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Biomedical subjects

M Korner

Publications and source records attributed to M Korner.

33 records · Page 2Linked to original sources
PubMed1988

Monoclonal antibodies with high affinity for spiroperidol.

A diverse panel of monoclonal antibodies was obtained from BALB/c mice immunized with two haptens structurally related to spiroperidol (SPD). Bromoacetyl derivatives of aminospiroperidol (NH2SPD) and N-amino-phenethylspiroperidol (NAPS) were synthesized to couple the haptens covalently to a protein carrier for immunization, thereby maintaining the butyrophenone portion of the immunogen. Hybridomas were selected based on their ability to secrete antibody that binds [3H]SPD with high affinity. Equilibrium dissociation constants for these antibodies ranged from 0.2 to greater than 100 nM. The antigen binding sites of the anti-NH2SPD and anti-NAPS antibodies were characterized in studies of the inhibition of the binding of [3H]-SPD by a series of ligands that are either (a) structurally related to SPD or (b) structurally unrelated to the butyrophenones but known to be selective antagonists of the D2 subtype of dopamine receptor. Based on the patterns of inhibition of the binding of [3H]SPD by these compounds, 12 classes of antibody combining sites were identified. Most of these antibodies bound butyrophenones with high affinity. One anti-NH2SPD and four anti-NAPS antibodies also bound domperidone, a nonbutyrophenone that has a high affinity for D2 receptors. None of the antibodies bound clebopride or sulpiride, D2-selective antagonists of the benzamide class, or the agonist dopamine.

Animals↗
PubMed1988

Polyclonal and monoclonal antibodies directed against SK & F 94461, a specific H1 histamine receptor ligand.

SK & F 94461, an aminopentyl analogue of mepyramine, is a recently described H1 receptor antagonist. At variance with the other available H1 receptor ligands, SK & F 94461 offers the possibility of coupling to a protein carrier to render the molecule immunogenic. SK & F 94461 coupled to succinylated bovine serum albumin was used as an immunogen to raise polyclonal antibodies in rabbits and BALB/c mice. In parallel, spleen cells from immunized mice were used to produce hybridomas by somatic cell fusion. Thus, six different murine monoclonal antibodies sharing anti-SK & F 94461 specificity were selected for further detailed characterization of their binding properties. Pharmacologic studies of competitive inhibition using a set of 11 histaminergic agents allowed analysis of the fine specificity of anti-SK & F 94461 antibodies. Both polyclonal and monoclonal anti-SK & F 94461 antibodies showed very high affinity for the immunizing molecule (i.e., Ka values for monoclonal antibodies 8 and 12 were, respectively, 3 X 10(10) and 1.4 X 10(10) M-1). Both types of antibodies bound with high affinity (IC50 ranging from 10(-10) to 10(-12) M) to mepyramine, which has a chemical structure closely resembling that of SK & F 94461. Moreover, these antibodies displayed clear-cut stereoselectivity inasmuch as they bound the d-configuration of chlorpheniramine with significantly higher affinity than the l-form. Thus, all six monoclonal antibodies showed IC50 values 1 to 6 log units lower for d- than for l-chlorpheniramine. For some monoclonal antibodies, spectroscopic and fluorescence spectra studies showed that their different binding capacities correlated with their optical properties. Similarly, polyclonal anti-SK & F 94461 antibodies showed a 500-fold lower affinity for l- than for d-chlorpheniramine. All these results indicate that the polyclonal and the majority of monoclonal anti-SK & F 94461 antibodies recognized with high affinity structural configurations known to be important for the pharmacologic activity of H1 ligands, namely the presence of the dimethylaminoethyl side chain and, with stereochemical selectivity, the d-configuration of chlorpheniramine. These data extend for the first time to an H1 histamine receptor ligand results reported in other hormone systems.

Aminopyridines↗
PubMed1986

[125I]Iodobolpyramine, a highly sensitive probe for histamine H1-receptors in guinea-pig brain.

[125I]Iodobolpyramine is a novel 125I-ligand for histamine H1-receptors, synthesised using the 125I-Bolton Hunter reagent (2000 Ci/mmol) for acylation of an aminopentyl analogue of mepyramine. Its specific binding varied linearly with the concentration of guinea-pig cerebellar membranes and represented about 80% of the total. Selective interaction with H1-receptors was demonstrated by estimation of Ki values of known agonists and antagonists and confirmed by the low affinity of histamine H2- and H3-receptor antagonists and of non-histaminergic agents. At 25 degrees C, [125I]iodobolpyramine exhibited a slow association rate (180-240 min to reach equilibrium) and a slow dissociation rate (t1/2 = 201 min). Kinetic and saturation data yielded KD values of 0.05 and 0.15 nM, respectively, indicating that it is among the most potent H1-receptor antagonists known. The sensitivity for detecting H1-receptors in guinea-pig cerebellum using [125I]iodobolpyramine was increased 50-fold relative to use of [3H]mepyramine. Well-contrasted autoradiograms of guinea-pig brain, obtained after a short exposure time, confirmed previous H1-receptor localisation established with [3H]mepyramine and revealed new localisations, e.g. in cerebral cortex and nucleus accumbens.

Aminopyridines↗
PubMed1986

Metabolic pools of ATP in cultured bovine adrenal medullary chromaffin cells.

Cultured bovine adrenal chromaffin cells contain a pool of ATP sequestered within the chromaffin vesicles and an extravesicular pool of ATP. In a previous study it was shown that the turnover of ATP in the extravesicular pool was biphasic. One phase occurred with a t1/2 of 3.5-4.5 h whereas the second phase occurred with a t1/2 of several days. The studies described here were undertaken to characterize further the vesicular and extravesicular pools of ATP by examining the effects of metabolic inhibitors, adenosine, and digitonin on ATP utilization and subcellular localization immediately after and 48 h after labeling with [3H]adenosine and 32Pi. Immediately after labeling a combination of cyanide, 2-deoxy-D-glucose, the beta-glucono-1,5-lactone resulted in a 90-95% depletion of the labeled ATP but only a 25% depletion of the endogenous ATP within 30 min. Forty-eight hours after labeling, addition of the inhibitors resulted in a 70% depletion of the [3H]ATP but only a 25% depletion of the [32P]ATP and endogenous ATP. Addition of 10 microM adenosine to the media resulted in a similar loss of [3H]ATP in cells examined immediately after or 48 h after labeling. Adenosine increased the amounts of [32P]ATP when added immediately after labeling but had no effect on the [32P]ATP content when added 48 h after labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1986

Properties and roles of the three subclasses of histamine receptors in brain.

In brain, histamine (HA) is the transmitter of a neuronal system resembling other monoaminergic systems. It is also present in mast cells from which it may control vascular and inflammatory processes. Its various actions are mediated not only by the two well known H1- and H2-receptor subclasses but also by the recently discovered H3-receptors, with distinct localization and pharmacology. H1-receptors mediate a series of biochemical responses which have several features in common: they require intact cells to be observed and largely depend upon the availability of Ca2+. H1-receptor-mediated responses include glycogenolysis, stimulation of cyclic GMP formation, potentiation of cyclic AMP formation. Recent studies indicate that H1-receptors are linked with phosphatidylinositol breakdown and generation of two intracellular signals which both contribute to the final response (e.g. in the cyclic AMP generation). H2-receptors seem to be directly linked with an adenylate cyclase and their stimulation results in enhanced electrophysiologically recorded responses to excitatory agents. Finally whereas H1- and H2-receptors appear to be postsynaptically located, a novel subclass (H3) of HA receptors was recently revealed with a presynaptic localization. H3-receptors are autoreceptors mediating inhibition of HA release from and biosynthesis in histaminergic nerve terminals in the CNS. The physiological and pharmacological implications of three distinct receptor subclasses for HA will be discussed.

Adenylyl Cyclases↗
PubMed1983

Trapping of the beta-adrenergic receptor in the hormone-induced state.

Isoproterenol and other agonists readily dissociate from the beta-adrenergic receptor in turkey erythrocyte membranes. However, when a low concentration of deoxycholate is added, the receptor locks the prebound agonist; i.e., the rate of dissociation of the prebound agonist decreases drastically. The dissociation of prebound antagonists is slightly increased by deoxycholate. Locking, which is thus agonist specific, occurs in the cold, is reversed when detergent is removed from the membranes, and appears not to require the guanyl nucleotide binding protein of the adenylate cyclase system. It is suggested that this induced fit of a receptor to an agonist represents the specific conformational response that normally propagates in the receptor molecule in its interaction with the next component along the pathway of signal transmission.

Animals↗
PubMed1982

Locking of hormone in the beta-adrenergic receptor by attack on a sulfhydryl in an associated component.

Isoproterenol incubated with turkey erythrocyte membranes causes exposure of a specific --SH in a component associated with the beta-adrenergic receptor, presumably in the guanyl nucleotide binding protein. Addition of a reagent which interacts with that specific --SH results in trapping of the hormone in the receptor. As a consequence, the number of hormone binding sites and the function of the receptor are both drastically reduced. Extended incubation at alkaline pH or addition of GDP or GTP at high concentration reactivate the beta-adrenergic receptor. Labeled antagonist binding as well as function of the receptor in activating an adenylate cyclase system are restored. The findings suggest that the normal interaction of the hormone-receptor complex with the guanyl nucleotide binding protein involves a conformational change which transiently locks the hormone in the receptor. GTP releases the tight interaction while addition of an --SH reagent traps the ternary complex of hormone-receptor-guanyl nucleotide binding protein in the locked conformation. Since the components of different hormone-activated adenylate cyclase systems were shown to be interchangeable, it seems likely that the hormone-receptor interaction with the guanyl nucleotide binding protein, as revealed in the present study, is not limited to beta-adrenergic receptor systems.

Animals↗
PubMed1980

Functional implantation of a solubilized beta-adrenergic receptor in the membrane of a cell.

When the beta-adrenergic receptor of turkey erythrocytes was solubilized by deoxycholate, it retained its potential to activate an adenylate cyclase system. Electron microscopy showed that true solubilization had apparently been achieved; no residual membrane or vesicle structure was found. After removal of deoxycholate and addition of phospholipid, the reprecipitated beta-adrenergic receptor was implanted in the cell membrane of Friend erythroleukemia cells by using a chemical fusion method recently developed. Membranes prepared from the cells demonstrated 30-fold stimulation of the Friend cell adenylate cyclase by the implanted beta-adrenergic receptor. The function of the indigenous prostaglandin E(1) receptor of the Friend cells was not much affected by the implantation of large amounts of the foreign receptor. Activity mediated by the beta-adrenergic receptor reached 60% of the activity obtained with fluoride. The implanted receptor is therefore considered to be efficiently coupled to the adenylate cyclase system. The major difficulties hitherto preventing solubilization of hormone receptors and subsequent reconstitution of their function have been overcome by the approach developed in the present work. Conditions of solubilization need preserve only the receptor because all other components, even those unidentified as yet, can be supplied in excess by the adenylate cyclase system of the cell in which the receptor will be implanted. Subsequent recoupling of the receptor to the adenylate cyclase is performed in the native insoluble state of these molecules. Thus, the components need not be subjected to the hazards of solubilization in a common detergent as is usually required in reconstitution procedures. The importance of using implantation as an assay for a functional receptor in the course of purification and the likelihood that the procedure can be adapted to other receptors for hormones and neurotransmitters are discussed.

Adenylyl Cyclases↗
PubMed1976

Central hyperglycemic effect of carbachol in rats.

Intraventricular injection of carbachol produces hyperglycemia in rats at doses which are ineffective when given subcutaneously. This effect is suppressed by intraventricular administration of small amounts of atropine, further supporting the suggestion that the effect of carbachol is due to its action on central cholinergic receptors. Carbachol-induced hyperglycemia is not abolished by adrenalectomy and hypophysectomy, or pretreatment with reserpine.

Adrenal Glands↗
PubMed1975

Evidence of central influences on blood glucose level: malathion hyperglycemia.

To suppress the hyperglycemic effect of malathion in rats, a smaller amount of atropine was required when the drug was injected by intraventricular (i. vent.) than s.c. Pentobarbital, but not diazepam, blocked the hyperglycemic response. The results suggest that central accumulation of acetylcholine was the mediator of the response. Since hyperglycemia was not abolished by adrenalectomy and/or hypophysectomy, a hypothesis is presented to explain how central accumulation of acetylcholine might cause hyperglycemia.

Acetylcholine↗