Adenylyl Cyclase VI Mediates Vasopressin-Stimulated ENaC Activity

Abstract

Vasopressin modulates sodium reabsorption in the collecting duct through adenylyl cyclase-stimulated cyclic AMP, which exists as multiple isoforms; the specific isoform involved in vasopressin-stimulated sodium transport is unknown. To assess this, we studied mice deficient in adenylyl cyclase type VI specifically in the principal cells of the collecting duct. Knockout mice had increased urine volume and reduced urine sodium concentration, but regardless of the level of sodium intake, they did not exhibit significant alterations in urinary sodium excretion, arterial pressure, or pulse rate. Plasma renin concentration was elevated in knockout mice, however, suggesting a compensatory response. Valsartan significantly reduced arterial pressure in knockout mice but not in controls. Knockout mice had decreased renal cortical mRNA content of all three epithelial sodium channel (ENaC) isoforms, and total cell sodium channel isoforms a and g were reduced in these animals. Patch-clamp analysis of split-open cortical collecting ducts revealed no difference in baseline activity of sodium channels, but knockout mice had abolished vasopressinstimulated ENaC open probability and apical membrane channel number. In summary, these data suggest that adenylyl cyclase VI mediates vasopressin-stimulated ENaC activity in the kidney. Arginine vasopressin (AVP) modulation of water flux across the renal collecting duct (CD) is well known; however, it is becoming increasingly apparent that AVP stimulation of epithelial Na channel (ENaC)-mediated Na transport is of substantial importance in Na and water homeostasis. Recent studies indicate that AVP-induced Na reabsorption is important in the hormone's stimulation of water reabsorption (reviewed in Stockand 1 ). Exogenous AVP enhances renal expression of all ENaC subunits in Brattleboro rats, 2 increases ENaC open probability (Po) in MDCK cells, 3 and stimulates Na flux in isolated perfused CD. 4 A 2-to 3-minute exposure to AVP was shown to increase ENaC Po in the split-open isolated mouse cortical CD (CCD); in addition, 30-minute exposure to AVP increased the ENaC apical membrane channel number (N). 5 Recent studies by our group showed that adrenalectomy surprisingly did not reduce renal ENaC abundance, whereas adrenalectomy and AVP V2 receptor antagonism markedly decreased renal ENaC levels. 6 The stimulatory effects of AVP on ENaC are mediated by cAMP. In the split-open mouse CCD, the AVP effect was dependent upon adenylyl cyclase (AC) activity, whereas AVPenhanced apical membrane Na conductance in perfused rat CCD was mediated by cAMP. 4 cAMP increases ENaC Po and N in pulmonary epithelial cells, transfected oocytes, CCD cell lines, cultured CDs, and MDCK cells. [7][8][9][10][11][12] The effect of cAMP on ENaC trafficking is due, at least in part, to inhibition of Nedd4-2-induced ENaC ubiquitination via phosphorylation of Nedd4-2 on Ser 327 , Ser 221 , and Thr 246 ; notably, these are the same residues that are phosphorylated by serum and glucocorticoid-inducible

Adenylyl Cyclase 6 Deficiency Ameliorates Polycystic Kidney Disease

“…Another point is that a given agonist may activate more than one adenylyl cyclase isoform, resulting in differing biologic effects. For example, CD AC6 deficiency prevents AVP-stimulated epithelial Na channel activity in the CD 15 ; however, CD water reabsorption is only mildly impaired by CD AC6 absence. 12 Although these studies were conducted under different conditions (epithelial Na channel activity was studied in vitro, and water reabsorption was studied in vivo), they raise the possibility that AVP might exert different effects on the CD depending on which adenylyl cyclase isoforms are activated.…”

Renal Phosphate Wasting in the Absence of Adenylyl Cyclase 6

“…27,28 In a collecting duct-specific AC6 knockdown model, vasopressin-stimulated epithelial sodium channel open probability was abolished. 29 Based on studies indicating that vasopressin V 2 receptors use the AC6-cAMP signaling pathway [26][27][28][29][30] and the relatively high abundance of AC6 in the proximal tubule, 23,30,31 we proposed that AC6 may contribute to PTH-stimulated cAMP formation, P i excretion, and thus, P i homeostasis. In the present study, we examined P i homeostasis, PTH-induced renal excretion of P i and cAMP, and trafficking and expression of Npt2a and Npt2c in mice lacking AC6 (AC6 2/2 ).…”

Characteristics and Pharmacological Regulation of Epithelial Na+ Channel (ENaC) and Epithelial Na+ Transport

“…However, it is still unclear how knock-out of AC-VI decreases mRNA expression of all a, b, and gENaC subunits and protein expression of a and gENaC. Interesting points of the observation reported in the study (49) are that: 1) the knock-out of AC-VI down-regulates mRNA expression of a, b, and gENaC; 2) although the expression level of bENaC mRNA is decreased by the knock-out, the knockout of AC-VI has no effect on the expression level of bENaC protein; and 3) the knock-out has no effects on number (N) or open probability (P o ) of ENaCs located at the apical membrane without application of vasopressin. The molecular mechanism of AC-VI participating in the above-mentioned phenomena is unfortunately still unknown.…”

“…In mice deficient in adenylyl cyclase type VI (AC-VI), mRNA contents of all three subunits of ENaC expressed in renal cortical collecting duct cells decrease, and expression of a and gENaC proteins is also lower compared with that in wild-type mice (49). Further, this study (49) indicates that NP o of ENaC detected by a single channel current recording technique is not altered under the basal condition without application of vasopressin in the AC-VIknock-out mice, but no stimulatory action of vasopressin on NP o of ENaC is observed these mice. However, it is still unclear how knock-out of AC-VI decreases mRNA expression of all a, b, and gENaC subunits and protein expression of a and gENaC.…”