Bicarbonate transport (JHCO3) was studied in rat proximal convoluted tubules by luminal and peritubular microperfusion, and the effects on tubular bicarbonate transport of selective changes in luminal and peritubular bicarbonate concentrations and of changes in luminal flow rate were evaluated. A pH glass electrode was used to measure [HCO3(-)] and gave results similar to those of a microcalorimetric method. Increasing the tubular and peritubular [HCO3(-)] at constant luminal perfusion rate (10 nl.min-1) augmented JHCO3, but JHCO3 increased more when pH changes were prevented by PCO2 adjustments (constant peritubular pH) than when pH was allowed to rise with the increase in [HCO3(-)] (constant PCO2). Elevation of the tubular HCO3(-) load by raising [HCO3(-)] stimulated JHCO3 more than when the HCO3(-) load was raised by enhancing luminal perfusion rate at constant [HCO3(-)] An increase in PCO2 at constant peritubular pH increased JHCO3. Diamox and benzolamide inhibited JHCO3 at luminal concentrations of 2-4 X 10(-4) M, yet a small but significant fraction of JHCO3 remained intact. Capillary perfusion with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (5 X 10(-4) M) depressed JHCO3 by 70%. Acute changes in luminal and peritubular potassium concentrations (range, 2-6 meq/liter) had no effect on JHCO3, but JHCO3 increased moderately but significantly in severe dietary hypokalemia.

Bicarbonate transport was studied in vivo by separate microperfusion experiments of early and late distal tubules. Total CO2 was measured by microcalorimetry and fluid absorption by 3H-inulin. Significant bicarbonate absorption was observed in all experimental conditions. Bicarbonate transport was loaddependent upon increasing the luminal bicarbonate concentration from 15 to 50 mM in both early and late distal tubule segments and remained constant at higher concentrations at a maximum rate of 100-110 pmol/min per mm. At low lumen bicarbonate concentrations (15 mM), higher rates of bicarbonate absorption were observed in early (32.9±4.57 pmol/min per mm) as compared to late distal tubules (10.7±3.1 pmol/ min per mm). Amiloride and ethyl-isopropylamiloride both inhibited early but not late distal tubule bicarbonate absorption whereas acetazolamide blocked bicarbonate transport in both tubule segments. Fluid absorption was significantly reduced in both tubule segments by amiloride but only in early distal tubules by ethyl-isopropylamiloride. Substitution of lumen chloride by gluconate increased bicarbonate absorption in late but not in early distal tubules. Bafilomycin Al, an inhibitor of HATPase, inhibited late and also early distal tubule bicarbonate absorption, the latter at higher concentration. After 8 d on a low K diet, bicarbonate absorption increased significantly in both early and late distal tubules. Schering compound 28080, a potent H-K ATPase inhibitor, completely blocked this increment of bicarbonate absorption in late but not in early distal tubule. The data suggest bicarbonate absorption via Na+-H+ exchange and H-ATPase in early, but only by amiloride-insensitive H' secretion (H-ATPase) in late distal tubules. The study also provides evidence for activation of K+-H+ exchange in late distal tubules of K depleted rats. Indirect evidence implies a component of chloride-dependent bicarbonate secretion in late distal tubules and suggests that net bicarbonate transport at this site results from bidirectional bicarbonate movement. (J. Clin.

The relationship between net sodium and bicarbonate transport was studied by microperfusion of proximal convoluted tubules and peritubular capillaries. Bicarbonate absorption was unchanged as long as the sodium concentration remained above 40 meq/liter, despite reduction of sodium transport to 10% of its control value. At a sodium concentration of 5 meq/liter, fluid absorption was completely abolished but bicarbonate transport was reduced to 39% of its control value. Even at reduction of luminal and peritubular sodium concentrations to nominally zero, bicarbonate transport continued at 23% of its control value. Amiloride, at a sodium concentration of 5 meq/liter, inhibited bicarbonate absorption in a dose-dependent manner. Elevating peritubular pH to 8.4 drastically reduced net bicarbonate transport, whereas fluid absorption was only slightly inhibited. These results are consistent with a dual mechanism of acidification: a sodium-hydrogen exchange that saturates at low extracellular sodium concentrations and an additional sodium-independent mechanism of hydrogen ion secretion.

Using continuous microperfusion techniques, we studied the load dependence of bicarbonate reabsorption along cortical distal tubules of the rat kidney and their bicarbonate permeability. Net bicarbonate transport was evaluated from changes in tracer inulin concentrations and total CO2 measurements by microcalorimetry. Bicarbonate permeability was estimated from the flux of total CO2 along known electrochemical gradients into bicarbonate-and chloride-free perfusion solution containing l0'-M acetazolamide.

This study was designed to examine the intracellular mechanism of inhibitory action of high concentration of angiotensin II (ANG II) on proximal tubular transport in rat kidneys by microperfusion methods. Perfusion of ANG II (10(-6) M) to peritubular capillaries caused a reduction of both fluid and HCO3- transport (Jv and JHCO3-, respectively) by 33 and 26%, respectively. These inhibitory effects were blocked by the ANG II-receptor antagonist [Sar1, Ile8]ANG II (10(-5) M). Similar degrees of inhibition on Jv and JHCO3- were observed when ionomycin (10(-7) and 10(-6) M), a Ca2+ ionophore, was added to capillary perfusate. Moreover, there was no additive effect when both ANG II and ionomycin were perfused together through capillaries, suggesting that both agents work via the same mechanism, presumably by increasing cytosolic Ca2+ concentration ([Ca2+]i). Inhibitory effects of ANG II on proximal tubular transport were still observed in a Ca2(+)-free perfusate containing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, indicating that these effects do not require influx of Ca2+ from extracellular medium. Furthermore, the observation that TMB-8, an agent that prevents intracellular Ca2+ mobilization, completely eliminated the effect of ANG II strongly suggests that intracellular Ca2+ rather than Ca2+ influx mediates effects of ANG II on proximal tubular transport. Direct measurement of [Ca2+]i by use of fura-2 in isolated proximal tubular cells showed slight but statistically significant increases in [Ca2+]i. Taken together, these observations support the idea that intracellular Ca2+ serves as a second messenger in the inhibitory effect of high concentrations of ANG II on Jv and JHCO3- in proximal tubule of kidney.