Calmodulin is required for vasopressin-stimulated increase in cyclic AMP production in inner medullary collecting duct

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.

Fig. 1

CaM inhibitors significantly reduce AVP-stimulated cAMP accumulation in isolated IMCD suspensions. A: Preincubation of tubules with MDC (200 μM) completely blocked the elevation of cAMP by vasopressin (AVP) (0.1 nM). MDC also significantly reduced baseline cAMP levels in the absence of AVP. Levels of cAMP are expressed as fmol cAMP per μg of total protein (n ≥ 3; P < 0.05). B: Preincubation of tubules with either TFP or W-7 resulted in a dose-dependent inhibition of AVP-stimulated cAMP (n ≥ 3; *, P < 0.01).

Fig. 2

Effect of CaM inhibitors on CTX and forskolin-stimulated cAMP accumulation. A: IMCD suspensions were preincubated for 10 min in the presence or absence of two different CaM inhibitors, W-7 (25 μM) or TFP (30 μM), and then incubated with CTX (1 μg/ml) for 45 min and cAMP measured (n = 4; * P < 0.01 vs. CTX alone). CaM inhibitors blocked elevation of cAMP produced by CTX. B: IMCD suspensions were preincubated with either DMSO (−) or MDC (+) (200 μM) for 10 min followed by a 5 min incubation in the presence or absence of forskolin (1 μM). MDC blocked forskolin-stimulated cAMP accumulation (n = 3; P < 0.01).

Fig. 3

Structural formulas of the three CaM inhibitors used in this study. There is high structural similarity between MDC and W-7 (Differences are boxed). Both MDC and W-7 have been reported to inhibit transglutaminase.

Fig. 4

Effect of MDC on cAMP accumulation in wildtype (WT) and transglutaminase 2 (TG2) knockout mice. MDC inhibits AVP-stimulated cAMP accumulation in both WT and TG2 (−/−) mice. (*, P < 0.001 vs. no AVP; Δ, P < 0.001 vs. AVP alone).

Fig. 5

Effect of CaM inhibitor W-7 on urea permeability of isolated perfused IMCD segments. After initial equilibration of tubules at 37°C for 30 min, P_urea was measured in three experimental periods: Control, AVP, and AVP+ W-7 (Top panel); W-7, W-7+AVP, and AVP (washout of W-7) (Bottom panel). Urea permeability was determined at 20 min after incubation with the reagent(s) in each period. AVP was used at 10⁻¹⁰ M concentration and W-7 was used at 25 μm concentration. All reagents were added into the peritubular bath solution. Values are mean ± SE, n = 3 in each group; *, significantly different from previous experimental period (P < 0.01).

Fig. 6

RT-PCR analysis of adenylyl cyclase isoforms in rat brain and IMCD. Total RNA was extracted from both brain and enriched IMCD suspensions and used for RT-PCR analysis with specific primers to each adenylyl cyclase (AC) isoform (–9). Reactions without reverse transcriptase (RT −). Brain was chosen as a positive control for all primer sets. The CaM-sensitive isoform (AC3) is present in IMCD cells, while the other CaM-sensitive isoforms, AC1 and 8, are absent.

Fig. 7

AC3 is enriched in rat IMCD fractions. Rat inner medulla was processed according to Methods and differential centrifugation was performed to isolate IMCD and non-IMCD fractions. Immunoblotting followed by quantitation of band density was performed to determine level of enrichment. A. Immunoblot of AQP1 showing 5.4-fold enrichment in non-IMCD vs. IMCD fractions (n = 3; P < 0.001). B. Immunoblot of AC3 showing 2.2-fold enrichment in IMCD vs. non-IMCD fractions.

Fig. 8

Immunoperoxidase labeling of AC3 and AC6 in rat inner medulla. Rat inner medulla was stained using antibodies to AC3 (A), AC3 preadsorbed with blocking peptide (B), AC6 (C), and AQP2 (D). AC3 and AC6 are both present in IMCD cells. AQP2 was used as a collecting duct-specific marker. Representative collecting ducts are indicated with an asterisk (*).

Fig. 9

Both AC3 and AC6 expression are decreased with long-term dDAVP treatment. Brattleboro rats were given dDAVP (20 ng/hr) or saline (control) via osmotic minipumps for 7 days, followed by isolation of inner medulla and immunoblotting (n = 4). Blots were probed with antibody to AC3 (top panel), AC6 (middle panel), or AQP2 (bottom panel).