Inhibition of connexin 43 in cardiac muscle during intense physical exercise.

Endurance training is accompanied by important adaptations in both cardiovascular and autonomic nervous systems. Previous works have shown that the main component of gap junctions in the ventricular myocardium (connexin 43 (Cx43) can be regulated by adrenergic stimulus. On the other hand, training raises vagal and decreases sympathetic tone, while augmenting myocardial sensitivity to sympathetic stimulation during exercise. We therefore evaluated the regulation of Cx43 expression by sympathetic tone during exercise in trained and sedentary mice. Training induced an increase in the protein level of Cx43 by 45-70% under resting conditions. The expression of Cx43 was inhibited in trained but not in untrained mice in response to a 60 min exercise bout. Normal basal expression was restored after 60 min of resting. Cx43 reached a minimum that was not different from basal expression in untrained mice. In accordance, electrocardiography and action potential analysis did not reveal major electrophysiological implications for the drop in Cx43 abundance in trained-exercise mice. We prevented Cx43 inhibition using propranolol, and observed increased basal mRNA levels of ß-adrenergic receptors without significant changes in the ratio ß1 to ß2. In conclusion, we showed that Cx43 expression is transiently inhibited by ß-adrenergic stimulus in trained mice during acute exercise.

Effect of chronic inhibition of converting enzyme on renal handling of salt and water: a study on a pediatric population.

BACKGROUND/AIMS: The effect of angiotensin-converting enzyme inhibition (ACEi) is amply documented in several pathological conditions. However, there are few reports about the effect of chronic ACEi on salt and water balance.The present work evaluates the effect of chronic ACEi on salt and water balance in a population of children receiving enalaprilchronically in order to reduce albuminuria elicited by auremic hemolytic syndrome. METHODS: Nine children aged from 9 to 19 years with normal glomerular filtration rate, normotension and with urinary concentration capacity preserved were treated with enalapril with doses ranging between 0.1 and 0.30 mg/kg/day. Diuresis, urinary absolute and fractional excretion of Na(+), K(+) and urea, creatinine clearance,osmolal clearance and tubular water reabsorption were measured under three experimental procedures: (1)with free access to water; (2) with a water load and (3) with water restriction. In the last group urinary antidiuretic hormone(ADH) was measured. These tests were performed ina paired way, just before starting ACEi treatment and after 6 months of enalapril treatment. RESULTS: Enalapril treatment diminished the urinary concentration capacity without affecting Na(+) and K(+) urinary excretion. Creatinine clearance was not modified except in the condition of water load where a fall in it was found after ACEi. ADH increased after enalapril treatment in children under water restriction. CONCLUSION: In these children chronic ACEi decreases urinary concentration capacity.

In vitro and in vivo evaluation of proximal tubular acidification in aging rats.

The normal aging process is accompanied by a progressive deterioration of renal function. We studied the kinetics of proximal tubular acidification of young (3 mo) and aging (22 mo) rats using in vivo and in vitro techniques. Blood acid-base parameters were similar in both groups. The maximum velocity of the Na(+)/H(+) exchange (NHE) in brush-border membrane vesicles (BBMV) showed a 72% decrease in aging compared with young rats, whereas the Michaelis constant remained unchanged. The NHE3 isoform of the Na(+)/H(+) exchanger was detected in BBMV by Western blot in both groups, and a decrease of 90% in the abundance was observed in aging rats. Micropuncture experiments with simultaneous luminal and peritubular perfusion with phosphate Ringer and continuous measurement of intratubular pH showed an acidification rate constant 34% smaller in aging compared with young rats. Proton flux was 48% lower in aging than in young rats. The present results suggest that proximal tubular acidification is impaired with aging.

Peritubular fluid viscosity modulates H+ flux in proximal tubules through NO release.

We evaluated the effects of increasing the viscosity (eta) in peritubular capillary perfusates (PCP; 20 mM HNaPO4--Ringer, pH 7.4) on proximal convoluted tubule (PCT) acidification. Micropuncture experiments were performed with simultaneous luminal and peritubular perfusion. Changes in pH of a 20 mM HNaPO4--Ringer (pH 7.4 at t = 0) droplet placed in PCT lumen were measured with H+-sensitive microelectrodes. By adding neutral dextran (molecular wt 300,000-400,000) to the PCP, eta was increased. The effect of 10(-5) M ATP added to normal-eta PCP was evaluated. High eta increased H+ flux (85 and 97% when eta was increased 20 and 30%, respectively, above the control value). This increase was abolished by adding the nitric oxide antagonist N(omega)-nitro-L-arginine (L-NNA; 10(-4) M) or the purinoreceptor antagonists suramin (10(-4) M) and reactive blue 2 (3 x 10(-5) M). Addition of 5 x 10(-3) M L-arginine to the peritubular perfusate overcame the inhibitory effect of L-NNA on high-eta-induced increase in H+ flux. ATP increased H+ flux (80%), and this effect was blocked by L-NNA. These results suggest that changes in eta can modulate proximal H+ flux, at least in part, through ATP-dependent nitric oxide release from the endothelial cells of the peritubular capillaries.

Intrarenal renin-angiotensin system contributes to tubular acidification adaptation following uninephrectomy.

BACKGROUND/AIMS: Reduction in renal mass by uninephrectomy induces a functional compensation in the remnant kidney. The activity of the angiotensin-converting enzyme (ACE) as well as renin mRNA in the proximal convoluted tubule (PCT) of uninephrectomized (UNx) rats increases. The aim of this work was to determine whether the increased activity of the local renin-angiotensin system (RAS) participates in the adaptation of renal function after uninephrectomy. METHOD: We utilized normal two-kidney (2K) and 3-week UNx rats to study the activity of the ACE in vesicles obtained from luminal membranes of proximal tubular cells and the acidification kinectics in PCTs using micropuncture techniques. RESULTS: The converting enzyme activity was significantly larger in UNx (5.87+/-0.69 nmol x min(-1) x mg protein(-1)) than in 2K rats (2.43+/-0.13 nmol x min(-1) x mg protein(-1); p<0.05). The acidification rate constant (kappa) in PCT of 2K rats was 0.18+/-0.02 s(-1) and in UNx rats 0.30+/-0.04 s(-1) (p<0.001). In UNx rats, microperfusion with 10(-5) M ramipril or 10(-5) M losartan decreased kappa to 0.19+/-0.02 and 0.18+/-0.02 s(-1), respectively, but had no effect on 2K rats. Luminal steady-state pH (pH(infinity)) was the same in 2K and UNx rats, and was not modified by addition of 10(-5) M ramipril or 10(-5) M losartan in both groups. The proximal H(+) flux (J(H(+))), calculated from pH(infinity) and kappa, was 1.12 nmol x cm(-2) x s(-1) in 2K rats and, 1.77 nmol. cm(-2). s(-1) in UNx rats (p<0.001). In 2K rats, this value was not changed by 10(-5) M ramipril or 10(-5) M losartan, but in UNx rats J(H(+)) decreased 25 and 30% with ramipril or losartan, respectively (p<0.001). CONCLUSIONS: These data suggest that the increase in the local RAS activity could be an adaptive change that contributes to maintain the homeostasis of body fluids after uninephrectomy.

Role of kinins in the renoprotective effect of angiotensin-converting enzyme inhibitors in experimental chronic renal failure.

The aim of this study was to investigate whether the renoprotective effect of angiotensin-converting enzyme inhibitors (ACEIs) following 5/6 renal mass reduction is due in part to the potentiation of kinins. Three groups of rats with 5/6 renal mass reduction were studied during the 14 weeks following surgery. One group received no therapy (control); the second group was treated from the beginning with the ACEI ramipril (1 mg/kg/day) added to the drinking water, and the last group received ramipril plus a beta2-bradykinin antagonist, HOE 140 (500 microg/kg/day) via osmotic minipumps. Plasma creatinine did not change in any group during the study. Urinary protein excretion rose in the controls from 9.18+/-1.6 to 45.0+/-5.6 mg/24 h at the end of the study. In ramipril group proteinuria was prevented (initial 7.5+/-1.0 and final 8.6+/-0.8 mg/24h). The effect of ramipril was abolished by HOE 140 (initial 11.6+/-2.0 and final 38.9+/-11 mg/ 24 h). The systolic blood pressure of the controls increased from 106+/-2 to 144+/-5 mm Hg at the 14th week. Ramipril abolished the increase in systolic blood pressure. The effect of ramipril was reverted by HOE 140 (initial 108+/-2 and final 140+/-9 mmHg). Control rats had more severe histopathologic changes. Those animals receiving ramipril + HOE 140 displayed less severe glomerular changes, while rats treated only with ramipril had mild alterations. Thus the glomerular injury score was 2.11+/-0.32 for controls, 1.53+/-0.52 for rats treated with ramipril + HOE 140, and 0.06+/-0.04 for rats treated only with ramipril. The glomerular area was 20,886+/-1,410, 19,693+/-2,200 and 14,352+/-3,200 microm2, respectively, for the 3 groups. These results suggest that the protective effect of ACEIs in the development of chronic renal failure is partially mediated by kinins.

Extracellular ATP and bradykinin increase cGMP in vascular endothelial cells via activation of PKC.

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.

Results of the biocompatible osteoconductive polymer (BOP) as an intersomatic graft in anterior cervical surgery.

Eighty-two patients operated on in our Department between 1989 and 1995 with an anterior cervical approach for soft and hard cervical disc herniations and cervical stenosis were included in this study. In 41 cases a heterologous intersomatic bovine graft (Surgibone) was used. Another 41 patients underwent surgery with a biocompatible osteoconductive polymer (BOP) as intervertebral graft. Both groups were retrospectively reviewed and compared with the objectives of evaluating the biodynamic behaviour of the grafts in the intersomatic space, the complications which appeared (specially those related to the grafts), the bone fusion rate achieved and the clinical outcome of the patients. The results of our study show that the BOP group presented a higher tendency to intersomatic space collapse 6 months after discectomy. There were no differences in the general surgical complications between both groups, but those related directly to the graft were significantly higher in the BOP group. The vast majority of the graft complications recorded had no clinical correlation. Without a strict radiological follow-up such complications would never have been discovered. Bone fusion in the BOP group was significantly slower and worse. Finally, the clinical outcome in both groups did not show any significant difference.

Control of proximal tubule acidification by the endothelium of the peritubular capillaries.

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.

Converting enzyme inhibition and renal function of conscious uninephrectomized rats [correction of enzyme].

We studied the effect of a Converting Enzyme Inhibitor, Captopril, on renal function of conscious, chronically instrumented uninephrectomized rats three weeks after surgery and normal two kidney rats. Captopril increased glomerular filtration rate from 9.28 +/- 0.50 to 14.23 +/- 1.07 ml/min.kg and effective renal plasma flow from 31.6 +/- 2.4 to 45.8 +/- 3.7 ml/min.kg in two kidney rats. Glomerular filtration rate and effective renal plasma flow of uninephrectomized rats did not change after acute converting enzyme inhibition (from 6.96 +/- 0.61 to 7.16 +/- 0.39 and 24.5 +/- 2.2 to 27.9 +/- 1.5 ml/min.kg respectively). Plasma renin activity was lower in uninephrectomized rats (0.72 +/- 0.15 ng AngI/ml.h) than in intact rats (1.41 +/- 0.20 ng AngI/ml.h). Acute converting enzyme inhibition increased urinary sodium excretion, fractional sodium excretion and plasma renin activity in both, two kidney and uninephrectomized rats without changes of mean arterial pressure. Present data suggest that the Angiotensin II does not participate in the control of glomerular filtration rate and effective renal plasma flow of uninephrectomized rats but it is implicated in the control of Na homeostasis.

Converting enzime inhibition and renal function of conscious uninephrectomized rats

We studied the effect of a Converting Enzyme Inhibitor, Captopril, on renal function of conscious, chronically instrumented uninephrectomized rats three weeks after surgery and normal two kidney rats. Captopril increased glomerular filtration rate from 9.28 ñ 0.50 to 14.23 ñ 1.07 ml/min.kg and effective renal plasma flow from 31.6 ñ 2.4 to 45.8 ñ 3.7 ml/min.kg in two kidney rats. Glomerular filtration rate and effective renal plasma flow of uninephrectomized rats did not change after acute converting enzyme inhibition (from 6.96 ñ 0.61 to 7.16 ñ 0.39 and 24.5 ñ 2.2 to 27.9 ñ 1.5 ml/min.kg respectively). Plasma renin activity was lower in uninephrectomized rats (0.72 ñ 0.15ng AngI/ ml.h) than in intact rats (1.41 ñ 0.20 ng AngI/ml.h). Acute converting enzyme inhibition increased urinary sodium excretion, fractional sodium excretion and plasma renin activity in both, two kidney and uninephrectomized rats without changes of mean arterial pressure. Present data suggest that the Angiotensin II does not participate in the control of glomerular filtration rate and effective renal plasma flow of uninephrectomized rats but it is implicated in the control of Na homeostasis.

Converting enzime inhibition and renal function of conscious uninephrectomized rats

We studied the effect of a Converting Enzyme Inhibitor, Captopril, on renal function of conscious, chronically instrumented uninephrectomized rats three weeks after surgery and normal two kidney rats. Captopril increased glomerular filtration rate from 9.28 ñ 0.50 to 14.23 ñ 1.07 ml/min.kg and effective renal plasma flow from 31.6 ñ 2.4 to 45.8 ñ 3.7 ml/min.kg in two kidney rats. Glomerular filtration rate and effective renal plasma flow of uninephrectomized rats did not change after acute converting enzyme inhibition (from 6.96 ñ 0.61 to 7.16 ñ 0.39 and 24.5 ñ 2.2 to 27.9 ñ 1.5 ml/min.kg respectively). Plasma renin activity was lower in uninephrectomized rats (0.72 ñ 0.15ng AngI/ ml.h) than in intact rats (1.41 ñ 0.20 ng AngI/ml.h). Acute converting enzyme inhibition increased urinary sodium excretion, fractional sodium excretion and plasma renin activity in both, two kidney and uninephrectomized rats without changes of mean arterial pressure. Present data suggest that the Angiotensin II does not participate in the control of glomerular filtration rate and effective renal plasma flow of uninephrectomized rats but it is implicated in the control of Na homeostasis. (AU)

Changes in intracellular pH caused by high K in normal and acidified frog muscle. Relation to metabolic changes.

We examined the effect of depolarization on intracellular pH (pHi) of normal (pHi approximately 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% O2, pH 7.35) containing either 2.5 or 25 mM K+. An NH4Cl prepulse was used to lower pHi. At normal pHi, depolarization usually produced a slight (0.04) alkalinization, followed by a fall in pHi of approximately 0.2. In contrast, in all 25 acidified bundles pHi rose by 0.1-0.7. The rise was greater the lower the initial pHi. 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 min) showed no change. We postulated that low pHi slows glycolysis with its associated ATP formation by reducing glycogenolysis and particularly by reducing conversion of fructose-6-phosphate to fructose-1,6-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-1,6-diphosphate also increased significantly; 3-phosphoglycerate was not affected. This suggests an additional acid-induced bottleneck between the latter two substrates. We measured the intrinsic buffering power, beta, of frog semitendinosus muscle with small pulses of NH4Cl. It was found to vary with pHi according to beta = 144.6 - 17.2 (pHi).

Direct vascular effects in the rat of the vehicles used for the intravenous and oral administration of cyclosporin A.

1. The contraction and relaxation responses to the polyoxyethylated vehicles currently used for the intravenous and oral administration of cyclosporin A in allograft recipients were studied in isolated rat aorta. The results were compared with those obtained with commercially available cyclosporin A for intravenous administration. 2. None of these compounds affected resting tension, noradrenaline-induced contraction or endothelium-independent relaxation produced by sodium nitroprusside or bumetanide. However, they all reversed the relaxation induced by acetylcholine, carbamylcholine or adenosine 5'-triphosphate, by a factor of approximately 66%. 3. This reversal of relaxation was unaffected by indomethacin and did not require the presence of cyclosporin A in the vehicles, and was completely abolished by L-arginine (3 x 10(-5) mol/l). 4. It is concluded that vehicles used for commercial preparations of cyclosporin A interfere with the synthesis of endothelium-derived relaxing factor at an early stage during which L-arginine is made available for enzymatic degradation.

Natriuretic response to saline load in one-kidney/one-clip hypertensive rats.

Parameters of renal function were studied in conscious and anesthetized one-kidney (1K) and one-kidney/one-clip (1K-1C) rats. Effective renal blood flow (ERBF) was significantly lower in anesthetized 1K-1C rats than in conscious ones (12.1 +/- 1.6 vs. 16.4 +/- 1.2 ml/min). Renal function was evaluated in two-kidney (2K), 1K and 1K-1C unanesthetized rats. ERBF was lower in 1K and 1K-1C animals than in 2K rats. Glomerular filtration rate (GFR) and urinary sodium excretion (UNa.V) were not affected by uninephrectomy with or without clipping the renal artery. In 1K-1C rats, mean arterial pressure (MAP) increased from 100 +/- 2 to 140 +/- 1 mm Hg. Subsequently, the renal ability of unanesthetized rats to handle Na was studied by a sustained extracellular fluid volume expansion (EFVE) in all groups. During EFVE, MAP remained unchanged in the 2K and 1K groups and decreased significantly in the 1K-1C group, ERBF did not change and GFR increased to the same extent in all groups. The increase in UNa.V was 40% higher in 2K than in 1K or 1K-1C rats. These findings indicate that the relatively smaller natriuretic response to a saline load of 1K rats with or without a clip in the renal artery, as compared with 2K rats, could be ascribed to renal mass reduction. Finally, the study shows the advantage of performing studies of renal function in hypertension in conscious rather than anesthetized rats.

Amiloride prevents the metabolic acidosis of a KCl load in nephrectomized rats.

1. Counter movements of K+ and H+ across cell membranes were studied in nephrectomized KCl-loaded rats. In one group of animals, the movements of K+ and H+ were determined during and after a KCl load, and in another group, amiloride was used in order to evaluate Na+ participation in K+/H+ exchange. 2. After a KCl load at constant PCO2, 79% of infused K+ left the inulin space, half of which was in exchange for H+. As a result, blood pH fell from 7.40 +/- 0.01 to 7.30 +/- 0.01 (mean +/- SEM; P less than 0.001). 3. During KCl infusion, the K+/H+ exchange ratio varied between 1.3 and 6.8, showing that the coupling ratio is not fixed. 4. Amiloride did not change blood pH and plasma [K+], but prevented the metabolic acidosis produced by the KCl load without affecting K+ entry into the non-inulin space. Therefore, K+ and H+ movements became completely dissociated. 5. The results indicate that KCl activates an amiloride-sensitive H+ extrusion from the cells. This finding is compatible with the view that Na+/H+ exchange participates in the metabolic acidosis produced by a KCl load.

Solvent drag of sucrose during absorption indicates paracellular water flow in the rat kidney proximal tubule.

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.

Transfer of base across the basolateral membrane of cortical tubules of rat kidney.

The transfer of HCO3-/OH- across the peritubular membrane of rat cortical tubules was studied by measuring capillary pH during stopped-flow microperfusion of peritubular capillaries with electrolyte solutions containing 3 mM HCO3-. The rate of alkalinization of these solutions was significantly delayed when 10(-4) M acetazolamide, 5 X 10(-4) M SITS or 2 mM Ba2+ were added, as well as when chloride was substituted by gluconate. Under these conditions, stationary capillary pH was slightly but significantly increased. In another series, the lumen of proximal tubules was perfused with alkaline solutions while pH was measured in adjacent capillaries. During perfusion, capillary pH rose to a level 0.4 units higher than in free-flow conditions, returning after filling the lumen with oil; the rate of capillary pH return to baseline is a measure of base extrusion from cells, in the absence of influx from the lumen. This rate is also significantly delayed by acetazolamide. The data show that peritubular base extrusion is dependent on carbonic anhydrase, on basolateral membrane voltage, and on interstitial chloride, and delayed by the anion exchange inhibitor SITS; they are compatible with both basolateral HCO3-/Cl- exchange and conductive bicarbonate transfer.

Factors affecting proximal tubular acidification of non-bicarbonate buffer in the rat.

The effect of peritubular PCO2 and pH changes within the physiological range on proximal tubular acidification of non-bicarbonate (phosphate) buffer was evaluated with and without carbonic anhydrase inhibition by stopped-flow microperfusion and Sb micro-electrode techniques. 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. After acetazolamide a marked, although attenuated, effect of CO2 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 CO2, uncatalysed CO2 hydration and H2CO3 recirculation. 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. 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. In conclusion, both peritubular CO2 changes at constant pH and pH changes at 0% CO2 were effective to modify JH+, in the presence and absence of carbonic anhydrase activity. In the studied range, capillary CO2 induced larger changes in JH+ than pH. The data show substrate (H ion) is a limiting factor for tubular H ion secretion.