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P. Fidzinski, M. Salvador-Silva, J. Geibel, M. Coca-Prados; Natriuretic Peptides Inhibit Na+/H+ - Exchanger Type 1 (NHE-1) in Bovine Nonpigmented Ciliary Epithelial (NPE) Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3206.
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Purpose: Natriuretic peptides (NPs), ANP, BNP and CNP, influence intraocular pressure (IOP) in experimental animals. The ocular ciliary epithelium (CE) in conjunction with the trabecular meshwork regulates IOP and both tissues predominantly express the natriuretic peptide receptor subtype NPR-B. CE is also known to locally synthesize ANP and BNP, which upon their release could serve autocrine/paracrine functions. NPs have been observed to influence NHE-1, which in CE may serve a key role for IOP and pH regulation. In this study we investigated the effects of NPs on intracellular pH (pHi) in NPE cells in vivo. Methods: Strips of ciliary processes were isolated from bovine eyes and incubated in serum-free medium at 37°C and 5% CO2 for up to 72h. Following incubation with pH sensitive dye (BCECF), pHi from NPE cells was monitored under HCO3- - free conditions using a digital imaging system. After an acid load (20mM NH4Cl) followed by Na+ - free solution pHi recovery was recorded during subsequent return to a Na+ - containing solution. In a separate series tissues were preincubated with ANP, BNP, CNP (100nM-1nM), +/- EIPA (100nM) or Amiloride (10µM), respectively. Heptanol (2mM) or 18-glycerrhitinic acid (100µM) were applied 4min before Na+ readdition post acid load. Results: Under control conditions the rate of pHi recovery in NPE cells was 0.169±0.01 pH/min (n=61). Application of EIPA or Amiloride completely inhibited pHi recovery, which indicated NHE-1 as the functional isoform. In contrast, no influence on pHi recovery was observed after exposure to gap junction blockers heptanol and 18-glycerrhitinic acid, respectively. Addition of NPs (100nM) significantly inhibited rate of pHi recovery in the following order of potency: CNP>ANP>BNP (CNP: 0.051±0.003, n=73; ANP: 0.076±0.004, n=29; BNP: 0.094±0.03, n=76; values in pH/min). These effects were concentration-dependent between 100nM and 1nM. Conclusions: Our results demonstrate that: 1) NPs inhibited NHE-1 in NPE cells, 2) the inhibition was mediated by NPR-B receptors, and 3) the changes in pHi are likely confined to the NPE layer. This data provide new insights on the signal pathways mediated by NPs and their hypotensive effects.
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