Activation of the vasopressin‐sensitive water permeability pathway in the toad bladder by N‐ethyl maleimide

Abstract

SUMMARYVasopressin stimulates transepithelial water flow in the toad urinary bladder. We report here that N-ethyl maleimide (NEM) (0-1 mM) produces a similar increase in osmotic water flow when applied to the mucosal surface of the tissue. NEM-induced water flow is sensitive to inhibitors of hormone-induced water flow, including serosal acidification, or exposure to quinidine or cytoskeleton-disruptive drugs. NEM-induced water flow is additive with that induced by a submaximal, but not a maximal, dose of vasopressin. The response to mucosal NEM is not reversed on removal of the reagent, but established NEM-induced water flow can be inhibited by serosal acidification or quinidine. Like vasopressin, mucosal NEM induces the appearance of fusion profiles and intramembranous particle aggregates (putative water channels) in the apical plasma membrane of the granular cells, and the incidence of particle aggregates correlates with water flow. NEM does not cause an increase in intracellular cAMP.Our data suggest that NEM stimulates transepithelial water flow by irreversibly activating cellular mechanisms normally triggered by vasopressin, hence causing the insertion of water channels.

N-ethylmaleimide causes aquaporin-2 trafficking in the renal inner medullary collecting duct by direct activation of protein kinase A

“…The response was increased when suspensions were incubated with 0.1 mM NEM (210 Ϯ 23% of control; n ϭ 6, P Ͻ 0.001, wrt control). The response to 0.1 mM NEM most closely resembled the response to AVP, in agreement with the findings of both Rasmussen et al (29) and Marples et al (23). The NEM-induced increase in the PM-to-ICV ratio was lower than that produced by 0.1 mM NEM when suspensions were treated with either 1 mM (1 mM NEM: 178 Ϯ 11% of control; n ϭ 5, P Ͻ 0.001 wrt control) or 5 mM NEM (5 mM NEM: 143 Ϯ 25% of control; n ϭ 6, P ϭ 0.05 wrt control).…”

“…This work is in agreement with previous studies in the toad urinary bladder, where NEM was shown to increase transepithelial water flow and induce the appearance of intramembranous particles thought to correspond to the water channels in this tissue (1,23,29). The response to NEM has similar characteristics to AVP-induced trafficking of AQP2, consistent with the view that NEM induces trafficking of AQP2 via activation of a process normally stimulated by AVP.…”

“…Marples et al (23) reported that the effects of 0.1 mM NEM are additive with a submaximal dose of AVP but not with a maximal one. An earlier study (29) reported that higher doses of NEM (e.g., 1 mM) inhibited the increase in transepithelial water flow caused by AVP in the toad urinary bladder.…”

“…However, when it became apparent that NEM was able to activate specific membrane-related processes in other systems (3,19,19). Marples et al (23) performed a detailed characterization of the water flow response to NEM in toad urinary bladder. NEM produced a concentration-dependent, irreversible increase in transepithelial water flow in urinary bladders isolated from Bufo marinus toads.…”

“…NEM had no effect on intracellular cAMP levels, yet the NEM-induced increase in transepithelial water flow was sensitive to PKA inhibition and microtubule disruption. This led to the speculation that NEM might activate PKA directly (23).…”