Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors.

Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.

Hormonal regulation of glycogen synthase: insulin decreases protein kinase sensitivity to cyclic AMP.

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1--2 microM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 microM. Insulin (0--100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.

Muscarinic receptors and their regulation of cyclic GMP in corneal endothelial cells.

Particulate fractions from fresh bovine corneal endothelium exhibited high affinity, specific binding by a potent muscarinic cholinergic radioligand, [3H]QNB. Particulate fraction binding sites exhibited half maximal binding at approximately 0.3 nM [3H]QNB and reached a maximal binding capacity of 820 fmoles/mg of protein at 3 nM [3H]QNB. Muscarinic cholinergic antagonists and agonists competed with [3H]QNB when incubated concurrently with the tissue, showing relative potencies expected of these agents when binding to muscarinic cholinergic receptors. Particulate fractions prepared from cultured bovine corneal endothelium exhibited qualitatively similar [3H]QNB binding characteristics, but maximal binding capacity was only about one-fifth of its fresh-tissue counterpart. Intact cultured cells showed 3-fold more specific [3H]QNB binding than did their particulate fractions. Incubation of intact corneal endothelial cells with muscarinic cholinergic agonists such as carbachol stimulated cyclic [3H]GMP 3-fold from endogenous [3H]GTP within 1 min of incubation. The effect diminished rapidly and returned to control levels within 8 min. Carbachol stimulation of cyclic [3H]GMP was concentration-dependent, reaching half maximal stimulation at 1 microM. Atropine was a potent, competitive inhibitor of carbachol stimulation of cyclic [3H]GMP in endothelial cultures, requiring only 1 microM to completely block the carbachol response. These experiments demonstrate the existence of muscarinic cholinergic receptors in bovine corneal endothelium and their control of cyclic GMP levels in this tissue.

Muscarinic cholinoceptor regulation of cyclic guanosine monophosphate in human corneal epithelium.

Cultured human corneal epithelial cells showed high-affinity, specific binding to the muscarinic cholinergic antagonist, 3H-quinuclidinyl benzilate (3H-QNB). The binding sites had a dissociation constant of 3.9 nM and a maximal binding capacity of 880 fmol bound/mg protein. Other muscarinic antagonists (cyclopentolate and atropine) effectively competed for binding with 3H-QNB at low concentrations (IC50 = 10 nM). The muscarinic cholinergic agonist carbachol also competed for binding with 3H-QNB at somewhat higher concentrations (IC50 = 0.5 microM), and the nicotinic cholinergic agonist, nicotine, was at least 400-fold less potent than carbachol. Carbachol stimulated cyclic guanosine monophosphate (GMP) levels in these cells up to threefold over control. This stimulation was sensitive to atropine inhibition, requiring only 2 nM atropine for 50% inhibition and 100 nM of atropine to block the carbachol effect completely. A 90% decrease in specific 3H-QNB binding was observed if cultured cells were homogenized and fractionated before assay. Significant levels of specific 3H-QNB binding could also be observed when intact human corneas were incubated with 3H-QNB and their epithelium subsequently isolated before measurement of bound radioligand. These studies indicate the presence of muscarinic cholinoceptors in human corneal epithelium which are associated with control of cyclic GMP levels in this tissue.

Adenylate cyclase activity in bovine and human corneal endothelium.

Adenylate cyclase activity was measured in particulate fractions prepared from bovine and human corneal endothelium. Bovine enzyme activity was stimulated by fluoride, 5'-guanylylimidodiphosphate (GppNp) and beta-adrenergic agonists. Drug stimulation patterns of the human enzyme appeared qualitatively similar, but activities were generally higher than those of the bovine enzyme. This study demonstrates the existence of a receptor-mediated adenylate cyclase system in bovine and human corneal endothelium and suggests a possible role for cycli AMP in corneal endothelial physiology.

Corneal function after storage in dexsol or optisol.

Rabbit corneas were stored in Dexsol or Optisol (Chiron, Irvine, CA) for up to 2 wk at 4 degrees C. The thickness of corneas placed in Dexsol decreased 10 microns after they were placed initially in Dexsol, then increased approximately 8 microns/d for 7 d and 3 microns/d thereafter. Corneas placed in Optisol decreased 35 microns in thickness initially, then increased 2 microns/d thereafter. Human corneas showed similar thickness changes to those of the rabbit when stored in these media. After 5.5, 10, and 14 d in storage, rabbit corneas from each medium were cultured to assess their net deturgescence ability. Identical groups were cultured in media containing 20 microM ouabain to monitor the corneas' passive swelling characteristics. Corneas stored in either medium showed similar net deturgescence and passive swelling patterns after each storage period. Deturgescence rates decreased with increasing storage time, primarily because the rates of passive corneal swelling increased with storage time. Knowledge of the net deturgescence and passive swelling rates allowed an estimation of the total deturgescence activity of corneas after removal from Dexsol or Optisol. The total deturgescence activity of corneas stored in Dexsol for 5.5, 10, and 14 d was 85%, 68%, and 63% of control corneas, which were processed identically but not stored before culture. Corneas stored in Optisol exhibited 87%, 71%, and 69% of control deturgescence activity, respectively. These experiments show that Optisol was not significantly better than Dexsol in retaining poststorage corneal deturgescence activity but was superior to Dexsol in preventing corneal swelling during storage.

Alpha 1-adrenoceptors in human corneal epithelium.

Specific binding of the potent, selective alpha 1-adrenoceptor antagonist 3H-prazosin was demonstrated in cultured human corneal epithelial cells. Specific binding of the radioligand was concentration-dependent between 0.5 and 6 nM, with apparent saturation of receptor sites seen at higher concentrations. The cells exhibited a maximum binding capacity for 3H-prazosin of 225 fmol/mg of cellular protein and a dissociation constant of 2 nM. The binding of 3H-prazosin was competitive with known alpha 1-adrenoceptor ligands and was reversible. Epithelium of intact human corneas also exhibited specific 3H-prazosin binding, as did cultures of bovine and rabbit corneal epithelium. The alpha-adrenergic agonist methoxamine significantly stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, measured as myoinositol trisphosphate accumulation in cultures of human corneal epithelium. This stimulation was inhibited by the presence of prazosin during the assays. These findings indicate the existence of specific, reversible, high-affinity receptors for alpha 1-adrenoceptors that regulate inositol phosphate turnover in human, rabbit, and bovine corneal epithelial cells.

Corneal function after storage in commercial eye bank media.

Rabbit corneas were stored in commercially prepared media (K-Sol, M-K, CSM, or Dexsol) for 3, 6, 9, or 12 days. Corneas stored in each medium showed decreased post-storage deturgescence with increased storage time. This effect was most pronounced in corneas stored in K-Sol, followed by those in M-K, CSM, and Dexsol. Corneas stored in K-Sol also exhibited the fastest swelling rate when cultured in the presence of 20 microM ouabain, followed by those in M-K, Dexsol, and CSM. An estimate of the active ion transport capacity of each experimental group was made by determining the area between each group's corneal thickness profiles in the presence and absence of ouabain. Corneas stored in M-K, K-Sol, or CSM retained approximately 70% of control activity after 3 days storage and 65% after 6 days storage. Corneas stored in Dexsol had 92% of control activity after 3 days of storage, 78% after 6 days, and 44% after 12 days.

Characterization of adenylate cyclase activity in bovine and human corneal epithelium.

The properties of adenylate cyclase from bovine and human corneal epithelium were investigated. Adrenergic drugs were the most effective stimulatory agents tested in bovine tissue, causing greater activation than did fluoride. Isoproterenol was the most potent agonist, followed by epinephrine and norepinephrine. Phenylephrine and dopamine also stimulated adenylate cyclase through beta-adrenergic receptors at relatively high concentrations. Enzyme stimulation by all the adrenergic drugs tested was completely inhibited by 1 microM propranolol or 0.1 microM timolol. The GTP analogue, GppNp, produced considerable activation and caused an augmented response when combined with isoproterenol, but not with fluoride. Prostaglandins E1, E2, or F2 alpha produced a small but significant stimulation over control which was not sensitive to propranolol inhibition. Adenylate cyclase from human corneal epithelium exhibited qualitatively similar characteristics to those of the bovine enzyme. Fluoride was the most effective stimulatory agent, followed by isoproterenol, phenylephrine, and dopamine. Prostaglandins failed to stimulate adenylate cyclase activity in human corneal epithelial preparations.

Atrial natriuretic peptide receptors on the corneal endothelium.

PURPOSE: To ascertain whether the corneal endothelium exhibits specific receptors for atrial natriuretic peptide (ANP) and to characterize partially any binding activity. METHODS: Most experiments employed radioligand-binding techniques to characterize 125I-ANP binding activity. Guanylate cyclase and corneal deturgescence assays were used in an attempt to correlate 125I-ANP binding activity with physiologic processes. RESULTS: Corneal endothelial cells reversibly bound 125I-ANP with high affinity and exhibited a finite number of 125I-ANP binding sites. These binding sites also bound several ANP fragments but did not show binding affinity for the peptide hormone, vasopressin. Autoradiograms of affinity labeled ANP receptors separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed specifically radiolabeled proteins of several molecular weights in nonreduced samples, but only one major radiolabeled protein of 65 kd when samples were chemically reduced before separation. CONCLUSIONS: Corneal endothelial cells from several species exhibit specific binding of 125I-ANP. The binding characteristics of these receptors are similar to physiologic ANP receptors identified in other tissues. Several lines of evidence indicate that corneal endothelial ANP receptors are the "clearance" (ANP-C) type.

Effect of cyclic AMP on collagen production by corneal fibroblasts.

Subcultured bovine corneal fibroblasts accumulated cyclic AMP intracellularly and in the culture media in response to incubation with 3-isobutyl-1-methylxanthine, isoproterenol, cholera toxin or forskolin. The duration of the intracellular cyclic AMP effect was rather short using isoproterenol (less than three hours) but cholera toxin and forskolin maintained cyclic AMP stimulations for 24 hours at greater than 3-fold and 27-fold over control, respectively. Agents which stimulated adenylate cyclase by different mechanisms or mimicked the action of cyclic AMP decreased collagen secretion by these cells. In general, the degree of inhibition of collagen production was consistent with the magnitude and duration of each drug's cyclic AMP response.

Corneal deturgescence during organ culture.

A simple method is described to monitor rabbit and human corneal deturgescence while the tissue is suspended in tissue culture media. The composition of the media is such that the cornea slowly thickens at 4 degrees but exhibits the classic "temperature reversal" thinning phenomenon when incubated at 23 degrees-37 degrees. Each cornea is cultured in a closed eye bank corneal viewing chamber during swelling and deswelling phases of an experiment, minimizing direct handling of the tissue. Optimal conditions for observing corneal deturgescence are described. This protocol has several advantages over corneal perfusion techniques and could be further developed for routine use in eye banks as a functional test of donor suitability for keratoplasty.

The effects of UW solution and its components on corneal thickness during and after storage.

The ability of rabbit corneas to undergo energy-dependent deturgescence was examined after the corneas were stored at 4 degrees C in UW solution, M-K media, or selected modifications of these media. All corneas slowly increased in thickness during storage, despite the presence of colloidal osmotic agents. Corneas stored for 2.5 days in M-K became slightly thinner when cultured over a 24-hour period. Corneas stored in UW swelled quickly in culture and became too opaque to measure within three hours. Corneas stored in UW with 1.8 mM CaCl2 swelled transiently, then maintained their thickness and exhibited deturgescence in the latter stages of the culture period. Deturgescence of corneas stored for 7 days in M-K was only slightly worse than those stored for 2.5 days. Corneas stored for 7 days in UW, however, became opaque almost immediately in culture and those stored in calcium-supplemented UW became opaque within 4.5 hours. Replacement of the dextran in M-K with hydroxyethyl starch produced a slower rate of corneal swelling during storage and a substantially better corneal deturgescence profile during culture. Use of high concentrations of potassium ion in the M-K formulation had no significant effect on post-storage deturgescence. Replacement of glucose in M-K with the impermeable sugar, raffinose had a slight deleterious effect on corneal deturgescence in subsequent culture. Use of the impermeable anions gluconate or lactobionate to replace chloride ion caused profound corneal swelling during culture, compared with those stored in M-K. These experiments show that UW solution is inferior to M-K at preserving post-storage corneal function.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic cholinoceptors in native and cultured human corneal endothelium.

Specific and high affinity binding of the potent muscarinic cholinergic antagonist, [3H]quinuclidinylbenzilate ([3H]QNB) was observed using intact native and cultured adult human corneal endothelium (HCE). Specific binding was proportional to radioligand concentration between 0.03 and 5 nM, indicating a maximal binding capacity (Bmax) of 130 fmol of [3H]QNB/mg protein and a dissociation constant (Kd) of 0.3 nM. Atropine competed effectively with [3H]QNB for binding sites; requiring 3 nM to inhibit 50% of the binding of 1 nM [3H]QNB. Carbachol also competed with [3H]QNB at higher concentrations, but nicotine did not affect [3H]QNB binding at levels up to 1 nM. [3H]QNB binding was also observed in cultured cells of adult human, rabbit, and bovine corneal endothelium. Native and cultured HCE were affinity labelled using tritium-labelled propylbenzilylcholine mustard (PBCM). Separation of the proteins in affinity labelled native and cultured tissue by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that only one protein in each preparation, of 60 and 55 kilodaltons (kDa), respectively, was specifically radiolabelled. These data indicate that the corneal endothelium of human and several animal species exhibit muscarinic cholinoceptors.

Desensitization of beta-adrenergic receptors in corneal endothelium.

Corneal endothelial cells in primary culture responded to isoproterenol (Iso), epinephrine (Epi), nor-epinephrine (NE), Prostaglandins E1 and E2 (PGE1, PGE2) and forskolin by increasing cyclic AMP 61, 52, 28, 14, 10, and 176-fold, respectively, over control within a 10 minute incubation period. However, when cells were preincubated with Iso for one hour, they lost 76% of their responsiveness to a subsequent Iso challenge. Iso preincubation also decreased the effectiveness of other beta-adrenergic agonists (Epi and NE), but responses to PGE1, PGE2 and forskolin remained unchanged. The decreased cyclic AMP response was associated with a loss of beta-adrenergic binding sites. The affinity of the remaining receptors was unchanged as determined by propranolol displacement of [125I] pindolol binding. This study shows that corneal endothelial cells not only respond to beta-adrenergic stimuli, but can also regulate the magnitude and duration of their response to the hormone.

Adenosine regulation of cyclic AMP in corneal endothelium.

Adenosine receptor agonists increased cyclic AMP in cultures of bovine corneal endothelium up to 12-fold over control. Effects were dose-dependent between 1 microM and 0.5 mM adenosine. N6-methyladenosine produced a greater maximal effect but was approximately 10-fold less potent. Isoproterenol and N6-methyladenosine produced an additive response. Propranolol blocked the isoproterenol, but not the adenosine effect on cyclic AMP. Adenine-9-beta-D-arabinofuranoside had no effect on cyclic AMP alone, but inhibited stimulations by N6-methyladenosine, isoproterenol and forskolin. These data indicate the presence of specific adenosine receptors which stimulate adenylate cyclase in cultured bovine corneal endothelium.

Reversible inhibition of cyclic AMP-dependent protein kinase by insulin.

Extracts of fasted rat diaphragms, previously treated with or without insulin were assayed for glycogen synthase, protein kinase and cyclic [3H]-AMP binding. Treatment with insulin produced an elevation in the % of glycogen synthase I and a concurrent decrease in cyclic AMP-dependent protein kinase activity and cyclic [3H]-AMP binding. Analysis of extracts by disc gel electrophoresis demonstrated the inhibition of cyclic [3H]-AMP binding to involve the Type I protein kinase holoenzyme. Inhibition of protein kinase activity was most apparent in the presence of 0.2 micrometer cyclic AMP, with enzymatic activity of the insulin-treated extracts typically 60--65% of control. Higher assay concentrations diminished the difference between control and insulin-treated extracts and concentrations greater than 20 micrometer abolished it. The inhibition of cyclic AMP-dependent protein kinase activity after insulin was a transient and labile phenomenon. The effect was independent of ATP concentration in the assay, but was sensitive to the pH of tissue extraction, requiring a pH of 7.0 to 8.4 to be observed. Insulin-mediated inhibition of protein kinase activity was reversed upon preincubation of extracts at 0--2 degrees. Relatively concentrated homogenates (less than 4 microliter buffer/mg tissue) yielded extracts which exhibited little or no inhibition of protein kinase activity compared to extracts prepared from more dilute (6--10 microliter/mg) homogenates. A model for the inhibition of the cyclic-AMP dependent protein kinase by an insulin-generated inhibitor which becomes directly associated with the Type 1 holoenzyme is proposed.

Characterization of beta-adrenergic receptors in fresh and primary cultured bovine corneal endothelium.

Homogenates of fresh bovine corneal endothelium and of cells from primary cultures exhibited high affinity binding of [3H]-dihydroalprenolol, a specific beta-adrenergic antagonist. The binding was rapid and reversible. Specific binding of the radioligand in each preparation was saturable with half-maximal binding occurring at 0.5 nM. Homogenates of fresh tissue consistently showed a higher maximal binding capacity than did those from cell cultures. Both homogenates bound adrenergic agents in a manner consistent with the labelling of beta-adrenergic receptors. The relative affinities of epinephrine and norepinephrine indicated that beta-adrenergic receptors in both preparations are of the beta 2 subtype. The beta-adrenergic agonist, isoproterenol, increased the cAMP content of intact, cultured endothelial cells 27-fold over control. This effect was completely blocked by the beta-adrenergic antagonist, propranolol. Bovine corneal endothelial cells in primary culture contain beta-adrenergic receptors which are essentially identical to those from in vivo sources and appear to be linked functionally to cAMP synthesis in these cells.

Characteristics of beta-adrenergic receptors in bovine corneal epithelium: comparison of fresh tissue and cultured cells.

The characteristics of the beta-adrenergic receptors in homogenates of fresh tissue and cultured bovine corneal epithelium were compared using [3H]dihydroalprenolol. High affinity, specific binding sites were observed in both preparations. Fresh tissue exhibited a higher binding site density (165 fmol/mg protein) than did cells in culture (57 fmol/mg protein). Studies with various beta-adrenergic agonists and antagonists indicated that binding characteristics were typical of beta-adrenergic receptors, predominantly of the beta 2 subtype. These results demonstrate that beta-adrenergic receptors exist in both fresh and cultured bovine corneal epithelium and that these receptors are qualitatively and quantitatively similar.

Alpha 1-adrenoceptors in the corneal endothelium.

The potent alpha 1-adrenoceptor antagonist, [3H]prazosin, exhibited high affinity, specific and reversible binding to intact rabbit, bovine and human corneal endothelial cells in culture. The binding of 1 nM [3H]prazosin to rabbit cells reached a steady-state level within 10 min at 37 degrees C. Under these conditions, approximately 50% of the [3H]prazosin bound was specific. The level of specific [3H]prazosin binding was concentration-dependent, but Rosenthal analysis indicated that [3H]prazosin bound to at least two sites. One site exhibited a high affinity for [3H]prazosin (Kd = 0.2 nM), but a relatively low binding capacity (Bmax = 175 fmol bound mg-1 protein); the other site showed a relatively low affinity for the radioligand (Kd = 85 nM), but a much higher binding capacity (1280 fmol mg-1). Several known alpha 1-adrenoceptor antagonists and agonists competitively inhibited [3H]prazosin binding at the high affinity site when incubated with the radioligand. The relative potencies of these competing ligands were generally consistent with their binding affinities for alpha 1-adrenoceptors in other tissues. Phenylephrine stimulated the rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate by 63% in these cells. This stimulation was inhibited by 52% if phentolamine was also present during the incubation. These data indicate that corneal endothelial cells have alpha 1-adrenoceptors which can modulate polyphosphoinositide turnover in this tissue.