The mechanisms involved in urate and p-aminohippurate (PAH) transport in the rabbit renal brush-border membrane were investigated through study of membrane vesicles. Transport of [14C]urate and [3H]PAH was measured by a rapid filtration method. As previously reported by others, no OH(-)-PAH exchanger could be demonstrated by imposing an outwardly directed OH- gradient (pHin 7.4, pHout 6). In contrast, an OH(-)-lactate exchanger (or H(+)-lactate cotransport) was demonstrated. In the presence of valinomycin and an inwardly directed K+ gradient, both [14C]urate and [3H]PAH vesicle uptake were stimulated, demonstrating a potential-driven transport of these two anions. Probenecid, PAH, or cold urate decreased potential-driven urate uptake, suggesting that this transport was facilitated by a specific transport mechanism. The potential-driven urate transport described here may play a role in the second step of urate secretion in rabbits, because rate (or PAH) is transported across the brush-border membrane from the negative interior of the cell to the more positive omen.
The present studies were designed to test our previous suggestion that Na+/H+ exchange was activated by muscarinic stimulation of rat parotid acinar cells. Consistent with this hypothesis, we demonstrate here that intact rat parotid acini stimulated with the muscarinic agonist carbachol in HCO3- -free medium show an enhanced recovery from an acute acid load as compared to similarly challenged untreated preparations. Amiloride-sensitive 22Na uptake, due to Na+/H+ exchange, was also studied in plasma membrane vesicles prepared from rat parotid acini pretreated with carbachol. This uptake was stimulated two-fold relative to that observed in vesicles from control (untreated) acini. This stimulation was time dependent, requiring approximately 15 min of acinar incubation with carbachol to reach completion, and was blocked by the presence of the muscarinic antagonist atropine (2 x 10(-5) M) in the pretreatment medium. The effect of carbachol was dose dependent with K0.5 approximately 3 x 10(-6) M. Stimulation of the exchanger was also seen in vesicles prepared from acini pretreated with the alpha-adrenergic agonist epinephrine, but not with the beta-adrenergic agonist isoproterenol, or with substance P. Kinetic analysis indicated that the stimulation induced by carbachol was due to an alkaline shift in the pH responsiveness of the exchanger in addition to an increased apparent transport capacity. Taken together with previous results from this and other laboratories, these results strongly suggest that the Na+/H+ exchanger and its regulation are intimately involved in the fluid-secretory response of the rat parotid.
The sections in this article are: Overall Renal Handling Mono‐, Di‐, and Tricarboxylates Sulfate and Thiosulfate Tubular Handling as Studied by Micropuncture and Microperfusion Monocarboxylates Di‐ftricarboxylates Sulfate/Thiosulfate Transport in Isolated Membrane Vesicles Monocarboxylates Di‐ltricarboxylates Sulfate/Thiosulfate Characterization of Proteins Involved in Transmembrane Transport Reconstitution of Mono‐, Di‐, and Tricarboxylate Transport in Artificial Liposomes Partial Purification of a Dicarboxylic Acid‐Binding Protein Identification of a Protein Involved in Basolateral Sulfate Transport Cellular Models Monocarboxylates Di‐/tricarboxylates Sulfate/Thiosulfate
pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H+ gradient (pHin = 6.0, pHout = 8.0) 22Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pHin = pHout = 6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K1 = 1.6 microM) while the transport inhibitors furosemide (1 mM), bumetanide (1 mM), SITS (0.5 mM) and DIDS (0.1 mM) were without effect. This transport activity copurified with the basolateral membrane marker K+-stimulated p-nitrophenyl phosphatase. In addition, 22Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H+ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration consistent with the existence of a single transport system with KM = 8.0 mM at 23 degrees C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na+/H+ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.
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