Intracellular pH in isolated rat renal papillary thin limbs of Henle's loop.

Intracellular pH (pHi) was measured in isolated, nonperfused and perfused rat papillary thin limbs of Henle's loops in N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES)- or HEPES/bicarbonate-buffered medium at pH 7.4 using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Resting pHi was about 6.7 in descending thin limbs (DTL) and about 6.9 in ascending thin limbs (ATL), even with a medium pH of 7.4. These values appeared to reflect the acid pH of the blood in the neighboring vasa recta found in vivo. The resting pHi did not differ whether or not the medium contained bicarbonate although the total buffering capacity of the tubule cells was increased in the presence of bicarbonate. In nonperfused DTL and ATL, pHi was further acidified following an NH4Cl pulse. The rate of recovery of pHi from this level to the resting pHi was reduced by Na+ removal from the bath in both DTL and ATL and by the addition of ethylisopropylamiloride (EIPA) to the bath in the presence of Na+ in DTL. The rate of recovery was not affected by Cl- removal from the bath or K+ (75 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) addition to the bath in either DTL or ATL. These results suggest that the common, amiloride-sensitive, basolateral Na+/H+ exchanger plays a role in the regulation of pHi in rat papillary DTL but that a different basolateral Na+/H+ exchanger or a luminal Na+/H+ exchanger is important in rat papillary ATL.

Mixed descending- and ascending-type thin limbs of Henle's loop in mammalian renal inner medulla.

Previous studies have generally indicated that the entire descending (DTL) and ascending thin limbs (ATL) of Henle's loops in the mammalian inner medulla exhibit structurally and functionally distinct properties. In the present study, we found that about 50% of Munich-Wistar rat inner medullary thin limbs, lying at positions distinctly above the bend, had segments exhibiting structural characteristics of DTL located immediately adjacent to segments exhibiting structural characteristics of ATL. Multiple DTL-type and ATL-type segments of variable length existed along a single straight portion of these mixed tubules. Inner medullary thin limbs with repeating, sequential expression of DTL-type and ATL-type regions were also numerous in Sprague-Dawley rats, mice, and rabbits with no evidence of sexual dimorphism. RT-PCR of microdissected segments showed that the water channel aquaporin-1 (AQP1) and the urea transporter UT-A2 were expressed in pure DTL, but not in pure ATL, and in DTL-type, but not in ATL-type, regions of mixed-type thin limbs. Immunocytochemistry revealed expression of AQP1 in cells of pure DTL, but not pure ATL, and in cells of DTL-type, but not ATL-type, regions of mixed-type thin limbs. In contrast, the chloride channel ClC-K1 was expressed in pure ATL, but not pure DTL, and in ATL-type, but not DTL-type, regions of mixed-type thin limbs. Discontinuous axial expression of AQP1, UT-A2, and ClC-K1 along the straight portion of single thin limbs indicates that these nephrons possess a more heterogeneous structure than previously recognized.

Amino acid fluxes in rat thin limb segments of Henle's loop during in vitro microperfusion.

Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes of L-[(14)C]alanine and [(14)C]taurine in thin limbs of Henle's loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle's loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route.

Basolateral regulation of pHi in proximal tubules of avian loopless and long-looped nephrons in bicarbonate.

In isolated, nonperfused chicken proximal tubules from both loopless reptilian-type and long-looped mammalian-type nephrons, resting intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), was approximately 7.1 under control HCO3- conditions [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/5 mM HCO3(-)-buffered medium with pH 7.4 at 37 degrees C] and was reduced to approximately 6.8 in response to NH4Cl pulse. The rate of recovery of pHi (dpHi/dt) from this level to the resting level in proximal tubules from both nephron types was (1) significantly reduced by the removal of Na+ or both Na+ and Cl- from the bath, and (2) unaffected by the removal of Cl- from the bath or the presence of a high K+ concentration or Ba2+ in the bath. In proximal tubules from long-looped mammalian-type, but not loopless reptilian-type, nephrons, dpHi/dt was significantly reduced by the addition of either 5-(N-ethyl-N-isopropyl) amiloride (EIPA) or 4,4'-diisothiocyanostilbene-2,2'disulfonate (DIDS) to the bath. These data suggest that a Na+/H+ exchanger and most likely a Na(+)-dependent Cl-/HCO3- exchanger are involved in basolateral regulation of pHi in mammalian-type nephrons whereas none of the commonly identified basolateral acid-base transporters appear to be involved in regulation of pHi in reptilian-type nephrons.

Basolateral regulation of pHi in isolated snake renal proximal tubules in presence and absence of bicarbonate.

Intracellular pH (pHi) and its basolateral regulation were studied in isolated proximal-proximal and distal-proximal segments of garter snake (Thamnophis spp.) renal tubules with oil-filled lumens in HEPES-buffered and in HEPES-HCO-3-buffered media (pH 7.4 at 25 degrees C). pHi was measured with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) under resting conditions and in response to NH4Cl pulse. Resting pHi (approximately 7.1-7.2) and its response to and rate of recovery (dpHi/dt) from an NH4Cl pulse were not affected by the presence or absence of HCO-3 in either segment. Rate of recovery was depressed by Na+ removal in distal-proximal segments only and only in HEPES buffer. It was not affected by removal of Cl- or of both Na+ and Cl- or by reduction in membrane potential through addition of Ba2+ (5 mM) or high K+ (75 mM) in either segment in either HEPES or HEPES-HCO-3 buffer. The Na+/H+ exchange inhibitor ethylisopropylamiloride (EIPA) (100 microM) and the anion exchange inhibitor DIDS (100 microM) reduced dpHi/dt in the distal-proximal segments only and only in HEPES-HCO-3 buffer. The H+-ATPase inhibitor bafilomycin (1 microM), H+-K+-ATPase and K+/NH+4 exchange inhibitor Schering 28080 (10-100 microM), organic cation efflux inhibitor tetrapentylammonium (25 microM-20 mM), and K+ channel blocker tetraethylammonium (20 mM) had no effect on dpHi/dt in either segment. These data do not clearly support basolateral regulation of pHi in snake proximal renal tubules by commonly recognized Na+-dependent or Na+-independent acid or base transporters.

Regulation of intracellular pH in proximal tubules of avian long-looped mammalian-type nephrons.

In nonperfused proximal tubules isolated from chicken long-looped mammalian-type nephrons, intracellular pH (pHi), measured with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, was approximately 7.3 under control conditions (HEPES-buffered medium with pH 7.4 at 37 degrees C) and was reduced to approximately 7.0 in response to NH4Cl pulse. The rate of recovery of pHi from this level to the resting level was 1) significantly reduced by the removal of Na+ from the bath, 2) significantly increased by the removal of Cl- from the bath, 3) unchanged by the removal of both Na+ and Cl- from the bath, 4) significantly reduced by the addition of either ethylisopropylamiloride or DIDS to the bath, 5) significantly increased by a high bath K+ concentration, and 6) unchanged by the addition of Ba2+ to the bath. These data suggest that both Na+-coupled and Cl--coupled basolateral acid-base fluxes are involved in determining the rate of recovery of pHi after acidification. The most likely ones to be important in regulating pHi are a Na+/H+ exchanger and a Na+-coupled Cl-/HCO-3 exchanger. In birds, long-looped mammalian-type nephrons resemble short-looped transitional nephrons but differ markedly from superficial loopless reptilian-type nephrons.

Regulation of intracellular pH in avian renal proximal tubules.

In proximal tubules isolated from chicken transitional nephrons, intracellular pH (pHi), measured with the pH-sensitive fluorescent dye 2'.7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), was approximately 7.3-7.4 under control conditions [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium with pH 7.4 at 39 degrees C] and was reduced to approximately 6.8 in response to NH4Cl pulse. The rate of recovery of pHi (dpHi/dt) from this acid level to the resting level and the resting pHi were 1) significantly reduced by the removal of Na+ from the bath, 2) significantly increased by the removal of Cl from the bath, and 3) unchanged by the removal of both Na+ and Cl from the bath. The addition of either amiloride or 4,4'-diisothiocyanostilbene-2,2'-disulfonate to the bath reduced dpHi/dt to about the same extent as the removal of Na+. These data suggest that both Na(+)-coupled and Cl-coupled acid-base fluxes at the basolateral membrane are involved in determining the resting pHi and the rate of recovery of pHi after acidification. The most likely possibilities appear to be a basolateral Na+/Hi exchanger, a basolateral Na(+)-coupled Cl/HCO3 exchanger, a basolateral Na(+)-HCO3(-)CO(3)2 cotransporter, and a basolateral Na(+)-independent Cl-/HCO3 exchanger.

Regulation of intracellular pH in proximal tubules of avian loopless reptilian-type nephrons.

In proximal tubules isolated from chicken superficial loopless reptilian-type nephrons, intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, was approximately 7.1-7.2 under control conditions (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium with pH 7.4 at 37 degrees C), and was reduced to approximately 6.9 in response to NH4Cl pulse. The rate of recovery of pHi (control value approximately equal to 5 x 10(-3) pH U/s) from this acid level was 1) significantly decreased by removal of Na+ or both Na+ and Cl- from the bath or addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.25 mM) to the bath, 2) significantly increased by high bath K+ (75 mM), and 3) unchanged by removal of Cl- alone from the bath or addition of ethylisopropylamiloride (1 mM) or Ba2+ (5 mM) to the bath. Resting pHi was 1) significantly decreased by Na+ or simultaneous Na+ and Cl- removal, 2) significantly increased by high K+, and 3) unchanged by Cl- removal alone or addition of Ba2+. The data do not fit the concept of pHi regulation by the most commonly suggested basolateral transporters (Na+/H+ exchanger, Na(+)-dependent and Na(+)-independent Cl-/HCO3- exchangers, or Na(+)-HCO3(-)-CO3(2-) cotransporter).

Transport of PAH, urate, TEA, and fluid by isolated perfused and nonperfused avian renal proximal tubules.

Transport of organic anions [p-aminohippurate (PAH) and urate] and organic cations [tetraethylammonium (TEA)] and reabsorption of fluid were studied for the first time in individual renal proximal tubules isolated from avian kidneys. In isolated nonperfused tubules, PAH and urate uptake occurred against electrochemical gradients, whereas TEA uptake appeared to result from the electrical gradient. Radiolabeled PAH uptake and radiolabeled urate uptake were inhibited to an equal extent by high concentrations of unlabeled PAH and probenecid, suggesting that they might share the same transport system. However, the rate of uptake of radiolabeled PAH was significantly stimulated by preloading with alpha-ketoglutarate (alpha-KG), suggesting PAH/alpha-KG countertransport as in mammals and reptiles, whereas uptake of radiolabeled urate was not clearly stimulated. In isolated perfused tubules, net fluid reabsorption averaged approximately 2 nl.min-1.mm-1 and was inhibited by ouabain with or without bicarbonate in the perfusate and bathing medium. In these perfused tubules, the unidirectional bath-to-lumen fluxes of PAH and urate exceeded the unidirectional lumen-to-bath fluxes, indicating net secretion of both compounds. During the bath-to-lumen fluxes the uptake across the basolateral membrane was against an electrochemical gradient for both compounds. However, for PAH the steady-state intracellular concentration was about half that observed in nonperfused tubules, as generally expected during net secretion, whereas for urate the steady-state intracellular concentration was about twice that observed in nonperfused tubules, suggesting stimulation of uptake during net secretion. During the PAH lumen-to-bath flux, the steady-state intracellular concentration was significantly above that in the perfusate, suggesting that this flux involved transport into the cells from the lumen against an electrochemical gradient. However, during the urate lumen-to-bath flux, there was no urate in the cells, suggesting that this flux, as in reptiles, occurred by a paracellular route.

Basolateral tetraethylammonium transport in intact tubules: specificity and trans-stimulation.

To examine the specificity of proximal renal basolateral organic cation transport, the effects of unlabeled organic cation substrates in the bathing medium on the rate of uptake [14C]tetraethylammonium ([14C]TEA) by intact nonperfused proximal tubules and isolated basolateral membrane vesicles (BLMV) from rabbit kidneys were explored. The pattern of inhibition of transport by a battery of unlabeled organic cations was similar in intact tubules and BLMV. To determine if trans-stimulation could be demonstrated across the basolateral membrane of intact tubules, the effects of preloading tubules with unlabeled substrates on the rate of uptake of [14C]TEA and the effects of unlabeled substrates in the bathing medium on the rate of efflux of [14C]TEA from tubules preloaded with this labeled substrate were examined. Trans-stimulation was clearly demonstrated for the first time in intact tubules. However, of the compounds that significantly inhibited [14C]TEA uptake (TEA, amiloride, tetrapropylammonium, mepiperphenidol, isopropyl pyridinium, and choline), only TEA itself and choline produced a trans-stimulation of [14C]TEA uptake. Moreover, choline appeared to be at least as effective as TEA itself as a counter ion for TEA transport. Such trans-stimulation could play a physiological role in the net reabsorption of choline and the net secretion of most other organic cations.

Tetraethylammonium transport by snake renal brush-border membrane vesicles.

Transport of tetraethylammonium (TEA) by snake (Thamnophis spp.) renal brush-border membrane vesicles (BBMV) was studied. An outwardly directed proton gradient (pH 6.0 in, pH 7.5 out) stimulated uptake of TEA into BBMV and supported concentrative accumulation. Uptake of radioactively labeled TEA was also stimulated by outwardly directed gradients of unlabeled TEA and choline. The initial rate of TEA uptake was a saturable process that was adequately described by Michaelis-Menten kinetics. TEA uptake was not influenced by changes in the electrical potential difference across the membranes. Although uptake of TEA was stimulated by an outwardly directed Na+ gradient and inhibited by an inwardly directed Na+ gradient, these effects were probably secondary to the generation of proton gradients via a Na+/H+ exchanger demonstrated in these same BBMV. In agreement with previous studies with intact snake renal tubules, the present results indicate that TEA transport across the brush-border membrane involves electroneutral countertransport for protons or organic cations.

Brush-border TEA transport in intact proximal tubules and isolated membrane vesicles.

The efflux of the organic cation, tetraethylammonium (TEA), across proximal cell luminal membranes was studied using intact, perfused rabbit proximal tubules and isolated rabbit cortical brush-border membrane vesicles (BBMV). Increases of either the extravesicular H+ concentration (from pH 7.5 to pH 6.5) or the extravesicular concentration of unlabeled TEA (from 0.1 to 0.5 mM) increased the rate of efflux of radioactively labeled TEA from BBMV. Similarly, when proximal tubules were preloaded with labeled TEA and then submerged in a mineral oil bath, a rapid increase in either the H+ concentration (from pH 7.5 to pH 5.8) or the TEA concentration (from 0 to 1 mM) of the tubular perfusate produced an acute increase in efflux of the labeled TEA across the luminal brush-border membrane. These results with intact tubules and isolated membranes are consistent with previous suggestions that TEA transport across the brush border of proximal tubule cells involves a carrier-mediated countertransport process and represent the first demonstration with intact proximal tubules that the secretory flux of TEA occurs by an exchange with H+.

TEA transport by snake renal tubules: choline effects, countertransport, H+-TEA exchange.

Tetraethylammonium (TEA) transport by isolated perfused snake (Thamnophis spp.) proximal renal tubules was examined for effects of choline and evidence of countertransport of organic cations and H+-TEA exchange. Choline in perfusate significantly inhibited unidirectional lumen-to-bath flux of TEA and accumulation of labeled TEA by tubule cells, and choline in bath significantly inhibited unidirectional bath-to-lumen flux of TEA and accumulation of labeled TEA by tubule cells. These data indicate that choline inhibits transport of TEA into the cells across both the luminal and peritubular membranes and suggest that it may share the TEA transporter. To examine transport at single membranes in intact tubules, we examined the efflux of labeled TEA across the luminal membrane of tubules covered with oil and across the peritubular membrane of tubules with oil-filled lumens. Unlabeled TEA, choline, and low pH in the perfusate stimulated efflux of labeled TEA across the luminal membrane. These data suggest that TEA transport across the luminal membrane can involve countertransport of organic cations and H+-TEA exchange. Unlabeled TEA, choline, and, to a small extent, low pH in the bath stimulated the efflux of labeled TEA across the peritubular membrane. These data suggest that TEA transport across the peritubular membrane can involve countertransport of organic cations and, possibly, H+-TEA exchange. The efflux data across both membranes further support the idea that choline may share the TEA transporter.

NMN transport by snake renal tubules: choline effects, countertransport, H+-NMN exchange.

N1-methylnicotinamide (NMN) transport by isolated perfused snake (Thamnophis spp.) proximal renal tubules was examined for effects of choline and evidence of countertransport of organic cations and H+-NMN exchange. Choline had no effect on NMN transport, supporting the concept of multiple organic cation transport processes. A transconcentration of unlabeled NMN had no effect on unidirectional transepithelial fluxes of labeled NMN. Low pH in the bath enhanced the lumen-to-bath flux of labeled NMN and, at the same time, reduced the intracellular concentration of labeled NMN, but low pH in the perfusate had no effect on the bath-to-lumen flux of labeled NMN. To examine transport at single membranes more directly in intact tubules, we examined efflux of labeled NMN across the luminal membrane of tubules covered with oil and across the peritubular membrane of tubules with oil-filled lumens. Neither unlabeled NMN nor low pH in the perfusate had any effect on efflux across the luminal membrane, providing no evidence of countertransport or H+-NMN exchange across this membrane. However, unlabeled NMN and low pH in the bath both significantly stimulated efflux across the peritubular membrane, providing evidence that both countertransport of organic cations and H+-NMN exchange may play a role in transport of NMN across this membrane.

Morphological changes with Na+-free fluid absorption in isolated perfused snake tubules.

Snake (Thamnophis spp.) proximal renal tubules were perfused and bathed in vitro either with medium containing sodium or with medium in which the sodium was replaced with choline. Net fluid absorption was measured by changes in volume marker concentration, and cell volumes and cell membrane surface areas were measured by ultrastructural morphometric methods. Net fluid absorption did not differ significantly in the presence or absence of sodium. However, during the 20-25-min perfusion in the absence of sodium, significant morphological changes took place. The volume of the cells, doubled and the volume of the intercellular spaces nearly quintupled. The areas of the lateral and apical cell membranes approximately doubled, but their surface densities remained essentially constant. Therefore the larger cells in the absence of sodium had proportionally enlarged surface areas, so that the volume-to-surface area ratio remained constant. These morphological changes occurred concomitantly with the maintenance of net fluid absorption and might play a permissive role in such maintenance in the absence of sodium.

N1-methylnicotinamide transport by isolated perfused snake proximal renal tubules.

N1-methylnicotinamide (NMN) transport was studied in isolated perfused snake (Thamnophis spp.) proximal renal tubules. Unidirectional lumen-to-bath (Jl----bNMN) and bath-to-lumen (Jb----lNMN) fluxes saturated. Although Jl----bNMN and Jb----lNMN were similar, mean Jl----bNMN tended to exceed mean Jb----lNMN at all concentrations studied. Direct measurements confirmed a net reabsorptive flux equal to the difference between the unidirectional fluxes. This transport, opposite in direction to tetraethylammonium (TEA) transport, was not inhibited by TEA. Transport into the cells across both the luminal and peritubular membranes during flux measurements was apparently down an electrochemical gradient by a mediated process that was sodium dependent. Inhibition with NMN analogues suggested that transport into the cells across the luminal membrane during Jl----bNMN was more specific than transport into the cells across the peritubular membrane during Jb----lNMN. Transport out of the cells across both the luminal and peritubular membranes during flux measurements was apparently against an electrochemical gradient.

Lack of effect of low [Ca2+], La3+, and pyrazinoate on urate transport by isolated, perfused snake renal tubules.

Effects of low medium calcium concentration, of lanthanum, and of pyrazinoate on urate transport by isolated, perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Removal of calcium from perfusate with 0.18 mmol/l calcium in bathing medium had no effect on net urate secretion (J net urate) or on net fluid absorption (Jv). In the presence of calcium (1.8 mmol/l), lanthanum (2.0 mmol/l) in perfusate alone, in bathing medium alone, or in both perfusate and bathing medium had no effect on J net urate. These findings suggest that urate transport, in contrast to para-aminohippurate (PAH) transport, is not sensitive to calcium entry into the cells and support the concept that urate and PAH are transported by separate mechanisms in these renal tubules. Pyrazinoate (1.0 mmol/l) in the bathing medium had no effect on J net urate or Jv. These findings do not support the idea of a primary urate secretory process uniquely sensitive to pyrazinoate among the vertebrates.

Effects of low [Ca2+] and La3+ on PAH transport by isolated perfused renal tubules.

Effects of low medium calcium concentration and of lanthanum on p-aminohippurate (PAH) transport by isolated perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Nominal removal of calcium from bath produced rapid, irreversible disruption of cells and depression of net PAH secretion (JPAH). However, nominal removal of calcium from lumen with calcium in bath reversibly depressed JPAH and apparent PAH permeability of luminal membrane without altering net fluid absorption (Jv) or intracellular PAH concentration [( PAH]cell). In the presence of calcium (1.8 mM), lanthanum (2 mM) in perfusate alone, in bath alone, or in both perfusate and bath reversibly depressed JPAH, apparent PAH permeabilities of luminal and peritubular membranes, and initial rate of PAH transport into cells without altering [PAH]cell or Jv. Variations in calcium concentration had little effect on depression of JPAH in sodium-free bath. These data suggest that calcium entry into cells, but possibly not cytosolic calcium concentration alone, is important for PAH transport from bath into cells and from cells into lumen and for normal passive permeability of peritubular membrane to PAH and that increased cytosolic calcium concentration is not primarily responsible for depression of JPAH in sodium-free medium.

Verapamil and quinidine effects on PAH transport by isolated perfused renal tubules.

The effects of verapamil and quinidine on p-aminohippurate (PAH) transport by isolated perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Addition of 5 X 10(-5) M verapamil or 1 X 10(-4) M quinidine to the bath reversibly depressed net PAH secretion (JnetPAH), apparent permeabilities of luminal (PL) and peritubular membranes (Pp) to PAH, and initial rate of PAH transport into cells at the peritubular membrane without affecting net fluid absorption (Jv), but simultaneously produced a threefold increase in cellular PAH concentration ( [PAH]cell). Addition of 5 X 10(-5) M verapamil to the perfusate reversibly depressed JnetPAH and PL without affecting [PAH]cell or Jv. Tetraethylammonium (1 X 10(-3) M) did not alter the effects of verapamil on PAH transport. The data indicate that verapamil and quinidine in the bath inhibit JnetPAH by inhibiting uptake into cells across peritubular membrane and efflux from cells across luminal and peritubular membranes and possibly by producing intracellular binding, verapamil in the perfusate inhibits JnetPAH by inhibiting efflux from cells across the luminal membrane and probably uptake into cells across the peritubular membrane, and the verapamil effects on JnetPAH do not depend on its entry into cells. The effects of verapamil may result primarily from inhibition of calcium entry into cells, but both verapamil and quinidine may have effects on PAH transport independent of effects on calcium.