Characterization of M1- and M2-muscarinic receptors in calf retina membranes

Muscarinic receptors are identified in bovine retina membranes by the specific binding of 1-[benzilic-4,4'-3H]-quinuclidinyl benzilate [3H]-QNB. Binding occurs to one population of non-cooperative binding sites: KD = 0.11 +/- 0.02 nM and Bmax = 0.61 +/- 0.07 pmol/mg protein. Competition binding curves of the M1-selective antagonist pirenzepine are shallow. Computer-analysis reveals the presence of 45 +/- 1% M1-receptors (high affinity sites for pirenzepine, Ki = 31 +/- 10 nM). The remaining low affinity sites (Ki = 1.0 +/- 0.3 microM) are denoted as M2-receptors. Competition binding curves with the agonist carbachol are shallow as well. 1 mM GTP causes a rightward shift and a steepening of the carbachol curve, whereas 1 mM N-ethylmaleimide (NEM) provokes a leftward shift and also a steepening of the curve. The GTP effect is abolished by NEM. Binding of the antagonists [3H]-QNB, atropine or pirenzepine is not modulated by GTP nor by NEM.     

GABA(A) receptor binding and localization in the tiger salamander retina

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photoreceptor development and regeneration. Although the tiger salamander is a major model system for the study of retinal circuitry and regeneration, our understanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharmacological binding properties of GABA(A) receptors and their anatomical localization in the tiger salamander retina. Radioligand-binding studies showed that specific 3H-GABA binding to GABA(A) receptors was dominated by a single high-affinity binding site (Kd = 15.6+/-6.9 nM). Specific binding of 3H-GABA was almost completely eliminated by muscimol (Ki = 105+/-62 nM) and bicuculline (Ki = 14.3+/-2.2 microM); however, SR-95531 only displaced about 40% of specific 3H-GABA binding (Ki = 35.0+/-3.8 nM). These data indicate that there are at least two subtypes of GABA(A) receptors present in the salamander retina that can be distinguished by their antagonist binding properties: one sensitive to both bicuculline and SR-95531, and one sensitive to bicuculline but insensitive to SR-95531. Because localization of GABA receptors in the salamander retina by immunocytochemistry is problematic, GABA(A) receptors were localized by fluorescent ligand binding combined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA(A) receptors was present in both plexiform layers and on photoreceptor cell bodies. GABA(A) receptors in the outer plexiform layer were localized to both photoreceptor terminals and horizontal cell processes.     

Characterization of adenosine A1-receptor binding sites in bovine retinal membranes

Using retinas prepared from freshly dissected bovine eyes, we have characterized the binding of the A1-selective agonist, [3H]PIA (N6-R-[3H](2-phenylisopropyl)adenosine). Specific binding was linear over a range of membrane protein concentrations from 0.10 to 1.0 mg, and accounted for an average of 80-90% of the total binding. At room temperature (24 degrees C), binding reached equilibrium at 60 min, and was reversible upon addition of an excess of cold ligand. Saturation analysis and Scatchard transformation revealed two apparent populations of receptor binding sites. The higher affinity site exhibited a Kd of 0.134 +/- 0.007 nM and Bmax of 26.18 +/- 3.06 fmol-1 mg protein. The lower affinity site exhibited a Kd of 21.83 +/- 4.39 nM and Bmax of 53.94 +/- 15.80 fmol mg-1 protein. Kinetic analysis of association and dissociation rates, performed at a low concentration of [3H]PIA, yielded a calculated affinity constant for the high affinity site of 0.2 nM, in agreement with saturation studies. Competition experiments with a number of purine nucleoside agonists and antagonists were performed, using radioligand concentrations of 1 nM or less to examine binding at the high affinity site, and revealed a rank order of potency consistent with the reported pharmacology of A1 receptors. We have also assayed for adenylate cyclase activity in this same preparation and determined that PIA inhibited forskolin-activated adenylate cyclase in a dose-dependent manner. Maximum inhibition (40%) was observed with 1 nM PIA, while 10 microM 8-cyclopentyl-1,3-dipropylxanthine, an A1 selective antagonist, completely inhibited this modulation by PIA.     

Pharmacological characterization of [3H]-Ifenprodil binding to polyamine binding sites on rabbit and rat retinal homogenates: role in neuroprotection?

Polyamine binding sites (PBS) represent one of the modulatory sites on the N-methyl-D-aspartate (NMDA) receptor-channel complex. We have characterized [3H]-ifenprodil binding to the PBS on washed homogenates of rabbit and rat retinas. Specific binding of [3H]-ifenprodil (2 nM) (in the presence of 3 microM 1,3-Di [2-tolyl] guanidine HCl and 10 microM GBR12909 to block sigma sites) comprised 47-56% of the total binding. Scatchard analyses indicated interaction with apparent high- and low-affinity sites: dissociation constants (K(d)s) = 0.5-0.6 microM and apparent density of sites (Bmax) = 1.5-4.3 pmol/mg protein and K(d)s = 2.0-2.9 microM, and Bmax values = 15.8-17.8 pmol/mg protein (n = 3). Ifenprodil (Ki = 0.4-0.8 microM), eliprodil (Ki = 0.7-0.8 microM), spermine (Ki = 72-79 microM), spermidine (Ki = 283-330 microM), putrescine (Ki > 650 microM) and MK-801 (Ki > 1 mM) (n = 3-5) differentially competed for [3H]-ifenprodil binding. The biphasic competition curves for ifenprodil were resolved into two binding components: rat retinas, IC50high = 0.19 +/- 0.13 microM and IC50low = 8.7 +/- 1.3 microM; rabbit retinas, IC50high = 0.1 +/- 0.01 microM and IC50low = 16.0 +/- 7.8 microM. These studies have shown the presence of specific PBS labeled by [3H]-ifenprodil in the rabbit and rat retinas which may, in part, be responsible for mediating the neuroprotective effects of eliprodil and ifenprodil.     

Specific receptor sites for PAF in iris and ciliary body of the rabbit eye

The existence of specific binding sites by using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF) was investigated on iris and ciliary body tissue of pigmented rabbit eyes. The binding was saturable, specific, time-dependent and reversible. Scatchard's analysis indicated the presence of two types of binding sites with a Kd1 4.90 +/- 0.47 nM, a Kd2 11.60 +/- 0.33 nM, a Bmax1 3.17 +/- 0.50 pmol/mg protein and a Bmax2 12.45 +/- 2.30 pmol/mg protein for iris tissue, and a Kd1 5.71 +/- 0.09 nM, a K2d 24.40 +/- 0.91 nM, a Bmax1 3.41 +/- 1.00 pmol/mg protein and a Bmax2 16.60 +/- 0.51 pmol/mg protein for ciliary body. The binding was fully displaced by unlabelled PAF in both iris and ciliary body preparations, and partially inhibited by the PAF antagonist BN 52021 in iris tissue.     

Ocular hypotensive FP prostaglandin (PG) analogs: PG receptor subtype binding affinities and selectivities, and agonist potencies at FP and other PG receptors in cultured cells.

Natural prostaglandins (PGs) such as PGD2, PGE2, PGF2(2alpha), and PGI2 exhibited the highest affinity for their respective cognate receptors, but were the least selective agents when tested in receptor binding assays. Travoprost acid ([+]-fluprostenol) was the most FP-receptor-selective compound, exhibiting a high affinity (Ki = 35 +/- 5 nM) for the FP receptor, and minimal affinity for DP (Ki = 52,000 nM), EP1 (Ki = 9540 nM), EP3 (Ki = 3501 nM), EP4 (Ki = 41,000 nM), IP (Ki > 90,000 nM), and TP (Ki = 121,000 nM) receptors. Travoprost acid was the most potent PG analog tested in FP receptor functional phosphoinositide turnover assays in the following cell types: human ciliary muscle (EC50 = 1.4 nM), human trabecular meshwork (EC50 = 3.6 nM), and mouse fibroblasts and rat aortic smooth muscle cells (EC50 = 2.6 nM). Although latanoprost acid exhibited a relatively high affinity for the FP receptor (Ki = 98 nM), it had significant functional activity at FP (EC50 = 32-124 nM) and EP1 (EC50 = 119 nM) receptors. Bimatoprost acid was less selective, exhibiting a relatively high affinity for the FP (Ki = 83 nM), EP1 (Ki = 95 nM), and EP3 (Ki = 387 nM) receptors. Bimatoprost acid exhibited functional activity at the EP1 (EC50 = 2.7 nM) and FP (EC50 = 2.8-3.8 nM in most cells) receptors. Bimatoprost (nonhydrolyzed amide) also behaved as an FP agonist at the cloned human FP receptor (EC50 = 681 nM), in h-TM (EC50 = 3245 nM) and other cell types. Unoprostone and S-1033 bound with low affinity (Ki = 5.9 microM to > 22 microM) to the FP receptor, were not selective, but activated the FP receptor. In conclusion, travoprost acid has the highest affinity, the highest FP-receptor-selectivity, and the highest potency at the FP receptor as compared to the other ocular hypotensive PG analogs known so far, including free acids of latanoprost, bimatoprost, and unoprostone isopropyl ester.     

Levobetaxolol (Betaxon) and other beta-adrenergic antagonists: preclinical pharmacology, IOP-lowering activity and sites of action in human eyes.

The pharmacological characteristics of levobetaxolol, a single active isomer of betaxolol, were determined and compared with activities of other beta-adrenoceptor antagonists. Levobetaxolol (43-fold beta1-selective) exhibited a higher affinity at cloned human beta1 (Ki = 0.76 nM) than at beta2 (Ki = 32.6 nM) receptors, while dextrobetaxolol was much weaker at both receptors. Levobetaxolol potently antagonized functional activities at cloned human beta1 and beta2 receptors, and also at guinea pig atrial beta1, tracheal beta2 and rat colonic beta3 receptors (IC50s = 33.2 nM, 2970 nM and 709 nM, respectively). Thus, levobetaxolol was 89-times beta1-selective (vs beta2). Levobetaxolol (Ki = 16.4 nM) was more potent than dextrobetaxolol (Ki = 2.97 microM) at inhibiting isoproterenol-induced cAMP production in human non-pigmented ciliary epithelial cells. Levobunolol and (l)-timolol had high affinities at beta1 and beta2 receptors but were considerably less beta1-selective than levobetaxolol. Levo-, dextro- and racemic-betaxolol exhibited little or no affinity, except at sigma sites and Ca2+-channels (IC50s > 1 microM), at 89 other receptor/ligand binding sites. Levobetaxolol exhibited a micromolar affinity for L-type Ca2+-channels. In conscious ocular hypertensive cynomolgus monkeys, levobetaxolol was more potent than dextrobetaxolol, reducing intraocular pressure by 25.9+/-3.2% at a dose of 150 microg/eye (n = 15-30). Quantitative [3H]-levobetaxolol autoradiography revealed high levels of binding to human ciliary processes, iris, choroid/retina, and ciliary muscles. In conclusion, levobetaxolol is a potent, high affinity and beta1-selective IOP-lowering beta-adrenoceptor antagonist.     

Effects of carbamylcholine on nuclear cyclic nucleotide-dependent protein kinase activity in cultured corneal epithelial cells of the rabbit

Cyclic nucleotide-dependent protein kinase activity and cyclic nucleotide binding were assayed in cell homogenates and subcellular fractions of cultured rabbit corneal epithelium, and effects of carbamylcholine (1 mM) on activity in each fraction were evaluated. In cell homogenates and in nuclei, carbamylcholine significantly elevated cGMP-dependent kinase activity and [3H]cGMP binding, and reduced cAMP-dependent kinase activity and [3H]cAMP binding. In the cytosol, the drug significantly reduced cAMP kinase and cAMP binding but did not alter cGMP binding or kinase activity. In both mitochondria/lysosomes and microsomes, cGMP binding was significantly enhanced and cAMP binding reduced, but differences in protein kinase activity were not significant. The drug did not alter cyclic nucleotide-independent protein kinase activity. All observed effects were blocked by atropine. Kinase activity in the purified nuclear fraction also was assayed over a range of carbamylcholine, substrate, and cyclic nucleotide concentrations. Vmax for cGMP kinase in control nuclei was 295 +/- 18 pmol/mg protein/min (KM = 367 +/- 70 micrograms/ml histone) vs. Vmax = 846 +/- 6 pmol/mg/min (KM = 131 +/- 13 mu/ml histone) in carbamylcholine-treated nuclei. Vmax for cAMP kinase in control nuclei was 282 +/- 12 pmol/mg/min (KM = 172 +/- 7 micrograms/ml histone); 1 mM carbamylcholine abolished nuclear cAMP-dependent kinase activity. Cyclic nucleotide concentrations at half-maximal binding were 5.4 +/- 0.9 nM cGMP and 9.3 +/- 0.4 nM cAMP, as compared to 15.4 +/- 5.0 nM cGMP and 36.2 +/- 6.2 nM cAMP for half-maximal protein kinase activity. Regression analysis of Hill plots for variation of nuclear cyclic nucleotide binding and kinase activity as a function of carbamylcholine concentration indicated half-maximal drug effects on activity of both enzymes at approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative autoradiographic visualization and pharmacology of FP-prostaglandin receptors in human eyes using the novel phosphor-imaging technology.

Quantitative autoradiographic visualization of FP prostaglandin receptors was determined in postmortem human eye sections using [3H]PGF2alpha and the novel phosphor-imaging technology coupled with computerized image analysis. Densitometric analysis of digital images obtained after a 19-day screen-exposure revealed FP receptors to be highly concentrated in the longitudinal ciliary muscle (12,741 digital light units [DLU/mm2]), iris sphincter muscle (19,261 DLU/mm2) and retina (9,544 DLU/mm2), with lesser amounts (1,558-3,776 DLU/mm2) in five other ocular structures (n = 3-4 donors). The highest percentage specific binding was in the iris sphincter and longitudinal ciliary muscle (78-73%) and lowest in the lens (12%). Binding of [3H]PGF2alpha to the longitudinal ciliary and iris sphincter muscles and retina was concentration-dependently and potently displaced by known FP-receptor-selective compounds such as cloprostenol (Ki = 13-37 nM), fluprostenol (Ki = 56-98 nM), PHXA85 (Ki = 181-206 nM) and latanoprost (isopropyl ester of PHXA85; Ki = 0.43-4.5 microM) (from up to 4 donor eyes). These quantitative phosphor-imaging autoradiography data provide further evidence for the presence of FP receptors in human longitudinal ciliary muscle, one of the tissues involved in the intraocular pressure lowering effects of FP-class prostaglandins. The quantitative localization of FP receptors in the human iris, iris sphincter muscle, and retina represent interesting new observations.     

Taurine binding by rat retinal membranes

Retinal membrane preparations contain endogenous taurine which is difficult to remove. By repeatedly washing the membranes in buffer the taurine content was reduced from 11.7 +/- 2.5 to 2.5 +/- 0.5 nmol/mg protein. However, complete elimination of the endogenous taurine from the membrane preparation was not achieved. Binding of [3H]-taurine to rat retinal membrane preparations revealed both high and low affinity binding sites. A Hill coefficient of 0.71 suggested that cooperativity may be involved in the taurine binding process. Taurine binding was sodium dependent with maximum binding achieved at 118 mM. At suboptimal concentrations of sodium ions (30 mM) only one binding site was observed which appears to be the high affinity binding site. Analogues of taurine were tested for their effectiveness in displacing taurine from its binding site. Hypotaurine, beta-alanine, gamma-aminobutyric acid, and guanidinoethanesulfonic acid were the most potent displacers.     

Insulin and IGF-1 binding in chick sclera

The sclera of embryonic (days 10 and 14) and young adult (2-week posthatching chicks) contains distinct binding sites for insulin and for insulin-like growth factor-1 (IGF-1). Since there is a nearly 50% decrease in insulin and IGF-1 binding between embryonic day 10 and the 2nd week posthatching, it is clear that these sites are developmentally regulated. The affinity of each binding site for its ligand is stable across development. This suggests that the developmental decrease in binding is the result of a decrease in the number of binding sites. The insulin binding site in the sclera is specific for insulin since it has a high affinity for insulin and a lower affinity for IGF-1 (IC50 for unlabeled insulin = 0.4 nM; unlabeled IGF-1 = 5.0 nM). The embryonic chick sclera also contains two high-affinity IGF-1 binding sites. One of these sites exhibits poor binding specificity since it has an equal affinity for insulin and IGF-1. However, the specificity of this site increases in the young adult. The second IGF-1 binding site exhibits a more conventional specificity in that it has a higher affinity for IGF-1 than for insulin. The specificity of this binding site also improves in the young adult. The presence of insulin and IGF-1 receptor binding site subtypes is not correlated with structurally different receptor binding subunits since only a single population of binding subunits is observed (apparent molecular weight of 125 +/- 2.7 kD) in embryonic and adult sclera.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding affinities of ocular hypotensive beta-blockers levobetaxolol, levobunolol, and timolol at endogenous guinea pig beta-adrenoceptors.

The current study determined the relative affinities and selectivities of numerous beta-adrenoceptor antagonists at the endogenous beta(1)- and beta(2)-adrenoceptors in guinea pig heart and lung, respectively, using [(3)H]-CGP12177. Specific binding of [(3)H]-CGP12177 comprised 80 +/- 0.2% (n = 11) and 94 +/- 0.2% (n = 16) of the total binding in washed heart and lung homogenates, respectively. Concentration-dependent displacement of [(3)H]-CGP12177 binding from beta-adrenoceptors in both preparations was observed with nine different beta-adrenoceptor antagonists. Levobetaxolol, betaxolol, CGP-20712A, levobunolol, and timolol yielded bi-phasic (two-site-fit) competition curves in the heart, while CGP-20712A, ICI-118551 and levobunolol produced bi-phasic curves in the lung preparation. The high-affinity component of [(3)H]-CGP12177 binding in the heart and lung reflected binding to beta(1)-receptors and beta(2)-receptors, respectively. The binding inhibition parameters (IC(50)s) for displacement of [(3)H]-CGP12177 from these predominantly high-affinity sites were: levobetaxolol (24.9 +/- 1.6 nM heart, 4810 +/- 367 nM lung), racemic betaxolol (37.9 +/- 8.7 mM heart; 8840 +/- 424 mM lung), CGP-20712A (4.6 +/- 0.9 nM heart; 171,000 +/- 109,000 nM lung), ICI-118551 (9230 +/- 3240 nM heart; 2.9 +/- 0.6 nM lung), levobunolol (42 +/- 15 nM heart, 0.3 +/- 0.2 nM lung), (l)-timolol (3.1 nM heart, 2.9 +/- 1.5 nM lung), ICI-215001 (5840 +/- 114 nM heart; 26100 +/- 3200 nM lung), BRL-37344 (83,300 +/- 2660 nM heart; 13,200 +/- 1250 lung). These data indicated that while levobetaxolol and betaxolol possessed a 193-233-fold selectivity for beta(1)-receptors, levobunolol exhibited a 140-fold beta(2)-receptor selectivity and (l)-timolol was essentially nonselective.     

Characterization of serotonin receptors in the iris + ciliary body of the albino rabbit

Experiments were undertaken to determine if serotonin (5-HT) radioligand binding sites were present in a membrane fraction of iris + ciliary body from adult, albino rabbits. The total binding of 3H-5-HT, 3H-spiroperidol (SPI) and 3H-ketanserin (KET), all at 2 nM, was determined in the absence and in the presence of ketanserin, mianserin, methysergide or 5-methoxytryptamine (5-MT), all at 1 microM. Except for ketanserin, which displaced 15% of 3H-KET binding, none of the agents altered 3H-SPI or 3H-KET binding. Reductions of 12% and 14% in 3H-5-HT binding were achieved by ketanserin and mianserin, respectively. In contrast, methysergide and 5-MT displaced 3H-5-HT binding by 57% and 56%, respectively. 5-HT (1 microM) also displaced 3H-5-HT binding by approximately 60% and this was used to measure nonspecific binding. Specific binding of 3H-5-HT was saturable and of high affinity with one population of binding sites being labelled. Kd and Bmax values of 1.1 nM and 57.8 fmoles/mg protein were obtained. Seven 5-HT antagonists possessing various affinities for 5-HT1 and 5-HT2 binding sites were assessed for their ability to displace specific 3H-5-HT binding. Ketanserin was the least potent (Ki greater than 3 microM). In contrast, the respective Ki values for metergoline and methysergide were 13 nM and 20 nM. These observations indicate the presence of 5-HT1 binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-mediated phosphoinositide hydrolysis in human ocular ciliary epithelial cells

The hydrolysis of phosphoinositides (PI) in peripheral tissues can be stimulated by a number of putative neurotransmitters and this stimulation can be blocked by specific antagonists. Epithelial cells derived from the nonpigmented layer of the ocular ciliary epithelium were transfected by simian virus 40 and grown in culture to semiconfluency. The cells were incubated in 3 microCi/ml of (3H)-myoinositol for 2 days. The accumulation of inositol phosphates in response to several agonists (carbachol, 1 mM; ATP, 100 microM; arginine vasopressin, 1 microM; and phenylephrine, 100 microM) was determined for times ranging from 5 sec to 15 min. In the presence of 10 mM LiCl, the maximum net production of the (3H)-inositol phosphates (expressed as a percent of conversion of (3H)-phospholipids) was approximately 7.5% for inositol-1 phosphate, 0.5% for inositol-1,4 bisphosphate, and 1% for inositol-1,4,5 trisphosphate. Carbachol elicited PI hydrolysis with an EC50 value of 39 +/- 9 microM. The EC50 values obtained for arginine vasopressin and ATP-initiated PI breakdown were 32 +/- 10 nM and 11.9 +/- 1 microM, respectively. Phenylephrine alone failed to stimulate the production of (3H)-inositol phosphates in these cells. The production of all (3H)-inositol phosphates in response to carbachol (1 mM) was inhibited by atropine (Ki = 0.3 nM) and the selective muscarinic antagonists 4-DAMP (Ki = 4.2 nM), pirenzepine (Ki = 102 nM) and AFDX-116 (Ki = 1.49 microM). Thus the muscarinic receptors that are coupled to PI hydrolysis in these cells have the pharmacologic characteristics of the M3 subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E2 binding sites in bovine iris-ciliary body

In the eye, prostaglandins (PGs), in particular PGE2 and PGF2 alpha, may induce vasodilation, disruption of the blood-aqueous barrier, and biphasic effects on intraocular pressure, depending on the species. The initial event leading to many of these physiologic responses is the interaction between the PG and a receptor. We have explored the specificity and selectivity of PGE2 receptors in bovine iris-ciliary body (ICB) membrane preparations. Pigment-free bovine ICB membranes were prepared by high-speed sucrose density-gradient centrifugation. Membranes were incubated with 1 nM 3H-PGE2 in the presence or absence of varying concentrations of unlabeled PGE2 or F2 alpha. Binding of 3H-PGE2 to membranes at 37 degrees C increased linearly with protein concentration, and binding reached equilibrium in 30 min. Specific PGE2 binding represented 80% of total 3H-PGE2 binding. Studies with unlabeled PGE2 or F2 alpha, as competing ligands, showed a dose-dependent inhibition of 3H-PGE2 specific binding. The IC50 for unlabeled PGE2 and F2 alpha was 3 and 379 nM, respectively, which suggests a 100-fold greater selectivity of the binding sites for PGE2 over F2 alpha. Scatchard analysis of saturation data revealed a mean Kd value of 13.3 nM with a Bmax of 156 fmoles bound/mg protein. The general linearity of our Scatchard plots tends to suggests a single class of binding sites for PGE2, although more than a single binding site could be present. These results indicate that binding sites selective for PGE2 exist in the bovine ICB.     

Agonist activity of bimatoprost, travoprost, latanoprost, unoprostone isopropyl ester and other prostaglandin analogs at the cloned human ciliary body FP prostaglandin receptor.

We have determined the agonist activity of a number of natural prostaglandins and prostaglandin analogs at the FP prostaglandin receptor cloned from a human ciliary body cDNA library using phosphoinositide (PI) turnover assays. Travoprost acid (EC50 = 3.2 +/- 0.6 nM) was the most potent agonist in these cells followed by bimatoprost free acid (17-phenyl-trinor PGF2alpha; EC50 = 5.8 +/- 2.6 nM), fluprostenol (EC50 = 6.1 +/- 1.5 nM), and latanoprost free acid (PHXA85; EC50 = 54.6 +/- 12.4 nM) which was 17-fold weaker (p < 0.001) than travoprost acid. Unoprostone and S-1033 were significantly (p < 0.001) weaker than travoprost acid. The amide prodrug, bimatoprost (EC50 = 694 +/- 293 nM), activated this FP receptor with an intermediate potency. The isopropyl ester prodrugs, travoprost (EC50 = 42.3 +/- 6.7 nM), latanoprost (EC50 = 126 +/- 347 nM) and unoprostone isopropyl ester (EC50 = 9,100 +/- 2,870 nM), also exhibited FP agonist activity. However, other compounds such as PGI2, bradykinin, histamine, and serotonin were inactive. The agonist activities of bimatoprost, unoprostone (UF-021), fluprostenol and acids of travoprost and latanoprost were antagonized by AL-8810 (11beta-fluoro- 15-epi-15-indanyl-PGF2alpha), an FP-receptor-selective antagonist (Ki = 1.0 - 2.1 microM; n = 3). These studies have demonstrated, for the first time, agonist activities of the currently known and marketed ocular hypotensive prostaglandin analogs at the cloned human ciliary body FP prostaglandin receptor.     

Biochemical and functional evidence for the presence of dopamine D1 receptors in the bovine ciliary body

The present paper reports both functional and biochemical evidence for the presence of dopamine D1 receptors in the bovine ciliary body. Dopamine (DA) and dopamine D1 agonists (such as SKF 38,393) but not D2 agonists (such as LY 141,865) produced a concentration-related decrease in the tone induced by a maximally active concentration of carbachol (1 x 10(-4)-5 x 10(-4) M). The maximal relaxation obtained was 100% of the carbachol response using 10(-5) M dopamine or 5 x 10(-6) M SKF 38,393. SCH 23,390, a D1 antagonist, but not (-)-sulpiride, antagonized the effect of DA and SKF 38,393. In accordance with the functional data, radioreceptor binding experiments revealed the existence of a high affinity saturable [3H]SCH 23,390 binding to membranes prepared from ciliary body (Bmax: 344 fmol mg protein-1; Kd: 0.87 nM). The binding was specifically displaced by SCH 23,390, dopamine and dopamine D1 agonists, but not by norepinephrine, D2 agonists, or antagonists such as LY 141,865 and sulpiride. No specific binding was found when using dopamine D2 ligands, such as tritiated spiroperidol.     

Human retinal pigment epithelial cells possess V1 vasopressin receptors.

Membrane preparations of cultured human retinal pigment epithelial (RPE) cells were incubated with various concentrations of [3H]arginine vasopressin (AVP) in the presence and absence of 10 microM nonradioactive AVP. Saturable, specific binding to a single site with a Kd of 6.2 nM and Bmax of 111 fmol/mg protein was detected. Vasopressin had no effect on RPE cyclic AMP levels measured by radioimmunoassay. Intracellular calcium fluxes were measured by spectrofluorometry of RPE cell suspensions preloaded with quin 2. The baseline cytosolic calcium level was 217 +/- 20 nM, and AVP caused a concentration-dependent increase in this level with a 3.5-fold maximal response at 10(-6) M and an EC50 of 120 nM. The production of inositol phosphates was measured in RPE preloaded with [3H]myoinositol, and AVP caused a concentration-dependent increase in their production with a 2.1-fold maximal response at 10(-5) M and an EC50 of 80 nM. A specific vasopressin receptor antagonist, SKF 101926, prevented the AVP-induced increase in calcium mobilization and inositol phosphate production in RPE. These data suggest that RPE cells possess V1 AVP receptors coupled to calcium mobilization and inositol phosphate metabolism.     

Functional characterization of insulin and IGF-I receptors in chicken lens epithelial and fiber cells.

Insulin and insulin-like growth factor I (IGF-I) play a role in lens cell growth and development. The binding of these hormones to their respective receptors with its concomitant signal transduction is an important step in these cellular processes. Hormone binding to adult chicken lens insulin and IGF-I receptors, partially purified from epithelial and fiber cells, was studied to examine this activity in lens. The associated stimulation of receptor-mediated tyrosine kinase by the hormones was also studied. At an insulin concentration of 0.02 nM, specific binding was similar for epithelial and fiber receptor preparations (Epi = 0.23 +/- 0.03 fmol, Fib = 0.19 +/- 0.02 fmol). Displacement studies revealed that there was also no difference between epithelial and fiber receptor preparations in the concentration of insulin necessary for half maximal displacement of specific [125I]-insulin binding (IC50: Epi = 0.32 nM +/- 0.07 nM, Fib = 0.31 nM +/- 0.05 nM). Comparison of IGF-I (0.02 nM) binding to receptor preparations from epithelial and fiber cells demonstrated that specific binding was similar in the two preparations (Epi = 0.50 +/- 0.05 fmol, Fib = 0.42 +/- 0.05 fmol). Also, there was no difference in the concentration of IGF-I necessary for half maximal displacement of specific [125I]-IGF-I binding (IC50 = Epi: 0.27 +/- 0.05 nM, Fib: 0.28 +/- 0.04 nM). The ability of IGF-I to displace bound [125I]-insulin was also examined. The IC50 for IGF-I binding to the insulin receptors isolated from epithelial and fiber cells was 37.4 +/- 2.4 nM, and 35.4 +/- 2.8 nM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)