Abstract
Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin. However, specific targets for calmodulin action have not been thoroughly addressed. In the present study, we investigated whether Ca2+/calmodulin regulates adenylyl cyclase activity in the renal inner medullary collecting duct. Rat inner medullary collecting duct suspensions were incubated in the presence or absence of 0.1 nM vasopressin and the calmodulin inhibitors, monodansylcadaverine, W-7, and trifluoperazine, followed by measurement of cAMP. Vasopressin-stimulated cAMP elevation was significantly attenuated in the presence of calmodulin inhibitors. Analysis of transglutaminase 2 knock-out mice confirmed that these compounds were not acting through inhibition of transglutaminase 2 activity. Calmodulin inhibitors also blocked both cholera toxin- and forskolin-stimulated cAMP accumulation. In isolated perfused tubules, W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane. These results suggest that calmodulin is required for vasopressin-stimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Reverse transcription-PCR, immunoblotting, and immunohistochemistry revealed the presence of the calmodulin-sensitive adenylyl cyclase type 3 in the rat collecting duct, an isoform previously not known to be expressed in the collecting duct. Long-term treatment of Brattleboro rats with a vasopressin analog markedly decreased adenylyl cyclase type 3 protein abundance, providing an explanation for long-term down-regulation of vasopressin response in the collecting duct. These studies demonstrate the importance of calmodulin in the regulation of collecting duct adenylyl cyclase activity and transport function.
Highlights
Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin
Following a 10-min preincubation period with these compounds in the presence of the phosphodiesterase inhibitor isobutyl methylxanthine (0.5 mM), 0.1 nM arginine vasopressin (AVP) or vehicle was added to the tubules for 5 min. cAMP content was subsequently measured by a non-radioactive enzyme immunoassay as described under “Experimental Procedures.” cAMP levels were significantly increased 4 –5-fold in inner medullary collecting duct (IMCD) suspensions incubated with AVP alone (Fig. 1)
CaM Inhibitors Act at the Level of Adenylyl Cyclase— our results indicated that CaM was required for elevation of cAMP by vasopressin, it was unclear at what level CaM was affecting the signaling pathway (e.g. V2 receptor, Gs, or adenylyl cyclase)
Summary
Vol 280, No 14, Issue of April 8, pp. 13624 –13630, 2005 Printed in U.S.A. Calmodulin Is Required for Vasopressin-stimulated Increase in Cyclic AMP Production in Inner Medullary Collecting Duct*. W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane These results suggest that calmodulin is required for vasopressinstimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Further analysis suggested that CaM is acting at the level of adenylyl cyclase This is the first demonstration of CaM-dependent cAMP accumulation in response to AVP in intact IMCD tubules, which supports prior conclusions from studies in cultured LLC-PK1 cells [8] and mouse outer medulla [9]. We present evidence showing that CaM is required for AVP-mediated urea permeability in isolated perfused IMCD, another process that is cAMP-dependent [10], suggesting that CaM may play a broader regulatory role in the collecting duct than initially thought. AC3 may act as the target cyclase for Ca2ϩ/CaM-dependent cAMP accumulation in response to vasopressin
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