Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: Localization along the nephron and effect of corticosteroids

Abstract

To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO3-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO3-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO3-ATPase in kidney membrane fractions. HCO3-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO3-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO3-ATPase to vanadate indicates that it belongs to the F0-F1 class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO3-ATPase activity at the sites of urinary acidification.

Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

“…Whether the difference is due to a greater number of copies of plasma membrane H+-ATPase in the S3 segment, a greater flux ofprotons per ATPase, or simply a greater number of fusion events is not presently known. The results of the present study complement the study ofAbdelkhalek et al (12), which localized extramitochondrial microsomal HCO-ATPase along the rabbit nephron. In that study, the highest enzyme activity was found in the S3 proximal straight tubule and the late distal convoluted tubule.…”

“…Abbreviations used in this paper: BCECF, 2',7'-bis(carboxyethyl-5,6-carboxyfluorescein; BCECF-AM, BCECF-acetoxymethyl ester; DCCD, NN'-dicyclohexylcarbodiimide; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid; GTP, guanosine 5'-triphosphate; NEM, N-ethylmaleimide; pH,, intracellular pH. function to absorb luminal bicarbonate and regulate pH, (6)(7)(8)(9)(10)(11)(12).…”

Morphology of the Loop of Henle, Distal Tubule, and Collecting Duct

“…The luminal membrane of intercalated cells displays a much lower conductivity for both sodium and potassium than that of CNT or CO cells (33, 34, 63a, 218,309). Biochemical as well as immunocytochemical (432a) data suggest that the intercalated cells possess a specific K+-ATPase similar to the K+-H +-ATPase described in the gastric mucosa (1,97,206). It is possible that intercalated cells reabsorb potassium (384a), as formerly suggested on the basis of several structural findings in medullary intercalated cells after hypokalemia (314,324,395).…”

“…The DTLs of long loops are heterogeneous in several respects. (1) They differ in length: "short" long loops have short DTLs, and "long" long loops have long DTLs. (2) Based on a pronounced axial heterogeneity, an upper part and a lower part are distinguished.…”

Ouabain-insensitive K-adenosine triphosphatase in distal nephron segments of the rabbit.

“…Ouabain was added to inhibit Na-KATPase, which is a major fraction oftotal ATPases in the renal tubules (13). Sodium azide was added to decrease the background by inhibiting mitochondrial and other nonspecific ATPases present in the renal tubules (15). For determination of K-ATPase, one-half of the number ofsamples were incubated in the imidazole buffer, which contained 2.5 mM KCI, and the other half were incubated in the buffer without any KCI.…”