Item

Abstract

Rectal gland tubule (RGT) segments of the spiny dogfish (Squalus acanthias) were perfused in vitro. The effects of inhibitors of known mode of action on transepithelial PD (PD_te resistance (R_te), the PD across the basolateral membrane (PD_bl), the fractional resistance of this membrane (FR_bl), and intracellular activities of Na⁺, Cl⁻, K⁺ (a cell) were examined. Furosemide (5·10⁻⁴ mol·l⁻¹) reduced PD_te from −12±0.7 to −2.3±0.2 mV (n=63), hyperpolarized PD_bl from −71±1.3 to −79±0.9 mV (n=59), FR_bl decreased from 0.2±0.03 to 0.13±0.01 (n=21),a cellCl⁻ fell from 38±4 to 11±2 mmol·l⁻¹ (n=14),a cellNa⁺ fell from 37±4 to 17±2 mmol·l⁻¹ (n=12) anda cellK⁺ was constant [113±14 vs. 117±15 mmol·l⁻¹ (n=6)]. Furosemide exerted its effects within some 20–40 s. Its action was completely reversible. Analysis of the time courses revealed that the furosemide induced initial fall ina cellCl⁻ was approximately twice as rapid when compared to that ofa cellNa⁺ . Ba²⁺ 0.5 mmol·l⁻¹ (bath) reduced PD_te from −7.4±1.2 to −4.1±0.6 mV (n=24), increased R_te from 18±2 to 22±2.5, Ωcm2 (n=14). PD_bl depolarized from −75±2 to −48±2 mV (n=42), FR_bl increased from 0.2±0.02 to 0.34±0.04 (n=14) anda cellK increased from 143±28 to 188±40 mmol·l⁻¹ (n=4). Ouabain (50·10⁻⁶ mol·l⁻¹, bath) reduced PD_te from −12±2 to −3±0.5 mV (n=9), R_te increased from 18±3 to 21±3 Ωcm2 (n=5), PD_bl depolarized from −67±4 to −26±3 mV (n=14), FR_bl increased from 0.23±0.04 to 0.45±0.05 (n=6),a cellK fell only slightly from 135±15 to 112±30 mmol·l⁻¹ (n=4), buta cellCl⁻ increased from 35±12 to 111±37 mmol·l⁻¹ (n=3). These effects of ouabain were slow when compared to those exerted by furosemide or Ba²⁺. The ouabain effects on PD_te and PD_bl were completely prevented if furosemide was applied first. Amiloride (≤10−3 mol·l⁻¹, both sides), and anthracene-9-carboxylate (≤10⁻³ mol·l⁻¹, lumen) were devoid of effects on PD_te and PD_bl. The stilbene disulfonate derivate SITS (≤10⁻³ mol·l⁻¹, both sides) led to a 36±9% inhibition of PD^te. The present data, apart from supporting the cell model proposed in a preceding report, allow the following conclusions: The transient analysis of the furosemide effects is suggestive for a 2 Cl⁻∶1 Na⁺ stoichiometry of the carrier. The furosemide induced reduction in FRbl suggests that the fall ina cellCl⁻ leads to a marked reduction in the apical Cl⁻-conductance. Furosemide apparently drivesa Cl⁻/cell to passive distribution and PD_bl to the K⁺ equilibrium potential.a cellNa⁺ then approaches a minimal value which is close to that present in nonstimulated RGT segments. Ba²⁺ leads to an increase ina cellK⁺ because it blocks the conductive recycling pathway for this ion. Ouabain does not only inhibit the (Na⁺+K⁺)-ATPase, it also reduces the K⁺-conductance of the basolateral membrane. This explains whya cellK⁺ stays high. The cell PD then is dominated bya cellCl⁻ . The Na⁺ 2Cl−K⁺-carrier increasesa cellCl⁻ further and the cell continues to depolarize. Thus, the depolarization of the cell is not a sign of K⁺ loss but indicative of Cl⁻ gain. In the presence of furosemide ouabain is devoid of any effect on PD_bl, suggesting that the Na⁺2 Cl⁻K⁺-carrier is the only quantitatively important source of Na⁺ entry into the RGT cell.