The tissue distribution of the human beta3-adrenoceptor studied using a monoclonal antibody: direct evidence of the beta3-adrenoceptor in human adipose tissue, atrium and skeletal muscle.

OBJECTIVE: To develop a monoclonal antibody that recognises an epitope of the native beta3-adrenoceptor expressed on the extracellular surface of human cells and tissues. DESIGN: A high affinity monoclonal antibody, Mab72c, was raised against the human beta3-adrenoceptor expressed on a transfected mammalian cell line. RESULTS: In CHO (Chinese hamster ovary) cells transfected with beta3-adrenoceptor cDNA, antibody labelling was found to be proportional to receptor density measured by the binding of the radiolabelled beta-adrenoceptor antagonist, [125I]-iodocyanopindolol. The use of Mab 72c has demonstrated the expression of the beta3-adrenoceptor in a variety of human tissues, including gall bladder, prostate and colon, where a mRNA signal had been detected previously. This study also provides the first direct demonstration of the expression of beta3-adrenoceptors in human skeletal muscle, atrium and adipose tissue. CONCLUSION: The development of this antibody represents an important addition to the armentarium of reagents that are available to study the localisation of beta3-adrenoceptors in human tissues.

The contribution of classical (beta1/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities.

The role of beta3- and other putative atypical beta-adrenoceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta3-adrenoceptor (beta3AR) agonists with varying intrinsic activities and selectivities for human cloned betaAR subtypes. The ability to demonstrate beta1/2AR antagonist-insensitive (beta3 or other atypical betaAR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta3AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta1/2AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta3AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta1/2AR antagonism, despite it having very low efficacies at cloned beta1- and beta2ARs. A component of the response to another phenylethanolamine selective beta3AR agonist (SB-215691) was insensitive to beta1/2AR antagonism in some experiments. Because no [corrected] novel aryloxypropanolamine had a beta1/2AR antagonist-insensitive inotropic effect, these results establish more firmly that beta3ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta4ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned betaARs which betaARs will mediate responses to agonists in tissues that have a high number of beta1- and beta2ARs or a low number of beta3ARs.

Potential acuity meter versus scanning laser ophthalmoscope to predict visual acuity in cataract patients.

PURPOSE: To compare the relative abilities of the Potential Acuity Meter (PAM) and the Scanning Laser Ophthalmoscope (SLO) to predict the postoperative visual acuity in patients with mild to dense cataract. SETTING: Gimbel Eye Centre, Calgary, Alberta, Canada. METHODS: In this single-masked prospective study of 31 consecutive eyes with mild to dense cataract and a best corrected visual acuity (BCVA) of 20/40 or less, patients had predictive visual acuity (PVA) testing using the PAM and SLO. Preoperative and postoperative BCVA was recorded using Snellen charts. Differences between PVA and postoperative BCVA results were assessed by a two-tailed t-test and correlation analysis. RESULTS: The SLO was correct within two lines of outcome in 30 of 31 eyes and the PAM, in 19 of 31 eyes. A two-tailed dependent t-test showed a significant difference between predicted and final visual acuity for the PAM (P = .0001) and a nonsignificant difference for the predicted and final visual acuity for the SLO. A correlation analysis showed a mild correlation for the PAM (r = .49) and a good correlation for the SLO (r = .81). For a prediction of better or worse than 20/40, the SLO showed a sensitivity of 0.964 and specificity of 1.0 and the PAM, of 0.67 and 1.0, respectively. CONCLUSION: The SLO was more accurate than the PAM in predicting visual acuity after cataract surgery. The relative cost of the SLO is potentially restrictive to its widespread clinical application. However, the cost/benefit in terms of patient satisfaction with predictable surgical results should be considered.

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.

Agonist potency at the cloned human beta-3 adrenoceptor depends on receptor expression level and nature of assay.

The cloned human beta-3 adrenoceptor was expressed in Chinese hamster ovary cells at three different levels (130, 400 and 3000 fmol/mg). The potency and intrinsic activity of a range of agonists in functional assays with these cell lines rose as a function of increasing receptor density. Operational analysis of concentration-response data allowed calculation of functional affinity and efficacy of agonists at the human beta-3 adrenoceptor. The data highlighted the low efficacy of BRL 37344 ¿(RR,SS)-(+/-)-4-[(2-(2-(3-chlorophenyl)-2-hydroxyethyl)amino)-propyl] phenoxyacetate¿ for the human beta-3 adrenoceptor, which may explain its lower potency at the human receptor despite its higher affinity relative to isoprenaline. The potency of catecholamines at the human beta-3 adrenoceptor was found to be 1 to 2 orders of magnitude higher when determined in an intact cell cAMP accumulation assay compared with a membrane-based adenylyl cyclase activation assay. The reason for this enhanced sensitivity is not clear, but the result is that the potency of the natural agonist noradrenaline in the intact cell is considerably higher than predicted either from its ligand binding affinity, or from its potency in membrane-based assays. Much smaller enhancements in sensitivity were observed for compounds of the aryloxypropanolamine class such as CGP 12177 [(+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one], with the result that the rank order of potency of such agonists at the beta-3 adrenoceptor was altered. In particular, CGP 12177 exhibited high relative potency in the cyclase assay, but low relative potency in intact cell assays. These findings highlight the importance of selecting appropriate expression levels and appropriate assay methodology when cloned receptors are used to characterize agonists.

Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells.

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (approximately 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti(-)[insulin receptor substrate 1 (IRS1)] and anti-alpha-p85 antibodies show the same level of insulin-stimulated activity, approximately 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of alpha-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

Synthesis of a selective alpha-2A adrenoceptor antagonist, BRL 48962, and its characterization at cloned human alpha-adrenoceptors.

The chiral synthesis of the potent and selective alpha-2A antagonist, BRL 48962, is described. Evaluation of BRL 48962 at cloned human alpha-adrenoceptors indicates that this antagonist has a selectivity in the order of 30-fold for the alpha-2A subtype.

Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling.

(+/-)-5-([4-[2-Methyl-2(pyridylamino)ethoxy]phenyl]methyl) 2,4-thiazolidinedione (BRL 49653) is a new potent antidiabetic agent that improves insulin sensitivity in animal models of NIDDM. In C57BL/6 obese (ob/ob) mice, BRL 49653, included in the diet for 8 days, improved glucose tolerance. The half-maximal effective dose was 3 mumol/kg diet, which is equivalent to approximately 0.1 mg/kg body wt. Improvements in glucose tolerance were accompanied by significant reductions in circulating triacylglycerol, nonesterified fatty acids, and insulin. The insulin receptor number of epididymal white adipocytes prepared from obese mice treated with BRL 49653 (30 mumol/kg diet) for 14 days was increased twofold. The affinity of the receptor for insulin was unchanged. In the absence of added insulin, the rates of glucose transport in adipocytes from untreated and BRL 49653-treated obese mice were similar. Insulin (73 nmol/l) produced only a 1.5-fold increase in glucose transport in adipocytes from control obese mice, whereas after BRL 49653 treatment, insulin stimulated glucose transport 2.8-fold. BRL 49653 did not alter the sensitivity of glucose transport to insulin. The increase in insulin responsiveness was accompanied by a 2.5-fold increase in the total tissue content of the glucose transporter GLUT4. Glucose transport in adipocytes from lean littermates was not altered by BRL 49653. To establish the contribution of changes in glucose transporter trafficking to the BRL 49653-mediated increase in insulin action, the cell-impermeant bis-mannose photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos++ +-4-yloxy) -2-[2-3H]-propylamine was used to measure adipocyte cell-surface-associated glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.

Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.

Intraocular lens implantation in developing countries: an ophthalmic surgical dilemma.

Rapid and remarkable development of intraocular lens technology during the past 10 years has made intraocular lens (IOL) implantation the standard of practice with cataract surgery in wealthy industrialized nations. However, although visual results of IOL implantation are vastly superior to aphakic spectacle correction after cataract surgery, several factors mitigate against the widespread implantation of IOLs in most developing nations: the high cost of surgical equipment, ancillary drugs, and intraocular lenses; difficulty in postoperative follow-up of cataract surgical patients; and a dearth of surgical personnel properly trained in IOL technology in developing nations. While many of these problems could be addressed through national initiatives, a major area of concern remains that of manpower, since the number of unoperated cataract patients is immense. One strategy for alternative surgical manpower development is the training of medical assistants to perform cataract surgery, as is presently being carried out in several African nations. The question arises, however, as to whether medical assistants, as opposed to ophthalmologists, are qualified to select patients and implant IOLs.

"Early" vitrectomy for vasoproliferative retinopathy in patients with insulin-dependent diabetes mellitus.

Early vitrectomy was carried out in 22 eyes with proliferative diabetic retinopathy and evidence of partial vitreous detachment in 17 patients with insulin-dependent diabetes. All the eyes had undergone panretinal laser photocoagulation, and all had a visual acuity of 6/12 or better, but 13 had had a vitreous hemorrhage. After a mean follow-up period of 29 months 19 of the eyes had retained the same visual acuity, 18 had been free of renewed vitreous hemorrhage for 6 months, there had been no macular retinal detachments and all the anterior segments were normal. The three instances of persistent visual loss were due to recurrent vitreous hemorrhage.

Pars plasma vitrectomy in the management of the Irvine-Gass syndrome.

Twelve eyes of 10 patients with type I Irvine-Gass syndrome underwent pars plana vitrectomy after cystoid macular edema had been present a mean of 13 months and showed no evidence of resolving spontaneously or, in some cases, of responding to laser photocoagulation or steroid therapy. The disorder appears to resolve spontaneously in an average of 16 months. Following vitrectomy the edema resolved clinically in 10 of the 12 eyes, in an average of 3.7 months, and the visual acuity improved significantly in 8 of the 12 eyes--in five instances from legal blindness to 20/50 or better. The results suggest that pars plana vitrectomy is of value in the treatment of this disorder.

Acetylcholine stimulates growth hormone secretion, phosphatidyl inositol labelling, 45Ca2+ efflux and cyclic GMP accumulation in bovine anterior pituitary glands.

Acetylcholine (25 micromol/l) in the presence of the choline esterase inhibitor physostigmine (67 micromol/l) increased the release of growth hormone and efflux of 45Ca2+ from perifused bovine pituitary slices; the time taken for the maximal response to occur was the same. In batch incubations, acetylcholine (1 micromol/l--1 mmol/l) increased pituitary cyclic GMP concentrations in the pituitary gland within 2 min, and increased incorporation of [3H]inositol and [32P]phosphate into pituitary phosphatidyl inositol within 15 min. Cyclic AMP concentrations were not significantly changed 2 or 5 min after acetylcholine addition. All the tissue responses were inhibited by prior exposure of the tissue to atropine (1 micromol/l) but not by tubocurarine (10 micromol/l--1mmol/l), indicating that the responses were mediated by receptors of the muscarinic type. The similarities between these responses and those to known hypothalamic hypophysiotrophic hormones are discussed.

Pars plana vitrectomy.

We describe our first 127 vitrectomies through the pars plana performed on 120 eyes for a variety of indications. The success rate in anterior segment disorders was 45% and in those of the posterior segment 34%. While in some cases the results were encouraging, in others they were disastrous. Some 12 eyes (10%) went on to lose perception of light.