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ABSTRACT: Anuran amphibians do not drink orally but absorb water osmotically through the highly permeable ventral skin. In this cutaneous water absorption, roles of the putative cerebral osmoreceptors and functions of arginine vasotocin (AVT) were examined in the central nervous system of the Japanese treefrog, Hyla japonica. Intracerebroventricular (ICV) or intralymphatic sac (ILS) administration of various hypertonic solutions (NaCl, mannitol and urea) significantly extended the residence time in water in a dose-dependent manner, suggesting facilitation of water absorption in frogs. ICV injection of AVT also increased significantly the residence time in a dose-dependent manner. The water absorption effect of AVT was significantly inhibited by pretreatment of ICV OPC-21268, a vasopressin V(1) receptor antagonist. But pre-ICV injection of OPC-31260, a vasopressin V(2) receptor antagonist, did not block the water absorption effect of AVT. Extension of the residence time induced by hyperosmotic NaCl (1000 mOsm) ICV injection was significantly inhibited by pretreatment of ICV OPC-21268. The present results showed that increases of osmotic pressure in plasma and/or cerebrospinal fluid stimulate water absorption response, suggesting that osmoreceptors are certainly present in the central nervous system and AVT may directly stimulate water absorption in the treefrog. It is also suggested that AVT activates cellular mechanisms via V(1)-like but not V(2)-like receptors in the central nervous system and facilitates water absorption response in the treefrog.
General and Comparative Endocrinology 07/2008; 157(2):196-202. · 3.27 Impact Factor
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ABSTRACT: Physiological function of arginine vasotocin (AVT) and effect of receptor antagonists of vasopressin were electrophysiologically investigated on transepithelial transport of ions in the abdominal skin of Hyla japonica and Rana nigromaculata by means of the Ussing chamber system. Administrations of AVT and forskolin (adenylate cyclase activator) in the serosal side of normal Ringer's solution significantly increased transepithelial potential difference (PD) and short-circuit current (Isc) accounting for Na(+) influx, mucosal to serosal direction, across the skin of H. japonica. In contrast, AVT administrations significantly decreased PD but not Isc on the skin of R. nigromaculata in a concentration-dependent manner ranging from 10(-11) to 10(-8)M. Administration of 10(-5)M forskolin also significantly decreased PD in normal and low Na(+) Ringer's solution and in the presence of amiloride (Na(+) channel blocker) on the mucosal side of normal Ringer's solution. On the other hand, forskolin significantly increased PD and Isc in the Cl(-) free Ringer's solution. These results suggested that AVT and forskolin stimulated mainly Cl(-) influx across the skin of R. nigromaculata. In two frog species, the AVT actions on ion transports were inhibited by pretreatment of OPC-31260 (a vasopressin V(2) receptor antagonist) but not OPC-21268 (a vasopressin V(1) receptor antagonist). These results suggested that AVT activates adenylate cyclase via V(2)-like receptor and stimulates actively net Na(+) and net Cl(-) transports in the abdominal skin of H. japonica and R. nigromaculata, respectively.
General and Comparative Endocrinology 06/2008; 157(1):63-9. · 3.27 Impact Factor
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ABSTRACT: In our previous study, it was suggested that ANP and cGMP may increase Na(+) absorption in the urinary bladder of the Japanese tree frog, Hyla japonica. Thus, Na(+) transport activated by ANP was investigated electrophysiologically by using a cell-attached patch-clamp technique in freshly isolated cells from the urinary bladder. A predominant channel expressed was a low conductance Na(+) channel in the epithelial cells. The channel exhibited conductance for inward currents of 4.9 +/- 0.2 pS, long open and closed times (c.a. 190 ms), and positive reversal potential. The channel activity was decreased under the pipette solution including 10(-6) M amiloride. These characteristics were similar to those of amiloride-sensitive Na(+) channels (ENaC). Addition of 10(-9) M ANP activated and significantly increased the ENaC activity from 0.58 +/- 0.09 to 1.47 +/- 0.34. On the other hand, mean amplitudes and conductance of single channel did not change significantly after the addition of ANP. Addition of 10(-5) M 8-Br-cGMP also activated the ENaC and significantly increased the channel activity from 0.56 +/- 0.10 to 2.00 +/- 0.33. The addition of ANP failed to activate the ENaC in the presence of 10(-6) M amiloride. These results suggested that ANP and cGMP activate Na(+) transport via ENaC in the epithelial cells of frog urinary bladder.
Journal of Comparative Physiology B 08/2007; 177(5):503-8. · 1.97 Impact Factor
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ABSTRACT: The amiloride-sensitive epithelial sodium channel (ENaC) has previously been shown to be involved in the maintenance of body fluid volume and in Na(+) absorption across the skin and urinary bladder in amphibians. However, the function and distribution of ENaC have not been clearly described in amphibian kidney. We therefore cloned the ENaC alpha-subunit cDNA from kidney of the marine toad, Bufo marinus. The ENaC mRNA and protein were abundantly expressed in the kidney and in the urinary bladder and ventral pelvic skin. In an immunohistochemical study, the ENaC alpha-subunit protein was specifically localized to the apical membrane of the principal cells but not the intercalated cells from the late distal tubule to the collecting duct in the kidney or in the apical area of cells of urinary bladder epithelia. When toads were acclimated to dry and hyper-saline environments, the levels of ENaC mRNA expression in the kidney and urinary bladder decreased under hyper-saline acclimation, but not under dry conditions. Immunohistochemical observations indicated that the levels of ENaC protein expression were much lower in the apical area of renal distal tubules and urinary bladder epithelia of hyper-saline acclimated toad compared with controls. The present study suggests that Bufo ENaC is significantly expressed and functions during Na(+) reabsorption in the apical membrane domain in the distal nephron of normal and desiccated toads. Natriuresis may be caused by decreases in ENaC expression and its trafficking to the cell surface in the distal nephron, a response to prevent excessive Na(+) reabsorption in hyper-saline-acclimated toads.
Cell and Tissue Research 07/2007; 328(3):583-94. · 3.11 Impact Factor
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ABSTRACT: The effects of atrial natriuretic peptide (ANP) and cGMP on transepithelial ion transport were examined in the urinary bladder of the Japanese tree frog, Hyla japonica, using Ussing chamber voltage-clamp and whole-cell patch-clamp techniques. When the bladders were exposed to 4.4 x 10(-11) to 10(-6) M ANP or 10(-7) to 3 x 10(-4) M 8-Br-cGMP, both the transepithelial potential difference (PD) and the short-circuit current (Isc) were significantly increased in a concentration-response manner. The cGMP-dependent responses were inhibited in a Na+-free bath solution and in the presence of amiloride. The cGMP-dependent increases in Isc were significantly inhibited by specific PKA inhibitors (5 x 10(-7) M KT-5720 and >10(-5) M H-89), but not by a specific PKG inhibitor (5 x 10(-7) M KT-5823). ANP-dependent increases in Isc were also significantly inhibited by KT-5720. In the patch-clamp study, ANP and cGMP significantly increased in inward currents involving Na+ uptake. These results suggest that a cross-talk mechanism exists between cAMP and cGMP signaling pathways, which leads to Na+ transport in the frog urinary bladder. In addition, the cGMP-dependent increases in Isc were partially inhibited by 10(-4) M l-cis-diltiazem, a specific inhibitor of cyclic nucleotide-gated (CNG) channels. These results also suggest a relation between CNG channels and the cGMP-dependent increases in Na+ absorption of the frog urinary bladder.
Journal of Comparative Physiology B 03/2006; 176(3):203-12. · 1.97 Impact Factor
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ABSTRACT: We examined the mechanism of amiloride-sensitive Na(+) channels (ENaC) activated by fANP in epithelial cells of frog urinary bladder by using a cell-attached patch-clamp technique. ENaC activities in the epithelial cells were significantly increased following administration of both 10(-9)M fANP and 10(-5)M 8-Br-cGMP. Both fANP and 8-Br-cGMP, however, failed to activate the ENaC in the presence of 10(-6)M amiloride. In addition, 8-Br-cGMP failed to activate the ENaC in the presence of a PKA inhibitor, KT-5720. In the next experiment, we measured both cGMP and cAMP production levels after treatment of fANP on the frog urinary bladder cells. Frog ANP significantly increased cGMP production, but not the cAMP production. Taken together, these results suggest that fANP activates ENaC through increases in cGMP production and activation of PKA.
General and Comparative Endocrinology 152(2-3):286-8. · 3.27 Impact Factor
