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.     

Ca2+ and Ba2+ effects on basolateral tetraethylammonium transport in isolated snake renal proximal tubules

 Previous work on snake renal tubules suggested that basolateral transport of tetraethylammonium (TEA) is symmetrical. To examine regulation of this transport step more closely, we determined the effects of (1) reductions in the extracellular Ca²⁺ concentration and basolateral Ca²⁺ entry, and (2) the presence of extracellular Ba²⁺ on TEA uptake and efflux across the basolateral membrane of isolated snake renal proximal tubules. Removal of extracellular Ca²⁺ reduced initial TEA uptake and enhanced TEA efflux. Blocking Ca²⁺ entry also reduced initial TEA uptake. Extracellular Ba²⁺ depolarized the basolateral membrane and reduced both TEA uptake and efflux. Inhibition of basolateral TEA uptake with a reduced membrane potential supports previous data indicating that uptake involves potential-driven facilitated diffusion. Inhibition of basolateral TEA efflux by Ba²⁺ even with a reduced membrane potential not only supports previously obtained data indicating that efflux is not influenced by the potential difference and that basolateral TEA transport is asymmetrical, but also suggests that TEA uptake and efflux may occur by separate pathways. Received: 22 May 1997 / Received after revision: 24 July 1997 / Accepted: 28 July 1997     

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.     

Effects of SITS on urate transport by isolated,perfused snake renal tubules

Effects of an inhibitor of membrane anion-exchange transport processes, 4-acetamido-4-isothiocyano-2,2-disulfonic stilbene (SITS), on urate transport by isolated, perfused snake (Thamnophis spp.) proximal renal tubules were studied. SITS (10^–4 mol/l) in the luminal perfusate had absolutely no effect on net urate secretion (J _urate ^net ) or on net fluid absorption (J _v). This observation is compatible with other data that give no support to the concept of a mediated transport step for urate from the cells to the lumen. SITS (10^–4 mol/l) in the bathing medium reversibly inhibitedJ _urate ^net without affectingJ _v. At the time of maximum inhibition ofJ _urate ^net , the concentration of urate in the cell water was increased and the apparent permeability of the luminal membrane to urate was decreased, but the urate efflux across the peritubular membrane and the apparent permeability of the peritubular membrane to urate were unchanged. There was no evidence of significant intracellular binding or trapping of urate. Although an increase in the initial rate of urate transport into the cells across the peritubular membrane could not be demonstrated conclusively in nonperfused tubules, the results still suggest that SITS in the bathing medium may inhibitJ _urate ^net by inhibiting urate movement from the cells to the lumen while actually enhancing transport from the bathing medium into the cells.     

Effects of prostaglandins on Na transport in isolated collecting tubules

Direct tubular effects of prostaglandins (PG's) on Na transport were examined in isolated cortical and medullary collecting tubules of rabbits perfused in vitro. The animals were treated with deoxycorticosterone acetate (DOCA, 1 mg kg^–1 day^–1, i.m.) for 3–6 days before experiments. In the cortical collecting tubules PGE₂ (1.2×10^–7–2.5×10^–5 M), E₁ (1.2×10^–5 M) and F₂(1.2×10^–5 M) added to the bath caused reversible decreases in transtubular potential difference (PD_t). But neither PGE₂ (1.2 ×10^–5 M) added to the perfusate nor PGA₂ (1.2 ×10^–5 M) added to the bath had an effect on PD_t. The net Na absorption was decreased with PGE₂ (1.2×10^–5 M) added to the bath from 8.6±1.36 to 1.5±1.04 pEq cm^–1 s^–1 (P<0.02). In rabbits not pretreated with DOCA, the net Na absorption was reduced from 2.73±0.74 to 1.02±0.74 pEq cm^–1 s^–1 (P<0.01). In the outer medullary collecting tubules PGE₂ (1.2×10^–5 M) added to the bath also caused a reversible decrease in PD_t. It is concluded that PGE₂, F₂ and E₁ inhibit Na absorption in the collecting tubules by acting on the peritubular membrane.     

Basolateral choline transport in isolated rabbit renal proximal tubules

 Choline can undergo both net secretion and net reabsorption by renal proximal tubules, but at physiological plasma levels net reabsorption occurs. During this process, choline enters the cells at the luminal side down an electrochemical gradient via a specific transporter with a high affinity for choline. It appeared likely that choline was then transported out of the cells against an electrochemical gradient at the basolateral membrane by countertransport for another organic cation. This possibility was examined by studying net transepithelial reabsorption and basolateral uptake and efflux of [¹⁴C]choline in isolated S2 segments of rabbit renal proximal tubules. Basolateral uptake, which was inhibited by other organic cations such as tetraethylammonium (TEA), appeared to occur by the standard organic cation transport pathway. However, the addition of TEA to the bathing medium not only failed to trans-stimulate net transepithelial reabsorption and basolateral efflux of [¹⁴C]choline but it actually inhibited transepithelial reabsorption by @60%. The results do not support the presence of a countertransport step for choline against an electrochemical gradient at the basolateral membrane. Instead, they suggest that choline crosses this membrane by some form of carrier-mediated diffusion even during the reabsorptive process. Received: 24 March 1998 / Received after Revision: 15 June 1998 / Accepted: 2 July 1998     

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 _urate ^net ) or on net fluid absorption (J _v). 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 onJ _urate ^net . 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 onJ _urate ^net orJ _v. These findings do not support the idea of a primary urate secretory process uniquely sensitive to pyrazinoate among the vertebrates.     

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.     

Procaine has opposite effects on passive Na and K permeabilities in frog skin

Procaine has opposite effects on the active transport of Na⁺ when applied on the mucosal side of the frog skin [where it produces a stimulation of the short-circuit current (I _sc)] or when added on the serosal side (where it produces an inhibition ofI _sc). In an attempt to reveal and localize the primary effect of procaine on either the apical or latero-basal membranes of the epithelial cells, we have tried to chemically dissect both membrane functions with inhibitors and ionophores. When applied on the apical side of the latero-basally depolarized epithelium, 25 mmol/l procaine increasesI _sc andV _oc (transepithelial open-circuit potential), while decreasing the transepithelial resistance. TheE ₁–E ₂ linearity domain of the I–V curves is narrowed. On the serosal side of the depolarized epithelium, the same concentration of procaine does not affectI _sc andV _oc (which are already inhibited) but it produces an increase in the transepithelial resistance (R _t). Procaine influence on the passive K⁺ permeability was studied by using the ionophore nystatin, which is assumed to form channels permeable to K⁺, when applied on the amiloride blocked apical membrane. In nystatin-treated epithelia, 25 mmol/l procaine on the apical side decreaseI _sc,V _oc andR _t. In parallel experiments during Cl^– substitution by SO ₄ ^2– , the procaine effects onI _sc andV _oc are no longer maintained, but transient. The results suggest that procaine positively influences the Na⁺ transient through the apical Na⁺-channels, and inhibits the epithelial permeability for K⁺, possibly by reducing K⁺-ions accessibility to the K⁺-channels.