Renal sulfate secretion is carbonic anhydrase dependent in a marine teleost, Pleuronectes americanus

Though chemical assays indicate that carbonic anhydrase (CA) activity is present in marine teleost nephrons, CA inhibitors have no effect on urine pH or bicarbonate excretion, parameters typically CA dependent in almost all vertebrate groups. Because marine teleost renal sulfate secretion is associated with bicarbonate anion exchange, we investigated the effect of CA inhibition on transepithelial sulfate transport by flounder renal tubule primary monolayer cultures (PTC) and on renal sulfate secretion (Q˙so 4) by intact flounder. Both methazolamide and ethoxzolamide (10 μM) inhibited PTC secretory flux by ∼50%; reabsorptive sulfate flux, Na-dependent glucose transport, and transepithelial electrical resistance were unaffected. A CA inhibitor restricted to the extracellular space (10 μM polyoxyethylene-aminobenzolamide, 3.7 kDa) had no effect on PTC sulfate transport. Intravenous administration of methazolamide reducedQ˙so 4almost 40% and had no effect on glomerular filtration rate (GFR), urine flow rate, or Pi excretion rate. Serum pH was significantly reduced 0.2 units, whereas urine pH was unchanged. Together, the in vitro and in vivo results indicate that CA facilitates renal sulfate secretion in the seawater teleost.

Effect of somatolactin and related hormones on phosphate transport by flounder renal tubule primary cultures

Winter flounder renal proximal tubule primary monolayer cultures mounted in Ussing chambers were used to determine the effect of salmon somatolactin (sSL) on transepithelial Pi and Ca2+ transport. sSL stimulated Pi reabsorption in a dose-dependent manner at physiological levels of the hormone (12.5 ng/ml). Net Pi transport was significantly altered by sSL (200 ng/ml) within 2 h after the initial exposure. Ca2+ fluxes were unchanged by the addition of 200 ng/ml sSL. The sSL-induced Pi reabsorption was abolished by 10 microM H-89, a highly specific protein kinase A inhibitor. Moreover the production and release of adenosine 3',5'-cyclic monophosphate were significantly increased after 1 and 2 h of exposure to sSL. The data indicate that sSL directly stimulates net renal Pi reabsorption by an adenosine 3',5'-cyclic monophosphate-dependent pathway. In addition to sSL, flounder SL and rat prolactin greatly, and salmon growth hormone (2.3 micrograms/ml) slightly, increased net Pi reabsorptive flux, whereas salmon prolactin had no effect.

Apical acidification induces paracellular injury in canine gastric mucosal monolayers

We used primary monolayer cultures of enzyme-dispersed canine oxyntic mucosal cells mounted in Ussing chambers to characterize the apical barrier to H+. [3H]mannitol flux (MF) and [14C]inulin flux (IF) were used as size probes for tight junctions. Apical H+ produced a three-phase effect. In phase 1, as the apical pH was decreased from 7 to about 2.5, resistance (R) increased, but short-circuit current (Isc) did not change. In phase 2, an increased paracellular permeability developed at pH below 2.5-1.7, evidenced by decreased R and increased MF but not IF. Size sieving and monolayer integrity were preserved, and this paracellular leak was either fully reversed or stabilized by apical neutralization, depending on the duration of the paracellular leak. In phase 3, after sustained exposure to an apical pH below approximately 2, transepithelial integrity was lost; R decreased to fluid R, and both MF and IF increased. Basolateral acidification below pH 5.5 produced rapid monolayer disruption. Low concentrations of cytochalasin D (CD) decreased R and increased MF but not IF; apical acidification to pH 4 after CD increased R and decreased the MF, indicating reduced paracellular permeability by apical H+. Apical amiloride did not alter Isc; however, after 48 h of treatment with hydrocortisone and insulin, an amiloride-sensitive Isc component became evident. Our data indicate that the increase in R observed with apical acidification reflects decreased paracellular permeability and that the earliest injury with apical acidification is a selective paracellular leak.

Stanniocalcin stimulates phosphate reabsorption by flounder renal proximal tubule in primary culture

The effects of several hormones on transepithelial Pi transport were determined in primary monolayer cultures of winter flounder proximal tubule epithelium in Ussing chambers. Salmon stanniocalcin (STC) had a dose-dependent stimulatory effect on net Pi reabsorption within the normal plasma hormone concentration range, 12.5-50 ng/ml (0.25-1.0 nM). Net Pi transport was significantly altered by STC (200 ng/ml) within 30 min and progressively increased from slight net secretion (0.26 +/- 0.744 nmol.cm-2.h-1) in untreated controls to net reabsorption (1.96 +/- 0.729 nmol.cm-2.h-1) after 3 h. The STC effect was mimicked by 10 microM forskolin, whereas 10 microM H-89, a highly specific protein kinase A inhibitor, significantly decreased both STC- and forskolin-induced Pi reabsorption. The release of adenosine 3',5'-cyclic monophosphate (cAMP) was increased more than twofold after a 1-h exposure to STC. This hormone had no effect on transepithelial Ca2+ transport. The results indicate that STC directly stimulates net renal Pi reabsorption by a cAMP-dependent pathway. In addition to STC, bovine parathyroid hormone (100 nM) and ovine prolactin (100 nM) significantly increased net Pi reabsorptive flux.