Calmodulin-sensitive adenylyl cyclases mediate AVP-dependent cAMP production and Cl⁻secretion by human autosomal dominant polycystic kidney cells

Abstract

In autosomal dominant polycystic kidney disease (ADPKD), binding of AVP to the V2 receptor (V2R) increases cAMP and accelerates cyst growth by stimulating cell proliferation and Cl(-)-dependent fluid secretion. Basal cAMP is elevated in human ADPKD cells compared with normal human kidney (NHK) cells. V2R mRNA levels are elevated in ADPKD cells; however, AVP caused a greater increase in global cAMP in NHK cells, suggesting an intrinsic difference in cAMP regulation. Expression, regulatory properties, and receptor coupling of specific adenylyl cyclases (ACs) provide temporal and spatial regulation of the cAMP signal. ADPKD and NHK cells express mRNAs for all nine ACs. Ca(2+)-inhibited ACs 5 and 6 are increased in ADPKD cells, while Ca(2+)/CaM-stimulated ACs 1 and 3 are downregulated. ACs 1, 3, 5, and 6 were detected in cyst cells in situ, and codistribution with aquaporin-2 suggests that these cysts were derived from collecting ducts. To determine the contribution of CaM-sensitive ACs to AVP signaling, cells were treated with W-7, a CaM inhibitor. W-7 decreased AVP-induced cAMP production and Cl(-) secretion by ADPKD cells. CaMKII inhibition increased AVP-induced cAMP, suggesting that cAMP synthesis is mediated by AC3. In contrast, CaM and CaMKII inhibition in NHK cells did not affect AVP-induced cAMP production. Restriction of intracellular Ca(2+) switched the response in NHK cells, such that CaM inhibition decreased AVP-induced cAMP production. We suggest that a compensatory response to decreased Ca(2+) in ADPKD cells switches V2R coupling from Ca(2+)-inhibited ACs 5/6 to Ca(2+)/CaM-stimulated AC3, to mitigate high cAMP levels in response to continuous AVP stimulation.

Adenylyl Cyclase 6 Deficiency Ameliorates Polycystic Kidney Disease

“…A recent study found that, although cAMP was elevated in PKD cells obtained from human kidneys (compared with normal human kidney [NHK] cells), AVP causes a greater increase in NHK cell cAMP, 16 suggesting that the total amount of cellular cAMP is less important than its localized effects within the cell. Inhibition of calmodulin, which reduces AC6 and AC3 activity, decreased AVP-induced cAMP in PKD but not NHK cells.…”

Phosphodiesterase Isoform Regulation of Cell Proliferation and Fluid Secretion in Autosomal Dominant Polycystic Kidney Disease

“…Although this observation was in agreement with rodent models of hereditary PKD, in which renal cAMP levels were shown to be higher than normal, [20][21][22]36 the cell culture studies suggest that there is also an intrinsic difference in cAMP regulation, leading to higher cAMP levels in ADPKD cells. This, together with our earlier work showing that global PDE inhibition increased cAMP to similar levels in ADPKD and NHK cells, 12 led us to explore specific PDE isoform expression levels and activities in NHK and ADPKD kidneys to determine the underlying basis for this effect. There are .50 isoforms in the PDE superfamily that regulate cAMP and cGMP levels.…”

“…ADPKD cells have higher basal cAMP levels compared with NHK cells (Table 1). 12 Vinpocetine and cilostamide significantly increased cAMP in NHK, but not ADPKD cells; by contrast, rolipram increased cAMP in both cells. The effect of 8-MeOM-IBMX, a potent PDE1 inhibitor, was similar to that of vinpocetine (Supplemental Figure 2).…”

“…5 AVP increased cAMP to a higher level in NHK cells than in ADPKD cells (Table 1), as shown previously. 12 Inhibition of PDE4 potentiated the AVP effect on cAMP in ADPKD cells, whereas inhibition of PDE1 and PDE3 had little effect. In NHK cells, vinpocetine, cilostamide, and rolipram increased AVP-induced cAMP levels compared with AVP alone.…”

“…[9][10][11] Elevated cAMP in ADPKD cells compared with normal human kidney (NHK) cells is consistent with a decreased rate of cAMP degradation by PDEs, as indicated by treatment with isobutyl methylxanthine (IBMX), a global PDE inhibitor, which eliminated this difference in cAMP levels. 12 ADPKD is caused by mutations in PKD1 or PKD2, genes that encode polycystin 1 (PC1) and 2 (PC2), respectively. PC1 and PC2 interact to form a multifunctional signaling complex that regulates intracellular Ca 2+ to maintain the differentiated phenotype of tubule epithelial cells.…”