Temperature Dependence of Vasopressin Action on the Toad Bladder

Abstract

Toad bladders were challenged with vasopressin at one temperature, fixed on the mucosa with 1% glutaraldehyde, and then subjected to an osmotic gradient at another temperature. Thus, the temperature dependence of vasopressin action on membrane permeability was distinguished from the temperature dependence of osmotic water flux. As the temperature was raised from 200 to 38 0 C, there was a substantial increase in the velocity of vasopressin action, but osmotic flux was hardly affected. In this range of temperature the apparent energy of activation for net water movement across the bladder amounted to only 1.2 kcal/mole, a value well below the activation energy for bulk water viscosity. It is suggested that osmotic water flux takes place through narrow, nonpolar channels in the membrane. When the temperature was raised from 40 to 20 0 C, both vasopressin action as well as osmotic water flux were markedly enhanced. Activation energies for net water movement were now 8.5 kcal/mole (4°-9C) and 4.1 kcal/mole (9 0 -20 0 C), indicating that the components of the aqueous channel undergo conformational changes as the temperature is lowered from 20 0 C. At 43°C bladder reactivity to vasopressin was lost, and irreversible changes in selective permeability were observed. The apparent energy of activation for net water movement across the denatured membrane was 6.6 kcal/mole. Approximately 1 puosmol of NaCl was exchanged for 1 1 of H 2 0 across the denatured membrane.

Metabolic dependence of the offset of antidiuretic hormone-induced osmotic flow of water across the toad urinary bladder

“…Thus, there is strong evidence to support the inference that cold temperature decreases the fluidity of the toad bladder. Despite this inference and despite the fact that lowering the temperature decreases the rate of osmotic flow of water in toad bladders with "fixed" water permeability (Eggena, 1972), in our studies the flow of water decreased more slowly at 2~ than at room temperature (Table 1, expt. A).…”

“…Earlier studies with inhibitors of energy production had also suggested that metabolic energy was necessary for the onset of action of ADH on water flow (Handler, Petersen & Orloff, 1966). In addition, the direct relationship between temperature and water flow has been studied in tissues where water permeability was "fixed" with glutaraldehyde (Eggena, 1972). In contrast, events involved in the offset of action of ADH have not been evaluated.…”

Inhibition of Vasopressin-Stimulated Urea Transport Across the Toad Bladder by Thiourea

“…Toads were doubly pithed and their urinary bladders were then resected and tied to the ends of glass cannulas with the mucosa on the inside and the serosa on the outside of the bladder sac (8,9 To measure the net mucosal-to-serosal movement of urea across the bladder wall (Tables I-V), bladders were filled with 7-ml urea solutions and suspended each into separate 10-ml serosal fluid baths that were changed at 15- Glutaraldehyde has been used previously to lock the water permeability barrier of the toad bladder in various stages of response to hormone (10)(11)(12). The same procedure was used here to preserve the effect of vasopressin on the permeability of the bladder to urea.…”

“…We had observed previously (10)(11)(12) that the water permeability barrier of the toad bladder could be virtually "locked" in various stages of response to vasopressin by exposing the mucosal surface of the bladder wall for 5 min to a 1 % solution of glutaraldehyde. These fixed bladders were found to retain about 78% of their prefixation permeability to water when removed to hormone-free solutions.…”

Hydrosmotic salt effect in toad skin: Urea permeability and glutaraldehyde fixation of water channels

“…The mild glutaraldehyde (E. Merck, Darmstadt) fixation [12][13][14] of the apical membrane structures was performed with 2% glutaraldehyde in water or KC1 solution without buffer, according to the experimental protocol, for a period of 2 rain followed by thoroughly rinsing the outer membrane surface with distilled H20 or KC1 solution to remove the fixative.…”