Water Permeability Of Toad Bladder Research Articles

Effects of potassium-free media on ADH action in toad urinary bladder.

We studied the effects of potassium-free media on processes related to the hydro-osmotic response of toad bladder to ADH (20 mU/ml). Exposure of bladders to potassium-free media did not affect base-line osmotic water flow, but it promptly attenuated the level of osmotic water permeability induced by ADH. Both the frequency of hormonally induced intra(luminal)membrane particle aggregates (presumed sites for transmembrane water flow) and the number of luminal membrane fusion events (associated with aggregate delivery from the cytoplasm) were also reduced. Potassium-free media had no measurable effect either on cytoplasmic microtubule integrity or on mean aggregate size. Potassium repletion reversed the inhibitory effect of potassium-free media on ADH-related osmotic water permeability. For bladders fully stimulated with ADH in the presence of potassium, subsequent bathing media depletion of potassium led to an inhibition of ADH-related water flow and to reductions in membrane fusion sites and aggregates. We confirmed that the inhibitory effect of potassium-free media on ADH-induced osmotic water permeability results from serosal bathing medium potassium depletion alone and occurs at a post-cyclic AMP site. In addition, we found that ADH-stimulated water permeability was attenuated in bathing media containing a low potassium concentration (0.5 mM). The data are consistent with the view that potassium-free media or media containing low levels of potassium inhibit ADH-enhanced osmotic water permeability in toad bladder by interfering with the process of or leading to membrane fusion required for the delivery of water-conducting structures to the luminal membrane. In addition, some of our results imply that aggregates may turn over during sustained ADH stimulation.

Pressure effects on the ADH-induced initiation of water flow in toad bladder.

Earlier studies employed colchicine to demonstrate the need for microtubules in the ADH-induced initiation of increased water permeability in toad bladder. We have used colchicine and hydrostatic pressure together to determine whether formed or growing microtubules are required for initiation of the ADH response in Bufo marinus. When ADH and 8,000 psi were administered simultaneously, the ADH-induced increase in water flow was inhibited while under pressure by 107 +/- 7% (n = 6). Application of 8,000 psi for 10 min before ADH administration resulted in an increased initiation of the ADH osmotic response over the non-pressure-treated control (average acceleration, rate of water flow increase during first 3 min after ADH stimulation, 1.25 +/- 0.27 vs. 0.43 +/- 0.12 mg X cm-2 X min-2, n = 6). In addition, the inhibition of the ADH response brought about by colchicine incubation was overcome with pretreatment of the colchicine-incubated bladders with 8,000 psi for 10 min (average 3-min acceleration, 0.18 +/- 0.04 vs. 1.13 +/- 0.06 mg X cm-2 X min-2, respectively). Repeating the experiments with dibutyryl cAMP gave similar results. We interpret these data as suggesting that growing microtubules are required for initiation. The proposed model is as follows. Pressure removes colchicine inhibition by introducing, through disassembly of formed microtubules, more colchicine-free tubulin subunits. These subunits are then available following decompression to reassemble when the tissue is challenged with hormone.

Nocodazole inhibition of the vasopressin-induced water permeability increase in toad urinary bladder

Nocodazole is a synthetic antitumor drug that binds rapidly to tubulin. When this drug is applied to toad bladder prior to vasopressin stimulation it inhibits the vasopressin response. A maximum inhibition (68%) is reached with a dose level of 10 μ/ml applied one-half hour prior to vasopressin stimulation (20 mU/ml). This compares with an inhibition of 50% seen with a 3-h exposure of the tissue to colchicine (0.1 mM) prior to stimulation with vasopressin. Application of nocodazole (1 μ/ml) 3 min after hormonal stimulation shows no inhibition of the response at one-half hour past stimulation. These data support the view that microtubules are involved in the vasopressin-induced increase in water permeability in toad bladder and also indicate that this involvement is limited to the period prior to or directly after stimulation.

Relation of ADH effects to altered membrane fluidity in toad urinary bladder.

Membrane fluidity, urea permeability, and osmotic water permeability in toad urinary bladder are regularly enhanced by antidiuretic hormone (ADH). In addition, organized intramembranous particle aggregates, which correlate specifically with hormonally stimulated water permeability, are found in granular cell luminal membranes consequent to ADH stimulation. In this investigation ADH-stimulated changes in urea and osmotic water permeability and luminal membrane aggregates at room temperature (24.8 +/- 0.4 degrees C) and in the cold 10.6 +/- 0.2 degrees) were compared with corresponding changes in membrane fluidity, as assessed by n-butyramide permeability. Although a critical level of membrane fluidity is undoubtedly required, the occurrence of aggregates in the luminal membrane is independent of an accompanying hormonally induced change of membrane fluidity. ADH-stimulated osmotic water permeability in toad bladder is also independent of the coincident change in membrane fluidity, and as a process almost certainly involves membrane channels, not a solubility-diffusion process through membrane lipids. For ADH-stimulated transbladder urea movement, channels seem to be involved as well, and the change induced in membrane fluidity by ADH could be an underlying factor in their formation.

Vasopressin and water permeability of artificial lipid membranes

Vasopressin markedly stimulated the water permeability of bilayer lipid membranes: a two-fold increase was measured at 25° in presence of 1.7·10 −9 M (50 μunits/ml) vasopressin. Oxytocin and a mixture of the amino acids comprising the vasopressin molecule could not substitute for vasopressin at comparable concentration. The experimental activation energy of water transport was reduced in the presence of vasopressin from 14 to 4 kcal/mole, in agreement with the effect of the hormone on water permeability of toad bladder.

Proposed mode of action of histamine.

SUTHERLAND and his collaborators have firmly established that the cyclic nucleotide, adenosine 3′,5′-monophosphate (CAMP), is a ubiquitous regulator of a variety of intracellular processes1. Its role in the activation of phosphorylase has been elucidated by Sutherland and Rall2. It is now almost certain that CAMP also participates in the activation of lipase in adipose tissue3, and may affect the activity of such key regulator enzymes as phosphofructokinase4,5. Furthermore, CAMP has been implicated as the intracellular mediator of a variety of hormonal effects ranging from steroidogenesis in the corpus luteum and adrenal cortex to increased water permeability in toad bladder and kidney tubules1.