JEZ 865 504 THEP.JOURNAL SILVA ET AL. OF EXPERIMENTAL ZOOLOGY 279:504–508 (1997) Transport Mechanisms That Mediate the Secretion of Chloride by the Rectal Gland of Squalus acanthias PATRICIO SILVA,* RICHARD J. SOLOMON, AND FRANKLIN H. EPSTEIN Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672 Division of Nephrology, Beth Israel Deaconess Medical Center and Joslin Diabetes Center, Boston, Massachusetts 02215 ABSTRACT The rectal gland of Squalus acanthias secretes chloride by a mechanism that has been termed “secondary active transport” because it depends on the activity of Na-K-ATPase. As currently described, chloride enters the cell across the basolateral cell membrane via the 2 chloride: sodium: potassium cotransporter. The energy for this electroneutral uphill movement of chloride and potassium is provided by the gradient for sodium directed into the cell. Present in the basolateral cell membrane is Na-K-ATPase that maintains the gradient for sodium. A potassium conductance, present as well in the basolateral cell membrane, recirculates the potassium. Chloride exits the cell across the luminal membrane via CFTR, the chloride conductance. This mechanism is widely distributed throughout vertebrates. This report reviews the experimental observations that led to the current definition of the mechanism of chloride transport in the rectal gland. J. Exp. Zool. 279:504508, 1997. © 1997 Wiley-Liss, Inc. Since the identification by Burger of the rectal gland as the organ responsible for the extrarenal excretion of salt in Squalus acanthias (Burger and Hess, ’59) its study has served to define the nature of chloride transport and of the transport proteins that mediate it. The finding by Stoff that the secretion of chloride by the rectal gland can be stimulated by cyclic AMP or theophylline allowed the study of the mechanism of chloride transpost and its regulation (Stoff et al., ’77). The basic mechanism for the secretion of chloride by the rectal gland was initially identified in isolated perfused rectal glands stimulated with cyclic AMP or theophylline. The observations that the secretion of chloride by the gland is inhibited by ouabain and furosemide, and is dependent on the sodium, chloride and potassium concentration in the perfusate provided the necessary evidence for the first description of the secretory mechanism (Silva et al., ’77; Solomon et al., ’77, ’78). Based on that evidence, a mechanism for the transport of chloride was proposed in which the uptake of chloride across the basolateral cell membrane is mediated by a sodium-chloride cotransporter. The energy for this uptake is provided by the sodium gradient directed into the cell and maintained by Na-K-ATPase (Silva et al., ’77). This initial model for the secretion of chloride by the rectal gland provided the first testable hypothesis for the © 1997 WILEY-LISS, INC. transport of chloride across epithelial tissues. In this review we summarize the data that support the role of each of the different transport proteins that mediate the secretion of chloride by the rectal gland. SODIUM, POTASSIUM ACTIVATED ADENOSINE TRIPHOSPHATASE (NA-K-ATPASE) The first description of Na-K-ATPase in the rectal gland was that of Bonting (’66). Na-K-ATPase of the rectal gland has been used by many investigators to examine the characteristics of the enzyme (Cornelius and Skou, ’84; Esmann et al., ’79; Hokin et al., ’73; Skou and Esmann, ’79). Hokin purified rectal gland Na-K-ATPase to 90–95% homogeneity, inserted it in phospholipid vesicles and determined that it transported sodium and potassium in a 3:2 stoichiometry, establishing, for the first time, a link between the biochemical activity of the enzyme and the transporting characteristics of the sodium pump (Hokin et al., ’73). Na-K-ATPase is localized on the basolateral membrane of the rectal gland cell (Eveloff et al., ’79; Goertmiller and Ellis, ’76). Stimulation of chlo- *Correspondence to: Patricio Silva, M.D., Division of Nephrology, Joslin Diabetes Center, Boston, MA 02215. RECTAL GLAND TRANSPORT MECHANISMS ride secretion by cAMP and theophylline in isolated perfused rectal glands results in a six-to seven-fold rise in ouabain-inhibitable oxygen consumption (Silva et al., ’79). Simultaneously, intracellular sodium concentration falls while intracellular potassium rises, suggesting activation of Na-K-ATPase (Lear et al., ’92; Silva et al., ’79). Stimulation with cyclic AMP and theophylline results in an increase in 86Rb uptake (Marver et al., ’86). Further support for activation of Na-K-ATPase during stimulation of chloride secretion comes from the observations that ouabain binding is increased in slices of rectal gland or isolated rectal gland cells (Marver et al., ’86; Shuttleworth and Thompson, ’80; Silva et al., ’83). Whether there is direct activation of Na-K-ATPase during stimulation or the effect is due to an initial rise in intracellular concentration of sodium has not been completely established. The increase in ouabain binding seen in response to stimulation is independent of transport activity; i.e., it is seen in the absence of sodium or in the presence of furosemide, suggesting that the enzyme is activated directly (Silva et al., ’83). In recent studies Cornelius et al. report the in vitro phosphorylation of reconstituted shark (no species identified) Na-K-ATPase by protein kinase A is associated with increased maximal activity of the enzyme in response to both sodium or potassium (Cornelius and Logvinenko, ’96). This finding suggests that activation of chloride transport by peptides that activate adenylate cyclase could directly activate the enzyme. However, this observation is contrary to the previous report that phosphorylation by protein kinase A inhibits the activity of the enzyme (Chibalin et al., ’92). SODIUM: POTASSIUM: 2 CHLORIDE COTRANSPORTER Studies in vesicles obtained from basolaterally enriched plasma membranes prepared from rectal gland provided direct evidence for furosemidesensitive coupled sodium and chloride fluxes across the plasma membrane of the rectal gland (Eveloff et al., ’78). Potassium was found to be needed for the uptake of sodium, and chloride for that of rubidium, indicating that the cotransporter for sodium and chloride required the presence of potassium (Hannafin et al., ’83). Further studies by these investigators demonstrated that the uptake of chloride into rectal gland plasma membrane vesicles required sodium and potassium and was inhibited by bumetanide, supporting the hypothesis that the uptake mechanism is a sodium- 505 potassium-chloride cotransporter (Hannafin and Kinne, ’85). Thus the model for the transport of chloride by the rectal gland included a sodium:potassium:chloride cotransporter. This cotransporter has recently been cloned, first by Xu et al. (’94) and then by Gamba et al. (’94). The stoichiometry of the cotransporter was thought to be 2 chlorides for every sodium because the gradient favoring the entry of sodium into the cell is three times greater than that opposing the entry of chloride (Silva et al., ’77), and because the efficiency of the transport system for chloride, where ~30 moles are transported for every mole oxygen consumed, suggested that 2 chlorides are transported for every sodium pumped out by Na-K-ATPase (Silva et al., ’80). This stoichiometry was confirmed by kinetic studies in isolated perfused rectal gland tubules (Greger and Schlatter, ’84) and in the isolated perfused rectal gland (Silva and Myers, ’86). The cotransporter is activated when the secretion of chloride is stimulated. Activation of isolated perfused rectal glands or isolated rectal gland tubules stimulated both the secretion of salt and the binding of [3H]benzmetanide, suggesting that the Na-K-Cl cotransport system is activated as part of the process of stimulation of secretion (Forbush et al., ’92; Lytle and Forbush, ’92). The activation of the cotransporter is the result of cAMP dependent phosphorylation at serine and threonine residues (Lytle and Forbush, ’92; Torchia et al., ’92), and appears to be triggered by a fall in intracellular chloride (Lytle and Forbush, ’92). POTASSIUM CONDUCTANCE The model for the secretion of chloride predicted that potassium recirculates across the basolateral membrane because the concentration of potassium in the secretion of the gland was very small (Silva et al., ’77). The importance of the recirculation of potassium across the basolateral membrane was evidenced by studies in which barium chloride in the perfusate inhibited the secretion of chloride. The effect of barium chloride was dose-dependent and reversible (Silva et al., ’81). The effect of barium was thought to be the result of depolarization of the rectal gland cell, a fact that was confirmed by the observations of Greger et al. in isolated perfused rectal gland tubules (Greger et al., ’87; Greger and Schlatter, ’84). CHLORIDE CONDUCTANCE The model also predicted that a chloride channel facilitated the diffusion of chloride across the 506 P. SILVA ET AL. luminal membrane. Indeed, stimulation of the secretion of chloride is associated with a fall in intracellular concentration of chloride both in isolated perfused rectal glands and isolated rectal gland cells in culture, suggesting an increase in the luminal permeability for chloride (Lear et al., ’92; Silva et al., ’79). It should be noted that the measurement of intracellular chloride concentration using chloride selective microelectrodes showed no change in intracellular chloride activity at a time when chloride secretion was enhanced by theophylline and 8-bromo-cAMP (Welsh et al., ’83). However, this observation suggests that stimulation is associated with an enhancement of net Cl movement across both the apical and basolateral membranes. In a series of experiments in isolated perfused rectal gland tubules Greger et al., were able to directly demonstrate the presence of cyclic AMP stimulated chloride conductances in the apical membrane of rectal gland cell (Gogelein et al., ’87; Greger et al., ’85, ’87). This cyclic AMP dependent conductance has also been demonstrated in cultured rectal gland cells (Devor et al.,’95; Moran and Valentich, ’91; ’93). Furthermore, injection of rectal gland mRNA into frog oocytes results in the expression of a cyclic AMP stimulated chloride conductance (Sullivan et al., ’91). Thus, chloride leaves the rectal gland cell via a chloride conductance present in the apical membrane in response to cyclic AMP. This conductance has recently been cloned and found to be 75% homologous with the CFTR found in the apical membrane of human respiratory epithelia (Marshall et al., ’91). CURRENT MODEL FOR CHLORIDE TRANSPORT BY THE RECTAL GLAND Figure 1 shows the current model for the secretion of chloride by the rectal gland. Chloride enters the cell across the basolateral cell membrane via the sodium: potassium: 2 chloride cotransporter. The energy for this electroneutral uptake step is provided by the gradient for sodium directed into the cell. The gradient for sodium is maintained by Na-K-ATPase present also in the basolateral cell membrane. Also present in the basolateral cell membrane is a potassium conductance that recirculates the potassium. Chloride leaves the cell across the luminal membrane via the chloride conductance CFTR. Thus, the transport of chloride by the rectal gland is similar to that of other chloride transporting cells present in vertebrate organisms. Fig. 1. Model for the secretion of chloride in the rectal gland. Chloride enters the cell across the basolateral cell membrane via the sodium: potassium: 2 chloride cotransporter. The energy for this electroneutral uptake step is provided by the gradient for sodium directed into the cell. The gradient for sodium is maintained by Na-K-ATPase present also in the basolateral cell membrane. Also present in the basolateral cell membrane is a potassium conductance that recirculates the potassium. Chloride leaves the cell across the luminal membrane via the chloride conductance CFTR. Adenylate cyclase activates the secretion of chloride by activating the apical chloride conductance, CFTR, the sodium: potassium 2 chloride cotransporter and possibly Na, K, ATPase. CONCLUSIONS The rectal gland has been a powerful tool in the elucidation of the different steps involved in the transepithelial transport of chloride. The model described for the transport of chloride in the rectal gland has been succesfully applied to all other furosemide-sensitive chloride-transporting epithelia. The characteristics of the transport of chloride by the rectal gland are in fact representative of those of transporting epithelia throughout the vertebrates, from the fish gill to the mammalian cornea. Many of fundamental steps in the transport of chloride were first described in this gland, to name a few remarkable observations: Na-K-ATPase purified from this gland was used to link for the first time the biochemical activity of the enzyme with its transport properties; the sodium: potassium: 2 chloride cotransporter was first described in membrane vesicles from this gland; the sodium: RECTAL GLAND TRANSPORT MECHANISMS potassium: 2 chloride cotransporter was first cloned from this gland. ACKNOWLEDGMENTS All work conducted in the authors laboratory was supported by grants from the USPHS NIH NIDDK, NIEHS; NSF; and the American Heart Association: Maine Affiliate. The authors acknowledge the help of all the dedicated assistants and of the many colleagues that made these studies possible. LITERATURE CITED Bonting, S.L. (1966) Studies on sodium-potassium-activated adenosinetriphosphatase. XV. The rectal gland of the elasmobranch. Comp. Biochem. Physiol., 17:953–966. Burger, J.W., and W.N. 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