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Biomedical subjects

M Keen

Publications and source records attributed to M Keen.

At least 55 records · Page 3Linked to original sources
PubMed1990

NaF and guanine nucleotides modulate adenylate cyclase activity in NG108-15 cells by interacting with both Gs and Gi.

1. NaF (10 mM) produced a 2-3 fold increase in adenylate cyclase activity in homogenates of NG108-15 cells incubated in the presence of 1 microM GTP. Higher concentrations of NaF suppressed adenylate cyclase activity. 2. In the presence of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA; 100 microM) or the prostacyclin receptor agonist iloprost (10 nM), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 microM), NaF only inhibited adenylate cyclase activity. 3. Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 microM iloprost-activated enzyme activity. In the presence of 10 microM forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration-effect curves indicated that NaF (2 mM) or Gpp(NH)p (100 microM) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost. 4. Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 microM) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. 5. In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost-activated adenylate cyclase was substantially lost in the absence of added GTP. These results suggest that NaF modulates adenylate cyclase activity in NG108-15 cell membranes by interacting with the alpha subunits of both G0 and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in GSO-O interaction following manipulations such as hormone receptor desensitization.

Adenosine↗
PubMed1989

A selective binding site for 3H-NECA that is not an adenosine A2 receptor.

In homogenates of NG108-15 cells, adenosine analogues activate adenylate cyclase with the following order of potency: N-ethylcarboxamidoadenosine (NECA) greater than 2-chloroadenosine greater than N6-(L-phenylisopropyl)-adenosine (PIA) = cyclohexyladenosine = 2-phenylaminoadenosine. Adenosine receptor antagonists inhibit NECA-stimulated adenylate cyclase activity with the order of potency 3-isobutyl-1-methyl-xanthine (IBMX) greater than theophylline greater than caffeine. These data suggest that these ligands act at an adenosine A2 receptor. There is an apparently homogenous population of saturable 3H-NECA binding sites in homogenates of NG108-15 cells. These sites have an affinity for 3H-NECA of approximately 1 microM, and are present at a density of approximately 10 pmol/mg protein. Unlabelled NECA, 2-chloroadenosine, IBMX and theophylline displace 3H-NECA binding, with an order of potency that suggests that the 3H-NECA binding site may represent an adenosine A2 receptor. However, PIA, cyclohexyladenosine and 2-phenylaminoadenosine produce no detectable displacement of 3H-NECA binding at concentrations that produce a maximal stimulation of adenylate cyclase activity. Pretreatment of NG108-15 cells with either NECA or PIA produces a homologous desensitization of subsequent responses to all the adenosine analogues, with no effect on subsequent responses to a prostacyclin receptor agonist or NaF. This suggests that all the adenosine analogues examined activate an adenosine A2 receptor. Therefore, the 3H-NECA site at which PIA is inactive cannot represent this receptor.

Adenosine↗
PubMed1989

Expression and desensitisation of A2 purinoceptors on cultured bovine aortic endothelial cells.

Purine nucleotides and their metabolites are important mediators of vascular tone. Adenosine promotes relaxation of vascular smooth muscle, acting on the A2 subclass of purinoceptors. There is some evidence that these receptors are also present on vascular endothelial cells. We have therefore examined cultured aortic endothelial cells for adenosine (A2) sensitivity. Bovine aortic endothelial cells (AG4762) were obtained from the Institute of Aging cell repository (USA), and cultured in monolayer flasks. Adenylate cyclase activity in homogenates of AG4762 cells was increased by 5'-(N-ethylcarboxamido)-adenosine (NECA) greater than 2-chloroadenosine greater than adenosine. NECA dependent activation of adenylate cyclase was inhibited by 3-isobutyl-l-methylxanthine greater than theophylline greater than caffeine. The rank order of potency of these compounds identified the responses as mediated by A2 purinoceptors. Prolonged exposure of AG4762 cells to NECA in culture resulted in loss of A2 purinoceptor responsiveness, and purinoceptor activity could be restored to non-dividing densensitized cells by further culture in the absence of the desensitising agent. The cyclic AMP dependent phosphorylation of specific sites in endothelial cells may trigger a number of important biological events which are yet to be determined.

Adenosine↗
PubMed1988

Toxicology screening in acute spinal cord injury.

The validity of self-reported intoxication at time of spinal cord injury (SCI) was examined for 88 cases at admission to an acute SCI center by comparing self-reports with serum and urine analyses. Serum ethanol greater than 50 mg/dl was the most frequently found substance (observed in 40% of the cases) followed by urine analysis evidence of cocaine (14%), cannabinoids (8%), benzodiazipines (5%), and opiates (4%). Evidence of substances with abuse potential was found in urine for 35% of the sample. While 62% of the sample had either serum ethanol greater than 50 mg/dl or a positive urine analysis, only 42% of the sample reported being under the influence of some substance at the time of SCI. Although the relationship between these two measures was statistically significant, self-report and toxicology analyses were discordant in 34% of the cases. These results suggest that routine drug testing at admission to an SCI center will produce both false-negative and false-positive results if substance presence alone is interpreted as evidence of intoxication.

Adolescent↗
PubMed1988

Muscarinic acetylcholine receptors linked to the inhibition of adenylate cyclase activity in membranes from the rat striatum and myocardium can be distinguished on the basis of agonist efficacy.

Muscarinic agonists produce an inhibition of adenylate cyclase activity in broken cell preparations from rat striatum and myocardium. We have attempted to determine the occupancy-response relationships of three muscarinic agonists (carbachol, arecoline, and pilocarpine) by comparing their dose-response curves in these preparations with occupancy curves obtained under the same conditions. These comparisons suggest that the occupancy-response relationships for all three agonists in myocardium and for arecoline and pilocarpine in striatum are linear, response being directly proportional to occupancy. However, there appears to be a considerable receptor reserve for carbachol in the striatum, with carbachol producing a 50% maximal response at concentrations that occupy only 3% of the striatal receptors. These postulated occupancy-response relationships have been tested by blocking different proportions of the muscarinic receptors with the irreversible antagonist benzilylcholine mustard. The effects of this blockade on the dose-response curves are as predicted from the occupancy-response relationships, suggesting that these relationships are correct. The relative efficacies of these muscarinic agonists can be determined from their occupancy-response relationships. The efficacies of arecoline and pilocarpine relative to carbachol are approximately 0.13 and approximately 0.03 in striatum and approximately 1.0 and approximately 0.45 in myocardium, respectively. This difference in relative efficacies in the two tissues is evidence of a real conformational difference between the muscarinic receptors linked to adenylate cyclase in these preparations.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Alcohol use by persons with recent spinal cord injury.

Detailed drinking histories, prevalence of alcohol abuse, and consequences of alcohol use were studied in 103 persons with recent spinal cord injury. Ninety-five percent of the sample acknowledge prior alcohol use. The mean weekday quantity of alcohol consumed was 5.9 drinks (SD = 4.7), with a range of one to 24 drinks per drinking episode during the six months before disability onset. The median frequency of alcohol use was one to two times per week. The sample's mean Michigan Alcoholism Screening Test (MAST) score was 6.8 (SD = 8.0), above the usual cutoff of 5.0, which is interpreted as indicating problematic alcohol use. Forty-nine percent of the sample had scores equal to or exceeding this cutoff. These results suggest that a significant number of individuals with recent spinal cord injuries have heavy drinking histories and experience behavioral problems resulting from alcohol use. The MAST proved to be an efficient method of assessing alcohol-related problems among those with recent spinal cord injuries. Finally, treatment and hospital policy implications are discussed.

Adolescent↗
PubMed1986

Structural microheterogeneity of the muscarinic cholinergic receptor is not related to functional diversity identified by differences in affinity for pirenzepine.

The muscarinic receptor for acetylcholine shows a diversity in ligand binding properties and effector mechanisms which have suggested the existence of two subtypes (M1 and M2), to which the selective antagonist pirenzepine binds with markedly different affinities. The receptor from rat brain, covalently labelled with the alkylating antagonist tritiated propylbenzilylcholine mustard, displays a structural microheterogeneity on electrophoresis, covering the region of apparent molecular weight 66,000-76,000, with dominant components at 68,000 and 73,000. Selective inhibition by pirenzepine of labelling of the M1 receptor with tritiated mustard has been analysed on fluorographs of sodium dodecyl sulphate-polyacrylamide gels and shown to cause a uniform reduction in radioactive labelling of the broad receptor peak, rather than selectively inhibiting either the high- or low-molecular-weight regions of the band. It is further shown that although this receptor microheterogeneity is found for each of four brain regions studied, it is not found for the heart receptor, which gives a discrete labelled band of apparent molecular weight 72,000. It is therefore suggested that the structural microheterogeneity is the result of tissue-specific, posttranslational modification of the molecule, such as glycosylation, and is not directly related to the functional diversity of the receptor.

Animals↗
PubMed1986

The binding of pirenzepine to digitonin-solubilized muscarinic acetylcholine receptors from the rat myocardium.

The binding of pirenzepine to digitonin-solubilized rat myocardial muscarinic acetylcholine receptors has been examined at 4 degrees C. Solubilization produced only small changes in the binding of N-methylscopolamine and atropine. In contrast to the low affinity binding of pirenzepine found to be present in in the membranes, high affinity binding was detected in the soluble preparation. In both preparations, pirenzepine binding was complex. High affinity pirenzepine binding (KD approximately 3 X 10(-8)M) to the soluble myocardial receptors could be monitored directly using [3H]-pirenzepine. [3H]-pirenzepine-labelled soluble myocardial receptors have a sedimentation coefficient of 11.1 s. This indicates that [3H]-pirenzepine binds predominantly to the uncoupled form of the receptor. However, [3H]-pirenzepine-agonist competition experiments indicated that the high affinity pirenzepine binding sites are capable of coupling with a guanosine 5'-triphosphate (GTP)-binding protein. Pirenzepine affinities for the soluble myocardial receptors were unaffected by their state of association with the GTP-binding proteins found in the heart. The equilibrium binding properties of the soluble cortical and myocardial receptors were very similar. However, the binding kinetics of the myocardial receptor were much slower. It appears that the membrane environment can affect the affinity of pirenzepine for the rat myocardial muscarinic receptor. Removal of the constraint by solubilization allows the expression of high affinity pirenzepine binding.

Animals↗
PubMed1986

Soluble and membrane-bound muscarinic acetylcholine receptors.

Three muscarinic acetylcholine receptor subtypes may be defined on the basis of functional and binding studies using selective antagonists. The subtypes may be solubilized in a stable form in digitonin. In solution, the subclasses still exhibit different structure-binding relationships but these have been perturbed by solubilization. The binding of the selective antagonist, pirenzepine, to the purified cortical receptor is complex and similar to that found in membranes. The muscarinic receptor subclasses thus appear to be different molecular entities. Possible explanations for the molecular heterogeneity are discussed. It has also been possible to solubilize receptor-GTP binding protein complexes which have higher sedimentation coefficients (13.4 S) than the apparently monomeric receptor (11.6 S).

Animals↗
PubMed1986

Pseudotumor of the pterygomaxillary space presenting as anesthesia of the mandibular nerve.

Pseudotumor is a term used to describe a space occupying inflammatory lesion which mimics a true neoplastic process in its clinical presentation. In this paper, we report a pseudotumor of the pterygomaxillary space that presented with anesthesia and paralysis of the mandibular nerve. The importance of this case is to call attention to pseudotumors as a clinical entity and to document a case of an extraordinarily aggressive variant that required a surgical approach usually reserved for malignant tumors.

Cranial Nerve Diseases↗
PubMed1985

Detection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis.

An immunodiagnostic test for the detection of a soluble nonprotein mycobacterial antigen by reverse passive haemagglutination with IgM murine monoclonal antibody was developed. The test was used to analyse the cerebrospinal fluid of 89 patients with tuberculous meningitis (TBM) from India and 127 control subjects from India and the UK. The antigen was demonstrable in 88% of culture-positive and 73% of culture-negative TBM patients. However, it was also detected in 21% of Indian patients with pyogenic meningitis, and in 8% of Indian and 1% of UK control subjects. Antibodies binding to a soluble mycobacterial extract were detected at low titre in 68% of all subjects with TBM and in 37% of Indian cases of pyogenic meningitis. Because patients with TBM had raised levels of the antigen and of antibodies to the antigen, the possible role of immune complexes in the pathogenesis of the disease is briefly discussed.

Antibodies, Bacterial↗