Binding of a glaucoma-associated myocilin variant to the αB-crystallin chaperone impedes protein clearance in trabecular meshwork cells.

Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.

Full-length myocilin protein is purified from mammalian cells as a dimer.

Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells. One method involved identification of a cell line (HeLa S3) that would secrete full-length protein (15 mg/L) while the second method involved a purification approach from 293 cells requiring identification and modification of an internal MYOC cleavage site (Glu214/Leu215). MYOC protein yield from 293 cells was improved by mutation of two MYOC N-terminal cysteines (C47 and C61) to serines. Analytical size exclusion chromatography of our full-length MYOC protein purified from 293 cells indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. We hope that by providing methods to obtain MYOC protein, researchers will be able to utilize the protein to obtain new insights into MYOC biology. The ultimate goal of MYOC research is to better understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life.

Preclinical pharmacology, ocular tolerability and ocular hypotensive efficacy of a novel non-peptide bradykinin mimetic small molecule.

We sought to characterize the ocular pharmacology, tolerability and intraocular pressure (IOP)-lowering efficacy of FR-190997, a non-peptidic bradykinin (BK) B2-receptor agonist. FR-190997 possessed a relatively high receptor binding affinity (Ki = 27 nM) and a high in vitro potency (EC50 = 18.3 ± 4.4 nM) for inositol-1-phosphate generation via human cloned B2-receptors expressed in host cells with mimimal activity at B1-receptors. It also mobilized intracellular Ca2+ in isolated human trabecular meshwork (h-TM), ciliary muscle (h-CM), and in immortalized non-pigmented ciliary epithelial (h-iNPE) cells (EC50s = 167-384 nM; Emax = 32-86% of BK-induced response). HOE-140, a selective B2-receptor antagonist, potently blocked the latter effects of FR-190997 (e.g., IC50 = 7.3 ± 0.6 nM in h-CM cells). FR-190997 also stimulated the release of prostaglandins (PGs) from h-TM and h-CM cells (EC50s = 60-84 nM; Emax = 29-44% relative to max. BK-induced effects). FR-190997 (0.3-300 µg t.o.) did not activate cat corneal polymodal nociceptors and did not cause ocular discomfort in Dutch-Belted rabbits, but it was not well tolerated in New Zealand albino rabbits and Hartley guinea pigs. A single topical ocular (t.o.) dose of 1% FR-190997 in Dutch-Belted rabbits and mixed breed cats did not lower IOP. However, FR-190997 efficaciously lowered IOP of conscious ocular hypertensive cynomolgus monkey eyes (e.g., 34.5 ± 7.5% decrease; 6 h post-dose of 30 µg t.o.; n = 8). Thus, FR-190997 is an unexampled efficacious ocular hypotensive B2-receptor non-peptide BK agonist that activates multiple signaling pathways to cause IOP reduction.

FR-190997, a nonpeptide bradykinin B2-receptor partial agonist, is a potent and efficacious intraocular pressure lowering agent in ocular hypertensive cynomolgus monkeys.

Preclinical Research FR-190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnaminoacetyl]-N-methylamino]benzyloxy]-2-methyl-4- (2-pyridylmethoxy) quinoline), a nonpeptide bradykinin (BK) B2-receptor-selective agonist, represents a novel class of ocular hypotensive agents. FR-190997 exhibited a high affinity for the human cloned B2-receptor (Ki = 9.8 nM) and a relatively high potency (EC50 = 155 nM) for mobilizing intracellular Ca(2+) ([Ca(2+)]i) in human ocular cells from nonpigmented ciliary epithelium; trabecular meshwork [h-TM]; ciliary muscle [h-CM] that are involved in regulating intraocular pressure (IOP). Unlike BK, FR-190997 behaved as a partial agonist (Emax = 38-80%) in these cells and its [Ca(2+)]i-mobilizing effects were blocked by the B2-receptor-selective antagonists (HOE-140, Ki = 0.8-7 nM; WIN-64338, Ki = 157-425 nM). FR-190997 stimulated the production of prostaglandins (PGs) in h-CM and h-TM cells (EC50 = 15-19 nM; Emax = 27-33%); an effect that was reduced by the cyclooxygenase-2 inhibitor bromfenac, and by HOE-140. FR-190997 also induced pro-matrix metalloproteinase (MMP)-1 and MMP-3 release from h-CM cells. FR-190997 significantly lowered IOP (37% [P < 0.001] with 30 µg, 24 h post-topical ocular dosing) in ocular hypertensive eyes of conscious Cynomolgus monkeys. This effect was reduced by bromfenac and completely blocked by a B2-antagonist. FR-190997 primarily stimulated uveoslceral outflow (UVSO) of aqueous humor (2.6 to 3.9-fold above baseline). In conclusion, FR-190997 is a B2-receptor selective partial agonist that activates phospholipase C, mobilizes [Ca(2+)]; induces PG and pro-MMP production, and that profoundly lowers IOP by promoting UVSO in ocular hypertensive Cynomolgus monkey eyes.

Human non-pigmented ciliary epithelium bradykinin B2-receptors: receptor localization, pharmacological characterization of intracellular Ca(2+) mobilization, and prostaglandin secretion.

PURPOSE: To characterize the bradykinin (BK) receptor system in human non-pigmented ciliary epithelium (NPCE) using immunohistochemistry and functional cell-based techniques. METHODS: B2-receptor protein expression was studied in sections of human donor eyes and in Cynomolgus monkey eyes using immunohistochemical methods. The pharmacological characteristics of intracellular Ca(2+) ([Ca(2+)]i) mobilization in response to BK and related peptides, and blockade by two antagonists, was studied in primary human (p-h-NPCE) and in immortalized human NPCE (imh-NPCE) cells. Prostaglandins (PGs) release induced by BK was also studied in both cell-types using ELISA assays. Limited studies on primary human ciliary muscle (h-CM) cells and human trabecular meshwork (h-TM) cells and Chinese hamster ovary cells expressing human cloned B2-receptors (CHO-B2) were performed to compare with responses in both the NPCE cell-types. RESULTS: B2-receptor immunoreactivity was observed on human and Cynomolgus monkey NPCE cells on eye sections from both species. BK and related analog peptides differentially activated signaling mechanisms in NPCE cells by mobilizing [Ca(2+)]i, and the BK-evoked responses were blocked by B2-receptor-selective antagonists, HOE-140 and (S)-WIN-64338. Relative agonist potencies (EC50, nM) in p-h-NPCE cells [and in imh-NPCE cells] were: BK=3.4 ± 0.4 [6.3 nM]; Hyp(3)-BK EC50=1.7 ± 0.2 [6.0 nM], Lys-BK EC50=7.0 ± 0.3 [19.8 nM]; Met-Lys-BK EC50=106 ± 57.8 [125 nM]; Des-Arg(9)-BK EC50=>10,000 [16 µM]. The antagonist potencies for attenuating BK-induced mobilization of [Ca(2+)]i in these cells were: HOE-140 (Ki=7.9 ± 1.8 nM, n=4) and (S)-WIN-64338 (Ki=451 ± 44 nM, n=4). These NPCE cell data correlated well with those obtained for h-CM and h-TM cells, and with B2-receptor binding (r=0.99, p<0.0001). However, BK failed to stimulate total PGs production in both NPCE cell-types even though 10% bovine serum increased PG release (by 4.9-fold above baseline), and even though BK stimulated PG release from h-CM, h-TM and in CHO-B2 cells. BK (1 µM) also failed to increase nitric oxide (NO) levels in NPCE cells even though sodium nitropruside increased NO production by 3-fold. CONCLUSIONS: Human and monkey NPCE express immunoreactive B2-receptor proteins. These proteins were functionally active, since BK and related peptides potently stimulated mobilization of [Ca(2+)]i in p-h-NPCE and imNPCE cells that was blocked by two B2-selective antagonists. Down-stream signaling from B2-receptor activation did not appear to involve PG synthesis/release (or NO production) in NPCE cell-types under the present conditions, even though h-CM, h-TM and CHO-B2 cells exhibited robust PG synthesis and release in response to BK.

Trabecular meshwork bradykinin receptors: mRNA levels, immunohistochemical visualization, signaling processes pharmacology, and linkage to IOP reduction.

PURPOSE: To localize mRNA and protein of bradykinin (BK) receptors, BK precursor polypeptide (kininogen) mRNA, and to study functional biochemical pharmacology of the signal transduction processes mediated by B2-receptors in isolated human trabecular meshwork (h-TM) cells. Intraocular pressure (IOP) lowering effects of 2 kinins were also investigated. METHODS: Previously documented procedures were utilized throughout these studies. RESULTS: Kinninogen mRNA was most abundant in TM, ciliary body (CB), and optic nerve head and appeared elevated in glaucomatous h-TM tissue. High levels of B2-receptor mRNA were found in the sclera, iris, TM, and CB. B2-receptor subtype protein was localized in cells of the monkey and h-TM, and the treatment of isolated h-TM cells with transforming growth factor-ß2 (5 ng/mL) caused significant (P<0.04) downregulation of B2-receptor mRNA. In isolated primary h-TM cells, BK (EC50=0.8±0.2 nM; n=19) and Met-Lys-BK (EC50=6.5±1.5 nM) mobilized intracellular Ca(2+) and induced the release of prostaglandins (PGs) that was blocked by 2 B2-receptor antagonists [HOE-140; (S)-WIN-64338]. The cyclooxygenase inhibitor, bromfenac, abolished BK-induced PGs production. BK concentration dependently increased cell impedance, and it significantly (P<0.05) decreased h-TM cell volume in vitro. Intravitreal (ivt) administration of BK (50 µg), but not a B1-agonist (Sar-[D-Phe(9)]-Des-Arg(9)-BK; also at 50 µg), efficaciously lowered IOP (22.9% to 37% from baseline) of Dutch-Belted rabbits that naturally have high IOPs (27-28 mmHg). CONCLUSIONS: BK activates multiple signal transduction pathways in h-TM cells via B2-receptors that also mediate IOP reduction as observed in rabbits following ivt administration of BK. These ocular hypotensive effects of BK may be physiologically important and suggest a novel therapeutic potential of BK-related B2-agonists.

Protein expression, biochemical pharmacology of signal transduction, and relation to intraocular pressure modulation by bradykinin B2 receptors in ciliary muscle.

PURPOSE: To examine the bradykinin (BK) B2-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied. METHODS: Previously published procedures were used throughout these studies. RESULTS: The human and monkey ciliary bodies expressed high levels of B2-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca²âº](i) mobilization in primary h-CM cells (BK EC50=2.4±0.2 nM > Hyp³,ß-(2-thienyl)-Ala5,Tyr(Me)8-(®)-Arg9-BK (RMP-7) > Des-Arg9-BK EC50=4.2 µM [n=3-6]), and this was blocked by B2-selective antagonists, HOE-140 (IC50=1.4±0.1 nM) and WIN-63448 (IC50=174 nM). A phospholipase C inhibitor (U73122; 10-30 µM) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca²âº](i) mobilization. Total prostaglandin (primarily PGE2) secretion stimulated by BK and other kinins in h-CM cells was attenuated by the cyclooxygenase inhibitors bromfenac and flurbiprofen, and by the B2-antagonists. BK and RMP-7 (100 nM) induced a twofold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and BK (0.1-1 µM; at 24 h) caused a 1.4-3.1-fold increase in promatrix metalloproteinases-1-3 release. Topical ocular BK (100 µg) failed to alter IOP in cynomolgus monkeys. However, intravitreal injection of 50 µg of BK, but not Des-Arg9-BK, lowered IOP in rabbit eyes (22.9±7.3% and 37.0±5.6% at 5 h and 8 h post-injection; n=7-10). CONCLUSIONS: These studies have provided evidence of a functional endogenously expressed B2-receptor system in the CM that appears to be involved in modulating IOP.

NPR-B natriuretic peptide receptors in human corneal epithelium: mRNA, immunohistochemistochemical, protein, and biochemical pharmacology studies.

PURPOSE: To demonstrate the presence of natriuretic peptide receptors (NPRs) in primary human corneal epithelial cells (p-CEPI), SV40-immortalized CEPI cells (CEPI-17-CL4) and in human corneal epithelium, and to define the pharmacology of natriuretic peptide (NP)-induced cGMP accumulation. METHODS: NPR presence was shown by RT-PCR, western blot analysis, and indirect immunofluoresence. cGMP accumulation was determined using an enzyme immunoassay. RESULTS: p-CEPI and CEPI-17-CL4 cells expressed mRNAs for NPR-A and NPR-B. Proteins for both NPRs were present in these cells and in human corneal epithelium. C-type NP (CNP), atrial NP (ANP) and brain NP (BNP) stimulated the accumulation of cGMP in a concentration-dependent manner in p-CEPI cells (potency; EC(50s)): CNP (1-53 amino acids) EC(50)=24+/-5 nM; CNP fragment (32-53 amino acids) EC(50)=51+/-8 nM; ANP (1-28 amino acids) EC(50)=>10 microM; BNP (32 amino acids) EC(50)>10 microM (all n=3-4). While the NPs were generally more potent in the CEPI-17-CL4 cells than in p-CEPI cells (n=4-9; p<0.01), the rank order of potency of the peptides was essentially the same in both cell types. Effects of CNP fragment in p-CEPI and CEPI-17-CL4 cells were potently blocked by HS-142-1, an NPR-B receptor subtype-selective antagonist (K(i)=0.25+/-0.05 microM in CEPI-CL4-17; K(i)=0.44+/-0.09 microM in p-CEPIs; n=6-7) but less so by an NPR-A receptor antagonist, isatin (K(i)=5.3-7.8 microM, n=3-7). CONCLUSIONS: Our studies showed the presence of NPR-A and NPR-B (mRNAs and protein) in p-CEPI and CEPI-17-CL4 cells and in human corneal epithelial tissue. However, detailed pharmacological studies revealed NPR-B to be the predominant functionally active receptor in both cell-types whose activation leads to the generation of cGMP. While the physiologic role(s) of the NP system in corneal function remains to be delineated, our multidisciplinary findings pave the way for such future investigations.

Cabergoline: Pharmacology, ocular hypotensive studies in multiple species, and aqueous humor dynamic modulation in the Cynomolgus monkey eyes.

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT(2A/B/C)), 5HT(1A), 5HT(7), alpha(2B), and dopamine-2/3 (D(2/3)) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT(2), 5HT(1A) and D(2/3) receptors but an antagonist at 5HT(7) and alpha(2) receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC(50)=19+/-7 nM in TM; 76 nM in h-CM) and intracellular Ca(2+) ([Ca(2+)](i)) mobilization (EC(50)=570+/-83 nM in h-TM; EC(50)=900+/-320 nM in h-CM). Cabergoline-induced [Ca(2+)](i) mobilization in h-TM and h-CM cells was potently antagonized by a 5HT(2A)-selective antagonist (M-100907, K(i)=0.29-0.53 nM). Cabergoline also stimulated [Ca(2+)](i) mobilization more potently via human cloned 5HT(2A) (EC(50)=63.4+/-10.3 nM) than via 5HT(2B) and 5HT(2C) receptors. In h-CM cells, cabergoline (1 microM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1 microM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300-500 microg) in Dutch-belted rabbit eyes yielded 4.5 microMM and 1.97 microM levels in the aqueous humor 30 min and 90 min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6 microg at 4h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6+/-3.6% with 50 microg at 3h post-dose; 30.4+/-4.5% with 500 microg at 7h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5h after the second topical ocular dose (300 microg) of cabergoline by 23% (p<0.02) and 35% (p<0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69+/-0.7 microL/min-1.61+/-0.97 microL/min, n=13; p<0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT(2), 5HT(1A), and D(2/3) receptors. Since 5HT(1A) agonists, 5HT(7) antagonists, and alpha(2) antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT(2) and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.

Pharmacological characterization of a serotonin receptor (5-HT7) stimulating cAMP production in human corneal epithelial cells.

PURPOSE: To study the mRNA and pharmacology of a serotonin (5-HT) receptor positively coupled to adenylyl cyclase in normal, primary (P-CEPI), and immortalized human corneal epithelial cells (CEPI-17-CL4), by using numerous 5-HT agonists and antagonists. To determine and compare cloned human 5-HT7 receptor binding affinities of compounds with their functional potency data. METHODS: RT-PCR was used to detect the presence of an mRNA for the human 5-HT7 receptor in CEPI-17-CL4 cells. Receptor-mediated production of cAMP in cultured cells was measured using an enzyme immunoassay. Compound binding affinities were determined using [3H]-lysergic acid diethylamide ([3H]-LSD) binding to cell membranes of human embryonic kidney (HEK-293) cells expressing the cloned human 5-HT7 receptor. RESULTS: RT-PCR revealed the presence of a 5-HT7 receptor mRNA in CEPI-17-CL4 cells. Normal P-CEPI cells generated cAMP in response to 5-HT (-log EC50; pEC50=7.6), 5-carboxamidotryptamine (5-CT; pEC50=7.8), 5-methoxy-tryptamine (pEC50=7.0) and 5-methoxy-dimethyl-tryptamine (pEC50=5.7). In CEPI-17-CL4 cells, serotonergic agonists also stimulated cAMP production with different potencies (pEC50): 5-CT (7.4)>5-HT (6.5)> or =5-methoxy-tryptamine (6.1)>5-methoxy-dimethyl-tryptamine (5.4)> or =8-OH-DPAT (<5.0)=alpha-methyl-5-HT (<5.0). Various 5-HT receptor antagonists inhibited cAMP production induced by 5-CT in CEPI-17-CL4 cells with different potencies (pKi): methiothepin (8.5)>mesulergine (8.1)=metergoline (8.0)>spiperone (7.4)> or =clozapine (7.2)=SB-258719 (7.2)>mianserin (6.9)>ketanserin (6.3). Antagonist pKi values in P-CEPI cells were methiothepin (8.7), spiperone (7.4) and SB-258719 (6.6). The rank order of affinity for displacement of [3H]-LSD from the cloned human 5-HT7 receptor was: methiothepin>ritanserin>mesulergine=clozapine> or =metergoline=5-HT>SB-258719> or =spiperone>mianserin> or =ketanserin. The functional agonist and antagonist potency data obtained from CEPI-17-CL4 cells correlated well with cloned human 5-HT7 receptor binding affinity data (r=0.69), with P-CEPI cell functional data (r=0.85), and with functional potency data in the literature for the cloned human 5-HT7 receptor (r=0.88). CONCLUSIONS: These collective data support the presence of a pharmacologically defined, adenylyl cyclase-coupled 5-HT7 receptor in the CEPI-17-CL4 cells that may have relevance to physiological and/or pathologic functions of 5-HT7 receptors in the human cornea.