Sharon E. Bryson

NaCl transport and ultrastructure of opercular epithelium from a freshwater-adapted euryhaline teleost, Fundulus heteroclitus

We adapted killifish to defined freshwater (FW: 1.0 mM Na, 1.0 mM Cl, + 0.1 mM Ca) and by fluorescence light microscopy and scanning and transmission electron microscopy found that the opercular epithelium retained mitochondria rich (MR) cells that were significantly larger but less numerous than in SW-acclimated tissues. Opercular epithelia mounted in vitro with FW bathing the mucosal surface take up Cl against a large negative inside transepithelial potential (Vt, grand mean –64.1 mV) and concentration gradient; the observed flux ratio was significantly different (P < 0.001) from that predicted for passive ion distribution but the net flux was consistently negative. The Na flux ratio suggested that Na was passively distributed. Vt was largely a Na diffusion potential, based on unilateral manipulations of [Na]. Cl unidirectional uptake was unaffected by mucosally added SITS (0.1 mM) but was inhibited by SCN (1.0 mM) and by anaerobiosis. Killifish transferred from SW to FW for 48 h had reduced Cl secretion by the opercular epithelium compared to SW controls but had not yet developed Cl uptake, indicating a slow adaptive process for development of Cl absorptive transport. Opercular epithelia of FW adapted fish, if bathed with isotonic saline on both sides, has a modest net Na and Cl uptake, unlike SW opercular epithelium that strongly secretes Cl under similar conditions. FW killifish opercular epithelium may provide a model to study ion regulation by euryhaline fish. J. Exp. Zool. 277:23–37, 1997.

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost, Fundulus heteroclitus

We examined transepithelial transport of Ca2+ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l−1 Ca2+) bathing the mucosal side, actively transported Ca2+ in the uptake direction; net flux averaged 20–30 nmol·cm−2·h−1. The rate of Ca2+ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca2+ uptake was saturable, apparent K1/2 of 0.348 mmol·l−1, indicative of a multistep transcellular pathway. Ca2+ uptake was inhibited partially by apically added 0.1 mmol·l−1 La3+ and 1.0 mmol·l−1 Mg2+. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l−1)+0.1 mmol·l−1 3-isobutyl-l-methylxanthine inhibited Ca2+ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca2+, thapsigargin (1.0 μmol·l−1, serosal side), ionomycin (1.0 μmol·l−1, serosal side) and the calmodulin blocker trifluoperazine (50 μmol·l−1, mucosal side) all partially inhibited Ca2+ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca2+ flux, indicating Ca2+ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 μmol·l−1, mucosal side), a Ca2+ channel blocker, had no effect. Results are consistent with a model of Ca2+ uptake by mitochondria rich cells that involves passive Ca2+ entry across the apical membrane via verapamil-insensitive Ca2+ channels, intracellular complexing of Ca2+ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca2+ invoke a downregulation of transcellular Ca2+ transport, implicating Ca2+ as a homeostatic mediator of its own transport.

Gonadotropin stimulation of K+ secretion and Na+ absorption by brook trout (Salvelinus fontinalis) sperm duct epithelium

The sperm duct epithelium from mature spermiating brook trout (Salvelinus fontinalis) was mounted in vitro to examine control of Na+ absorptive and K+ secretory transport. Na+ absorption (measured as the short-circuit current) and K+ secretion (measured using 86Rb+ as tracer) were stimulated by 3-isobutyl-1-methylxanthine and cyclic AMP while unstimulated tissues had no net ion transport. Purified chum salmon (Oncorhynchus nerka) Con AII carbohydrate-rich gonadotropin produced a rapid, sustained rise in Rb+ secretion and Na+ uptake in a log linear dose-dependent manner. Addition of gonadotropin to either apical (mucosal) or basolateral (serosal) sides evoked the response, but addition to the apical side produced the more rapid effect, indicating that receptors for the hormone are present on both sides of the transporting cells and suggesting that subepithelial tissue may slow the response to serosally added hormone. This is the first indication that gonadotropin may directly regulate ion transport functions of the blood-testis barrier of vertebrates and in this way regulate seminal plasma ionic composition.

Na(+)-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus.

Abstract It is concluded that Ca2+ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na+/Ca2+-exchange mechanism that is driven by the (transmembrane) Na+ gradient established by Na+/K+-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na+/Ca2+-exchanger next to the Ca2+-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca2+ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na+ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca2+ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca2+ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na+/K+-ATPase activity was two times higher in SW adapted fish.

Control of ion transport by the sperm duct epithelium of brook trout (Salvelinus fontinalis).

The sperm duct epithelium of brook trout (Salvelinus fontinalis), mountedin vitro in Ussing-style epithelial chambers actively absorbs Na+ (measured as the short-circuit current, Isc) and secretes K+ (measured using86Rb+ as tracer). Dibutyryl-cyclic-adenosine monophosphate (db-cAMP) and 3-isobutyl-1-methylxanthine (IMX) produce a rapid, sustained stimulation of both ion transport processes, but the hormone connected to the response is unknown. Purified sockeye salmon CON A2 gonadotropin (GtH) produces a dose-dependent, rapid and sustained rise in Na+ uptake and K+ secretion. The time course, electrophysiological and transport characteristics are similar to those evoked by IMX. Carbohydrate-poor (chum salmon CON A1) GtH is ineffective. Pretreatment of fish with 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P) significantly increases milt volume but is without effect on resting or stimulated (IMX + db-cAMP) levels of sperm duct ion transport. This is the first indication of a direct, rapid action of GtH on ion transport by the vertebrate blood-testis barrier. The results suggest direct involvement of GtH in control of later stages of sperm maturation.

Volume regulation in glutathione-treated brook trout (Salvelinus fontinalis) erythrocytes.

Brook trout erythrocytes that were washed with and suspended in Ringers solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringers at pH 6.8), hypoosmotic solutions (or both) and intracellular K+ and Cl− concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K+ but had significantly elevated intracellular Cl− Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K+ and Cl− In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K+ release were dependent on Cl− implying a direct or indirect coupling of K+ to Cl− transport in volume regulation. RVD was partially blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na+/H+ exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na+/H+ and Cl−/HCO3− exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K+ channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C−-dependent K+ release and the resultant regulatory volume decrease in osmotically-swollen cells.