Topical epinephrine causes a decrease in density of beta-adrenergic receptors and cathecholamine-stimulated chloride transport in the rabbit cornea.

A single administration, or twice daily administration for 4.5 days, of topical 2% epinephrine to the rabbit eye in vivo causes a 30-40% decrease in the density of beta-adrenergic receptors on membranes prepared from the cornea. Such treatment also causes complete loss of the ability of excised corneas to respond to epinephrine in vitro with enhanced active chloride transport. These findings indicate that stimulation with a high concentration of catecholamine depresses the entire pathway from receptor to physiological response.

Cellular localization of neuronal nitric oxide synthase (NOS-1) in the human and rat retina.

The neuronal form of nitric oxide synthase (NOS-1) has been localized to several cell types in the retinas of experimental animals; however, localization in the human retina has not been definitive. By using in situ hybridization and immunohistochemistry, we have compared the cellular expression and localization of NOS-1 in the rat and human retinas. In both rat and human retinas, NOS-1 is expressed in the inner segments of photoreceptors, cells in the inner nuclear layer, particularly amacrine cells, and retinal ganglion cells. In human cones, NOS-1 is abundantly present in the outer segments. In the rat, optic nerve transection caused a loss of cells that were positive for NOS-1 in the ganglion cell layer. Although a retinal ganglion cell localization has not been reported consistently in the literature, our data clearly localize NOS-1 to the retinal ganglion cells of the rat and human retinas.

A tissue culture assay of corneal epithelial wound closure.

Experimental assays have been developed using cultured tissue derived from rabbit corneal epithelium to study migration of epithelial sheets during wound closure and cell-substrate adhesion. To study wound closure, epithelial defects, 6 mm in diameter, were produced in vitro in 24 well multiplates by a local freezing technique, and the size of the remaining defect was quantitated over time by staining. To study adhesion, cultured cells were labeled with 3H-leucine, suspended, and added to fresh culture plates. At various times, adherent cells were lysed and the radioactivity of the lysate was determined. Serum enhances the closure of experimental defects, but laminin and fibronectin have no effect. Agents which alter mitotic rate, such as epidermal growth factor and 5-fluorouracil, do not influence the rate of wound closure in this assay. Compounds which elevate intracellular levels of cyclic AMP inhibit wound closure but promote cell-substrate adhesion. Thus, cultured corneal epithelial cells may be used to assay for influences on the migratory events governing closure of superficial epithelial wounds.

The rabbit cornea lacks cholinergic receptors.

Cholinergic receptors were studied in membranes prepared from rabbit cornea, iris-ciliary body, and retina, using 3H-quinuclidinyl benzilate (3H-QNB) to identify muscarinic receptors and 125I-alpha-bungarotoxin (125I-BGT) to identify nicotinic receptors. Muscarinic cholinergic receptors were not found in the cornea. As a positive control, muscarinic cholinergic receptors were characterized in preparations of the iris-ciliary body. Specific binding of 3H-QNB to iris-ciliary body membrane preparations was saturable, with a Kd of 1.3 nM QNB. Specificity of the assay for muscarinic receptors was confirmed by the relative abilities of the following compounds to displace 3H-QNB: atropine greater than pilocarpine greater than hexamethonium. Nicotinic cholinergic receptors were not found in the cornea. As a positive control, nicotinic cholinergic receptors were characterized in preparations of the retina. Specific binding of 1252-BGT to retinal membrane preparations was saturable with both high and low affinity receptors (Kd values of 1.0 nM and 93 nM BGT, respectively). Specificity of the assay for nicotinic receptors was confirmed by the relative abilities of the following compounds to prevent 125I-BGT binding: curare greater than or equal to nicotine greater than hexamethonium greater than atropine. The lack of cholinergic receptors in the cornea, which has high levels of acetylcholine and related enzymes, suggests either an extraordinary use or a lack of function for acetylcholine in this tissue.

Beta-adrenergic and serotonergic responsiveness of rabbit corneal epithelial cells in culture.

Rabbit corneal epithelial cell cultures were established from Dispase-treated anterior corneas. In culture medium containing cholera toxin, insulin and epidermal growth factor, these cells proliferated in vitro in the absence of any contaminating cells. Following subculture, cells retained epithelial morphology and the ability to synthesize cAMP in response to beta-adrenergic stimulation, but lacked the ability to respond to serotonergic stimulation. Retention of the beta-adrenergic system in culture serves as a functional epithelial cell marker; whereas expression of serotonergic responsiveness may be regulated by developmental or extrapithelial systems that are absent in these cell cultures.

In vitro determination of the ability of drugs to bind to adrenergic receptors.

Alpha- and beta-adrenergic receptors were studied by measuring the binding of 3H-dihydroergocryptine and 3H-dihydroalprenolol, respectively, to membranes prepared from homogenized rabbit iris--ciliary bodies. The binding of 3H-dihydroergocryptine appears to be specific for alpha-adrenergic receptors, since adrenergic agents displace this radioligand with the following order of potency: phentolamine greater than epinephrine greater than or equal to norepinephrine greater than or equal to isoproterenol = propranolol. The binding of 3H-dihydroalprenolol appears to be specific for beta-adrenergic receptors, since adrenergic agents displace this radioligand with the following order of potency: propranolol greater than or equal to isoproterenol greater than or equal to epinephrine greater than norepinephrine greater than or equal to phentolamine. Clonidine and dopamine bind to the alpha-adrenergic receptor but have little activity at the beta-adrenergic receptor. Timolol, d-isoproterenol, and dipivalyl epinephrine bind to the beta-adrenergic receptor but have little activity at the alpha-adrenergic receptor. The results demonstrate that in vitro binding assays for alpha- and beta-adrenergic receptors are useful for studying the mechanism of drug action.

Study of central regulation of intraocular pressure using ventriculocisternal perfusion.

The ability of hypoosmotic solution, prostaglandin E1 (PGE1) and clonidine to influence intraocular pressure (IOP) by a central mechanism was studied using the technique of ventriculocisternal perfusion in conscious rabbits. IOP remained unchanged during the perfusion of 150 mOsm artificial cerebrospinal fluid. IOP rapidly increased by 15 mmHg during the perfusion of PGE1 at the dose of 1 or 3 micrograms/min. However, when PGE1 was perfused intravenously at the dose of 1 microgram/min, a similar IOP response was observed. Furthermore, during the ventriculocisternal perfusion of PGE1 a significant systemic absorption occurred. These observations indicate that the ocular hypertension during the ventriculocisternal perfusion of PGE1 is primarily due to the peripheral action of systematically absorbed PGE1. IOP gradually decreased by 3 mmHg during the ventriculocisternal perfusion of clonidine at the dose of 0.1 or 0.33 micrograms/min. Intravenous perfusion of clonidine at the same doses did not change the IOP. These results indicate that clonidine can lower IOP by a centrally mediated mechanism. Ventriculocisternal perfusion of clonidine (0.1 micrograms/min) in rabbits with unilateral superior cervical ganglionectomy lowered IOP in both eyes, indicating that ocular adrenergic innervation does not participate in this centrally mediated IOP response. However, the cardiovascular parameters of anesthetized rabbits were altered by the ventriculocisternal perfusion of clonidine (0.1 micrograms/min), suggesting that a change in systemic hemodynamics is involved in the central IOP effect of clonidine.

Cell culture of the human lamina cribrosa.

The extracellular matrix of the lamina cribrosa may be important in the changes in the optic nerve head associated with glaucoma. To investigate the cell biology of this tissue, human lamina cribrosa was explanted in tissue culture and two cell types grown from this tissue were characterized. The most common cell type obtained was a large, flat, polygonal cell which was negative for glial fibrillar acidic protein (GFAP) and could be serially subcultured. This cell type synthesized collagens type III and type IV, fibronectin and elastin. Much less commonly grown was a cell type with conspicuous long processes and which was positive for GFAP. This presumed astrocyte synthesized collagen type IV and fibronectin. Fibroblastic cells were not obtained from this tissue but were easily grown from sclera. The cells that we have cultured from the human lamina cribrosa may produce the extracellular matrix present in the cribriform plates of this tissue and be important in the glaucomatous process.

Isoforms of nitric oxide synthase in the optic nerves of rat eyes with chronic moderately elevated intraocular pressure.

PURPOSE: To investigate the hypothesis that nitric oxide (NO) in the optic nerve heads of rats with chronic moderately elevated intraocular pressure (IOP) contributes to neurotoxicity of the retinal ganglion cells, the presence of the three isoforms of nitric oxide synthase (NOS) was determined in the tissue. METHODS: Unilateral chronic moderately elevated IOP was produced in rats by cautery of three episcleral vessels. Histologic sections of optic nerves from eyes with normal IOP and with chronic moderately elevated IOP were studied by immunohistochemistry and by immunoblot analysis. Polyclonal antibodies to NOS-1, NOS-2, NOS-3, and glial fibrillary acidic protein (GFAP) were localized with immunoperoxidase. RESULTS: In the optic nerve of rat eyes with normal IOP, NOS-1 was constitutively present in astrocytes, pericytes and nerve terminals in the walls of the central artery. NOS-2 was not present in eyes with normal IOP. In these eyes, NOS-3 was constitutively present in the vascular endothelia of large and small vessels. Rat eyes treated with three-vessel cautery had sustained elevated IOP (1.6 fold) for at least 3 months. In these eyes, no obvious changes in NOS-1 or NOS-3 were noted. However, at time points as early as 4 days of chronic moderately elevated IOP, NOS-2 appeared in astrocytes in the optic nerve heads of these eyes and persisted for up to 3 months. Immunoblot analysis did not detect differences in NOS isoforms. CONCLUSION: The cellular distributions of constitutive NOS isoforms in the rat optic nerve suggest physiological roles for NO in this tissue. NOS-1 in astrocytes may produce NO as a mediator between neighboring cells. NO, produced by NOS-1 in pericytes and nitrergic nerve terminals and by NOS-3 in vascular endothelia, is probably a physiological vasodilator in this tissue. In eyes with chronic moderately elevated IOP, NOS-2 is apparently induced in astrocytes. The excessive NO production that is associated with this isoform may contribute to the neurotoxicity of the retinal ganglion cells in eyes with chronic moderately elevated IOP.

In vitro pharmacologic separation of corneal endothelial migration and spreading responses.

Repair of corneal endothelial wounds involves two forms of cell translocation: (1) "migration," in which individual cells at the wound edge break contacts with neighboring cells and move as individuals into the wound defect, and (2) "spreading," in which cells within the confluent monolayer adjacent to the wound move as a group into the wound area. The authors combined morphometric analysis of Giemsa-stained cultures, phase-contrast video microscopy, and Rh-phalloidin staining of actin filaments to study the effects of epidermal growth factor (EGF) and indomethacin on the migratory and spreading responses to wounding using an in vitro wound-closure model which mimics the amitotic state and general behavior of human corneal endothelium. They found that EGF stimulated the migration of individual cells from the wound edge, induced cellular elongation, and promoted a diffuse distribution of actin filaments. Indomethacin promoted spreading of the confluent monolayer into the wound defect, induced enlargement and flattening of cells, and promoted the formation of long, thick actin stress fibers. These results provide evidence that the migration and spreading responses of corneal endothelial cells to wounding can be pharmacologically separated. The findings suggest that migration of individual cells during wound repair may result from an endogenous form of EGF-like stimulation and that the elongated shape associated with this form of translocation results, at least in part, from an EGF-like alteration in actin-filament organization. Spreading of the confluent monolayer to cover the wound defect may result from a decrease in cyclic adenosine monophosphate induced by a transient reduction in prostaglandin E2 synthesis. This form of translocation may result, in part, from enlargement and flattening of corneal endothelial cells secondary to an enhancement of actin stress-fiber formation.

Localization of collagen types I and IV mRNAs in human optic nerve head by in situ hybridization.

Using in situ hybridization, individual cells expressing mRNAs for collagen types I and IV were localized in fixed-tissue sections of adult and fetal human optic nerve heads. Astroglial cells lining the cribriform plates and cells inside the cribriform plates of the lamina cribrosa had mRNA for collagen type IV. Cells in the glial columns, pial septa, and vascular wall also contained mRNA collagen type IV. Collagen type I mRNA was expressed by cells of the cribriform plates of the lamina cribrosa of adults. Few cells in the glial columns, pial septa, and blood vessels had mRNA for collagen type I. Scleral fibroblasts contained mRNA for collagen type I. These results indicated that the expression of mRNA for both collagen types I and IV paralleled the localization of these extracellular matrix proteins in the optic nerve head and suggested that both collagen types were synthesized in this tissue throughout life.

Adrenergic stimulation of ciliary process epithelium causes surface membrane internalization.

An ultrastructural change induced in the nonpigmented epithelium (NPE) of the ciliary processes by adrenergic stimulation in the albino rabbit was studied. Thirty min after topical treatment with 2% isoproterenol, an extensive intracellular membranous network, previously reported to be smooth endoplasmic reticulum, was revealed by electron microscopy. It was postulated that this network originated from the plasma membrane. Using cationized ferritin (CF) as an ultrastructural tracer, freshly isolated anterior segments were incubated in buffer containing 10(-5) M isoproterenol and 0.2% CF. As early as 10 min, and for at least 30 min, the isoproterenol-treated NPE cells contained a membranous network that was morphologically similar to that which occurs in vivo. CF particles were present within the network, indicating that the membranous network had originated at the cell surface. This labeling was prevented by pretreatment with the beta-adrenergic antagonist timolol maleate. In both treated and control ciliary processes, CF was present in the ciliary canals between the NPE and the underlying pigmented epithelium after 10 min incubation. This suggests that the NPE is able to transport CF from its basilar to apical surface. These experiments imply that the NPE is able to internalize rapidly large amounts of plasma membrane in response to adrenergic stimulation. This response may be part of the mechanism of adrenergic receptor desensitization, alteration of aqueous humor production, or another adrenergic response.

Effects of stimulation of the ocular sympathetic nerves on IOP and aqueous humor flow.

Ocular sympathetic nerves were stimulated chronically in awake rabbits using electrodes unilaterally implanted on the cervical sympathetic trunk. IOP was measured by pneumatonometry and aqueous inflow was measured by fluorophotometry. In each animal, continuous trains of 1 msec pulses were delivered by means of a portable electrical stimulator. Experiments were spaced by 1 week recovery periods. Stimulation was varied over a range of amplitudes (5-15 V) and frequencies (3-12 Hz). Continuous sympathetic stimulation produced an immediate sharp decrease in IOP followed by a gradual rise to pre-stimulation values which were attained 60-90 min after onset. A rebound increase in IOP occurred when stimulation was terminated. The magnitude of the initial IOP drop, the delay in the return to pre-stimulation IOP, and the rebound rise in IOP subsequent to termination of electrical stimulation were proportional to the stimulation frequency. Maximal effects were observed at 12 Hz, and stimulation with 8-10 Hz for 180 min caused a sustained reduction in anterior chamber aqueous humor flow. Topical 2% phentolamine 1 hr before stimulation markedly reduced IOP and abolished the acute IOP changes observed in untreated stimulated animals. Topical 1% timolol did not affect either the initial IOP drop or the rebound; however, the IOP recovered during stimulation to values greater than pre-stimulation IOP. We conclude that in rabbits the beta-adrenergic effect of prolonged sympathetic nerve stimulation is to decrease aqueous flow. Chronic electrical stimulation in awake animals provides an experimental model for studying the role of the ocular sympathetic nerves.

Pharmacological regulation of morphology and mitosis in cultured rabbit corneal endothelium.

Cultured rabbit corneal endothelial cells elongate when grown in the presence of epidermal growth factor (EGF) and indomethacin (INDO); whereas maintenance of the differentiated polygonal cell shape is apparently dependent upon endogenous synthesis of prostaglandin E2 (PGE2). In the current study, the authors demonstrate morphological changes in phenotypically altered cells and identify two intracellular pathways which interdependently regulate endothelial cells. Morphometric and mitotic analyses of cultures treated with a variety of pharmacological agents indicate that both protein kinases A- and C-dependent pathways regulate cell shape and cell division in corneal endothelial cells. Marked intracellular reorganization is associated with the morphological changes in the endothelial cells. When stained with rhodamine conjugated phallicidin, polygonal endothelial cells have circumferential bands of f-actin at their borders. EGF and/or INDO induce elongation and redistribution of f-actin into a diffuse cytoplasmic reticulum. Transmission electron microscopy demonstrates loss of several characteristic morphological markers for endothelial cells in response to pharmacologically induced elongation. The elongated cells lose intracellular junctions, apical/basal polarity and rough endoplasmic reticulum. These ultrastructural markers and circumferential f-actin bands are restored in cultures supplemented with exogenous PGE2. Modulation of these pathways in vivo may regulate cellular migration and mitosis during wound closure, stress, trauma and with age.

Effects of l- and d-timolol on cyclic AMP synthesis and intraocular pressure in water-loaded, albino and pigmented rabbits.

Topical 2% l- or d-timolol reduced the elevation of intraocular pressure induced by water-loading in conscious rabbits. This drug effect appeared on the peak elevation (in pigmented eyes) and on the down-phase (in albino and pigmented eyes) of elevated intraocular pressure. The contralateral eye and the treated eye responded similarly. In urethane anesthetized, water-loaded rabbits, a greater inhibitory effect of l-timolol was observed in pigmented eyes than in albino eyes. Two per cent l-timolol caused alterations of heart rate and arterial blood pressure in water-loaded anesthetized rabbits, but time courses of these alterations did not correlate with the inhibitory effect on the elevation of intraocular pressure. The beta-adrenergic antagonistic activity of l-timolol and d-timolol were compared by their ability to inhibit l-isoproterenol-stimulated cyclic AMP synthesis in the rabbit iris-ciliary body preparation in vitro. The I50S for l- and d-timolol differ by about 1.5 log units. In our studies, d-timolol has little of the intraocular pressure lowering and the beta-adrenergic antagonistic activity of l-timolol. Thus, the conscious, water-loaded, pigmented rabbit can be used as a model for studying the effects of beta-adrenergic antagonists on intraocular pressure.

Modification by timolol of catecholamine stimulation of chloride transport in isolated corneas.

In the isolated frog cornea, 10(-5)M timolol completely blocked the stimulation of chloride transport by 10(-6)M isoproterenol. In this preraration, timolol inconsistently modified the response to epinephrine. In some experiments, epinephrine added to the bathing medium after timolol caused a smaller than normal increase in chloride transport, but in other instances, epinephrine caused a decrease in chloride transport. In the isolated rabbit cornea, 10(-5)M timolol totally blocked the stimulation of chloride transport by 10(-6)M epinephrine. In vivo, topical treatment of rabbit eyes with 0.5% timolol resulted in corneas from these eyes having an inhibited response to epinephrine when incubated in vitro. This inability to stimulate chloride transport persisted for several days following termination of topical treatment with timolol.

Cholera toxin stimulates adenosine 3',5'-monophosphate synthesis and epithelial wound closure in the rabbit cornea.

Rabbit corneas were treated in vitro and in vivo with cholera toxin (CTX), a specific and irreversible activator of adenylate cyclase. Tissue pieces incubated in vitro in the presence of 10 mug/ml CTX for 15 min continuously synthesized adenosine 3',5'-monophosphate (cyclic AMP) at an increased rate for 3 hr in the absence of CTX in the medium. Corneas exposed for 10 min to CTX topically in vivo and after various time intervals incubated in vitro had an increased ability to synthesize cyclic AMP for at least 30 hr after topical treatment. Epithelial wounds, 6 mm in diameter, were made by brief exposure of corneas in vivo to filter disks soaked in heptanol. Wounds in corneas pretreated with CTX closed at a faster rate and earlier than wounds in corneas pretreated with inactivated CTX. We postulate that cyclic AMP mediates the initial events governing the rate of closure of an epithelial defect.

Changes in responsiveness of the beta-adrenergic and serotonergic pathways of the rabbit corneal epithelium.

Adrenergic agonists stimulate the synthesis of cyclic AMP by incubated rabbit corneas with the following order of potency: isoproterenol greater than epinephrine greater than norepinephrine. These agonists have the same order of potency when displacing the specific, beta-adrenergic radioligand, 3H-dihydroalprenolol, from beta-adrenergic receptors on membranes prepared from corneal epithelium. At another locus, serotonin stimulates cyclic AMP synthesis. Inhibition of stimulation in vitro by lysergic acid diethylamide, methysergide, cyproheptadine, and spiroperidol demonstrates the specificity of this pathway for serotonin. Topical epinephrine causes subsensitivity or decreased responsiveness of the beta-adrenergic pathway. There is loss of approximately half the beta-adrenergic receptors from the cornea and a similar loss of epinephrine-stimulated cyclic AMP synthesis, both of which return to control levels in 96 hrs. There is no change in affinity for catecholamines and no loss of responsiveness to prostaglandin E2 or serotonin. Pretreatment with nialamide or subsequent treatment with additional epinephrine does not cause further loss of responsiveness. Supersensitivity or increased responsiveness of this pathway occurs following superior cervical ganglionectomy. Topical serotonin causes decreased responsiveness of the serotonergic pathway. When potentiated by nialamide, serotonin causes almost complete loss of serotonin-stimulated cyclic AMP synthesis for 24-48 hrs. There is no loss of responsiveness to epinephrine. Increased responsiveness of this pathway does not occur following superior cervical ganglionectomy. The authors conclude that the corneal epithelium has both beta 2-adrenergic and serotonin-2 pathways, and each pathway exhibits altered responsiveness by similar mechanisms. In response to exogenous or endogenous stimulation, the beta-adrenergic responsive cells and the serotonergic responsive cells apparently regulate the total number of pathway-specific receptors on their surfaces. Furthermore, the authors postulate that two populations of beta-adrenergic responsive cells exist; those on the apical surface of the epithelium that respond to catecholamine in the tears and those near the basal surface that respond to neuronal catecholamine.

Effects of intravitreal cholera toxin on adenosine 3',5'-monophosphate, intraocular pressure, and outflow facility in rabbits.

Catecholamines, prostaglandins, and various hormones may influence aqueous humor dynamics via the second messenger, adenosine 3',5'-monophosphate (cyclic AMP). To test this hypothesis in rabbit ocular tissues, we have investigated the effects of cholera toxin (CTX), a specific, irreversible activator of adenylate cyclase. CTX (5 x 10(-4) to 5 x 10(2) microgram/ml) in both the presence and absence of isobutylmethylxanthine (IBMX) increased cyclic AMP production in the isolated iris-ciliary body. The effects of CTX were dependent on its concentration, duration of exposure, and presence of IBMX. Furthermore, iris-ciliary bodies and scleral-trabecular rings exercised after intravitreal injection of 10 microgram of CTX and incubated in vitro produced significantly more cyclic AMP than contralateral control tissues. Thus significant binding of CTX to both iris-ciliary body and scleral-trabecular ring occurred within 5 hr after intravitreal injection. Intraocular pressure (IOP) and outflow facility were measured by intraocular cannulation. Five hours after intravitreal injection of CTX, the IOP was lower than in control eyes. At this time, the outflow facility was threefold greater in the CTX-treated eyes than in control eyes. On the basis of these results, we conclude that (1) CTX stimulates cyclic AMP production in iris-ciliary body and scleral-trabecular ring of rabbits, (2) IOP decreases and outflow facility increases after intravitreal injection of CTX, and (3) the hypotensive effect of CTX is apparently mediated, at least partially, by outflow mechanisms.

Effects of EGF and indomethacin on rabbit corneal endothelial wound closure in excised corneas.

Corneal endothelial cells of rabbit corneas stored in M-K medium at 37 degrees C were wounded by touching them lightly with a micropipet under video specular microscope observation. Three groups were studied: control, with EGF, and with EGF + indomethacin. The wound closure process (initial wound area about 8500 microns 2) was observed and recorded with time-lapse videography for 6 hr. The recorded video images were digitized and computer assisted morphometric analysis was performed. (1) Addition of either EGF (10 ng/ml) + indomethacin (1 microM), or EGF (10 ng/ml) alone to the M-K medium statistically significantly shortened the wound closure time as compared with the control group. (2) Both EGF + indomethacin and EGF alone resulted in a greater average percent relative change of the shape factor, more than three times greater with EGF + indomethacin and more than two times greater with EGF alone, than in the control group 150 min after wounding. (3) The maximum cell shape change occurred at about 150 min after wounding in the groups EGF + indomethacin and EGF alone, and at about 200 min in the control group. After this time in all three groups the cells began to approach a normal shape. (4) The cells near the wound boundary moved faster in the EGF + indomethacin and the EGF groups as compared with the control group. These results suggest that EGF and indomethacin may be of therapeutic value in promoting closure of traumatized human corneal endothelium.