Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Abstract

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a ^cell_Cl− ,a ^cell_K+ ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark “Ringers” solution. The bath perfusate contained in addition db-cAMP 10⁻⁴, forskolin 10⁻⁶, and adenosine 10⁻⁴ mol · l⁻¹. PD_te was −11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) Ω cm². PD_bl −75±0.4 (n=260) mV.a ^cell_K+ anda ^cell_Cl− were 109±22 (n=4) and 38±4 (n=36) mmol · l⁻¹ respectively. These data indicate that Cl⁻ secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl⁻ is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l⁻¹ Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl⁻ concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl⁻ concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl⁻ held at 276 mmol · l⁻¹, and as a function of symmetrical changes in Cl⁻ concentration, with Na⁺ held at 278 mmol · l⁻¹ I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l⁻¹,n=7) and also a saturable function of Cl⁻ concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l⁻¹,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl⁻ stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda ^cell_Cl− transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10⁻⁵ to 10⁻⁴ mol · l⁻¹) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl⁻ uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl⁻K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl⁻ conductance; and a Na⁺ conductive paracellular pathway.