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R Crook, D Hochgesand, JJ Dunn, MT Lai; VASOACTIVEINTESTINAL PEPTIDE STIMULATES NKCC1 BUT NOT NAK ATPASE VIA VIP I RECEPTORS ON FETAL HUMAN NPE CELLS . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3243.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide which results in a 40-50% increase in aqueous humor inflow in primates when given intracamerally or intravenously. Since the Na-K-Cl cotransporter (NKCC1) contributes to blood-to-aqueous chloride transport across ciliary epithelium and is stimulated in cultured cells by activation of protein kinase A, we asked whether VIP might influence NKCC1 activity. Methods: NKCC activity was measured by 86Rb uptake into confluent fetal human NPE and PE cells. Western blotting was by standard methods. Paraformaldehyde-fixed bovine eyes were sectioned into 8 micron sections and treated with a monoclonal antibody to mammalian cotransporter and a fluor-conjugated secondary antibody, then observed in a fluorescent microscope. Results:100nM VIP given to NPE for 5 min increased NKCC activity by 42%. The EC50 of this effect was 10nM. No effect of VIP on Na-K ATPase was seen. The PKA-specific inhibitor PKI 14-22 blocked NKCC stimulation, suggesting that activation of PKA is required. Analogues of VIP specific for either the VIP I receptor (VIPR-I) or VIP 2 receptor (VIPR 2) were tested. The VIPR1- specific peptide stimulated NKCC activity by 38%, whereas the VIPR 2-specific peptide did not stimulate. This suggests that the NPE VIP receptor is a type 1 receptor. In PE cells, neither NKCC1 nor NaK ATPase responded to VIP or peptides 1 and 2, although NKCC1 was stimulated by 1uM isoproterenol. Peptides related to VIP including glucagon, secretin and PHI27 stimulated NKCC1 in NPE similarly to their published potencies for stimulating cAMP. Polyclonal antisera to either VIPR1 or VIPR2 bound to different areas of bovine ciliary epithelium in fixed, frozen sections. VIPR 1 was concentrated on the PE basolateral membrane whereas VIPR2 was most concentrated on the NPE basolateral membrane. Conclusion:These results suggest that VIP stimulates NKCC activity in human NPE cells. Although NKCC is primarily located in the PE layer in vivo, this suggests that VIP increases aqueous humor formation in part by stimulating NKCC.
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