The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.
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Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies. (+info)
Brain renin-angiotensin system and ouabain-induced sympathetic hyperactivity and hypertension in Wistar rats.
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In Dahl salt-sensitive rats on a high salt diet or normotensive rats with chronic central infusion of sodium, increased brain "ouabain" results in sympathetic hyperactivity and hypertension, possibly by activating the brain renin-angiotensin system. In the present study, we tested whether the hypertension caused by exogenous ouabain also depends on activation of brain renin-angiotensin system. In Wistar rats, ouabain (50 micrograms/d) was infused subcutaneously for 14 days with the use of osmotic minipumps. Concomitantly, in one group, the angiotensin II type 1 receptor blocker losartan (1 mg/kg per day) was infused intracerebroventricularly. On day 15, mean arterial pressure, heart rate, central venous pressure, and renal sympathetic nerve activity were recorded in conscious rats at rest and in response to air-jet stress, intracerebroventricular injection of the alpha(2)-agonist guanabenz (25 and 75 micrograms) or angiotensin II (30 ng), acute volume expansion, and ramp changes of blood pressure by +/-50 mm Hg with phenylephrine and nitroprusside. Compared with control rats, in rats treated with ouabain, resting mean arterial pressure was significantly increased (111+/-4 versus 93+/-3 mm Hg; P<0.05), and increases or decreases in mean arterial pressure, heart rate, and renal sympathetic nerve activity in response to air stress or guanabenz were enhanced significantly. These effects of ouabain were prevented when losartan was given concomitantly. Maximal slopes of arterial baroreflex control of renal sympathetic nerve activity and heart rate tended to be decreased in ouabain-treated versus control rats and were significantly increased in ouabain-treated rats with versus without losartan. No differences in cardiopulmonary baroreflex function were detected. It seems that by day 14 to 15, the central effect of ouabain on baroreflex control prevails over its peripheral sensitizing effect on baroreceptors, leading to a tendency of desensitization. These results indicate that chronic administration of ouabain activates the brain renin-angiotensin system, resulting in decreased sympathoinhibition and increased sympathoexcitation, impairment of baroreflex function, and hypertension. (+info)
Na(+)-K(+) pump and metabolic activities of trout erythrocytes during anoxia.
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Metabolic activity in the red blood cells of brown trout was monitored under conditions of oxygen depletion and chemically induced anoxia. Although metabolic activity was reduced during anoxia to one-third of the normoxic value, these cells maintained their ATP contents stable and were viable for hours in the absence of oxygen. In addition, Na(+)-K(+) pump activity was not down-regulated when metabolic activity was reduced during anoxia. The compatibility of this finding with energy equilibrium and ion homeostasis was investigated. (+info)
Stimulation of both aerobic glycolysis and Na(+)-K(+)-ATPase activity in skeletal muscle by epinephrine or amylin.
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Epinephrine and amylin stimulate glycogenolysis, glycolysis, and Na(+)-K(+)-ATPase activity in skeletal muscle. However, it is not known whether these hormones stimulate glycolytic ATP production that is specifically coupled to ATP consumption by the Na(+)-K(+) pump. These studies correlated glycolysis with Na(+)-K(+)-ATPase activity in resting rat extensor digitorum longus and soleus muscles incubated at 30 degrees C in well-oxygenated medium. Lactate production rose three- to fourfold, and the intracellular Na(+)-to-K(+) ratio (Na(+)/K(+)) fell with increasing concentrations of epinephrine or amylin. In muscles exposed to epinephrine at high concentrations (5 x 10(-7) and 5 x 10(-6) M), ouabain significantly inhibited glycolysis by approximately 70% in either muscle and inhibited glycogenolysis by approximately 40 and approximately 75% in extensor digitorum longus and soleus, respectively. In the absence of ouabain, but not in its presence, statistically significant inverse correlations were observed between lactate production and intracellular Na(+)/K(+) for each hormone. Epinephrine had no significant effect on oxygen consumption or ATP content in either muscle. These results suggest for the first time that stimulation of glycolysis and glycogenolysis in resting skeletal muscle by epinephrine or amylin is closely linked to stimulation of active Na(+)-K(+) transport. (+info)
Structural equivalents of latency for lysosome hydrolases.
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1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane. (+info)
Mutations of Ser-23 of the alpha1 subunit of the rat Na+/K+-ATPase to negatively charged amino acid residues mimic the functional effect of PKC-mediated phosphorylation.
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The Na+/K+-ATPase is a target protein for protein kinase C (PKC). The PKC-mediated phosphorylation of the rat alpha1 subunit at Ser-23 results in the inhibition of its transport function. To understand the molecular basis of the inhibition by PKC, the Ser-23 in the rat alpha1 subunit has been replaced by negatively (Asp, Glu) or positively (Lys) charged, or uncharged (Gln, Ala) residues, and the mutants were expressed in Xenopus oocytes. Ouabain-specific 86Rb uptake and pump-generated current as well as sensitivity to ouabain and to external K+ have been investigated. When Ser-23 was replaced by the negatively charged residues, transport function was inhibited, and simultaneously synthesis of the alpha subunits was enhanced. In addition, if Ser-23 was substituted by Glu, the K(I) value for inhibition of transport by ouabain was drastically increased from 46.5 microM to 1.05 mM. The data suggest that insertion of a negative charge within the N-terminus of alpha subunit of the Na+/K+-ATPase due to phosphorylation of Ser-23 plays an important role in the PKC-mediated inhibition of transport function. (+info)
Block of rapid depolarization induced by in vitro energy depletion of rat dorsal vagal motoneurones.
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1. The ionic mechanisms contributing to the rapid depolarization (RD) induced by in vitro ischaemia have been studied in dorsal vagal motoneurones (DVMs) of brainstem slices. Compared with CA1 hippocampal neurones, RD of DVMs was slower, generally occurred from a more depolarized membrane potential and was accompanied by smaller increases in [K+]o. 2. RD was not induced by elevation of [K+]o to values measured around DVMs during in vitro ischaemia or by a combination of raised [K+]o and 2-5 microM ouabain. 3. Neither TTX (5-10 microM) nor TTX combined with bepridil (10-30 microM), a Na+-Ca2+ exchange inhibitor, slowed RD. Block of voltage-dependent Ca2+ channels with Cd2+ (0.2 mM) and Ni2+ (0.3 mM) led to an earlier onset of RD, possibly because [K+]o was higher than that measured during in vitro ischaemia in the absence of divalent ions. 4. When [Na+]o was reduced to 11.25-25 mM, RD did not occur, although a slow depolarization was observed. RD was slowed (i) by 10 mM Mg2+ and 0.5 mM Ca2+, (ii) by a combination of TTX (1.5-5 microM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D-2-amino-5-phosphonovalerate (AP5, 50 microM) and (iii) by TTX (1.5-5 microM) and AP5 (50 microM). 5. Ni2+ at concentrations of 0.6 or 1.33 mM blocked RD whereas 0.6 mM Cd2+ did not. A combination of Cd2+ (0.2 mM), Ni2+ (0.3 mM), AP5 (50 microM) and bepridil (10 microM) was largely able to mimic the effects of high concentrations of Ni2+. 6. It is concluded that RD is due to Na+ entry, predominantly through N-methyl-D-aspartate receptor ionophores, and to Ca2+ entry through voltage-dependent Ca2+ channels. These results are consistent with known changes in the concentrations of extracellular ions when ischaemia-induced rapid depolarization occurs. (+info)
Sodium-potassium pump current in smooth muscle cells from mesenteric resistance arteries of the guinea-pig.
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1. The Na+-K+ pump current was studied in smooth muscle cells from mesenteric resistance arteries of guinea-pigs by the use of the perforated patch-clamp technique in the presence of blockers for various ion channels and exchangers. 2. When the Na+ concentration in the pipette solution ([Na+]i) was 50 mM, an increase in the extracellular K+ concentration ([K+]o) from 0 to 10 mM caused an outward current. Both the removal of K+ from the bath solution and the application of 10 microM ouabain abolished this current. Thus, this K+-induced and ouabain-sensitive current was considered to be the Na+-K+ pump current. 3. The amplitude of the Na+-K+ pump current increased as the membrane potential was made more positive until around 0 mV, while the amplitude saturated at more positive potentials than 0 mV. 4. An increase in [K+]o or [Na+]i amplified the Na+-K+ pump current. For [K+]o, the binding constant (Kd) was 1.6+/-0.3 mM and the Hill coefficient (nH) was 1.1+/-0.2 (n = 6). For [Na+]i, Kd was 22+/-5 mM and nH was 1.7+/-0.5 (n = 4-19). 5. The presence of various monovalent cations other than Na+ in the bath solution also evoked the Na+-K+ pump current. The order of potency was K+ >= Rb+ > Cs+ >> Li+. 6. Ouabain inhibited the Na+-K+ pump current in a dose-dependent manner with a Kd of 0.35+/-0.03 microM and an nH of 1.2+/-0.1 (n = 6-8). 7. The Na+-K+ pump current increased as temperature increased. The temperature coefficient (Q10; 26-36 C) was 1.87 (n = 9). 8. In summary the present study characterized for the first time the Na+-K+ pump current in vascular smooth muscle cells by the use of the voltage-clamp method. The use of this method should provide essential information for Na+,K+-ATPase-mediated changes in the cell functions of vascular smooth muscle cells. (+info)