Vasopressin regulation of inner medullary collecting ducts and compensatory changes in mice lacking adenosine A1 receptors.

Activation of adenosine A(1) receptors (A(1)R) can inhibit arginine vasopressin (AVP)-induced cAMP formation in isolated cortical and medullary collecting ducts. To assess the in vivo consequences of the absence of A(1)R, we performed experiments in mice lacking A(1)R (A(1)R(-/-)). We assessed the effects of the vasopressin V(2) receptor (V(2)R) agonist 1-desamino-8-d-arginine vasopressin (dDAVP) on cAMP formation in isolated inner medullary collecting ducts (IMCD) and on water excretion in conscious water-loaded mice. dDAVP-induced cAMP formation in isolated IMCD was significantly greater ( approximately 2-fold) in A(1)R(-/-) compared with wild-type mice (WT) and, in contrast to WT, was not inhibited by the A(1)R agonist N6-cyclohexyladenosine. A(1)R(-/-) and WT had similar basal urinary excretion of vasopressin, expression of aquaporin-2 protein in renal cortex and medulla, and acute increases in urinary flow rate and electrolyte-free water clearance in response to the V(2)R antagonist SR121463 or acute water loading; the latter increased inner medullary A(1)R expression in WT. Dose dependence of dDAVP-induced antidiuresis after acute water loading was not different between the genotypes. However, A(1)R(-/-) had greater inner medullary expression of cyclooxygenase-1 under basal conditions and of the P2Y(2) and EP(3) receptor in response to water loading compared with WT mice. Thus vasopressin-induced cAMP formation is enhanced in isolated IMCD of mice lacking A(1)R, but the adenosine-A(1)R/V(2)R interaction demonstrated in vitro is likely compensated in vivo by multiple mechanisms, a number of which can be "uncovered" by water loading.

Gene expression signatures of cAMP/protein kinase A (PKA)-promoted, mitochondrial-dependent apoptosis. Comparative analysis of wild-type and cAMP-deathless S49 lymphoma cells.

The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA-promoted apoptosis by comparing wild-type (WT) S49 lymphoma cells and the S49 variant, D(-) (cAMP-deathless), which lacks cAMP-promoted apoptosis but has wild-type levels of PKA activity and cAMP-promoted G(1) growth arrest. Treatment of WT, but not D(-), S49 cells with 8-CPT-cAMP (8-(4-chlorophenylthio)-adenosine-3':5'-cyclic monophosphate) for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and SMAC, and increase in caspase-3 activity. Gene expression analysis (using Affymetrix 430 2.0 arrays) revealed that WT and D(-) cells incubated with 8-CPT-cAMP have similar, but non-identical, extents of cAMP-regulated gene expression at 2 h (approximately 800 transcripts) and 6 h (approximately 1000 transcripts) (|Fold| > 2, p < 0.06); by contrast, at 24 h, approximately 2500 and approximately 1100 transcripts were changed in WT and D(-) cells, respectively. Using an approach that combined regression analysis, clustering, and functional annotation to identify transcripts that showed differential expression between WT and D(-) cells, we found differences in cAMP-mediated regulation of mRNAs involved in transcriptional repression, apoptosis, the cell cycle, RNA splicing, Golgi, and lysosomes. The two cell lines differed in cAMP-response element-binding protein (CREB) phosphorylation and expression of the transcriptional inhibitor ICER (inducible cAMP early repressor) and in cAMP-regulated expression of genes in the inhibitor of apoptosis (IAP) and Bcl families. The findings indicate that cAMP/PKA-promoted apoptosis of lymphoid cells occurs via mitochondrial-mediated events and imply that such apoptosis involves gene networks in multiple biochemical pathways.