No abstract

No abstract

To study the regulatory role of atrial natriuretic peptide (ANP) on the Cl- transport activity of retinal pigment epithelial (RPE) cells, RPE cells from rabbits were cultured and exposed to ANP and other reagents under perfusion. The changes in intracellular Cl- concentration ([Cl-]i) were continuously recorded using a Cl(-)-sensitive fluorescent dye. The cGMP content was estimated by radioimmunoassay. ANP increased the cGMP content and the [Cl-]i in RPE cells. A guanylate cyclase activator, nitric oxide, and a cell permeable cGMP precursor, 8-Br-cGMP, also increased the level of cGMP and the [Cl-]i. A guanylate cyclase inhibitor, LY83583, an inhibitor of cGMP-dependent protein kinase, KT5823, and an inhibitor of Na+/K+/2Cl- cotransporter, bumetanide, diminished or abolished the ANP-induced increase in [Cl-]i. ANP facilitates Cl- accumulation in RPE cells, which is mediated by guanylate cyclase, cGMP-dependent protein kinase, and the Na+/K+/2Cl- cotransporter.

We tried to detect natriuretic peptide (NP) receptor (NPR-A and NPR-B) mRNAs in cultured rabbit retinal pigment epithelium (RPE) cells and examined the regulation of their expression in relation to subretinal fluid absorption or RPE cell proliferation. RPE cells from 2-4 passages were grown to confluence on microporous membranes and analyzed for levels of expression of receptor mRNAs by quantitative RT-PCR and Northern blotting. The expression of NPR-B mRNA was approximately tenfold higher than that of NPR-A mRNA. The expression of NPR-A mRNA was not affected by treatments that may change subretinal fluid transport, while that of NPR-B mRNA was inhibited by transmitters involved in light- and dark-adaptation such as dopamine and melatonin. Expression of NPR-B mRNA was also suppressed by platelet-derived growth factor and transforming growth factor-beta. Furthermore, atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), ligands for NPR-A and B, respectively, inhibited the proliferation of RPE cells, as analyzed by incorporation of [3H]thymidine. These findings suggest that ANP may be involved in constitutive absorption of subretinal fluid and that NPs form an important regulatory system of proliferation in RPE cells.

ABSTRACT-Adrenoceptor-mediated C1-transport in cultured rabbit corneal endothelium was examined using a Cl--sensitive fluorescent dye. The intracellular Cl-concentration ([C1-];) in the endothelial cells was estimated to be about 30 mM. Noradrenaline (0.001-0.1 mM) transiently decreased the [Cl-]; in a dose-dependent manner. Such a decrease in [Cl-]; was completely antagonized by pretreatment with the aadrenoceptor antagonist phentolamine (0.1 mM). The selective a2-adrenoceptor agonist UK 14304-18 (5-bromo-6-[(4H,5H-imidazol-2-yl)amino]quinoxaline, 0.1 mM) persistently decreased the [Cl-];, but neither the al-adrenoceptor agonist phenylephrine (0.1 mM) nor the ;3-adrenoceptor agonist isoproterenol (0.1 mM) had any effect. The a2-adrenoceptor agonist/antagonist yohimbine (0.1 mM) persistently and more strongly decreased the [Cl-]; than UK 14304-18 did. The yohimbine-induced decrease in the [C1-]; was not further altered by UK 14304-18 or phenylephrine, but partly reversed by noradrenaline, isoproterenol and an adenylate cyclase activator, forskolin (0.1 mM). The yohimbine-induced decrease in [C1-]; was inhibited by the carbonic anhydrase inhibitor acetazolamide (1 mM), and Cl-/HC03-exchange inhibitors, 4-acetamido-4 isothiocyanostilbene-2,2 disulfonic acid and 4,4 diisothiocyanostilbene-2,2 disulfonic acid, but not by the H+-ATPase inhibitor N,N dicyclohexylcarbodiimide.The forskolin-induced recovery in [Cl-]; was inhibited by the Na+/K+/C1-cotransport inhibitor bumetanide (0.1 mM), but not by the Clchannel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid. These findings suggest that corneal endothelial cells extrude Cl-upon a2-adrenoceptor stimulation and accumulate Cl-upon 13-adrenoceptor stimulation under low [C1-]; conditions, probably via acceleration of Cl-/HC03-exchange and Na+/K+/ Cl-cotransport, respectively.