Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes (␤1-4, ␥1-2, and ␦1-2), we have found PLC␤4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the ␣-subunits of the G q family of G proteins, which are known to activate PLC␤4, have also demonstrated G ␣11 -like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLC␤4 and anti-G ␣11 antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by in situ hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, G ␣11 or a G ␣11 -like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as G ␣q (another G q family member), are present elsewhere in the retina.Visual transduction in the retina takes place in the outer segments of rod and cone photoreceptors and is known to involve a cGMP signaling cascade (see ref. 1 for a recent review). In this process, light isomerizes the visual pigment into an active form, which, via the G protein transducin, stimulates a cGMP-phosphodiesterase to lead to cGMP hydrolysis. In darkness, cytoplasmic cGMP binds to and opens cGMP-activated cation channels on the plasma membrane of the outer segment. These open channels sustain an inward dark current to keep the cell partially depolarized and maintain a steady release of glutamate from the synaptic terminal of the photoreceptor. In the light, the hydrolysis of cGMP leads to the closure of these channels, producing a membrane hyperpolarization as the light response and reducing the glutamate release from the cell.Over the years, however, there have also been reports of a phosphoinositide signaling pathway in the rod outer segment. In this pathway, the membrane phospholipid phosphatidylinositol-4,5-bisphosphate is hydrolyzed by phospholipase C (PLC) enzymes to release the second messengers inositol-1,4,5-trisphosphate and diacylglycerol, with the former leading to intracellular Ca 2ϩ release and the latter activating the enzyme protein kinase C (PKC). In rod outer segment preparations, phosph...

No abstract

We have isolated and characterized a cDNA for IGF-binding protein-2 (IGFBP-2) and its gene from the chick embryo. Using primers from a conserved region of the mammalian IGFBP-2 sequence, a cDNA clone (1.6 kb) was isolated from an embryonic day-18 chick retina cDNA library. Although the clone was truncated at the 5' end, the complete coding sequence was obtained from 5' rapid amplification of cDNA ends and genomic sequencing. The open reading frame encoded a 311 amino acid precursor protein which contains a putative 36 residue signal peptide. The mature 275 amino acid protein had a predicted M(r) of 33,500 and exhibited 71, 68, 68 and 66% identity to rat, bovine, ovine and human IGFBP-2 cDNA respectively, with conservation of all 18 cysteines. The cDNA contained an RGD peptide but lacked a putative ATP-binding motif. A single transcript of approximately 2.3 kb was present in embryonic day-15 eye, brain, skeletal muscle, heart and intestine, but was virtually absent from embryonic day-15 liver. The chicken IGFBP-2 gene spanned approximately 38 kb, consisted of four exons, and was similarly organized to that of the rat and human. Southern blot analysis of chicken genomic DNA suggested that it is encoded by a single gene. The sequence information from the avian IGFBP-2 should be of value in examining the role of IGFBP-2 in vertebrate development.

Although patients with diabetic retinopathy have been reported to have elevated vitreal IGF-I levels, it is not known whether diabetes also affects the levels of vitreal IGF binding proteins (IGFBPs) which control IGF's bioavailability. To address this issue, vitreal IGFBP levels were assayed in human diabetics, rats with streptozotocin-induced diabetes and galactose-fed dogs with diabetic-like retinopathy. Using 125I-IGF-II ligand blots, it was found that human diabetics have a 4-fold increase in vitreal IGFBP levels. Also, western blots on human diabetic vitreous reveal increased levels of IGFBP-2 and proteolytic fragments of IGFBP-3. IGF binding assays on vitreous from streptozotocin-treated rats (three months in duration) also indicate a 5-fold increase in IGF binding activity. IGF ligand blots using vitreous from rats with a shorter duration of diabetes (one month) show a 63% increase in IGFBP binding and a marked decrease in serum IGFBP binding. IGF ligand blots and IGFBP-2 and -4 western blots using vitreous from galactose-fed dogs with diabetic-like retinopathy exhibit a 6-fold increase in vitreal IGFBPs. The observation that vitreal IGFBPs are elevated in diabetic humans and rats without overt retinopathy suggests that these increases are not the result of a preexisting end-stage retinopathy but rather are an early ocular event in the diabetic process. Increases in vitreal IGFBPs thus could participate in the proliferative aspects of diabetic retinopathy by virtue of their putative intrinsic bioactivity or their capacity to alter IGF bioavailability.

The expression and regulation of insulin-like growth factor-binding proteins (IGFBPs) in developing avian vitreous humor and serum were compared. Vitreal IGF-I-binding activity was highest on embryonic day 6 [E-6; bound/free ratio (B/F), 0.22 +/- 0.019/50 microliters), decreased 10-fold between E-6 and E-19, and then remained stable through the remainder of embryonic development. In contrast, serum IGF-I binding increased 2-fold over this period, from a B/F of 0.380 +/- 0.056 (E-6) to a B/F of 0.89 +/- 0.18 (E-19). After hatching, serum IGF-I-binding activity continued to increase through posthatching week 12, while vitreal IGF-I binding increased only slightly and then remained constant. Although IGF-II binding in the vitreous humor and serum is 2- to 3-fold higher than that of IGF-I, the same pattern of developmental regulation was observed as with IGF-I. Western ligand blots revealed a vitreal 24-kilodalton (kDa) IGFBP that was absent from both embryonic and adult sera. Likewise, posthatching serum was found to contain a 70-kDa IGFBP absent in vitreous humor. Deglycosylation of vitreal and serum IGFBPs followed by Western ligand blotting revealed unique glycosylation patterns for vitreal and serum IGFBPs. One of the IGFBPs that is differentially glycosylated in vitreous and serum is a 33-kDa IGFBP that is precipitated with human IGFBP-2 antiserum. Northern blot analysis revealed the presence of IGFBP-2 mRNA in several embryonic ocular tissues as well as liver. The observations that vitreal and serum IGFBP levels are independently regulated during development and that IGFBPs from these two compartments have different molecular weights and glycosylation patterns suggest that the vitreal IGFBPs are not derived from serum. The presence of IGFBP-2 mRNA in ocular tissue surrounding the vitreal chamber supports the view that certain vitreal IGFBPs may be synthesized locally.

Despite the high prevalence of age-related cataracts, there are currently no known therapies to delay or prevent their occurrence. Studies in humans and rodent models suggest that estrogen may provide protection against age-related cataracts. The discovery of ocular estrogen receptors (ERs) indicates that estrogen protection may result from direct interactions with its receptors in the eye, instead an indirect consequence from effects on another tissue. Studies in our transgenic mouse model validate the concept that estrogen is beneficial for the eye. These mice express ER⌬3, a dominant-negative form of ER␣ that inhibits ER␣ function. In the ER⌬3 transgenic mice, cortical cataracts spontaneously form in ER⌬3 females after puberty and progress with age. The cataracts initiate in the equatorial region of the lens where the epithelial cells differentiate into elongating fiber cells. Cataract formation can be prevented if the females are ovariectomized before, but not after, sexual maturity. Both male and female ER⌬3 mice develop cataracts after neonatal treatment with the potent estrogen diethylstilbestrol (DES). The incidence of spontaneous and DESinduced cataracts in ER⌬3 mice is 100%, yet these cataracts are absent from the wild-type mice. These data suggest that repression of estrogen action induces cortical cataract formation because estrogen is required to activate the ER⌬3 repressor. Evidence of DES-induced cataracts in the ER⌬3 males as well as the females suggests that estrogen is important in lens physiology in both sexes. The ER⌬3 mice provide a powerful model for assessing the role of estrogen in maintaining the transparency of the lens. Currently there is no known therapy that can delay or prevent age-related cataracts that plague the ever-growing numbers of aging men and women. The incidence of age-related cataracts is higher in women (1), and their onset coincides with estrogen deficiency that occurs after menopause. Until recently, the eye had not been considered to be an estrogen target tissue; however, recent studies have begun to correlate estrogen status with risk of cataracts (2-4). In addition, the estrogen receptor (ER) has been detected in ocular tissues (5, 6,).Estrogens acting through the ER modulate transcription of estrogen-responsive genes (7). The ER is a ligand-dependent transcription factor belonging to the nuclear receptor superfamily (8). Currently, two subtypes of the ER are known to exist, ER␣ and ER␤. The classic ER, which has been associated for decades with female reproductive responses, is now designated as ER␣. The recently discovered second gene codes for ER␤ (9). Expression of the ER␣ protein has been detected in the rat and bovine (5) and human (6) retina and in the rat lens ʈ ; ER␤ mRNA has been detected in the rat lens (10). The presence of these receptors indicates that the eye can respond directly to estrogens or antiestrogens, such as tamoxifen.Studies in women suggest that estrogens may protect against the development of senile cataracts. The three types of agerelat...

No abstract

The IGFs (-I and -II) are normally found in serum and other extracellular fluids complexed to specific binding proteins (IGFBPs). While several IGFBPs have been identified in vitreous and aqueous humors, the major serum carrier of IGF, IGFBP-3, is notably absent from these fluids. To determine if this paucity could be due to an IGFBP-3 proteinase (IGFBP-3ase), samples of bovine vitreous or aqueous humor were mixed with serum and incubated at 37 degrees C for 4 h followed by western ligand blotting. In these experiments, a distinct loss of the 46 kDa band representing IGFBP-3 was observed while other bands present at 35, 28 and 25 kDa were unaltered. The IGFBP-3ase activity is temperature sensitive, has a pH optimum of about 8.0 and is inhibited by EDTA. Acid treatment of serum to remove endogenously bound IGF does not affect the specificity or activity of the IGFBP-3 proteinase. Size exclusion chromatography of bovine aqueous indicates an approximate molecular weight of 260 kDa. Incubation of recombinant IGFBP-3 or serum with partially-purified IGFBP-3ase results in the appearance of low molecular weight fragments of approximately 30 kDa. These fragments are undetectable by western ligand blotting but are readily visualized using an IGFBP-3 specific antibody. Comparison of normal and diabetic vitreous humor reveals the presence of an increased amount of IGFBP-3 proteolytic fragments in the diabetic as compared to control. These findings indicate the presence of a IGFBP-3 proteinase in aqueous and vitreous humors that may be important in regulating ocular homeostasis.