by endoplasmic reticulum, accelerates and intensifies the extinction. The obtained results testify that the short-term cholinoreceptors plasticity of the above neurones is positively controlled by Ca2+ entering the cell by chemically controlled ion channels and mobilized from intracellular Ca-depot.     

The involvement of intracellular calcium ion concentration and calmodulin in the 25-hydroxylation of cholecalciferol in ovine and rat liver

The effect of Ca2+ ion concentration on the 25 hydroxylation of tritiated cholecalciferol (3HD3) was investigated using homogenates of ovine liver from vitamin D replete sheep. A significant decrease in the production of 25 hydroxycholecalciferol (25OHD3) was observed when the concentration of Ca2+ in the homogenate was raised above 0.68 mmol/l by the addition of calcium gluconate. Similarly, a final concentration of 37 mumol EGTA/1 (equivalent to a Ca2+ concentration of 26.5 nmol/l) was associated with a 50% reduction of 25OHD3 production. That is, a broad bell-shaped relationship was observed between the production of 25OHD3 and the Ca2+ concentration in the homogenate. These changes in the rate of production of 25OHD3 were reproduced with hepatocytes from vitamin D replete rats, prepared by collagenase perfusion, using the drugs dantrolene sodium (DaNa) to reduce (ED50 = 57 mmol/l) and veratridine to increase (ED50 = 550 mmol/l) the intracellular Ca2+ concentration. Hepatocytes from vitamin D replete rats also showed a reduction in 25 hydroxylation of D3 (ED50 = 6 ng/ml) in response to the addition of 1-25 dihydroxycholecalciferol (1-25 (OH)2D3). The calmodulin antagonists; W7, compound 48/80, trifluoperazine (TFP) and calmidazolium (R24571) were all found to effect a dose response inhibition of the 25 hydroxylation of cholecalciferol by homogenates of ovine liver. R24571 had a similar inhibitory effect (ED50 = 70 mumol/l) upon the 25 hydroxylase enzyme of rat hepatocytes. It is concluded that the 25 hydroxylation of cholecalciferol in liver of vitamin D replete rats and sheep is calcium sensitive and is reduced in the presence of increased concentrations of 1,25(OH)2D3. Calmodulin may also be involved in the regulation of hepatocyte 25-hydroxylase activity by Ca2+.     

Selective and reversible inhibition of heart sarcolemmal (Na+ + K+)-ATPase by p-bromophenyl isothiocyanate. Evidence for a sulfhydryl group in the ATP-binding site of the enzyme

Isothiocyanates are potent modifiers of thiol groups, and they have been successfully applied in studying the active site structure of renal (Na+ + K+)-ATPase. However, very little has been known on interactions of isothiocyanates with myocardial sarcolemmal ATPases. In the present study the mode of interaction and inhibitory effect of p-bromophenyl isothiocyanate (BPITC) on isolated rat heart sarcolemmal preparation ATPase activities not exhibiting (Mg-Ca)-ATPase activity was investigated. BPITC in concentrations of 10(-7)-10(-4) mol . l-1 inhibited selectively and non-competitively the (Na+ + K+)-ATPase activity in the sarcolemma with an ID50 around 2.10(-7) mol . l-1. The non-specific interaction of BPITC with bivalent cations, namely with Mg2+ and Ca2+, in the reaction system was eliminated by preincubation of membranes with BPITC keeping the ratio of inhibitor to membrane protein concentration constant. Under these conditions no considerable inhibitory effects were observed on Mg2+-ATPase or the low-affinity Ca2+-ATPase of sarcolemma. Preincubation of membranes with 2 mmol . l-1 ATP protected (Na+ + K+)-ATPase activity against inhibition by BPITC. The interaction of BIPTC with the sarcolemma proved to be reversible in the presence of beta-mercaptoethanol or dithiothreitol.     

Purification and characterization of an alpha-L-rhamnosidase from Aspergillus nidulans

An enzyme exhibiting alpha-L-rhamnosidase activity was purified by fractionating a culture filtrate of Aspergillus nidulans grown on L-rhamnose as the sole carbon source. The alpha-L-rhamnosidase was shown to be N-glycosylated and had a molecular mass of 102 kDa, of which approximately 7% was contributed by carbohydrate. The enzyme, optimally active at pH 4.5-6 and 60 degrees C, had an isoelectric point of 5. With rho-nitrophenyl-alpha-L-rhamnopyranoside as the substrate it showed Km and Vmax values of 0.27 mmol l-1 and 64.6 U mg-1, respectively. The enzyme was competitively inhibited by L-rhamnose (Ki 0.3 mmol l-1). Ca2+ (2 mmol l-1) stimulated the activity of the enzyme by 14%, whereas Mg2+ (2 mmol l-1) inhibited it by 63%. Substrate specificity studies showed the alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides.     

Possible role of Na+ ions in intracellular Ca2+ release in frog auricular trabeculae

Membrane potential-current and mechanical tension of frog atrial muscle were studied in a Ca and Mg-free solution containing 1 mmol/l EGTA (Ca-free solution). Exposure to Ca-free solution resulted in a shortening of action potential duration within 1.5 min and a subsequent lengthening which were paralleled by changes in magnitude and duration of the contraction. Similarly, the slow inward current quickly disappeared and progressively reappeared with a quite slower inactivation time-course. Its reversal potential varied with [Na]0 as for a pure Na current. By 12 min in Ca-free solution, the tension-voltage relation could be interpreted as the sum of two components correlated with the slow inward current and the membrane potential respectively. Contractures in response to sustained large depolarizations had similar time courses in Ca-free solution and Ringer's containing Na-Ca exchange blockers (Mn2+ 15 mmol/l or La3+ 3 mmol/l). Intracellular Na loading by voltage-clamp depolarizations (40 mV from the resting potential for 100 ms, at 0.2 Hz) in the presence of Veratrine (7.5 X 10(-6) g/ml) caused a large progressive increase in tonic tension. An intracellular Ca2+ release is invoked, partly related to Na+ entry and partly to membrane potential changes. The potential dependent part could be influenced by intracellular Na+.     

Hormonal regulation of potassium shifts during graded exhausting exercise

Serum potassium, aldosterone and insulin, and plasma adrenaline, noradrenaline and cyclic adenosine 3':5'-monophosphate (cAMP) concentrations were measured during graded exhausting exercise and during the following 30 min recovery period in six untrained young men. During exercise there was an increase in concentration of serum potassium (4.74 mmol.l-1, SEM 0.12 at the end of exercise vs 3.80 mmol.l-1, SEM 0.05 basal, P less than 0.001), plasma adrenaline (2.14 nmol.l-1, SEM 0.05 at the end of exercise vs 0.30 nmol.l-1, SEM 0.02 basal, P less than 0.001), plasma noradrenaline (1.10 nmol.l-1, SEM 0.64 at the end of exercise vs 1.50 nmol.l-1, SEM 0.05 basal, P less than 0.001), serum aldosterone (0.92 nmol.l-1, SEM 0.14 at the end of exercise vs 0.36 nmol.l-1, SEM 0.05 basal, P less than 0.01), and plasma cAMP (35.4 nmol.l-1, SEM 2.3 at the end of exercise vs 21.4 nmol.l-1, SEM 4.5 basal, P less than 0.05). While concentrations of serum potassium, plasma adrenaline and cAMP returned to their basal levels immediately after exercise, those of plasma noradrenaline and serum aldosterone remained elevated 30 min later (1.90 nmol.l-1, SEM 0.01, P less than 0.01; and 0.85 nmol.l-1, SEM 0.12, P less than 0.01, respectively). Serum insulin concentration did not change during exercise (6.47 mlU.l-1, SEM 0.58 at the end of exercise vs 5.47 mlU.l-1, SEM 0.41 basal, NS) but increased significantly (P less than 0.02) at the end of the recovery period (7.12 mlU.l-1, SEM 0.65).(ABSTRACT TRUNCATED AT 250 WORDS)