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M B Wax

Publications and source records attributed to M B Wax.

68 records · Page 4Linked to original sources
PubMed1990

Regulation of protein phosphorylation in ocular ciliary epithelial cells by A, C and Ca2+/calmodulin-dependent protein kinases.

We have studied protein phosphorylation events in a cell line (8-SVHCE) derived from the human ocular ciliary epithelium after transformation within Simian Virus-40. We have investigated the time-course and identification of intracellular phosphorylated protein substrates in response to isoproterenol, phorbol-12-myristate-13-acetate (PMA), and ionophore A23187, which are activators of protein kinase A, C and calcium/calmodulin-dependent protein kinase, respectively. Five major endogenous phosphoproteins were readily identified by two-dimensional polyacrylamide gel electrophoresis with the following molecular weights: 80, 57, 24 and 19 kDa. Tryptic peptide analysis and phosphoaminoacid composition were utilized to aid the identification of the phosphoproteins. From these studies we have observed the following: (a) the most prominent phosphorylation of the 80-kDa protein occurs rapidly (1 min) in response to PMA treatment and is potentiated by isoproterenol, (b) the phosphorylation of the 57-kDa substrate (vimentin) occurs preferentially with isoproterenol treatment and increases gradually from 1 to 30 min, (c) late phosphorylation (60 min) of the 80-kDa protein by PMA is potentiated by isoproterenol, and (d) late phosphorylation of 19-kDa and 24-kDa substrates occurs with PMA treatment and is potentiated by A21387. The desensitization of adenylate cyclase activity by PMA or isoproterenol in 8-SVHCE cells results in altered adenylate cyclase activity, which appears to be correlated with similar alterations in the phosphorylation of the 57-kDa substrate (vimentin).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1990

G protein complement of SV40-transformed ciliary epithelial cells.

Guanine nucleotide-binding proteins (G proteins) are a family of receptor-coupled signal-transducing proteins that regulate a variety of second-messenger systems and ion channels. The complement of G proteins in SV40-transformed pigmented and nonpigmented ciliary epithelial cells was determined by Western blot analysis utilizing peptide and holoprotein derived antisera to known G protein alpha and beta subunits and cholera toxin catalyzed ADP-ribosylation. The complement of alpha subunits found in both SV40-transformed NPE and PE cells includes Gs alpha and all three members of the Gi alpha family. Neither cell type contains Go alpha or Gz alpha. Both cell lines contain beta 35 and beta 36. Future studies will examine the functional involvement of these G proteins in the regulation of aqueous humor stimulus-secretion coupling.

Amino Acid Sequence↗
PubMed1990

Angiotensin binding sites in rabbit anterior uvea and human ciliary epithelial cells.

Angiotensin binding sites in membrane homogenates of rabbit iris/ciliary body and human nonpigmented ciliary epithelial (NPE) cells grown in culture were characterized using radioligand binding assays with (125I)-sarcosine1-isoleucine8-angiotensin II [(125I)-SARILE]. Scatchard analysis of the binding of (125I)-SARILE yielded linear plots with a Kd value of 0.55 +/- 0.1 nM and a Bmax of 98 +/- 23 fmol/mg protein in rabbit iris/ciliary body, and a Kd value of 0.63 +/- 0.1 nM, and a Bmax of 36.2 +/- 24 fmol/mg protein in NPE cells. Studies of the inhibition of the binding of (125I)-SARILE in rabbit iris/ciliary body were performed with a series of competing ligands, including the angiotensin receptor antagonist SARILE and the agonists angiotensin I, angiotensin II and angiotensin III. Inhibition curves for the antagonist resulted in Hill coefficients of approximately 1, consistent with the presence of a single class of binding sites with high affinity for (125I)-SARILE. Competition for the binding of (125I)-SARILE to binding sites with each of the agonists resulted in inhibition curves with Hill coefficients significantly less than 1 in the absence of GTP. However, in the presence of 100 microM GTP the Hill coefficients increased to approximately 1. The order of potencies of these agents was consistent with the pharmacologic profile of angiotensin II receptors. Thus rabbit iris/ciliary body homogenates, which include vascular tissue, contain a homogeneous population of angiotensin binding sites coupled to a guanine nucleotide binding protein. The presence of binding sites in cultured NPE cells indicates that at least some are located on the cells thought to be responsible for aqueous humor secretion.

Angiotensin I↗
PubMed1989

Alterations of intracellular calcium in human non-pigmented ciliary epithelial cells of the eye.

The ciliary epithelium of the eye is thought to be responsible for the active production of aqueous humor. The role of hormones and neuropeptides in mediating this process is unclear. Alterations in intracellular calcium in response to several hormones were assayed by fluorometric detection utilizing Quin2 in human SV-40 transformed non-pigmented ciliary epithelial cells grown in monolayer culture. A dose-dependent increase in intracellular calcium was found for the following drugs, which are given with their respective EC50 values: carbachol (15.7 +/- 4 microM), ATP (1.67 +/- 0.4 microM), arginine vasopressin (52 +/- 14 nM), bradykinin (2.4 +/- 0.7 nM), histamine (0.7 +/- 0.1 microM), and angiotensin II (6.4 +/- 1 nM). The following increases in calcium levels above typical resting levels of 45.9 +/- 4.6 nM were observed: 30% (0.1 microM angiotensin II), 50% (0.1 microM bradykinin, 2 microM arginine vasopressin), 100% (10 microM histamine), and 150% (1 mM carbachol, 10 microM ATP). Dopamine, KCl, phorbol esters, propranolol, epinephrine, and norepinephrine failed to increase intracellular calcium levels. The mobilization of intracellular calcium was unaffected by changes in the pH of the extracellular medium (over the pH range of 7.6 to 6.9) induced by glacial acetic, sulfuric or hydrochloric acids. Phosphatidic acid, however, did cause an elevation in intracellular calcium and is consistent with its putative role as an ionophore in other non-excitable exocrine tissues. These studies suggest a role for the hormonally induced mobilization of intracellular calcium which may underlie the secretion of aqueous humor by these cells.

Angiotensin II↗
PubMed1989

New form of pseudohypoparathyroidism with abnormal catalytic adenylate cyclase.

Patients with pseudohypoparathyroidism type Ia have resistance to multiple hormones because of deficient activity of the stimulatory guanine nucleotide-binding protein (Gs) that couples membrane receptors to activation of adenylate cyclase. However, in a subset of patients with pseudohypoparathyroidism who have resistance to multiple hormones yet possess normal erythrocyte membrane Gs activity, the biochemical abnormality responsible for hormone resistance has remained undefined. Cultured skin fibroblasts were derived from a patient with this atypical form of pseudohypoparathyroidism. In the patient's fibroblast membranes, adenylate cyclase stimulation mediated by Gs after fluoride ion treatment produced only 52% of normal activity, yet fibroblast membrane Gs activity measured by cyc- complementation was normal. Activation of the catalytic unit of adenylate cyclase with manganese produced 49% of normal activity; manganese plus forskolin produced 54% of normal adenylate cyclase activity. beta-Adrenergic receptor coupling to Gs and phosphodiesterase activity were normal. A defect in the catalytic unit of adenylate cyclase can account for these results and may be a mechanism for clinical resistance to multiple hormones that act through adenylate cyclase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Characterization of beta-adrenergic receptors in cultured human trabecular cells and in human trabecular meshwork.

The characterization of beta-adrenergic receptors on cultured human trabecular cells and trabecular meshwork from human autopsy eyes was carried out by radioligand binding utilizing (125I)-iodopindolol. In cultured cells, the observed binding of (125I)-iodopindolol was of high affinity (Kd = 43 pM) and saturable. Scatchard plots were linear and revealed a Bmax of 33 +/- 7 fmol/mg of protein. Competition studies with a series of agonists and antagonists revealed that human trabecular cells contain a single class of beta-adrenergic receptors of the beta 2 subtype. Similarly, the IC50 of ICI 89,406 (176 nM) in human trabecular meshwork from autopsy eyes supports the presence of beta 2-adrenergic receptors in this tissue.

Adult↗
PubMed1989

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)

Cells, Cultured↗
PubMed1988

Variability and reproducibility of optic disk topographic measurements with the Rodenstock Optic Nerve Head Analyzer.

Variability of optic disk topographic measurements obtained with the Rodenstock Optic Nerve Head Analyzer was determined by obtaining three separate images on one eye of ten normal subjects and nine subjects without glaucoma. Marking of the disk margin was performed in a random and masked fashion on each image three times by three independent observers. The overall variabilities of the measurements of the subjects with glaucoma were not statistically different from those of the normal subjects. Overall variability was about 0.2 mm2 for total disk area, 0.08 for cup/disk ratio, 0.2 mm2 for disk rim area, and 0.07 mm3 for cup volume. The largest component of the variability was the result of acquisition of separate images of the optic disk at different times. Observer inconsistency in marking the disk edge was relatively small. Based on the expected amount of random variability of the measurements, we proposed criteria for detecting significant change in the optic disk over time.

Adult↗
PubMed1988

Characterization of binding sites for 3H-spiroperidol in human retina.

Binding sites for the D-2-selective antagonist (3H)-spiroperidol were characterized in human retina. Nonspecific binding, measured in the presence of 2 microM (+)-butaclamol, made up 20% of total binding. Scatchard analysis of the binding of (3H)-spiroperidol resulted in linear plots and yielded a Kd value of 87 pM and a Bmax value of 1500 fmol/mg protein. In studies of the inhibition of the binding of (3H)-spiroperidol, (+)-butaclamol was approximately 1000-fold more potent than the (-)-stereoisomer. The inhibition curve for dopamine was shifted to the right and the Hill coefficient was increased by the addition of 300 microM GTP. This effect was agonist-specific and suggests that some of the receptors are coupled to stimulation or inhibition of the enzyme adenylate cyclase. The inhibition curves for most of the antagonists had Hill coefficients between 0.6 and 0.8. Hill coefficients were also consistently less than 1.0 for agonists even in the presence of GTP. Nonlinear regression analysis of untransformed data revealed that these shallow inhibition curves were best explained by the presence of two populations of binding sites, 40% of the sites having a high affinity for dopamine in the presence of GTP and domperidone and the remaining 60% having a lower affinity for these ligands. The larger population of sites had a higher affinity for sulpiride, fluphenazine, and N-propylnorapomorphine in the presence of GTP. The possibility that either of these classes of sites consisted of serotonin receptors was ruled out by the finding that the 5-HT2 antagonist ketanserin had a low affinity for both classes of sites.

Adolescent↗
PubMed1988

Effects of neuropeptide Y on the isolated rabbit iris dilator muscle.

The effects of neuropeptide Y (NPY) were studied on an in vitro preparation of rabbit iris dilator muscle. NPY by itself (10(-11) M to 10(-6) M) had no effect on the resting tension or on the maximal electrically-induced response (MER) of the dilator. Phenylephrine (10(-9) M to 10(-4) M) caused a dose-dependent contraction of the dilator muscle (7.8% to 40.6% of the MER). The addition of NPY 10(-6) M enhanced the phenylephrine-induced muscle contraction (8.8% to 76.8% of the MER) without altering the EC50 value (5 X 10(-6) M) of the phenylephrine dose-response curve. These findings support a modulatory role for NPY on the iris dilator muscle.

Animals↗
PubMed1987

Distribution and properties of beta-adrenergic receptors in human iris-ciliary body.

beta-Adrenergic receptors from the iris-ciliary body of human eyes removed shortly after death were studied using membranes prepared by isopycnic centrifugation of tissue homogenates. This procedure separates uveal melanin pigment from plasma membranes and reduces nonspecific binding of 125I-iodopindolol. The observed binding of 125I-iodopindolol was of high affinity (Kd = 78 +/- 6.6 pM), saturable, and fully reversible (t1/2 = 4.6 min). Scatchard plots were linear and revealed a Bmax of 134 +/- 20 fmol/mg of protein from the whole iris-ciliary body. The affinities of the receptors for a series of agonists and antagonists were determined. The order of potency for the inhibition of the binding of the radioligand by antagonists was ICI 118,551 greater than MK950 greater than propranolol greater than ICI 89,406 greater than metoprolol. This order of potency is characteristic of beta-adrenergic receptors of the beta 2 subtype. Preparations of iris-ciliary body were also subjected to microdissection prior to density gradient centrifugation to permit the study of beta-adrenergic receptors in the ciliary processes, ciliary body, and iris. Each of these regions was found to contain approximately one third of the total number of beta-adrenergic receptors in the human iris-ciliary body. The highest density of receptors was located in the ciliary processes (180 +/- 40 fmol/mg of protein), while the density of receptors in the iris (98 +/- 7.5 fmol/mg of protein) and ciliary body (less the processes) (42 +/- 17 fmol/mg of protein) was notably lower. Only beta 2-adrenergic receptors are detectable by competition experiments in the iris-ciliary body as a whole, or in the individual preparations of iris, ciliary processes, or ciliary body; however, microdissection and analysis of beta-adrenergic receptor subtypes in isolated ciliary muscle permitted detection of a small number of beta 1-adrenergic receptors. beta 1-Adrenergic receptors comprised about 10% of the total number of beta-adrenergic receptors in the whole iris-ciliary body. The finding that most of the beta-adrenergic receptors in the human iris-ciliary body are of the beta 2 subtype may be of significant therapeutic importance in the medical management of glaucoma.

Adolescent↗
PubMed1986

Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.

Ciliary epithelial cells derived from human eye were successfully propagated through many generations after transformation with simian virus 40. The cell clone 8-SVHCE was isolated and characterized by immunoprecipitation and pharmacological studies that demonstrated the presence of several functional properties observed in the parent cells of this tissue. Immunoprecipitation revealed the presence of large tumor (T) antigen, and Southern blot analysis showed the incorporation of viral DNA into high molecular weight ciliary epithelial cell DNA. The presence of beta-adrenergic receptors was demonstrated by direct binding of a radiolabeled antagonist, [125I]iodopindolol, to membrane preparations of 8-SVHCE cells (Kd = 41.8 pM and Bmax = 67.1 fmol/mg of protein). Competition experiments with [125I]iodopindolol and selective drugs suggested that the receptors are of the beta 2-adrenergic subtype. Studies of catecholamine-stimulated cellular cAMP production and of isoproterenol-dependent protein phosphorylation of vimentin in 8-SVHCE indicated the functional conservation of beta-adrenergic receptor-mediated processes that are thought to be important in the regulation of aqueous humor production by the ciliary epithelium in vivo.

Adenylyl Cyclases↗
PubMed1982

Reversal of retinal and optic disc ischemia in a patient with sickle cell trait and glaucoma secondary to traumatic hyphema.

A 14-year-old black boy with sickle cell trait, who sustained a traumatic hyphema, developed moderately elevated intraocular pressure that failed to respond to carbonic anhydrase inhibitors and osmotic agents. On the tenth postinjury day, a sudden increased cupping of the optic disc and partial central retinal artery obstruction caused painless loss of vision. Reversal of the cupping, the retinal ischemia, and the intraocular pressure was documented following anterior chamber paracentesis, and visual acuity returned to 6/6. Pathophysiology of the posterior ischemia is discussed. This case documents the potentially debilitating course of traumatic hyphema in "benign" sickle cell trait and its avoidance with proper management. The authors endorse recent suggestions for careful observation of any sickle cell patient with traumatic hyphema, and recommend anterior chamber paracentesis, supplemental oxygen, and avoidance of osmotic agents, if secondary glaucoma develops following the initial trauma.

Adolescent↗