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Monoclonal antibodies to maltase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from young adult and aged rats were prepared by the hybridoma technique. Four cell lines producing antibodies of the IgG1 subclass to maltase were established. Two, designated 1F12E1 and 8B1G6, produced monoclonal antibodies specific for the catalytically active form of the enzyme found predominantly in enzyme preparations from young animals. The other two clones designated 7G10H3 and 2E1C10 produced monoclonal antibodies that reacted exclusively with an enzymatically inactive form of maltase found mostly in enzyme preparations from aged rats. The increased prevalence of an inactive form of the enzyme in the old rat accounts for the decreased maltase-specific activity previously reported in the senescent rat. The active and inactive maltase species were separated by immunoaffinity chromatography by using the monoclonal antibodies as ligands. The separated forms of the enzyme were not distinguished by NaDodSO4/polyacrylamide gel electrophoresis, peptide mapping of the CNBr-cleaved proteins, and the NH2-terminal residues of these peptides. This study demonstrates the presence of an altered, antigenically distinct enzyme in senescent animals. Critical issues on the mechanism of the aging process may be addressed by application of these findings.

Renal cells isolated from vitamin D-deficient chicks had an increased Na+-dependent phosphate uptake when preincubated with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. Phosphate uptake in the absence of Na+ and methyl alpha-glucoside uptake dependent on Na+ were not affected. Phosphate uptake was stimulated 15% by 0.010 pM 1,25-(OH)2D3. Maximal enhancement of 30% was obtained with 100 pM. The uptake when fully stimulated by preincubation in vitro approximated the uptake of cells isolated from chicks that were previously repleted with 1,25-(OH)2D3 in vivo. Cells from repleted chicks were not stimulated additionally when preincubated with 1,25-(OH)2D3 in vitro. The increase in phosphate uptake could be measured after a 1-hr preincubation period; full response required at least 2 hr. Phosphate uptake induced by 1,25-(OH)2D3 was blocked by cycloheximide and actinomycin D. Enhancement of phosphate uptake was relatively specific for the 1,25-(OH)2D3 analog of vitamin D3. The potency order was 1,25-(OH)2D3 greater than 25-(OH)D3 = 1-(OH)D3 greater than 24,25-(OH)2D3 greater than D3. Kinetically, 1,25-(OH)2D3 increased the Vmax of the phosphate uptake system; the affinity for phosphate was unaffected. 3H-Labeled 1,25-(OH)2D3 was taken up by the isolated renal cells. It was estimated that the stimulation of phosphate uptake might be initiated by very few molecules of 1,25-(OH)2D3 per cell. It is proposed that 1,25-(OH)2D3 contributes importantly to the mechanisms by which phosphate transport is regulated in the kidney.

Amiloride-sensitive Na+ -H+ exchange activity in brush border membrane vesicles isolated from rat proximal tubule was increased in metabolic acidosis. The enhancement of exchange activity required an intact adrenal gland or glucocorticoid supplements. Ammonium and phosphate excretions were increased during acidosis and these were also largely dependent on an intact adrenal gland or glucocorticoid supplements. Amiloride-insensitive Na+ uptake and passive H+ permeability were not altered by acidosis or the glucocorticoid status of the animal. These findings are consistent with glucocorticoids having an important regulatory role in the kidney by orchestrating the proximal tubular adaptation to metabolic acidosis.

Pre-steady-state kinetic measurements of 22Na+ uptake by the amiloride-sensitive Na+-H+ exchanger in renal brush border membrane vesicles (BBMV) were performed at 0 degrees C to characterize the intermediate reactions of the exchange cycle. At 1 mM Na+, the initial time course of Na+ uptake was resolved into three separate components: (i) a lag phase, (ii) an exponential or "burst" phase, and (iii) a constant velocity or steady-state phase. Pulse-chase experiments using partially loaded BBMV showed no evidence for 22Na+ back-flux, suggesting that the decline in the rate of Na+ uptake rate following the burst represents completion of the first turnover of the exchanger. Gramicidin completely abolished Na+ uptake, indicating that the burst phase results from the translocation of Na+ rather than from residual Na+ binding to external sites. Raising the [Na+] from 1 to 10 mM at constant pH (internal pH 5.7; external pH 7.7) produced a sigmoidal increase in the amplitude of the burst phase without affecting the lag duration or the apparent burst rate. In contrast, Na+ uptake in the steady state obeyed Michaelis-Menten kinetics. These results suggest that a minimum of two Na+ transport sites must be occupied to activate Na+ uptake in the pre-steady state. The transition to Michaelis-Menten kinetics in the steady state can be explained by a "flip-flop" or alternating site mechanism in which the functional transport unit is an oligomer and only one promoter per cycle is allowed to form a translocation complex with Na+ after the first turnover.

The glucocorticoid dexamethasone, but not the mineralocorticoid aldosterone, increased amiloride-sensitive Na+-H+ exchange activity in rat proximal tubule brush border vesicles. Na+ uptake, independent of amiloride, was not affected. The glucocorticoid decreased the Na+ gradient-dependent phosphate uptake. Uptake in the absence of a Na+ gradient was not inhibited. Dexamethasone did not affect the Na+ gradient-dependent D-glucose uptake. These findings are consistent with the effects of glucocorticoids in stimulating acid secretion and causing phosphaturia in man and animals and may identify the locus of action and suggest the mechanisms by which the hormones act.

Na+-H+ exchange activity, i.e., amiloride-sensitive Na+ and H+ flux, in renal proximal tubule brush border (luminal) membrane vesicles was increased in the hyperthyroid rat and decreased in the hypothyroid rat, relative to the euthyroid animal. A positive correlation was found between Na+-H+ exchange activity and serum concentrations of thyroxine (T4) and triiodothyronine (T3). The thyroid status of the animal did not alter amiloride-insensitive Na+ uptake. The rate of passive pH gradient dissipation was higher in membrane vesicles from hyperthyroid rats compared to the rate in vesicles from hypothyroid animals, a result which would tend to limit the increase in Na+ uptake in vesicles from hyperthyroid animals. Na+-dependent phosphate uptake was increased in membrane vesicles from hyperthyroid rats; Na+-dependent D-glucose and L-proline uptakes were not changed by the thyroid status of the animal. The effect of thyroid hormones in increasing the uptake of Na+ in the brush border membrane vesicle is consistent with the action of the hormones in enhancing renal Na+ reabsorption. Further, the regulation of transtubular Na+ flux has now been shown to be concomitant with modulation of the entry of Na+ into the tubular cell across its luminal membrane, mediated by the exchange reaction, and with the previously reported control of the pumping of Na+ out of the cell across its basolateral membrane, mediated by the Na+,K+-ATPase.