Intramembranous particle aggregates in the luminal membrane of toad bladder granular cells after vasopressin stimulation have been found to correlate closely and specifically with induced alterations of water permeability. Roles for microtubules and microfilaments in mediating the latter response have been proposed on the basis of studies involving colchicine and cytochalasin B, respectively. In the present investigation the effects of these agents on both initiating and sustaining vasopressin-induced osmotic water flow and the particle aggregation phenomenon were studied. The results indicate that during initiation the aggregation and water flow responses to vasopressin are each colchicine- and cytochalasin B-sensitive and that these sensitivities can be wholly additive. However, after full vasopressin stimulation is established, the same responses demonstrate sensitivity only to cytochalasin B, not to colchicine. The findings, therefore, suggest that microtubules and microfilaments may be independently necessary for the initiation of the aggregation and water flow responses to vasopressin, and that microfilaments, but not microtubules, are required for their maintenance.

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Toad urinary bladders were subjected to sequential 30-min stimulation with antidiuretic hormone (ADH) followed by 30-min hormone washout over 4 h in the absence of a transmural osmotic gradient. Immediately thereafter, during test stimulation with hormone in the presence of a transmural gradient, transbladder water flow was profoundly inhibited, but intra(luminal)membrane particle aggregates, presumed markers of luminal membrane water permeability, were as numerous as in fully responsive controls. The protocol followed was designed to eliminate any distorting effect of prior water flow on cytoplasmic organization and to equalize, for both experimental and control tissues, the time of aggregate presence in the luminal membrane during final test stimulation. On the assumption that cytoplasmically stored and/or newly synthesized aggregates would be unaffected by the protocol followed, these observations appear to be consistent with the view that bladder refractoriness to prolonged ADH treatment may involve regulation of tissue water permeability at a resistance distal to the luminal membrane.