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Molecular cloning of the G-protein subunits in ocular ciliary epithelial cells
Thursday, Sep 24, 1992 La Palms/C EPlTHELIUM
X ICER Abstracts
SEWEMBER
CODEE-10
24/THURSDAY EXPRESSION OF AND FUNCTIONAL EPITHELIUM.
YOFT-
ERSONS;
NUMBER
TlME
Yale University School of Ophthalmology and Visual Science,
MIGUEL COCA-PRADOS (USA) MARTIN B. WAX (USA)
11:oo Wax
and M. Coca-Prados (USA)
S. Ghosh and J.M. Martin-
M. Coca-Prado%
Alonso (USA) Molecular
&nine of the G-Protein r Ctltarv v M.B. Wax and T.N. Tillis (USA)
DEFINE IN THE
Medicine, New Haven,
REGIONAL CILIARY
Department CT 06510.
of
In an effort to identify genes specifically expressed in the ciliary epithelium (CE), cDNA libraries were constructed in lambda gtll Sfi-Not and Uni-ZAP XR vectors from CE poly(A)+ RNA. lmmunoscreening of these libraries with polyclonal and monoclonal antibodies to the CE has resulted in the isolation of cDNA clones coding for known and unknown proteins. Among the cDNA clones identified are: a glutathione S-transferase (GST) class-lr, a glutathione peroxidase (GP), the cellular retinaldehyde-binding protein (CRALBP) and the CD9 antigen. Expression of GST-r, GP and CRALBP mRNAs, but not of CD9 mRNA, exhibited marked regional differences along the CE. These results, together with those of genes known to exhibit similar patterns of expression (Na+,K+-ATPase a and fi isoforms), suggest that some of these genes are involved in defining regional and functional boundaries within this tissue. Supported by NIH Grants EY04873 and EY08672.
PRESENTATION
M.B.
A GROUP OF GENES BOUNDARIES
Subunits
R.B. Crook (USA) Molecular
Bioloeical
Identification
MOLECULAR CLONING OF THE G-PROTEIN SUBUNITS . IN OCULAR CILIARY EPITHELIAL CELLS. Wax.,. and TIJ&
D. W. Gil, A.M. Bogardus, E. WoldeMussie and L.A. Wheeler (USA)
T& Depts. of Ophthalmology and Visual Science, Washington School of Medicine, St. Louis, MO.
Signal transducing GTP-binding proteins (G-proteins) may serve a key role in stimulus-secretion coupling in ocular ciliary epithelium and subsequent formation of aqueous humor. We have previously identified several of the known a subunits of gnanine nucleotide binding proteins in SV-40 aansfomxd ciliary epithelia. Bovine-derived pigmented ciliary epithelial cells and human-derived nonpigmented ciliary epithelial cells (ODM2) were grown in monolayer culture. PolyA mRNA was then obtained from total RNA extracted from these cells utilizing oligo-dT purification. Reverse transctiptase was then used to make cDNA and PCR was performed using primers previously shown to amplify specific G-protein a, 0 and y subunit cDNAs. Utilizing a PCR primer selective for they subunit, a DNA fragment of approximately 14Okb size and characteristic of mammalian y subunits, was amplified. The DNA fragment was inserted into a plasmid, transformed into E. coli and the nucleotide sequence was determined. DNA extracted and sequenced from clonal populations revealed the presence of the ‘/3 and @ subunits. In pigmented ciliruy epithelia, similar cloning pmcedurcs utilizing an initial PCR primer selective for the Cl protein a subunit has resulted in a sequence which appears similar but distinct from other known a subunits. The elucidation of specific G protein subunits in ciliary epithelia may provide a basis by which G-proteins may be genetically targeted for therapeutic modalities which alter the secretion of aqueous humor. (Supported by NE1 EYO6810 and Research to Prevent Blindness)
N.A. Delamere and T. Mito (USA) 6
12:30
7
12:45
Control of Transoort Mechanisms in the .c&aN Process &&&&ul T.W. Mittag, H. Mori and H. Kobayashi (USA) mn of Cvtokine mv Body N.Yoshimura
University
Genes in Rat lrlfc
(Japan)
PROTEIN
KINASE C REGULATES NEUROPEPTIDE RECEPTORS AND ION COTRANSPORT IN RUHAN NONP1GMENTF.D CILIARY EPITHELIAL CELLS. Be Crook, Department of ophthalmology, University of California, San Francisco, CA, 94143 USA. Neuropeptides sucbas atrionatriureticpeptide (ANP) andvasoactive intestinalpeptide (VIP) stimulate aqueous inflow in primates, presumably by affecting the ciliary epithelium. We have studied both ANP stimulation of CGMP and VIP stimulation of CAMP in human fetal NPE cells. Both of these functions are inhibited bv prior exuosure of cells to activators of protein kina% C (PKCj. For both ANP and VIP, inhibition of cyclic nucleotide
formation
is
due
to
a reduction
in
specific
receptor
binding sites. In the case of ANP, downregulation can be also be achieved by compounds which activate the inositol phosphate/diacylgiycerbl pathway, such a8 muecarinic aaonists. Reduction in AWP binding sites is due at least ii part to a‘ PKC-stimulated increase in turnover of endocytosed receptor-ligand complexes via a nonlyeosomal mechanism. PKC also regulates ion transport. Activation of PKC inhibits the activity of a Na*,K*,Cl-cotransporter in both NPE and PE ceJls but Ms no effect on the Na/K ATPaee or uotake of Rb' throuah K+ channels. Immunolwical ecidence suggests that ia+,K+,Cl-cotraneporters are-ale0 present in adult+ hFn-ciliary epithelium j,u&&~. The presence of Na ,K ,Clcotransporters in the CiliaZy epithelium is of in$er+est.-because, in secretory tissues such as kidney, Na ,K ,Clcotransport is necessary for transepithelial chloride movement and fluid flow.
S.168
X ICER Abstracts
SEWEMBER
CODEE-10
24/THURSDAY EXPRESSION OF AND FUNCTIONAL EPITHELIUM.
YOFT-
ERSONS;
NUMBER
TlME
Yale University School of Ophthalmology and Visual Science,
MIGUEL COCA-PRADOS (USA) MARTIN B. WAX (USA)
11:oo Wax
and M. Coca-Prados (USA)
S. Ghosh and J.M. Martin-
M. Coca-Prado%
Alonso (USA) Molecular
&nine of the G-Protein r Ctltarv v M.B. Wax and T.N. Tillis (USA)
DEFINE IN THE
Medicine, New Haven,
REGIONAL CILIARY
Department CT 06510.
of
In an effort to identify genes specifically expressed in the ciliary epithelium (CE), cDNA libraries were constructed in lambda gtll Sfi-Not and Uni-ZAP XR vectors from CE poly(A)+ RNA. lmmunoscreening of these libraries with polyclonal and monoclonal antibodies to the CE has resulted in the isolation of cDNA clones coding for known and unknown proteins. Among the cDNA clones identified are: a glutathione S-transferase (GST) class-lr, a glutathione peroxidase (GP), the cellular retinaldehyde-binding protein (CRALBP) and the CD9 antigen. Expression of GST-r, GP and CRALBP mRNAs, but not of CD9 mRNA, exhibited marked regional differences along the CE. These results, together with those of genes known to exhibit similar patterns of expression (Na+,K+-ATPase a and fi isoforms), suggest that some of these genes are involved in defining regional and functional boundaries within this tissue. Supported by NIH Grants EY04873 and EY08672.
PRESENTATION
M.B.
A GROUP OF GENES BOUNDARIES
Subunits
R.B. Crook (USA) Molecular
Bioloeical
Identification
MOLECULAR CLONING OF THE G-PROTEIN SUBUNITS . IN OCULAR CILIARY EPITHELIAL CELLS. Wax.,. and TIJ&
D. W. Gil, A.M. Bogardus, E. WoldeMussie and L.A. Wheeler (USA)
T& Depts. of Ophthalmology and Visual Science, Washington School of Medicine, St. Louis, MO.
Signal transducing GTP-binding proteins (G-proteins) may serve a key role in stimulus-secretion coupling in ocular ciliary epithelium and subsequent formation of aqueous humor. We have previously identified several of the known a subunits of gnanine nucleotide binding proteins in SV-40 aansfomxd ciliary epithelia. Bovine-derived pigmented ciliary epithelial cells and human-derived nonpigmented ciliary epithelial cells (ODM2) were grown in monolayer culture. PolyA mRNA was then obtained from total RNA extracted from these cells utilizing oligo-dT purification. Reverse transctiptase was then used to make cDNA and PCR was performed using primers previously shown to amplify specific G-protein a, 0 and y subunit cDNAs. Utilizing a PCR primer selective for they subunit, a DNA fragment of approximately 14Okb size and characteristic of mammalian y subunits, was amplified. The DNA fragment was inserted into a plasmid, transformed into E. coli and the nucleotide sequence was determined. DNA extracted and sequenced from clonal populations revealed the presence of the ‘/3 and @ subunits. In pigmented ciliruy epithelia, similar cloning pmcedurcs utilizing an initial PCR primer selective for the Cl protein a subunit has resulted in a sequence which appears similar but distinct from other known a subunits. The elucidation of specific G protein subunits in ciliary epithelia may provide a basis by which G-proteins may be genetically targeted for therapeutic modalities which alter the secretion of aqueous humor. (Supported by NE1 EYO6810 and Research to Prevent Blindness)
N.A. Delamere and T. Mito (USA) 6
12:30
7
12:45
Control of Transoort Mechanisms in the .c&aN Process &&&&ul T.W. Mittag, H. Mori and H. Kobayashi (USA) mn of Cvtokine mv Body N.Yoshimura
University
Genes in Rat lrlfc
(Japan)
PROTEIN
KINASE C REGULATES NEUROPEPTIDE RECEPTORS AND ION COTRANSPORT IN RUHAN NONP1GMENTF.D CILIARY EPITHELIAL CELLS. Be Crook, Department of ophthalmology, University of California, San Francisco, CA, 94143 USA. Neuropeptides sucbas atrionatriureticpeptide (ANP) andvasoactive intestinalpeptide (VIP) stimulate aqueous inflow in primates, presumably by affecting the ciliary epithelium. We have studied both ANP stimulation of CGMP and VIP stimulation of CAMP in human fetal NPE cells. Both of these functions are inhibited bv prior exuosure of cells to activators of protein kina% C (PKCj. For both ANP and VIP, inhibition of cyclic nucleotide
formation
is
due
to
a reduction
in
specific
receptor
binding sites. In the case of ANP, downregulation can be also be achieved by compounds which activate the inositol phosphate/diacylgiycerbl pathway, such a8 muecarinic aaonists. Reduction in AWP binding sites is due at least ii part to a‘ PKC-stimulated increase in turnover of endocytosed receptor-ligand complexes via a nonlyeosomal mechanism. PKC also regulates ion transport. Activation of PKC inhibits the activity of a Na*,K*,Cl-cotransporter in both NPE and PE ceJls but Ms no effect on the Na/K ATPaee or uotake of Rb' throuah K+ channels. Immunolwical ecidence suggests that ia+,K+,Cl-cotraneporters are-ale0 present in adult+ hFn-ciliary epithelium j,u&&~. The presence of Na ,K ,Clcotransporters in the CiliaZy epithelium is of in$er+est.-because, in secretory tissues such as kidney, Na ,K ,Clcotransport is necessary for transepithelial chloride movement and fluid flow.
S.168