Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: Implications for modulation of the membrane-associated protein kinase A

Intestinal Cl− secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl− secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl− secretion. FSK-stimulated Cl− secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl− secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl− secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl− conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl−>Br−>I− permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl− secretion, which is carried by a novel, previously undescribed Cl− channel.

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V1a receptor mRNA expression in mouse early proximal tubule (S1) cell line is regulated by protein kinase A

IUBMB Life ◽

10.1080/15216549800202022 ◽

1998 ◽

Vol 44 (5) ◽

pp. 961-969

Author(s):

Michio Takeda ◽

Mami Kobayashi ◽

Isao Shirato ◽

Hitoshi Endou

Keyword(s):

Protein Kinase ◽

Proximal Tubule ◽

Mrna Expression ◽

Protein Kinase A ◽

Cell Line ◽

Receptor Mrna ◽

V1a Receptor ◽

Kinase A ◽

Early Proximal Tubule

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Acid secretion in α-toxin-permeabilized gastric glands

Biochemistry and Cell Biology ◽

10.1139/o94-005 ◽

1994 ◽

Vol 72 (1-2) ◽

pp. 26-35 ◽

Cited By ~ 12

Author(s):

Alain Thibodeau ◽

Xuebiao Yao ◽

John G. Forte

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Basolateral Membrane ◽

Gastric Glands ◽

Paired Samples ◽

Atpase Inhibitors ◽

Dose Dependent Increase ◽

Dose Dependent ◽

Functional Responsiveness ◽

Kinase A

Rabbit gastric glands were treated with α-toxin to test for permeabilization of basolateral membrane and retention of functional activity of parietal cells. Treatment with up to 400 U α-toxin/mL resulted in a dose-dependent increase in permeabilization, as judged by nuclear uptake of trypan blue (960 daltons), while causing relatively little loss of cytoplasmic macromolecules in the size range of lactate dehydrogenase (134 000 daltons). In the presence of cAMP and ATP, α-toxin-permeabilized resting gastric glands were stimulated to accumulate aminopyrine by ~10-fold over glands incubated without added nucleotides. Aminopyrine accumulation in stimulated permeabilized glands was inhibited by specific H+,K+-ATPase inhibitors, omeprazole and SCH-28080, and by the selective inhibitor of protein kinase A, H-89 (IC50 = 7.17 ± 2.05 μM; n = 4). Aminopyrine accumulation in the α-toxin-treated glands was dependent on both exogenous ATP and cAMP; however, when no exogenous ATP was present, cAMP-activated aminopyrine accumulation reached ~50% of maximum, and at levels of ATP > 0.05 mM, maximal aminopyrine accumulation occurred without exogenous cAMP. In the presence of ATP alone, aminopyrine accumulation in permeabilized glands achieved 61.1 ± 3.2% (n = 10; range, 50–70%) of the values measured on paired samples of intact glands stimulated with histamine plus isobutylmethylxanthine. These results demonstrate the functional responsiveness of α-toxin-permeabilized resting gastric glands. The participation of a protein kinase A dependent pathway during activation of permeabilized parietal cell is proposed.Key words: aminopyrine accumulation, H+,K+-ATPase, H-89, protein kinase A, oxidative phosphorylation, digitonin.

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