Endothelium-dependent inhibition of Na(+)-K+ ATPase activity in rabbit aorta by hyperglycemia. Possible role of endothelium-derived nitric oxide.

Hyperglycemia has been shown to diminish Na(+)-K+ ATPase activity in rabbit aorta. To examine the basis for this effect, aortic rings were incubated for 3 h in Krebs-Henseleit solution containing 5.5 or 44 mM glucose, and Na(+)-K+ ATPase activity was then quantified on the basis of ouabain-sensitive (OS) 86Rb-uptake. Incubation with 44 mM glucose medium caused a 60% decrease in Na(+)-K+ ATPase activity in rings with intact endothelium (from 0.22 +/- 0.01 to 0.091 +/- 0.006 nmol/min per mg dry wt; P less than 0.01). Similar decreases (45%; P less than 0.01) in Na(+)-K+ ATPase activity were seen when rings incubated with 5.5 mM glucose were exposed to NG-monomethyl L-arginine (300 microM), an inhibitor of endothelium-derived nitric oxide (EDNO) synthesis or when the endothelium was removed (43% decrease). The decrease in Na(+)-K+ ATPase activity induced by hyperglycemia was totally reversed upon adding to the medium either L-arginine, a precursor of EDNO biosynthesis or sodium nitroprusside, which bypasses endothelium and directly activates the soluble guanylate cyclase in vascular smooth muscle. A decrease in Na(+)-K+ ATPase activity (42%; P less than 0.05), only seen in the presence of endothelium, was also observed in aortas taken directly from alloxan-induced diabetic rabbits. These studies suggest that the decrease in vascular Na(+)-K+ ATPase activity induced by hyperglycemia is related, at least in part, to a decrease in the basal release of EDNO. They also suggest that alterations in basal EDNO release and possibly Na(+)-K+ ATPase activity contribute to the impairment in vascular relaxation caused by hyperglycemia and diabetes.     

Mechanism of action of cobra cardiotoxin in the skeletal muscle.

Cobra cardiotoxin (CTX) is a potent polypeptide in inducing irreversible contracture of the chick biventer cervicis muscle. this polypeptide is about 2000 times more potent than caffeine in inducing contracture of this muscle preparation but the rate of CTX contracture is slower. Studies on the interaction on CTX with divalent cations showed that low Ca0++ (10(-31-2 mM)-Krebs' enhanced markedly while ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid-Krebs' or high Ca++ (12 mM) inhibited completely CTX contracture; 10 mM Mg++ enhanced but 20 mM Mg++ or 10 mM Zn++ inhibited CTX contracture. Neither Na+ nor tetrodotoxin affected CTX contracture. Comparison of CTX and caffeine contracture showed that ethylenediamine tetraacetic acid (1 mM) and Mn++ (5 mM) inhibited CTX contracture completely but inhibited caffeine contracture only partially; procaine did not inhibit CTX contracture but inhibited caffeine contracture competitively; by contrast, N-ethylmaleimide inhibited CTX but not caffeine contracture. Neither caffeine nor K+ contracture was inhibited by 12 mM Ca++. CTX could induce contracture in the depolarized muscle and the muscle with T-tubule destroyed or closed, whereas K+ failed to induce contracture in the latter. Caffeine contracture was inhibited in the muscle with a previous elicitation of CTX contracture. This antagonistic effect of CTX could be prevented by calcium. Moreover, CTX increased both 45Ca++ efflux and 45Ca++ uptake. Both Zn++ (0.6 mM) and Mg++ (10 mM) but not protamine and polylysine mimicked CTX to increase 45 Ca++ uptake. Zn++ (0.6mM) was also found to be effective in replacing Ca0++ to induce CTX contracture in 10(-6) M Ca0++. CTX increased Ca and Na but decreased K contents of the muscle. The binding of radioactive iodinated CTX was inhibited not only by unlabeled CTX but also by 10mM Ca++. All of these findings suggest that CTX may affect a membrane calcium binding site and may induce contracture by releasing the membrane calcium rather than by increasing Na+ permeability of the muscle membrane.     

Canrenone as a partial agonist at the digitalis receptor site of sodium-potassium-activated adenosine triphosphatase

Canrenone, a spironolactone metabolite, was tested for its possible effects on (Na+-K+) adenosine triphosphatase (ATPase) activity [Mg++-dependent, (Na+-K+)-activated ATP phosphohydrolase (E.C.3.6.1.3) and ouabain interaction with the enzyme. Canrenone competitively antagonized the binding of [3H]ouabain to (Na+-K+)ATPase and inhibited (Na+-K+)ATPase activity. The multiple inhibition technique was used to demonstrate that canrenone is a partial inhibitor of (Na+-K+)ATPase, mutually exclusive with respect to ouabain. Comparative studies of the effects of ouabain and canrenone on potassium-dependent p-nitrophenylphosphatase activity (E.C.9.6.1.7) and potassium activation of (Na+-K+)ATPase confirmed that ouabain and canrenone interacted with the same receptor site. The finding that canrenone is a partial agonist may explain the results of previous in vivo studies showing that spironolactone and the allied drug to potassium conrenoate have either a positive inotropic action or an antagonistic effect against digitalis toxicity.     

Pharmacological characterization of a new Ca(2+) sensitizer

The benzimidazole molecule was modified to synthesize a Ca(2+) sensitizer devoid of additional effects associated with Ca(2+) overload. Newly synthesized compounds, termed 1, 2, 3, 4, and 5, were evaluated in spontaneously beating and electrically driven atria from reserpine-treated guinea pigs. Compound 3 resulted as the most effective positive inotropic agent, and experiments were performed to study its mechanism of action. In spontaneously beating atria, the inotropic effect of 3 was concentration-dependent (3.0 microM-0.3 mM). Compound 3 was more potent and more active than the structurally related Ca(2+) sensitizers sulmazole and caffeine, but unlike them it did not increase the heart rate. In electrically driven atria, the inotropic activity of 3 was well preserved and it was not inhibited by propranolol, prazosin, ranitidine, pyrilamine, carbachol, adenosine deaminase, or ruthenium red. At high concentrations (0.1-1.0 mM) 3 inhibited phosphodiesterase-III, whereas it did not affect Na(+)/K(+)-ATPase, sarcolemmal Ca(2+)-ATPase, Na(+)/Ca(2+) exchange carrier, or sarcoplasmic reticulum Ca(2+) pump activities of guinea pig heart. In skinned fibers obtained from guinea pig papillary muscle and skeletal soleus muscle, compound 3 (0.1 mM, 1 mM) shifted the pCa/tension relation curve to the left, with no effect on maximal tension and no signs of toxicity. Compound 3 did not influence the basal or raised tone of guinea pig isolated aorta rings, whose cells do not contain the contractile protein troponin. The present results indicate that the inotropic effect of compound 3 seems to be primarily sustained by sensitization of the contractile proteins to Ca(2+).     

Block of 45Ca uptake into synaptosomes by methylmercury: Ca++- and Na+-dependence

Block of Ca++ influx into isolated nerve terminals by the neurotoxicant methylmercury (MeHg) was studied for its dependence on extracellular Ca++ and Na+. Depolarization-independent entry of 45Ca++ was determined in rat forebrain synaptosomes incubated in 5 mM K+ solution. 45Ca++ uptake was similarly measured after 1 ("fast" phase) or 10 sec ("total") of elevated K+ (41.25 mM)-induced depolarization or after 10 sec of elevated K+-induced depolarization after synaptosomes had been predepolarized for 10 sec in Ca++- and MeHg-free solutions ("slow" phase). In 5 mM K+ solutions, MeHg concentrations of 125 microM and greater significantly reduced synaptosomal 45Ca++ uptake measured during 1 or 10 sec of incubation. In K+-depolarized synaptosomes, the estimated IC50 for block of total, fast and slow 45Ca++ uptake by MeHg is 75 microM; 250 microM MeHg reduced uptake by approximately 90%. The reversibility of block by extracellular Ca++ was tested by increasing the extracellular Ca++ concentration from 0.01 to 1.15 mM. When compared to control, 50 microM MeHg reduced total uptake of 45Ca++ by greater than or equal to 70% and reduced fast uptake by 20 to 60% at all concentrations of extracellular Ca++ tested. At Ca++ concentrations of 0.01 to 0.15 mM, MeHg (50 microM) reduced slow uptake by 75 to 90%, but did not affect slow uptake at higher Ca++ concentrations (greater than or equal to 0.30 mM). When the dependence of block of 45Ca++ uptake on extracellular Na+ was tested, equivalent levels of inhibition were caused by MeHg (25 microM) for fast uptake by synaptosomes in Na+-containing and Na+-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of patulin on the kinetics of substrate and cationic ligand activation of adenosine triphosphatase in mouse brain.

Patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one), a carcinogenic lactone produced as a major metabolite by several fungi, inhibited the Mg++-dependent Na+-K+ activated adenosine triphosphatase (ATPase) activity of mouse brain microsomal fractions with an estimated IC50 of 3.0 X 10(-4) M. Inhibition was concentration dependent. Hydrolysis of ATP was linear with both time and enzyme concentration either with or without patulin in reaction mixtures. Altered pH and activity curves for Na+-K+ ATPase demonstrated comparable inhibition by patulin in buffered acidic ranges through an optimum of 7.5, followed by a reduction of toxicity to this system at higher alkaline pH. Kinetic studies of cationic-substrate activation of Na+-K+ ATPase indicated noncompetitive inhibition with respect to ATP (at low affinity nucleotide-directed sites) and Na+ (in the presence of low, noninterfering concentrations of K+). Competitive inhibition with respect to activation of the Na+-k+-stimulated activity and K+-stimulated p-nitrophenyl phosphatase activity of the enzyme system was indicated by altered binding site parameters without change in apparent Vmax in the presence of patulin. Activity was partially restored by washing. Preincubation of patulin with dithiothreitol or glutathione protected the enzyme from inhibition. Results suggest that patulin exerted its effect on Na+-K+ ATPase either directly by interfering with K+ binding or indirectly by inducing a conformational change in the enzyme.     

PD 122860: a novel dihydropyridine with sodium channel stimulating and calcium channel blocking properties

The cardiac and vascular activities of ethyl 5-cyano-1,4-dihydro-6-methyl-2-[(4-pyridinyl-sulfonyl)methyl]-4-[2- (trifluoromethyl)phenyl]-3-pyridine carboxylase (PD 122860), a novel dihydropyridine, were investigated in vitro using rat heart and rabbit aorta, and compared with reference inotropic and vasodilator agents. In the rat heart, PD 122860 increased left ventricular contractility, decreased coronary resistance and altered the shape of the electrocardiogram T-wave. All three effects were observed at comparable concentrations of PD 122860. The inotropic response to PD 122860 was reversed by the Na+ channel blocker tetrodotoxin and blunted by the Na+-Ca++ exchange inhibitor dichlorobenzamil. The effects of tetrodotoxin and dichlorobenzamil on the inotropic response to the reference Na+ channel stimulant veratridine were comparable to PD 122860, whereas tetrodotoxin and dichlorobenzamil had no inhibitory effect on the inotropic responses to the adenylate cyclase stimulator forskolin or the Ca++ channel stimulant BAY K 8644. PD 122860 selectively relaxed potassium-contracted aortic rings and inhibited [3H]nitrendipine binding to rat brain membranes, suggesting that the vasodilator activity of PD 122860 is due to Ca++ channel blockade. In contrast to BAY K 8644, PD 122860 did not contract partially depolarized aortic rings, suggesting an absence of Ca++ channel stimulant activity. PD 122860 is a racemic mixture and both the vasorelaxant and [3H]nitrendipine binding inhibitory activities selectively reside in the (+)-enantiomer [(+)-PD 122860]. In contrast, the inotropic response resides with both enantiomers of PD 122860. It is therefore concluded that PD 122860 represents a unique dihydropyridine derivative which possesses both Na+ channel stimulating and Ca++ channel blocking activities.     

Taurine modulation of calcium binding to cardiac sarcolemma

A sarcolemma-enriched membrane fraction (SL) was prepared from the hearts of Sprague-Dawley rats and its ability to bind Ca++ was measured by equilibrium dialysis. We found that the effect of taurine on SL Ca++ binding varied with the buffer and with Na+ concentration. In Tris, in the presence of Na+ (140 mM), taurine (10 mM) increased the affinity but decreased the maximal binding of Ca++ (0.5-7 mM). In the absence of Na+, taurine decreased the affinity without altering the maximal binding. These effects on Ca++ binding were absent in bicarbonate or Krebs-Henseleit buffers. However, incubations with A23187, a Ca++ ionophore, and lanthanum, a Ca++ antagonist, indicated that SL membranes incubated in Tris, but not in buffers containing bicarbonate, were sealed vesicles with internal environments low in Ca++. High-affinity binding of Ca++ (10(-6)-10(-4) M) was measured in modified Krebs-Henseleit buffers. Taurine decreased Ca++ binding in a high-Na+ (145 mM), low-K+ (4.7 mM) buffer. Taurine increased Ca++ binding in both 4.7 mM Na+-145 mM K+ and 25 mM Na+-4.7 mM K+ buffers. Taurine also increased Ca++ binding in the presence of ATP. Thus, taurine increased high-affinity Ca++ binding in "intracellular" buffers, but it did not affect low-affinity Ca++ binding in "extracellular" buffers. These results suggest taurine may exert its cardiotonic actions through modulation of the high-affinity Ca++ binding sites on the internal aspect of the SL.     

Inositol 1,4,5-trisphosphate may regulate rat brain Cai++ by inhibiting membrane bound Na(+)-Ca++ exchanger.

The role of inositol 1,4,5-trisphosphate (1,4,5-IP3) in regulating cytosolic Ca++ by stimulating Ca++ release from intracellular organelles is well established. However, other modes of intracellular Ca++ regulation by 1,4,5-IP3 have not been determined. To determine if 1,4,5-IP3 may regulate cell cytosolic Ca++ by acting on plasma membrane bound Na(+)-Ca++ exchanger, we investigated Ca++ transport in synaptosomes using 45Ca++ as tracer. In the presence of either an inhibitor of voltage gated Na+ channels (tetrodotoxin) or the K+ ionophore (valinomycin), Ca++ uptake was significantly inhibited (P less than 0.05) by 1,4,5-IP3 in a concentration dependent manner, with half-maximal inhibition occurring at submicromolar concentrations between 10(-9) M and 10(-10) M 1,4,5-IP3. Similarly, Ca++ efflux by the exchanger was significantly inhibited 40% by 1,4,5-IP3. The inhibitory effect of 1,4,5-IP3 on the Na(+)-Ca++ exchanger was observed in the presence of Ca++ channel blockers, and in vesicles pretreated with caffeine to deplete the 1,4,5-IP3-sensitive stores of Ca++. These results suggest that during signal transduction in brain, 1,4,5-IP3 may increase cytosolic [Ca++] in part by inhibiting the Na(+)-Ca++ exchanger and thus, Ca++ efflux from cell.     

Characterization of K+ channel-dependent as well as -independent components of pinacidil-induced vasodilation

The mechanisms of pinacidil-induced direct vasodilation were studied in vitro in RMA and RAO. In RMA, pinacidil produced dose-dependent relaxations of norepinephrine (5 microM)-induced contractions with an IC50 of 0.2 microM. This component of pinacidil relaxation appeared to be dependent on K+ conductance because pretreatment with tetraethylammonium (10 mM), Ba++ (0.5 mM), glyburide (1 microM) and 20 mM K+ all caused a rightward shift of the pinacidil dose-response curve (DRC) and a corresponding increase in the pinacidil IC50. However, additional relaxation effects of pinacidil were still evident in the presence of various K+ channel blockers. Pinacidil also showed a relaxation DRC under the condition of 80 mM K+ contraction in both RMA and RAO with IC50 values of 27 and 50 microM, respectively. Pinacidil could also produce maximal relaxation in RMA and RAO remained unaffected in 145 mM K+ (zero Na+) depolarizing solution suggesting a lack of dependence on Na(+)-Ca++ exchange mechanism for this action of pinacidil. Studies using 1 or 3 min pulse labeling with 45Ca showed an absence of an inhibitory effect of pinacidil (at 50 and 100 microM) on unidirectional 45Ca influx stimulated by high-K+. Net 45Ca uptake studies showed that pinacidil inhibited high-K+ stimulated 45Ca uptake at 100 but not at 50 microM. Ryanodine (10-100 microM) was used as a tool to investigate the role of sarcoplasmic reticulum (SR) in this action of pinacidil. Under the condition in which ryanodine (10-100 microM) treatment was found to cause the SR to be nonfunctional, pinacidil relaxation DRC remained unaltered, suggesting a lack of a stimulatory effect of pinacidil on SR Ca++ accumulation. These data thus show that the K+ channel-independent effect of pinacidil does not involve to any significant degree an effect of pinacidil on plasmalemmal voltage-sensitive Ca++ channels, SR Ca++ stores, Na(+)-Ca++ exchange or membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium-potassium-activated adenosine triphosphatase of brain microsomes: modification of sodium inhibition by diphenylhydantoins

Effects of diphenylhydantoins on (Na(+) + K(+))-ATPase activity in rat and cat brain microsomes were studied. 5,5-diphenylhydantoin (DPH) in concentrations of 5-20 mug ml(-1) produces an apparent stimulation of the rat brain (Na(+) + K(+))-activated ATPase of 55-65% in media containing 50 mM Na(+), 0.15 mM K(+), 3 mM Mg(++), and 3 mM ATP. No effects are found on the Mg-ATPase. At constant K(+) levels of 0.05 mmole/liter and 0.15 mmole/liter, increasing the Na(+) concentration activates the enzyme similarly with and without DPH. However, Na(+) concentrations greater than 5 mmoles/liter and 10 mmoles/liter, respectively, which are inhibitory in these low K(+) media, produce less inhibition in the presence of DPH. In media containing 10 mM Na(+), the K(+) activation, on the other hand, is potentiated by DPH. In preparations from cat brain qualitatively similar results are obtained. No effect of DPH is seen on the inhibition produced by high K(+) in low Na(+) media. DPH produces an approximately constant apparent stimulation of 45% in the (Na(+) + K(+)) increments when these ions are varied simultaneously at a fixed ratio of 150 Na(+):1 K(+) with cat brain extracts. 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH) has the same potency as DPH in reducing the Na(+) inhibition at high Na:K ratios. The hydantoins appear to act by decreasing the Na(+) inhibition that occurs at high Na:K ratios.     

Effect of steviol on para-aminohippurate transport by isolated perfused rabbit renal proximal tubule

An inhibitory effect of steviol, metabolite of the natural sweetener stevioside, on transepithelial transport of p-aminohippurate (J(PAH)) was observed in isolated S(2) segments of rabbit renal proximal tubules using in vitro microperfusion. Addition of steviol (0.01--0.25 mM) to the bathing medium significantly depressed J(PAH) (approximately 50--90%). This inhibitory effect was dose-dependent and was maximum at a concentration of 0.05 mM. To further examine this effect, a steviol concentration (0.01 mM) that produced approximately 50% inhibition of J(PAH), was chosen. Addition of 0.01 mM steviol to the bathing medium significantly depressed J(PAH) by about 50 to 60%. Steviol at the same concentration (0.01 mM), when present in the tubule lumen, had no significant effect on J(PAH). Addition of 0.01 mM steviol to lumen and bath simultaneously, produced a slightly greater inhibitory effect compared with addition to bath alone (60 versus 70%). A higher concentration of steviol, 0.05 mM (which maximally inhibited J(PAH) when on the basolateral side), was required on the luminal side than on the basolateral side before an inhibitory effect was observed. To further examine the mechanism by which steviol inhibited J(PAH), its effect on Na(+)-K(+) ATPase activity and ATP content was determined. Steviol at concentrations of 0.01 and 0.05 mM had no effect on Na(+)-K(+) ATPase activity or cell ATP content. Kinetic analyses indicated that steviol can competitively inhibit PAH transport at the basolateral membrane. The present study clearly showed that steviol can have a direct inhibitory effect on renal tubular transport by competitive binding with organic anion transporter.     

Action of bepridil, a new calcium channel blocker on oxidative phosphorylation, oligomycin-sensitive adenosine triphosphatase activity, swelling, Ca++ uptake and Na+-induced Ca++ release processes of rabbit heart mitochondria in vitro

Effects of bepridil [1-[3-isobutoxy-2]benzylphenyl-amino)propyl pyrrolidine) on oxidative phosphorylation, oligomycin-sensitive adenosine triphosphatase, swelling, Ca++ uptake and Na+-induced Ca++ release processes of mitochondria isolated from rabbit heart were investigated. Bepridil, in concentrations greater than 5 microM, produced uncoupling of oxidative phosphorylation and stimulated oligomycin-sensitive adenosine triphosphatase activity. At low concentrations it prevented inorganic phosphate-induced swelling and associated depression of oxidative phosphorylation. Its effectiveness in preventing swelling and depression of oxidative phosphorylation was found to be dependent on inorganic phosphate concentration. A concentration of 1 microM of bepridil was effective in producing 50% less depression of phosphorylating respiration in the presence of 10 mM inorganic phosphate. Concentrations of bepridil above 25 microM inhibited the rate of Ca++ uptake. A 50% inhibition of Ca++ uptake was observed at 93 microM bepridil. The rate of Na+-induced Ca++ release was also inhibited by bepridil. A 50% inhibition of the rate of Na+-induced Ca++ release occurred at 11 microM of bepridil. When the Na+-dependent Ca++ release process was about 80% inhibited by 25 microM bepridil, the uptake process still remained at the same level as the untreated control. Results suggest that in addition to reported effects on sarcolemma and sarcoplasmic reticulum, mitochondria are also affected by bepridil.     

Activation of heart sarcoplasmic reticulum Ca++-stimulated adenosine triphosphatase by insulin

Although insulin is known to elicit a positive inotropic effect in cardiac muscle preparations, very little is known concerning the mechanism of this action. In view of the crucial role played by the sarcoplasmic reticular (SR) calcium transport in cardiac contractile events, the effects of insulin on the pig heart SR were investigated. Insulin activated the SR Ca++-stimulated adenosine triphosphatase (ATPase) in a concentration-dependent manner (0.1 mU to 1 U/ml); maximal activation (125%) was seen at 0.1 to 1 U/ml of insulin. Kinetic studies revealed that the insulin-induced activation was due to an increase in the apparent Vmax of Ca++-stimulated ATPase without any alteration in the Km. Insulin was found to bind with SR membranes in a specific manner and this binding was rapid, saturable and displacable. The dose-related increase in the activation of Ca++-stimulated ATPase was related linearly (r = 0.98) to binding of insulin with SR membranes; 50% activation of Ca++-stimulated ATPase was found to occur at 13.5 fmol of insulin binding per mg of SR protein. When insulin was allowed to dissociate by a 100-fold dilution of the insulin-receptor complex, the activity of SR Ca++-stimulated ATPase also declined gradually. Furthermore, proteolytic digestion on the membrane with trypsin (3 micrograms/mg of protein) decreased both insulin binding as well as the increase in Ca++-stimulated ATPase activity by about 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid N-methylation-dependent alterations of cardiac contractile function by L-methionine

Isolated rat, rabbit and guinea pig hearts exhibited an initial negative inotropic (20-30%) effect followed by a positive inotropic response (60-80%) upon perfusion with 300 microM L-methionine. In contrast, frog hearts did not show any delayed positive inotropic effect, whereas initial negative inotropic effect (25%) of L-methionine was seen. In subsequent studies using rat hearts, methionine was found to induce a dose-dependent increase in contractile force which correlated linearly (r = 0.93) with incorporation of methyl groups into tissue N-methylated phospholipids. The presence of adenosine, L-homocysteine thiolactone and erythro-9-(2-hydroxy-3-nonyl) adenine mixture in the perfusion medium inhibited the contractile effects of L-methionine as well as the incorporation of 3H-methyl groups by about 75%. Cycloleucine, an inhibitor of S-adenosylmethionine synthase, and methyl acetimidate, a blocker of the phosphatidylethanolamine polar groups, inhibited phospholipid N-methylation and prevented the contractile changes due to L-methionine. The initial negative inotropic effect of methionine was attenuated by lowering the concentration of Na+, whereas the delayed positive inotropic effect was dependent on the concentration of Ca++ in the perfusion medium. Ryanodine, a blocker of the sarcoplasmic reticular Ca++ release, prevented the positive inotropic effect of methionine whereas verapamil, a well known Ca++ antagonist, blocked the initial depressant effect and reduced the delayed positive inotropic response. Marked alterations in the sarcolemmal and sarcoplasmic reticular calcium transport activities were seen upon perfusing the hearts with methionine.(ABSTRACT TRUNCATED AT 250 WORDS)     

中药补肾方对心力衰竭大鼠Na+-K+ATP酶、Ca2+-Mg2+ATP酶和琥珀酸脱氢酶的作用研究

目的研究补肾方对心力衰竭大鼠Na+-K+ATP酶、Ca2+-Mg2+ATP酶和琥珀酸脱氢酶(SDH)的作用。方法 60只大鼠随机分为模型组、假手术组、曲美他嗪组、补肾低、中、高剂量组,每组10只。采用结扎冠状动脉前降支法制备心力衰竭大鼠模型,术后2周开始灌胃,分别给予药物干预8周。8周后通过颈动脉插管记录大鼠血流动力学改变,包括左室收缩压(LVSP)、左室舒张压(LVEDP)、左室内压上升下降最大速率(±LVdp/dtmax);采用分光光度计检测心力衰竭大鼠心肌Na+-K+ATP酶、Ca2+-Mg2+ATP酶以及SDH水平。结果模型组大鼠心肌LVSP和+LVdp/dtmax较假手术组明显降低(P<0.01),而LVEDP和-LVdp/dtmax明显升高(P<0.05);补肾方干预后,中、高剂量大鼠心肌收缩、舒张功能明显优于模型组(P<0.01),以高剂量补肾方改善心力衰竭大鼠心功能明显。补肾方干预后,Na+-K+ATP酶、Ca2+-Mg2+ATP酶和SDH活力高于模型组,但低于假手术组,且随补肾方剂量增加,Na+-K+ATP酶、Ca2+-Mg2+ATP酶和SDH活力逐渐增强。结论补肾方可改善心力衰竭大鼠的心功能,可能与Na+-K+ATP酶、Ca2+-Mg2+ATP酶以及SDH活力增强相关。     

Adenosine Triphosphatases of Rat Pancreatic Islets: COMPARISON WITH THOSE OF RAT KIDNEY

Electrolyte fluxes are fundamental to normal endocrine pancreatic function. Adenosine triphosphatases (ATPases) are enzyme systems believed to modulate electrolyte movements across membranes in a number of cell types. This study was undertaken to measure cation-dependent ATPases of rat pancreatic islets. In addition, we compared effects of substances which influence endocrine pancreatic function upon ATPases in homogenates of islets and kidney, the latter being a tissue which would not be expected to have a stimulus-secretion response to substances which activate islets.Both tissues were generally similar with respect to apparent Michaelis constant (ATP) of Na(+)K(+)ATPase, Mg(++)ATPase, and Ca(++)ATPase. In islets and kidney, Na(+)K(+)ATPase specific activity was increased when the Na:K ratio was lowered from 250:1 (175:0.7 mM) to 5:1 (100:20 mM). Inhibition of Na(+)K(+)ATPase at either Na:K ratio by ouabain, an activator of secretion, and enhancement of the high-ratio Na(+)K(+)ATPase by diphenylhydantoin, an islet secretory inhibitor, were also common to both tissues.Because both inhibition and enhancement of Na(+)K(+)ATPase could be studied at the high Na:K ratio, we examined the effect of regulators of secretion upon the activity of this enzyme. Like ouabain, substances which induce or support islet secretion, glucose 16 mM or 3.3 mM, arginine 14.2 mM (with 3.3 mM glucose), or Ca(++) 1 mM, inhibited high-ratio islet Na(+)K(+)ATPase. Like diphenylhydantoin, the inhibitors of insulin secretion, diazoxide 0.22 mM, or NH(4)Cl 16 mM, enhanced this islet ATPase. Neither valine, which is non-secretogenic, nor arginine without glucose, which is a weak secretagogue, had any effect upon islet Na(+)K(+)ATPase. We examined the effect of these substances upon other cation-dependent islet ATPases. Ca(++) inhibited Mg(++)ATPase, and glucose inhibited Ca(++)ATPase. Leucine, 22.9 mM, which induces insulin secretion in the absence of glucose, suppressed islet Ca(++)ATPase and had no effect upon high-ratio Na(+)K(+)ATPase.In contrast to the observations in the islets, most substances which influence islet function had no effect on kidney ATPases, or effects which were different from those seen in islets. Except for ouabain, none of these substances influenced the three kidney ATPases in a manner similar to that seen with islets.These findings support the hypothesis that cation-dependent ATPases are involved in specificity of islet response to substances which influence endocrine pancreatic activity.     

Energetic aspects of the regulation of Ca++ sensitivity of permeabilized rabbit mesenteric artery: possible involvement of a second Ca++ regulatory system in smooth muscle contraction

The effects of phosphagen concentrations and adenosine-5'-O-(2-thiodiphosphate)(ADP beta S), a nonhydrolyzable ADP analog, on the pCa++ tension relationships were investigated, using alpha-toxin permeabilized rabbit mesenteric artery. The removal of creatine phosphate (CP) greatly affected the Ca++ sensitivity and induced a leftward shift of the pCa++ tension curve. Addition of ADP beta S (10-300 microM) also caused a leftward shift of the pCa++ tension curve. Ca++ solutions (0.3-10 microM) containing 0.1 mM ATP did not induce contraction. However, the addition of CP in the presence of 0.1 mM ATP dose-dependently increased force development which reached a maximum around 3 mM CP. A 10 microM Ca++ solution containing 0.1 mM ATP and 1 mM CP was much more effective in inducing contraction than a 10 microM Ca++ solution containing 1.1 mM ATP alone, although the total concentration of phosphagen (ATP + CP) was the same. Application of 0.1 mM ATP solution containing various concentrations of Ca++ after the maximal Ca+(+)-induced contraction relaxed the tissue, with the higher Ca++ concentrations inducing the faster relaxation. The same pattern of the relaxation was seen when the tissue was pretreated with adenosine-5'-O-(3-thiotriphosphate) beforehand. The contractile state observed in the Ca+(+)-free solution containing 0.1 mM ATP and 0.1 mM CP was completely relaxed by 1 mM vanadate, consistent with the idea that the sustained contraction was due to accumulation of the actomyosin-ADP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of caffeine-induced calcium overload in cardiac Purkinje fibers

In cardiac Purkinje fibers, caffeine initially increases and then decreases contractile force. The possible role of calcium overload in the negative inotropic effect of caffeine was studied in vitro under conditions that have been demonstrated to increase or decrease cellular calcium. The following results were obtained. Increasing [K]0 increased the initial positive and decreased the subsequent negative inotropic effect of 1 mM caffeine. At higher concentrations (2 and 4 mM), caffeine induced a larger negative inotropic effect which was reversed by high [K]0. Similarly, the positive inotropic effect was increased and the negative decreased by lowering [Ca]0. In high [Ca]0 or low [Na]0, caffeine had a predominantly negative inotropic effect which also was reversed by adding tetrodotoxin or by high [K]0. Reciprocally, in high [K]0, increasing [Ca]0 restored the negative inotropic effect of caffeine. During a brief exposure to zero [Ca]0, the force fell in the absence but less or not at all in the presence of caffeine. It is concluded that caffeine decreases contractile force in cardiac Purkinje fibers mostly by causing calcium overload.     

Stimulation of cardiac myofilament force, ATPase activity and troponin C Ca++ binding by bepridil

We report that bepridil, a Ca++ channel blocker and calmodulin antagonist, which has been shown to enter myocytes, stimulates the mechanical and biochemical activity of cardiac myofilaments. Bepridil increased significantly the level of Ca++-dependent actomyosin Mg++-ATPase activity of myofibrils and the submaximal force developed by chemically skinned trabeculae of pig heart. In the range of concentrations (10-100 microM) in which bepridil showed this stimulatory activity, diltiazem and verapamil were without effect. The effect of bepridil on myofilament force and ATPase activity was higher at relatively low free Ca++ concentrations, and myofibrils lacking troponin-tropomyosin were not affected by bepridil. Associated with the stimulation of force and ATPase activity by bepridil was an increase in the amounts of Ca++ bound to troponin C (TnC). That bepridil stimulates TnC Ca++ binding was also shown in experiments using pure TnC labeled with 2-(4'-iodoacetamidoanilo)naphthalene-6-sulfonic acid, a fluorescent probe that reports Ca++ bound to the single "regulatory" site. Effects of bepridil on the fluorescence of a felodipine-cardiac TnC complex indicate that bepridil binds to TnC over the same range of doses where it affects myofilament activity. Our results indicate that the inotropic action of bepridil may result from a net response of heart cells to influences on the delivery of Ca++ to the myofilaments and their response to Ca++.