Effects of sulfhydryl reagent, p-chloromercuribenzoate, on p-aminohippurate transport by isolated, perfused snake renal tubules.

Effects of mercaptide-forming sulfhydryl reagent, p-chloromercuribenzoate (PCMB), on transport of p-aminohippurate (PAH) by isolated, perfused snake (Thamnophis species) renal tubules were studied. PCMB (10(-7) mol/l) in bathing medium irreversibly inhibited net transepithelial transport of PAH (JPAH) without affecting net fluid absorption (Jv). PCMB (10(-8) mol/l) in perfusate irreversibly inhibited JPAH without affecting Jv and markedly reduced the apparent permeability of the luminal membrane to PAH. These data suggest that sulfhydryl groups may be involved in the PAH transport steps at both the peritubular and luminal membranes.

Effects of chloride substitutes on PAH transport by isolated perfused renal tubules.

Effects of substituting isethionate, methyl sulfate, or thiocyanate for chloride on p-aminohippurate (PAH) transport by isolated perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. In the perfusate, isethionate or methyl sulfate substitution irreversibly depressed net PAH secretion and the apparent PAH permeability of the luminal membrane by about 60-80%, whereas thiocyanate substitution had no effect. In the bathing medium, isethionate substitution reversibly stimulated net PAH secretion by about 35% without changing the apparent permeability of the peritubular membrane to PAH (Pp); thiocyanate substitution reversibly inhibited net PAH secretion by about 45% without affecting Pp; and methyl sulfate substitution had no effect. With simultaneous substitutions in perfusate and bath, isethionate depressed net PAH secretion irreversibly, whereas thiocyanate had no effect. Effects on PAH transport were not simply the result of changes in transepithelial potential or of the changes in net transepithelial fluid movement. These data strengthen the concept that net PAH secretion involves different mediated steps at the peritubular and luminal membranes. Since these tubules are highly permeable to thiocyanate and poorly permeable to isethionate and methyl sulfate, the data suggest that the mediated step from cells to lumen does not require chloride in the lumen but does require a highly permeant anion.

Bath and lumen effects of SITS on PAH transport by isolated perfused renal tubules.

Effects of 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS) in bath or lumen on p-aminohippurate (PAH) transport by isolated perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Addition of SITS to the bath in concentrations as low as 10(-10) M irreversibly depressed net PAH secretion. Addition of 10(-4) M SITS in the bath depressed net PAH secretion by about 60% without affecting apparent permeability of luminal membrane to PAH (PL) or net fluid absorption. Cellular PAH concentration was reduced by about 70% at this time but was still greater than that in the bath. Apparent permeability of peritubular membrane, determined from PAH efflux from tubules with oil-filled lumens, was unchanged even with 10(-3) M SITS in bath. These data indicate that SITS in the bath inhibits PAH transport from bath into cells without enhancing passive efflux from cells to bath. Addition of 10(-4) M SITS to the lumen alone depressed net PAH secretion and PL by about 60%, but this depression was reversed when SITS was removed from the lumen. These data suggest that PAH moves from cells to lumen by a mediated process distinctly different from that fro transport into cells from bath.

Effects of inhibitors in lumen on PAH and urate transport by isolated renal tubules.

Effects of the presence of unlabeled p-aminohippurate (PAH) or urate, probenecid, and phenol red in the lumen on labeled PAH or urate transport by isolated, perfused snake (Thamnophis spp.) proximal renal tubules were studied. Net secretion of labeled urate and luminal membrane permeability to urate were unaffected by the presence of unlabeled urate (up to 0.1 mM) or probenecid (up to 1.0 mM) in lumen only. The data are compatible with movement of urate from cells to lumen during urate secretion by a simple passive process. Net secretion of labeled PAH was rapidly and reversibly depressed to about 25-35% of control when unlabeled PAH (0.05 mM), phenol red (0.05 mM), or probenecid (0.1 mM) was added to the lumen only. During maximum depression of PAH transport, luminal membrane permeability to PAH was reduced by 60-70%. The data suggest that movement of PAH from cells to lumen down an electrochemical gradient during PAH secretion occurs by a readily inhibited, mediated process.

Fluid absorption with and without sodium in isolated perfused snake proximal tubules.

Net fluid absorption (JV) was studied in isolated, perfused snake (Thamnophis spp.) proximal renal tubules. With standard (150 mmol/liter Na+) bicarbonate-buffered Ringer in perfusate and standard Ringer plus dextran (4 g/100 ml) in bath, JV was about 0.9 nl min-1 mm-1. Removing dextran from bath reduced JV by about 20 percent. When sodium in perfusate was replaced with choline JV approached zero. However, when sodium in bath as well as perfusate was replaced with choline, JV returned to control level. Results were the same when sodium was replaced with tetramethyl-ammonium, sodium chloride was replaced with sucrose or lactose, or chloride was replaced with methyl sulfate. In contrast, replacing sodium in perfusate or in both perfusate and bath with lithium did not reduce JV. Fluid absorption was always isosmotic. Replacing bicarbonate with phosphate or Tris in sodium-containing media had no effect on JV, but the presence of buffer in sodium-free or low-chloride media may have been important for JV. Reducing temperature 10 degrees C reduced JV by about 35 percent with either sodium chloride or sucrose in both perfusate and bath. The results indicate that isosmotic fluid absorption can occur when lithium is substituted for sodium or when some other substitution is made for sodium, chloride, or both in perfusate and bath simultaneously.

Low Na+ effects on PAH transport and permeabilities in isolated snake renal tubules.

Effects of low sodium concentrations on p-aminohippurate (PAH) transport by isolated, perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Replacement of sodium in bath with choline led to significant depression of net PAH transport from bath to lumen in less than 10 min and to maximum depression (to 25-30% of control) in about 30 min, but transport still occurred against concentration gradient. In absence of sodium, PAH concentration in cell water was markedly depressed, but was still slightly greater than that in bath or lumen. Apparent permeability of peritubular membrane, determined from PAH efflux from tubules with oil-filled lumens, averaged about 0.5 X 10(-5) cm s-1 in 150 mM sodium and about 1.1 X 10(-5) cm s-1 in sodium-free medium. Data suggest that both decreased rate of active transport and increased apparent permeability of peritubular membrane contribute to depression of net transepithelial PAH transport and cell water PAH concentration in sodium-free medium. When sodium was restored to bath, net PAH transport nearly tripled in 15 min. Reduction of bath sodium concentration to one-half control or perfusion with sodium-free medium in lumen and control medium in bath had no effect on net PAH transport.

High K+ effects on PAH transport and permeabilities in isolated snake renal tubules.

Effects of high potassium concentrations on para-aminohippurate (PAH) transport by isolated, perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Increasing the potassium concentration in bath from 3 mM TO 10 mM OR 40 MM led to about 50% decrease in net PAH transport from bath to lumen in less than 10 min, but transport still occurred against concentration gradient. Cell water PAH concentration was not significantly depressed in 10 mMpotassium and was nearly double control level in 40 mM potassium. Apparent luminal membrane permeability to PAH, calculated from perfusion studies, averaged about 3.5 x 10(-5) cm sec(-1) in 3 mM potassium, 1.15 x 10(-5) cm sec(-1) in 10 mM potassium, and 0.48 x 10(-5) cm sec(-1) in 40 mM potassium. Apparent peritubular membrane permeability, determined from PAH efflux from tubules with oil-filled lumens averaged about 0.5 x 10(-5) cm sec(-1) in 3 mM potassium, 0.29 x 10(-5) cm sec(-1) in 10 mM potassium, and 0.17 x 10(-5) cm sec(-1) in 40 mM potassium. These data suggest that high potassium concentrations depress transepithelial PAH transport primarily by reducing luminal and peritubular membrane permeabilities. Effect of high potassium on PAH transport was reversed within 20 min after restoration of control potassium concentration.