Representational difference analysis of cDNA of genes expressed in embryonic kidney.

Representational difference analysis of cDNA (cDNA-RDA) is a PCR-based differential cloning method. It involves hybridization of two populations of cDNA with selective amplification of differentially expressed genes. To isolate the differentially expressed genes during renal development, mRNAs from embryonic kidneys at day 13 (E13) and postnatal kidneys from three-week-old (P3) mice were extracted, and double stranded cDNAs prepared. Double stranded cDNAs were digested with DpnII, adaptor-ligated, and amplified by PCR, using adaptor primer to generate "representative amplicons." These reflect the "representation" of most of the cDNA population. The term "amplicons" denotes amplified PCR product. Among the two populations of cDNA, E13 kidney cDNA was used as a "tester," containing target genes, and P3 kidney cDNA as a "driver," driving the process of subtraction, following which, they were subjected to cDNA-RDA under low stringency conditions. During the first round of cDNA-RDA embryonic globin genes were isolated. To competitively eliminate these genes, plasmid DNAs of globin genes were supplemented into driver, and subjected to the second round of cDNA-RDA. This resulted in the isolation of four cDNA clones: H19 gene, mesoderm-specific cDNA, COL2A1 gene, and a novel cDNA. By Northern blot analyses, the H19 gene and mesoderm-specific cDNA exhibited a high degree of developmental regulation, that is, they were abundantly expressed in E13 kidney, and their expression was barely detectable in P3 kidney. The differential developmental regulation of mesoderm-specific cDNA was confirmed by tissue in situ hybridization experiments. The COL2A1 and novel cDNA were rare transcripts in the embryonic Kidney. However, Southern blot analyses of representations indicated their up-regulated expressions in E13 kidneys. The novel gene was differentially expressed in 13-day embryonic lung, and Northern blot analysis revealed an approximately 10 Kb transcript. These results indicate that cDNA-RDA is a sensitive technique to identify rare transcripts with differential expression, and since there is a minimal chance to isolate false positive clones, cDNA-RDA may serve as a powerful tool for delineating up- or down-regulation of the genes involved in various pathological or physiological states of the kidney.

Na+/H+ antiporter (NHE-1 isoform) in cultured vascular smooth muscle from the spontaneously hypertensive rat.

An increase in Na+/H+ antiporter activity may be involved in hyperproliferation of vascular smooth muscle cells (VSMC) and possibly in the vascular hyperplasia characteristic of hypertension. The present study was designed to examine cell proliferation, Na+/H+ exchange activity, and mRNA levels of the NHE-1 isoform of the Na+/H+ antiporter in cultured aortic VSMC derived from the spontaneously hypertensive rat (SHR) and from normotensive controls, the Wistar/Kyoto rat (WKY). VSMC derived from the SHR grown in early (2 to 6), but not in later (7 to 10) sub-passages, exhibited an increase in [3H]-thymidine incorporation and shorter doubling times as compared to those derived from WKY rats. Na+/H+ exchange activity assayed in the nominal absence of HCO3-/CO2, as the rate of intracellular pH (pHi) recovery after cell acidification was significantly higher in cells from SHR than in those from WKY rats when cells were studied in early sub-passages, but not in cells studied in later sub-passages. In cells grown in early sub-passage, Na+/H+ exchange activity assessed as the initial rate of Na+i accumulation following acute cell acidification was also significantly higher in SHR than WKY cells both in the nominal absence (10.22 +/- 1.15 and 6.98 +/- 1.17 mmol Na+i/90 seconds, P < 0.05, respectively) and in the presence of HCO3-/CO2 (9.94 +/- 1.02 and 5.59 +/- 0.86 mmol Na+/90 seconds, P < 0.01, respectively). There were no detectable differences in the level of steady-state Na+/H+ antiporter (NHE-1) mRNA between VSMC from SHR and WKY rats. Our findings indicate that Na+/H+ exchange activity is increased in cultured aortic VSMC derived from SHR as compared to those derived from WKY rats. The higher functional activity of the Na+/H+ antiporter in VSMC from the SHR is due to a post-transcriptional event(s) and may be related to enhanced growth in culture.

Tissue renin angiotensin systems: theoretical implications for the development of hyperkalemia using angiotensin-converting enzyme inhibitors.

In patients with renal insufficiency, as the number of functioning nephrons is reduced, potassium balance is maintained by an increase in potassium excretion in the remaining nephrons. This adaptive response is, in part, mediated by an increase in aldosterone production by the adrenal gland. Use of angiotensin-converting enzyme (ACE) inhibitors in these patients can result in hyperkalemia by suppressing aldosterone production by the adrenal gland. Inhibition of aldosterone production depends on the degree of inhibition of angiotensin II formation in the circulation as well as the degree of inhibition of angiotensin II formed locally in the adrenal gland. Recent experimental evidence suggests that the latter process may be important for the tonic regulation of aldosterone production. Because the various ACE inhibitors exhibit different degrees of ACE inhibition at the tissue level, it is reasonable to postulate that angiotensin II-dependent aldosterone production will be inhibited to a lesser degree by agents that have low tissue specificity for the adrenal gland. This feature would be most advantageous in treating patients with chronic renal insufficiency and congestive heart failure who are at risk for hyperkalemia. Therefore, the ideal ACE inhibitor should not suppress aldosterone secretion in such patients.

Na+/H+ exchange and vascular smooth muscle proliferation.

Abnormal growth of vascular smooth muscle (VSM) is seen in various pathologic conditions such as hypertension and atherosclerosis. Many classic vasoconstrictors have now been shown to be mitogenic, either by themselves or in conjunction with other cofactors, such as insulin. The mitogenic effects of vasoconstrictors may be due, in part, to activation of similar second messenger pathways, including stimulation of the Na+/H+ antiporter. It has been suggested, therefore, that an enhanced proliferation rate may be, in part, the consequence of elevated Na+/H+ exchange. This hypothesis is supported by several observations of the close association between Na+/H+ exchange activity and DNA synthesis in some cell types including fibroblasts and VSM. Stimulation of Na+/H+ exchange may play a permissive role in optimal growth by preventing H+ accumulation (a fall in intracellular pH [pHi]) due to the increased metabolic activity during cell stimulation. Enhancement of Na+/H+ exchange activity increases Na+ influx into the cell, and secondarily increases K+ entry through activation of Na+/K+ ATPase activity. Although the Na+/H+ antiporter may influence cell proliferation through various ionic mechanisms, it is not clear that enhanced proliferation is the consequence of overactivity of this antiporter. In VSM, there are also differences in the pattern of activation of the Na+/H+ antiporter by hyperplastic and hypertrophic agents. Although pHi is increased in response to both acute and chronic stimulation by hyperplastic factors, such as platelet-derived growth factor, a hypertrophic agonist such as angiotensin II increases pHi acutely but lowers it chronically. Likewise, hyperplastic factors increase the Na+/H+ antiporter (NHE-1) mRNA levels, whereas angiotensin II does not.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic effects of converting enzyme inhibitors: focus on the reduction of cholesterol and lipoprotein(a) by fosinopril.

It is generally believed that the use of angiotensin-converting enzyme (ACE) inhibitors has no effect on the lipid profile. Our recent data show that in patients with proteinuric renal disease, serum levels of total cholesterol and lipoprotein(a) [Lp(a)] may be lowered during treatment with an ACE inhibitor, fosinopril sodium. During a 12-week randomized, placebo-controlled, double-blind study involving 26 patients with mild-to-moderate renal impairment, fosinopril administration was associated with significant decreases in both urinary protein excretion and serum total cholesterol levels, whereas placebo was not. During a 6-week washout phase, both parameters returned to baseline in fosinopril-treated patients and remained unchanged in placebo recipients. In addition, fosinopril-treated patients had a decrease in plasma levels of Lp(a), whereas this was not seen in placebo-treated patients. When data from a subset of 13 patients with proteinuric renal disease and hypertension were examined, a significant decrease in serum total cholesterol levels was observed; this decrease reversed after discontinuation of fosinopril. Analysis of the effect of fosinopril on plasma Lp(a) levels in a subset of patients who had type II diabetes mellitus and overt proteinuria revealed a significant decrease in plasma Lp(a) after administration of fosinopril. Moreover, fosinopril lowered plasma Lp(a) levels in blacks, whose pretreatment levels were higher than those of whites with comparable degrees of proteinuria and levels of serum total cholesterol. Thus, the reduction in serum Lp(a) levels may be related not only to amelioration of proteinuria, but also to another direct action of fosinopril on the metabolism of Lp(a).

Renal acid excretion and intracellular pH in salt-sensitive genetic hypertension.

Acid-base status and renal acid excretion were studied in the Dahl/Rapp salt-sensitive (S) rat and its genetically salt-resistant counterpart (R). S rats developed hypertension while on a very high salt diet (8%) and while on a more physiological salt diet (1%) and remained normotensive while on a very low salt diet (0.08%). Under the high salt diet, intracellular pH measured in freshly isolated thymic lymphocytes using 2',7'-bis (carboxyethyl)-5 (6)-carboxyfluorescein acetomethyl ester, a pH-sensitive dye, was lower in S than in R rats both when measured in the presence of HCO3/CO2 (7.32 +/- 0.02 vs. 7.38 +/- 0.02, respectively, P < 0.05) and in its absence (7.18 +/- 0.04 vs. 7.27 +/- 0.02, respectively, P < 0.05). Under the high salt diet, net acid excretion was higher in S than R rats (1,777 +/- 111 vs. 1,017 +/- 73 muEq/24 h per 100 g body wt, respectively, P < 0.001), and this difference was due to higher rates of both titratable acid and ammonium excretion. Directionally similar differences in intracellular pH and net acid excretion between S and R rats were also observed in salt-restricted animals. In S and R rats placed on a normal salt intake (1%) and strictly pair-fed to control food intake as a determinant of dietary acid, net acid excretion was also higher in S than in R rats (562 +/- 27 vs. 329 +/- 21 muEq/24 h per 100 g, respectively, P < 0.01). No significant difference in either blood pH or bicarbonate levels were found between S and R rats on either the 0.08%, 1%, or 8% salt diets. We conclude that renal acid excretion is augmented in the salt-sensitive Dahl/Rapp rat. Enhanced renal acid excretion may be a marker of increased acid production by cells from subjects with salt-sensitive hypertension.

Cytosolic free calcium regulation in response to acute changes in intracellular pH in vascular smooth muscle.

This study examined the mechanisms whereby alterations of intracellular pH (pHi) impact on free cytosolic calcium (Cai2+) in cultured rat aortic vascular smooth muscle cells (VSMC) assayed in the presence of HCO3/CO2. Rapid cell alkalinization, effected by the exposure to NH4Cl or removal of CO2 from the superfusate, produced a rapid increase in Cai2+. The rise in Cai2+ was markedly diminished when sarcoplasmic reticulum (SR) Ca2+ stores had been depleted by prior exposure to arginine vasopressin (AVP) in Ca(2+)-free media or when SR release and reuptake of Ca2+ were blocked by the addition of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), but was unaffected by the removal of external Ca2+ or inhibition of Ca2+ entry using NiCl2. Cell acidification also resulted in a rapid increase in Cai2+. This Cai2+ increase was most apparent when pHi was very low (< 6.6) and was unaffected by removal of external Ca2+ or NiCl2 addition. Unlike the effect of cell alkalinization, the increase in Cai2+ associated with cell acidification was not prevented by pretreatment with AVP or TMB-8. We conclude that, in cultured VSMC, acute intracellular alkalinization and, to a lesser extent, acidification result in release of Ca2+ from internal stores. Alkalinization increases Cai2+ by promoting its release from a store which is AVP and TMB-8 sensitive, most likely the SR. Cell acidification increases Cai2+ from an intracellular store(s) that is neither AVP nor TMB-8 sensitive. The increase in Cai2+ produced by cell acidification may be explained on the basis of cell buffering such that, as cytosolic H+ increases, it displaces Cai2+ from internal buffers with similar affinities for Ca2+ and H+.

Improvement of lipid abnormalities associated with proteinuria using fosinopril, an angiotensin-converting enzyme inhibitor.

OBJECTIVE: To examine whether reducing protein excretion in patients with proteinuric renal disease using an angiotensin-converting enzyme inhibitor, fosinopril sodium, would be accompanied by an amelioration of the associated hyperlipidemia. DESIGN: A randomized, placebo-controlled, double-blind study of 12 weeks, followed by 23 weeks of an open-label trial using fosinopril. SETTING: Outpatient renal clinics. PATIENTS: Twenty-six patients (age range, 28 to 70 years) with mild to moderate renal impairment and proteinuria associated with type II diabetes (15 patients) and other causes of nondiabetic renal disease (11 patients) completed the double-blind phase of the study. All patients except one were men. INTERVENTION: Fosinopril, 10 mg initial oral daily dose (randomized trial), and 20 mg orally once a day (open-label phase). MEASUREMENTS: Proteinuria and serum lipids (total cholesterol, high-density lipoprotein, and low-density lipoprotein [LDL] cholesterol, and lipoprotein(a) protein). RESULTS: In a group of 17 patients treated with fosinopril, protein excretion decreased from 5.56 to 4.28 g/d, a reduction of 1.28 (95% CI, -2.49 to -0.08). The reduction was associated with a decrease in serum total cholesterol from 6.39 to 5.82 mmol/L, a decrease of 0.58 mmol/L (CI, -1.01 to -0.15 mmol/L). In a group of nine patients treated with placebo, neither protein excretion (from 5.11 to 4.81 g/d, a change of -0.29 g/d [CI, -1.78 to +1.13 g/d]) nor serum total cholesterol (from 6.08 to 5.77 mmol/L, a change of -0.31 mmol/L [CI, -0.78 to +0.13 mmol/L]) change significantly. At the end of the double-blind phase, plasma lipoprotein(a) protein decreased in the fosinopril-treated group (from 3.94 to 3.33 mg/dL, a reduction of 0.60 mg/dL [CI, -1.02 to -0.18 mg/dL]) but not in the placebo group (from 2.85 to 3.19 mg/dL, a change of +0.34 mg/dL [CI, -0.53 to +1.2 mg/dL]). Dietary protein and fat intake were similar in the two groups throughout the study. In 16 patients who completed an extended open-label phase, fosinopril was associted with a decrease in protein excretion from 4.53 to 3.22 g/d, a reduction of 1.29 g/d (CI, -2.54 to -0.05 g/d), which was associated with a reduction in serum total cholesterol (from 6.37 to 5.54 mmol/L, a decrease of 0.84 mmol/L [CI, -1.59 to -0.08 mmol/L]), LDL cholesterol (from 4.38 to 3.72 mmol/L [a decrease of 0.68 mmol/L [CI, -1.33 to -0.03 mmol/L]), and plasma lipoprotein(a) protein (from 3.58 to 2.81 mg/dL, a reduction of 0.82 mg/dL [CI, -1.58 to -0.05 mg/dL]). CONCLUSION: The angiotensin-converting enzyme inhibitor, fosinopril, can result in a sustained reduction in serum total cholesterol, LDL cholesterol, and plasma lipoprotein(a) protein levels in conjunction with a partial reduction in proteinuria.

Altered blood pressure during sleep in normotensive subjects with type I diabetes.

This study was designed to examine the circadian pattern of blood pressure in children and young adults with type I diabetes who were completely normotensive by standard criteria. Forty-five patients and the same number of age- and sex-matched control subjects were studied. In diabetic children of 10-14 years of age, the nocturnal fall in systolic and diastolic blood pressures was intact. In diabetics of 15-20 years of age, the fall in systolic blood pressure was blunted; in diabetics of 21-37 years of age, the fall in both systolic and diastolic blood pressures during sleep was blunted. When data from all diabetic subjects were pooled and analyzed in a multiple linear regression model, mean blood pressure during sleep correlated best with urinary albumin excretion (r = 0.60). On the basis of this finding, we subdivided our patients into two groups: a microalbuminuric group (urinary albumin excretion > 30 mg per 24 hours; mean, 160.3 +/- 29.7; n = 11) and a normoalbuminuric group (urinary albumin excretion < 30 mg per 24 hours; mean, 6.6 +/- 6.5; n = 34). Both systolic and diastolic blood pressures during sleep were higher in microalbuminuric (121.1 +/- 3.3 and 69.3 +/- 2.5 mm Hg, respectively) than in normoalbuminuric diabetics (114.2 +/- 1.8 and 60.1 +/- 1.2 mm Hg, p < 0.05) or control subjects (113.3 +/- 1.2 and 60.1 +/- 1.2 mm Hg, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

On the mechanism of impaired distal acidification in hyperkalemic renal tubular acidosis: evaluation with amiloride and bumetanide.

It has been postulated that a distinctive type of hyperkalemic distal renal tubular acidosis (DRTA), referred to as voltage-dependent DRTA, results from diminished potassium and hydrogen ion secretion in the distal nephron, which is due to a suboptimal voltage (lumen negative) as a result of impaired sodium reabsorption. To test for the presence of a voltage-dependent DRTA, we used amiloride (20 mg oral, single dose) and bumetanide (2 mg oral, single dose) to inhibit and to stimulate voltage-dependent potassium and hydrogen ion secretion, respectively. Eighteen patients with hyperkalemic DRTA and seven controls with a comparable degree of renal impairment were studied. Patients were subdivided in two groups on the basis of their ability to lower their urine pH during spontaneous acidosis. Patients in Group I lowered their urine pH to the level of controls (5.29 +/- 0.06 and 5.37 +/- 0.11, respectively) whereas patients in Group II could not lower their urine pH below 5.5 (6.38 +/- 0.11). Patients in Group I and Group II had a similar degree of metabolic acidosis and hyperkalemia whereas controls had neither acidosis or hyperkalemia. Most patients in Group II and all patients in Group I had low plasma aldosterone levels. The administration of amiloride resulted in an increase in urine pH and a decrease in potassium excretion in all three groups. The finding that amiloride, presumably by obliterating the transtubular voltage as a result of blockade of sodium transport, inhibited potassium excretion to about the same extent in both groups of patients and in controls argues against the existence of a voltage-dependent defect. Bumetanide produced a fall in urine pH below 5.5 and an increase in potassium excretion in controls and Group I patients. In Group II patients, bumetanide failed to elicit a fall in urine pH below 5.5 but resulted in an increase in potassium excretion similar to that seen in controls and Group I patients. These findings suggest that a derangement other than a voltage-dependent defect is responsible for the inability, characteristic of Group II patients, to lower their urine pH. It was concluded that the impairment in urinary acidification observed in patients with this subtype of hyperkalemic DRTA is due to a defect in collecting tubule hydrogen secretion that results from H+ ATPase dysfunction rather than from a voltage-dependent defect.

The vasorelaxant effects of acetate: role of adenosine, glycolysis, lyotropism, and pHi and Cai2+.

The mechanism of acetate vasorelaxation is unknown. In the rat caudal artery, acetate has a vasorelaxant effect and also increases cyclic AMP. Here we evaluate the role of adenosine, of possible glycolysis inhibition by acetate, of the lyotropic properties of acetate and other anions, and of intracellular calcium and pH. Adenosine per se did not relax the caudal artery in the range of 10(-8) to 10(-2) M. Preincubation with adenosine deaminase (ADA, 5.0 U/ml) or with 8-phenyltheophylline (8-PT, 10(-6) to 10(-4) M) increased, rather than blocked the vasorelaxant effect of acetate. Oxypurinol (10(-3) M) or the nucleoside transport inhibitor NBMPR (10(-4) M) had no effect on acetate relaxation. Whereas acetate increased tissue cyclic AMP content, 10(-3) M adenosine or 10(-6) M PIA had no effect. In strips studied under conditions of inhibited glycolysis (no glucose, with 11 mM 2-deoxyglucose, 1.0 mM pyruvate, and 0.5 mM 5-iodoacetate), acetate-induced relaxation, as well as acetate-induced cyclic AMP generation, tended to be reduced but not significantly so. Other anions relaxed vascular strips in relation to their lyotropic number, but only at higher doses, and they did not stimulate cyclic AMP formation. Acetate (10 mM) caused a transient fall in Ca2+i followed by a slight, sustained rise. A concomitant decrease in pHi was seen. DIDS, which blocks the relaxant and cyclic AMP effects of acetate, had no effect on the pHi decrease, but did decrease the rate of pHi recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular Na+ dependency of free cytosolic Ca2+ regulation in aortic vascular smooth muscle cells.

This study examined contribution of Na(+)-dependent processes to the regulation of free cytosolic calcium (Ca2+i) in cultured vascular smooth muscle cells (VSMC) using fura-2. Removal of Na+ from superfusate (replacement with choline) resulted in an increment of Ca2+i that was greatly augmented by pretreatment with ouabain. Under both conditions, Ca2+i increase was followed by partial recovery to a new steady state that was still significantly higher than that seen before removal of external Na+ (Na+o). In ouabain-pretreated cells lowering of Na+o caused progressive increases in Ca2+i. Addition of NiCl2, a Na(+)-Ca2+ exchange inhibitor, completely blocked the increase in Ca2+i produced by removal of Na+o, indicating that the Na(+)-Ca2+ antiporter was responsible for observed Ca2+i changes. Ca2+i increase produced by reduction of Na+o was also seen after depletion of inositol trisphosphate-sensitive Ca2+ stores with repeated pulses of angiotensin II or after blockade of sarcoplasmatic reticulum Ca2+ release with TMB-8 but was not observed in the absence of external Ca2+. These observations indicate that the source of Ca2+i increase in response to changes in the transmembrane Na+ gradient is largely external, and potentiation of the Ca2+i surge by ouabain suggests Ca2+ influx via the Na(+)-Ca2+ exchanger operating in the reverse mode. The relative contribution of a Na(+)-dependent and -independent component of Ca2+i recovery was investigated by superfusing cells with ionomycin in a Na(+)-free medium and later adding Na+ to the medium. This Ca2+ ionophore increased Ca2+i to a peak, and this was followed by a rapid but partial recovery to a new steady state. Readdition of varying amounts of Na+ to the superfusate, in the continued presence of ionomycin, resulted in concentration-related decline in Ca2+i, thereby uncovering a substantial contribution of a Na(+)-dependent mechanism of Ca2+i regulation. Decline of Ca2+i produced by readdition of Na+ was blocked by addition of NiCl2 to the superfusate. Our findings thereby provide evidence for Ca2+i regulation in VSMC via a Na(+)-dependent mechanism, consistent with a Na(+)-Ca2+ exchanger, which acts as a Ca2+ efflux mechanism when Ca2+i is elevated. Na(+)-Ca2+ exchanger acts as a Ca2+ influx mechanism when intracellular Na+ is elevated by prior exposure to ouabain.

Extrarenal potassium tolerance in chronic renal failure: implications for the treatment of acute hyperkalemia.

The role of extrarenal potassium homeostasis is well recognized as a major mechanism for the acute defense against the development of hyperkalemia. The purpose of this report is to examine whether or not the various mechanisms of extrarenal potassium regulation are intact in patients with end-stage renal disease (ESRD). The available data suggest that with the development of ESRD and the uremic syndrome there is impaired extrarenal potassium metabolism that is related to a defect in the Na,K-adenosine triphosphatase (ATPase). The responsiveness of uremic patients to the various effector systems that regulate extrarenal potassium handling is discussed. Insulin is well positioned to play an important role in the regulation of plasma potassium concentration in patients with impaired renal function. The role of basal insulin may be even more important than previously appreciated, since somatostatin infusion causes a much greater increase in the fasting plasma potassium in rats with renal failure than in controls. Furthermore, stimulation of endogenous insulin by oral glucose results in a greater intracellular translocation of potassium in uremic rats than in controls. Under at least two common physiologic circumstances, feeding and vigorous exercise, endogenous catecholamines might also act to defend against acute increments in extracellular potassium concentration. However, it is important to appreciate that the response to beta 2-adrenoreceptor-mediated internal potassium disposal is heterogeneous as judged by the variable responses to epinephrine infusion. Based on the evidence presented in this report, a regimen for the treatment of life-threatening hyperkalemia is outlined. Interpretation of the available data demonstrate that bicarbonate should not be relied on as the sole initial treatment for severe hyperkalemia, since the magnitude of the effect of bicarbonate on potassium is variable and may be delayed. The initial treatment for life-threatening hyperkalemia should always include insulin plus glucose, as the hypokalemic response to insulin is both prompt and predictable. Combined treatment with beta 2-agonists and insulin is also effective and may help prevent insulin-induced hypoglycemia.

Intracellular H+ buffering power and its dependency on intracellular pH.

Intracellular hydrogen ion (H+) buffering power, conventionally defined as the amount of acid or base that would have to be introduced into the cell cytosol to decrease or increase ipH by one pH unit, is generally said to increase as intracellular pH (ipH) decreases. This implies that the cell has a lesser capability to resist acute acid or base perturbations at its steady state ipH than at any lower ipH. We re-examined this notion, reasoning that the logarithmic nature of the pH unit could limit the validity of the conventional expression of buffering power in imparting physiologic insight into the mechanisms of cellular H+ homeostasis. The mathematical derivation of the formula, delta i[NH4+]/delta ipH, conventionally used to estimate buffering power using the NH4Cl technique, revealed that this parameter is, by design, inversely proportional to the exponential of ipH. This a priori dependence on pH dictates an increase in buffering power with decreasing ipH, and thereby interferes with the assessment of the physiologic capability of the intracellular milieu to buffer protons at different ipH levels. To circumvent this problem, buffering power was defined as the amount of hydrogen ions that would have to be added to or removed from the cell to effect a change in the concentration of H+ in the cell cytosol of 1 mM (a term heretofore referred to as the cell H+ buffering coefficient). The mathematical derivation of the formula used to calculate the cell H+ buffering coefficient, delta i[NH4+]/delta[H+]i, does not suffer from an a priori dependence on ipH.(ABSTRACT TRUNCATED AT 250 WORDS)