Lal C. Garg

The rates of hydration of carbon dioxide and dehydration of carbonic acid at 37

Abstract The rates of hydration of CO 2 and dehydration of H 2 CO 3 were determined using a stopped-flow spectrophotometric apparatus. The hydration rate constant, k 1 , was found to be 0.145 sec −1 in imidazole buffer at 37° and the dehydration rate constant, k −1 , was calculated to be 49.6 sec −1 . These rate constants are compared with previously reported values in the literature. For comparison, the rate constants at 25° were also determined. At both temperatures, the ratio of k −1 and k 1 agrees reasonably well with the equilibrium constant, K , calculated from ionization constants.

Effect of Low Potassium-Diet on Na-K-ATPase in Rat Nephron Segments

Na−K-ATPase activity was determined in 10 segments of the rat nephron using a fluorometric microassay method [4]. The enzyme activity showed three peaks (>200 pmol ADP min−1 mm−1) along the nephron of normal rats. These peaks were in the S1 portion of the proximal tubule, the medullary thick ascending limb from the inner stripe and the distal convoluted tubule. Feeding the rats a low potassium diet for 8 weeks produced a significant decrease in Na−K-ATPase activity in the cortical collecting duct, but no significant change in this enzyme in any other segment. The low potassium diet did not produce a significant change in Mg-ATPase in any nephron segments. We conclude that Na−K-ATPase activity along the rat nephron shows a pattern that is qualitatively similar to that seen in the rabbit nephron [4]. However, quantitatively the Na−K-ATPase activity in the rat nephron is greater than in the corresponding segments of the rabbit nephron. The results are consistent with the greater rate of glomerular filtration and Na+ reabsorption per rat nephron. Furthermore, our results suggest that the decrease in potassium excretion during potassium deficiency is modulated, at leat in part, by the level of Na−K-ATPase activity in the cortical collecting duct.

Catalytic activity and inhibition of carbonic anhydrase of rat tissues.

Abstract Carbonic anhydrase activity was studied in stomach and kidney homogenates, and isoenzymes were purified from erythrocytes and livers of male and female rats. Two liver isoenzymes of male and female rats and one erythrocyte isoenzyme had low CO 2 hydration activity. The enzymes of stomach and kidney, one isoenzyme of erythrocytes and one of female rat liver had high CO 2 hydration activity. The esterase activity toward p -nitrophenyl acetate paralleled the CO 2 hydration activity of all the isoenzymes. However, the esterase activity toward β-naphthyl acetate was either absent or did not show any correlation with the CO 2 hydration activity of isoenzymes. Male rat liver carbonic anhydrases were 1000 times less sensitive to sulfonamides than female rat liver carbonic anhydrases for the inhibition both of CO 2 hydration and esterase activity. Male rat liver carbonic anhydrases were as sensitive to inhibition by monovalent anions as were the female rat liver carbonic anhydrases. It is concluded that the active site of carbonic anhydrases from male rat liver is more hydrophilic than the active site of carbonic anhydrase from female rat liver or other tissues of the rat.

Correlative evidence of hypertension and altered cochlear microhomeostasis: electrophysiological changes in the spontaneously hypertensive rat.

The spontaneously hypertensive rat model has been used to show that hypertension is an important pathophysiological risk factor in age-related hearing loss. In the present study, compound action potential (CAP), electrochemical potential (ECP), and potassium concentration (CK+) measurements were taken from the cochlea of genetically predisposed, spontaneously hypertensive rats (SHR) and from normotensive Wistar-Kyoto (WKY) rats. In the SHR model, as the duration of hypertension increased with the animals age (from 3 to 8 months), CAP thresholds increased, ECP increased in marginal cells only, and CK+ increased in both endolymph and marginal cells. Collectively, the data suggest that ionic alternations of cellular potentials are involved in hearing changes in the hypertensive state. Ultimately, such data may assist in understanding hearing loss in individuals who are diagnosed with hypertension.

Ca2 + -ATPases in the Cochlear Duct

Differing levels of the Ca(2+)-ATPase enzymes that reside on the plasma membrane (PM) and on the endoplasmic reticulum (ER) were identified in individual rat cochlear tissues by the use of a semi-quantitative enzyme-linked immunosorbent assay (ELISA). Unlike other studies, a specific antibody to PM Ca(2+)-ATPase was used to detect significantly greater levels (about 2x) of PM Ca(2+)-ATPase in the stria vascularis (SV) than that in the spiral ligament (SL) and organ of Corti (OC) tissues. Similarly, levels of ER Ca(2+)-ATPase were also significantly higher in the SV than in the SL and OC tissues. The presence of ER Ca(2+)-ATPase in the tissues of the SV has not been demonstrated previously. Given the importance of Ca2+ homeostasis in the inner ear, the statistically significantly higher densities of both PM and ER Ca(2+)-ATPase measured in the SV relative to the SL and OC regions would indicate tissue-specific responses to fluctuations in systemic and local Ca2+ concentrations.

Differences in renal tubular Na-K-adenosine triphosphatase in spontaneously hypertensive and normotensive rats

Summary: Na-K-adenosine triphosphatase (ATPase) activity in seven specific renal tubular segments of 8-week-old spontaneously hypertensive rats (SHR) was compared with age-matched normotensive Wistar-Kyoto rats (WKY). Systolic blood pressure in 8-week-old SHR were significantly higher than in age-matched WKY. Na-K-ATPase activity in proximal convoluted tubule and outer and inner medullary collecting ducts was significantly higher in SHR than in WKY. On the other hand, medullary thick ascending limbs had reduced Na-K-ATPase activity in SHR. The possible role of the abnormal pattern of renal tubular Na-K-ATPase in the development of hypertension in SHR remains to be determined.

Lack of effect of amphotericin B on urine-bloodpCO2 gradient in spite of urinary acidification defect

The alkaline urine of normal animals and humans has higher carbon dioxide tension (pCO2) than that of blood. On the other hand, the urine-bloodpCO2 gradient is decreased in some patients and experimental animals having urinary acidification defect. It has been proposed that the urine-bloodpCO2 gradient is dependent on H+ accumulation in the distal nephron to form H2CO3 which is not dehydrated to CO2 until it passes the nephrons. We produced a urinary acidification defect in rats by treating the animals with amphotericin B and studied their ability to excrete acute acid and alkali loads. The amphotericin-treated animals could not decrease their urine pH lower than 5.84, even with a plasma HCO3− of 10.0 mEq/l. On the other hand, control animals decreased their urine pH to 5.50 with a plasma HCO3− of 18.2 mEq/l. There was no significant differences in urine-bloodpCO2 gradient between amphotericin-treated and control rats. Thus, there was a dissociation between the pH of an acid urine andpCO2 of an alkaline urine in the amphotericin-treated rats. The possible mechanisms of this dissociation along with the mechanisms of urinarypCO2 formation are discussed. It is concluded, that the urine-bloodpCO2 gradient during alkaline infusion is not an accurate index of H+ secretory capability.

Synthesis and release of acetylcholine in the rabbit kidney cortex

Several cholinergic processes were demonstrated and partially characterized in rabbit kidney cortical minces: choline uptake, acetylcholine synthesis and calcium-dependent release. Minces took up labelled choline, acetylated it, and stored it in a pool that was not readily accessible to physostigmine-sensitive cholinesterase activity. [3H]Acetylcholine synthesis but not [3H]choline uptake was inhibited by the removal of sodium ions or incubation at 0 degrees C. The release of newly synthesized [3H]acetylcholine was increased by 300 mOsmol urea in a calcium-dependent manner, but not by potassium depolarization (300 mOsmol), vasopressin (10 microM), or bradykinin (10 microM). These results suggest that acetylcholine may be synthesized by non-neuronal rabbit kidney cortical cells and that this transmitter may be released in response to physiological levels of urea.

Effects of potassium bicarbonate on distal nephron Na−K-ATPase in adrenalectomized rabbits

Na−K-ATPase activity in the connecting tubule (CNT) and cortical collecting duct (CCD) has been shown to be influenced by KCl both in the presence and in the absence of aldosterone. To investigate if the aldosterone-independent effect of K+ on Na−K-ATPase can be produced by other K+ salts, we studied the effects of dietary KHCO3 on Na−K-ATPase and ouabain-insensitive Mg-ATPase activities in four nephron segments of adrenalectomized (ADX) rabbits. The segments examined were: the distal convoluted tubule (DCT), CNT, CCD and medullary collecting duct (MCD). All diets were similar in composition except their KHCO3 contents which were 100, 300, 500 and 700 meq/kg in groups 1 to 4 respectively. Increasing KHCO3 in the diet increased K+ excretion (7×) and urine pH (6.6 to 8.3). Na−K-ATPase activity in the CCD increased >200% as dietary KHCO3 was increased to 700 meq/kg. There was a linear relation between Na−K-ATPase activity in this segment and steady state plasma K+ as well as K+ excretion in the urine. However, Na−K-ATPase activity in the CCD was lower in KHCO3-fed ADX rabbits than the KCl-fed animals studied previously under similar conditions. There were no significant differences in Na−K-ATPase activities in DCT, CNT and MCD among the four groups given different KHCO3-diets. It is concluded that dietary intake of KHCO3 can also influence Na−K-ATPase activity in the CCD independent of aldosterone.

Failure of parathyroid hormone and cyclic AMP to inhibit renal carbonic anhydrase.

SummaryIt has been suggested that the parathyroid hormone and cyclic AMP produce their bicarbonaturic effects through inhibition of renal carbonic anhydrase. In the present study, the incubation of renal carbonic anhydrase with parathyroid hormone or cyclic AMP in presence of ATP, Mg++ and K+ ions, did not produce any inhibition of the enzyme when the pH of the solution was maintained above 7. It is concluded, that parathyroid hormone and cyclic AMP produced urinary bicarbonate excretion by a mechanism independent of carbonic anhydrase inhibition.