We examined the effect of depolarization on intracellular pH (pH.~ of normal (pH i ~7.37) and acidified (pHi 5.90-6.70) frog semitendinosus muscle using microelectrodes. A small bundle was superfused with a Na+-free buffered solution (10 mM HEPES, 100% O~, pH 7.35) containing either 2.5 or 25 mM K +. An NH4CI prepLdse was used to lower pH i. At normal phi, depolarization usually produced a slight (0.04) alkalinization, followed by a fall in pHi. of ~0.2. In contrast, in all 25 acidified bundles pH i rose by 0.1-0.7. The rise was greater the lower the initial pH i. It could be imitated by caffeine and blocked by tetracaine and thus was, most likely, initiated by release of calcium. We ascribed the alkalinization to hydrolysis of phosphocreatine (PCr); 2,4-dinitrofluorobenzene abolished it. Biochemical analysis on fibers at the peak of alkalinization showed PCr to be reduced by one-half, while PCr in normal fibers that had been depolarized for the same period (4-6 rain) showed no change. We postulated that low pH i slows glycolysis with its associated ATP formation by reducing glycogenolysis and particularly by reducing conversion of fructose-6-phosphate to fructose-l,6-diphosphate through inhibition of phosphofructokinase (PFK), an enzyme which is known to be highly pH sensitive. Thus PCr hydrolysis would be required to replace much of the hydrolyzed ATP. This postulated effect on PFK is in agreement with the finding that glucose-6-phosphate (in near-equilibrium with fructose-6-phosphate) was increased nearly fivefold in the depolarized acid fibers, but not in the depolarized normal fibers. However, fructose-l,6-diphosphate also increased significantly; 3-phosphoglycerate was not affected. This suggests an additional acid-induced botdeneck between the latter two substrates. We measured the intrinsic buffering power, fl, of frog semitendinosus muscle with small pulses of NH4C1. It was found to vary with pH i according to fl = 144.6 -17.2 (pH 0.Address reprint requests to Dr. A. Roos,

Guanosine 3',5'-cyclic monophosphate (cGMP), a nitric oxide mediator, stimulates Na+/H+ exchange in brush-border vesicles of the renal cortex. The aim of the present work was to test whether the endothelium of the peritubular capillaries modulated the rate of proximal luminal acidification through the release of endothelium-derived nitric oxide (EDNO). Perfusion of the tubule lumen with dibutyryl cGMP increased net proton flux (J(H)). Two agents that elicit EDNO production, bradykinin (BK) and carbamylcholine (Cch), increased J(H) when added to the peritubular capillary perfusate. Bradykinin did not affect J(H) when the peritubular capillaries and the lumen were perfused with Na-free solution. Methylene blue (MB) and N(G)-nitro-L-arginine methyl ester (L-NAME) blocked the elevation in J(H) by Cch and also decreased basal J(H). Bradykinin increased cGMP content of isolated proximal convoluted tubules, but only if they were coincubated with endothelial cells. This effect of BK was blocked by L-NAME. The results suggest that the endothelium of the peritubular capillaries affects proximal tubule acidification through changes of cGMP in proximal tubule cells, probably via stimulation of Na+/H+ exchanger.

SUMMARY1. The effect of peritubular PCO2 and pH changes within the physiological range on proximal tubular acidification ofnon-bicarbonate (phosphate) buffer was evaluated with and without carbonic anhydrase inhibition by stopped-flow microperfusion and Sb micro-electrode techniques.2. Luminal steady-state pH was reduced from 6-69 to 6-37 and H ion fluxes (JH+)increased from 0-63 to 1-57 nmol cm-2 s-1 by increasing capillary CO2 from 0 to 9-6 % at pH 7-2. 3. After acetazolamide a marked, although attenuated, effect ofCO2 on acidification was still observed; JH+ increased from 0-088 nmol cm-2 s-1 at 0 % CO2 to 0-78 at 9-6% CO2. Most of this effect can be explained by titration of luminal buffer by C02, uncatalysed CO2 hydration and H2CO3 recirculation.4. An increase in capillary CO2 reduced acidification half-times (t/2), which, according to an analogue circuit model, may be due to increased H ion access to the pump.5. Peritubular pH changes at 0 % CO2 also modified tubular acidification, increasing JH+ from 0-73 nmol cm-2 s-1 at pH 7-6 to 0 99 at pH 7 0. After acetazolamide, JH+ still increased from 0.11 nmol cm-2 s-1 at pH 7-6 to 0 57 at pH 7 0. 6. In conclusion, both peritubular C02 changes at constant pH and pH changes at 0 % C02 were effective to modify JH+, in the presence and absence of carbonic anhydrase activity. In the studied range, capillary C02 induced larger changes in JH+ than pH. The data show substrate (H ion) is a limiting factor for tubular H ion secretion.

Single convoluted proximal tubules of the rat kidney were lumen perfused in situ with isosmotic solutions containing C14-sucrose and H3-inulin as tracers, to evaluate whether the extracellular marker sucrose is entrained by water during proximal tubular reabsorption. Inulin was used as volume marker. The absorptive rate was varied by using as luminal perfusion fluids either a solution made up of (in mmole/l) 120 NaCl, 5 glucose, 25 NaHCO3 and altering the perfusion rate, or a solution containing 110 NaCl and 70 raffinose. Js, the net sucrose efflux is found to be a function of the net volume flow, Jv, such that at Jv = 0, Js is very small and at high rates of Jv, Js is over 60-fold the value observed at low Jv values. In addition, the transported to luminal sucrose concentrations decreased with Jv in a hyperbolic manner. Unstirred layers affect the diffusive component of Js, but only to a small extent. Therefore, the large remaining dependency of Js with Jv must be due to drag of sucrose by water, within the paracellular pathway. This leads to the conclusion that water flows through the paracellular pathway during absorption in the rat proximal tubule, in addition to transcellular water flow. Using equations for molecular sieving and the measured value of sigma s for sucrose of 0.76-0.91, it is calculated that the pathway where entrainment of solute by water occurs must be 1.0-1.1 nm wide. This calculation is only tentative since sigma s depends on the as yet unknown relative contribution of transcellular and paracellular pathways to transepithelial water osmotic permeability.

Vasodilation by agents such as bradykinin and ATP is dependent on nitric oxide, the endothelium-dependent relaxing factor (EDRF). The release of EDRF results in elevation of cGMP in endothelial and smooth muscle cells (9). The signaling pathway that leads to increases in cGMP is not completely understood. The role of protein kinase C (PKC) in the elevation of cGMP induced by ATP and bradykinin was studied in cultured porcine aortic endothelial cells, by measuring PKC phosphorylation of a substrate and by measuring cGMP levels by radioimmunoassay. Extracellular ATP and bradykinin simultaneously elevated cGMP levels and PKC activity. The PKC inhibitors staurosporine, calphostin C, and Cremophor EL (T. Tamaoki and H. Nakano. Bio/Technology 8: 732–735, 1990; F. K. Zhao, L. F. Chuang, M. Israel, and R. Y. Chuang. Biochem. Biophys. Res. Commun. 159: 1359–1367, 1989) prevented the elevation of cGMP elicited by ATP and reduced that produced by bradykinin. Cremophor did not affect the elevation of cGMP by nitroprusside, an agent that directly increases guanylate cyclase activity (9). The PKC activator phorbol 12-myristate 13-acetate, but not a phorbol ester analog inactive on PKC, also elevated cGMP levels. These results suggest that EDRF agonists elevate cGMP in endothelial cells via PKC stimulation.