Journal of Neuroolieinisirr Lippincoti—Raven Publishers, Ph ladetphia 996 International Society for Neurochemistry Identification of a Neuropeptide and Neuropeptide-Processing Enzymes in Aqueous Humor Confers Neuroendocrine Features to the Human Ocular Ciliary Epithelium Javier Ortego, Julio Escribano, *John Crabb, and Miguel Coca-Prados Department of Ophthalmology and Visual S ience, Yale University School of Medicine, New Haven, Con necticul, and ~‘W. Alton Jones Cell Science Center, Lake Placid, New York, U.S.A. Abstract: The ocular ciliary epithelium, the site of aque- ous humor secretion in the mammalian eye, is believed to play a key function in signaling mechanisms that regu- late the rate of secretion, and thus intraocular pressure. One possible way of mediating these signaling functions is through neuropeptides and hormones secreted into the aqueous humor and acting on target tissues. We recently identified a cDNA clone sharing 100% identity with car- boxypeptidase E (CPE), a neuropeptide-processing en- zyme. Utilizing polymerase chain reaction, we further identified and characterized another processing enzyme, the peptidylglycine cs-amidating monooxygenase (PAM), and the neuropeptide secretogranin II, a molecular marker restricted to neuroendocrine tissues. Using spe- cific probes, we found that the nonpigmented ciliary epi- thelial cells express CPE, PAM, and secretogranin II mRNA, and protein. We also found that CPE and secreto- granin II are abundant in aqueous humor. Treatment of cultured ciliary epithelial cells with veratridine and phorbol ester up-regulates CPE and PAM. Secretogranin II was found to be induced by veratridine, whereas phorbol ester had little effect, suggesting different mechanisms for se- cretion. The results demonstrate that secretogranin II, CPE, and PAM represent a specialized group of neuro- peptide and neuropeptide-processing enzymes secreted by the ciliary epithelial cells which may confer to them neuroendocrine functions in cell—cell communication or cell signaling. Key Words: Neuropeptide—Processing enzymes—Aqueous humor—Ciliary epithelium. J. Neurochem. 66, 787—796 (1996). plasma proteins from the stroma. located adjacent to the PE cell layer, to the posterior chamber of the eye (anterior segment) which contains the aqueous humor and is located on the NPE side. Among the functions ascribed to the ciliary epitheliurn are the secretion of aqueous humor fluid and the regulation of intraocular pressure (lOP), which, in pathological conditions, may rise to abnormal levels causing irreversible damage to the optic nerve and ganglion cells of the retina. The ciliary body, which is comprised of blood vessels and smooth muscle cells, in addition to the bilayer of the ciliary epithelium, is postsynaptically innervated by adrenergic fibers that are believed to release neuropep- tides to modulate important functions of the ciliary epithelium (i.e., lOP, secretion) (Osborne. 1993). To study the physiological, biochemical, and phar- macological properties of the human ciliary epithe- hum, we developed and characterized in vitro cell sys- tems of the secretory ciliary epithelia (Martin-Vasallo et al., 1989; Ghosh et al., 1990; Coca-Prados et al.. 1995). Most recently, we constructed and character- ized cDNA libraries from the human intact ciliary body tissue and cells derived from the ciliary epitheliurn (Escribano et al., 1994, 1995). These libraries have been instrumental in providing information on the tran- scriptional activity within the ciliary body and in iden- tifying many genes of biological interest expressed in The mammalian ciliary epithelium is a unique bi- layer of secretory neuroepithelial cells comprised of pigmented (PE) and nonpigmented (NPE) cells. NPE cells contain tight junctions at their apical plasma membrane and are coupled to the PE cells through gap junctions (Coca-Prados et al., 1992) and other complex junctions (Raviola and Raviola, 1978). PE cells lack tight junctions to their apical plasma mem- brane. This anatomical configuration makes the NPE cell layer a tight epithelium, establishing a blood— aqueous harrier that prevents the free passage of Received June 12. 1995: revised manuscript received August 29, 1995; accepted September 12, 1995. Address correspondence and reprint requests to Dr. M. Coca- Prados at Department of Ophthalmology and Visual Science. Yale University School of Medicine. 330 Cedar St.. New Haven. CT 06510, U.S.A. The present address of Dr. J. Escrihano is Secctdtt de Biotecno- logia, Instituto de Desarrollo Regional. Universtdad de Casttlla-La Mancha. 02071 Albacete, Spain. Ahhres’iution,s used: CPE, carhoxypeptidase E: lOP, intraocular pressure; NPE, nonpigmented ciliary epithelial: nt. nucleotide: PAM. peptidylglycine a-amidating nlonooxvgenase: PCR. polynierase chain reaction; PE. pigmented ctliary epithelial: pfu. plaque-forming units; PMA. phorhol I 2-myristate 13-acetate: SOS. sodium dodecyl sulfate. 787