Biomedical subjects

J W Bloom

Publications and source records attributed to J W Bloom.

At least 37 records · Page 2Linked to original sources

PubMed1992

Chimeric M1/M2 muscarinic receptors: correlation of ligand selectivity and functional coupling with structural modifications.

Chimeric M1/M2 receptors were expressed in murine fibroblasts (B82) transfected with recombinant m1/m2 receptor genes. The binding affinities of a number of muscarinic antagonists and the agonist carbachol for these chimeric receptors were compared with the ligands' affinities for the M1 and M2 receptors expressed in the B82 cells. The tricyclic compounds, namely pirenzepine (PZ), 11-([2-[(diethylamino)methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido-[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116) and himbacine, shared a binding site between transmembrane domains VI and VII. However, the selective interaction of pirenzepine with M1 and AF-DX 116 and himbacine with M2 involved different structural regions. The high-affinity binding for 4-diphenylacetoxy-N- methylpiperidine and hexahydrosiladifenidol was confined to within loop o2 and transmembrane domains V and VI, which were clearly distinguishable from those of the tricyclic compounds. These results support the hypothesis that the ligands' stereochemical features are critical in their optimal alignment within the ligand binding pocket. The cytoplasmic i3 loop modulated the binding of carbachol such that receptors which contained the i3 domain from the M2 receptor exhibited a single high-affinity state, whereas those with the i3 domain from the M1 receptor had an additional low-affinity state for the agonist. The i3 regions are essential for the differential functional coupling of the M1 and M2 receptors to second messenger systems; however, additional upstream regions seem to be essential for a potent and efficacious activation of phospholipase C by the M1 receptor. This study provides new insight into the molecular basis of ligand selectivity.

Amino Acid Sequence↗

PubMed1991

Respiratory effects of non-tobacco cigarettes: a longitudinal study in general population.

Data from four consecutive surveys of Tucson longitudinal study of airways obstructive disease were used to examine the relation of respiratory symptoms and pulmonary function to non-tobacco cigarette smoking. The surveys were conducted over a six-year period and provided data on 1802 subjects 15-60 years of age, with a total of 5659 individual questionnaires. Estimated odds ratio (OR) of current non-tobacco smoking for chronic cough was 1.73, for chronic phlegm: 1.53, and for wheeze: 2.01 (p less than 0.05). These estimates were adjusted for age, tobacco smoking and occurrence of the symptom in preceding survey. The increased risk of the symptoms was related to the habit continued for several years, and there was no immediate remission of the symptoms after quitting smoking. A significant (p less than 0.05) reduction in pulmonary function (FEV1, Vmax50 and their ratios with FVC) was found a year or more after current non-tobacco smoking was reported. Although the average consumption of non-tobacco cigarettes, believed to be marijuana smoking, was less than one per day, significant effects were still detectable in both pulmonary function and respiratory symptoms.

Adolescent↗

PubMed1991

The m2 muscarinic acetylcholine receptors are coupled to multiple signaling pathways via pertussis toxin-sensitive guanine nucleotide regulatory proteins.

The muscarinic m1 and m2 receptors are functionally coupled to multiple effectors via distinct guanine nucleotide regulatory proteins (G-proteins) defined by their pertussis toxin (PTX) sensitivity. Both receptors are coupled to the hydrolysis of phosphoinositides (PI), whereas only the m2 receptors inhibit cAMP formation. This study examines how the selective interactions of these two receptors with G-proteins may govern their specific functional coupling to the two second messenger pathways. Murine fibroblasts (B82) transfected with the rat m1 or m2 receptor genes were used to test the PTX sensitivity of the m1 and m2 receptor-mediated pathways. It was found that the stimulation of PI hydrolysis and inhibition of cyclic AMP mediated by m2 receptors had similar PTX sensitivity (IC50 = 0.14 ng/ml and 0.26 ng/ml), whereas the m1-mediated PI hydrolysis was PTX insensitive. The EC50 value for carbachol in the m1 receptor-mediated PI hydrolysis was 9.5 microM, whereas those for the m2 receptor-mediated PI hydrolysis and inhibition of cyclic AMP formation were 0.3 microM and 1.2 microM, respectively. The potency of carbachol correlated well with its binding affinities for the two receptor subtypes. These results suggest that the m2 receptors are coupled to multiple pathways via PTX-sensitive G-proteins, which are distinct from those that interact with the m1 receptor. The formation of functional receptor-G-protein complexes may be selective and governed by the efficiencies in coupling between receptors and G-proteins.

Adenylyl Cyclase Inhibitors↗

PubMed1990

Amplification of the rat M2 muscarinic receptor gene by the polymerase chain reaction: functional expression of the M2 muscarinic receptor.

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH beta APr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H] (-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411 +/- 50 fmol/10(6) cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H] (-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 microM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.

Alkaloids↗

PubMed1990

Characterization of receptors for platelet-activating factor in guinea pig lung membranes.

Platelet-activating factor (PAF) is a potent contractile agonist in guinea pig peripheral lung strips. Whether PAF acts via specific receptor sites in the lung has not been fully established. To determine if specific receptor sites could be demonstrated in guinea pig peripheral lung tissue, we performed direct radioligand binding studies in tissue homogenates with [3H]C16-PAF (1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine) and the PAF antagonists [3H]WEB 2086 and [3H]RP52770. These studies demonstrated binding sites for [3H]C16-PAF of high affinity (Kd of 2.6 nM from saturation isotherms and 0.9 nM from kinetic experiments) and a mean density of 200 fmol/mg protein. Binding was inhibited to the same degree with unlabeled C16-PAF, WEB 2086, and RP52770, all with pseudo-Hill slopes of unity. [3H]WEB 2086 binding demonstrated a receptor density similar to that of [3H]C16-PAF (226 fmol/mg protein) and was inhibited to the same degree by unlabeled C16-PAF, C18-PAF, WEB 2086, and RP52770. Although antagonist inhibition yielded pseudo-Hill slopes near unity, agonist inhibition slopes were shallow, suggesting two types or states for the PAF receptors. Direct binding studies with [3H]RP52770 revealed a much larger density of binding sites (1,200 fmol/mg protein), and this binding was not inhibited with C16-PAF, C18-PAF, WEB 2086, or lyso-PAF. These results indicate that guinea pig lung has specific binding sites for [3H]C16-PAF, that WEB 2086 is an effective antagonist of C16- and C18-PAF binding at these sites, and that RP52770 binds to the PAF site but, in addition, binds to another site with a much greater density.

Animals↗

PubMed1989

The interactions of factors X and IX with phospholipid.

The interactions of human factors X and IX with phospholipid were studied with an ELISA system. In the presence of calcium ions, factor X bound to phosphatidylserine with an affinity of 1.56 x 10(10) M-1 and to phosphatidylcholine with an affinity of 5.6 x 10(9) M-1. In the presence of calcium ions, factor IX bound to phosphatidylserine with an affinity of 8.4 x 10(8) M-1 and no binding to phosphatidylcholine was observed. No competition of factors X and IX for phosphatidylserine binding sites was observed.

Binding, Competitive↗

PubMed1989

Detection and reduction of protein A contamination in immobilized protein A purified monoclonal antibody preparations.

Protein A chromatography is an excellent technique for the purification of monoclonal antibodies. However, the presence of Protein A in therapeutic monoclonal antibody preparations due to leaching has been linked with toxicity in animals and humans. Two sandwich ELISAs were developed to monitor Protein A column leaching: (1) rabbit anti-Protein A for capture and anti-Protein A F(ab')2-HRP for detection; and (2) rabbit anti-Protein A for capture and anti-Protein A-biotin for detection. The biotin ELISA is sensitive to the subnanogram range. In addition, these assays were used to develop DEAE and gel filtration chromatography techniques that substantially reduce Protein A levels in monoclonal antibodies purified by Protein A chromatography.

Animals↗

PubMed1989

Subclinical effects of cigarette smoking. A five-year follow-up of physiologic comparisons of healthy middle-aged smokers and nonsmokers.

Measurements of ventilatory function, lung elastic recoil, diffusing capacity, and distribution of ventilation were obtained on healthy middle-aged cigarette smokers and nonsmokers on two occasions five years apart in order to assess the effects of smoking and the change which may occur over this five-year interval. Subjects were drawn from a randomly selected sample of the population of Tucson, AZ. Exactly the same protocol, methods, and equipment were employed in both studies. Although very few of these healthy subjects had abnormal function, there were significant differences in most indices of function between smokers and nonsmokers. However, we could discern no difference between smokers and nonsmokers in change in function over five years. It appears that, in smokers who remain free of serious respiratory trouble, there are subtle changes which accumulate over the years and which are too gradual to detect over a five-year interval.

Female↗

PubMed1988

Mycoplasma and ureaplasma in bronchoalveolar lavage fluids from immunocompromised hosts.

The significance of Mycoplasma spp. and Ureaplasma urealyticum infection in immunocompromised patients has not been clearly established. We identified mycoplasma or ureaplasma in bronchoalveolar lavage fluid from 12 of 61 (20%) immunocompromised patients with pulmonary infiltrates. A complete microbiological investigation was made on the bronchoalveolar lavage fluids, and Mycoplasma pneumoniae was the sole agent detected in three instances, suggesting that it may have been the cause of the infiltrates in these immunocompromised patients. Other Mycoplasma spp. and ureaplasma were detected in nine patients, but in eight of these patients other pulmonary pathogens were also recovered.

Bronchoalveolar Lavage Fluid↗

PubMed1988

A muscarinic receptor subtype modulates vagally stimulated bronchial contraction.

An in vitro preparation was developed to study vagus nerve-stimulated (preganglionic) and field-stimulated (post-ganglionic) contraction of the rabbit main stem bronchus and to compare the inhibitory effects of muscarinic antagonists on that contraction. The maximal contractile responses (20 V, 0.5 ms, 64 Hz) for either field or vagal stimulation were completely abolished by atropine (60 nM). Hexamethonium (0.1 mM) abolished the response to vagal stimulation but did not affect the field-stimulated response. To compare the effectiveness of atropine and pirenzepine as antagonists at the nerve-smooth muscle junction, inhibition studies of field-stimulated contractions were performed. Pirenzepine was 102- to 178-fold less potent than atropine when compared at the inhibitory concentration of antagonist that produced 25, 50, and 75% inhibition (IC25, IC50, and IC75, respectively), indicating that the muscarinic receptor at the nerve-smooth muscle junction is a muscarinic receptor with low affinity for pirenzepine (M2 subtype). Atropine had similar inhibitory effects on vagal- and field-stimulated contractions. In contrast, pirenzepine was more potent in inhibiting vagally stimulated contraction than field-stimulated contraction, especially at the IC25 where pirenzepine was only 8- to 22-fold less potent than atropine in inhibiting vagally stimulated contraction. These data suggest that an M1 subtype of muscarinic receptor modulates excitatory neurotransmission through bronchial parasympathetic ganglia.

Animals↗

PubMed1988

Risk factors in a general population for snoring. Importance of cigarette smoking and obesity.

In order to study risk factors associated with snoring in a general adult population, 2,187 subjects in the Tucson Epidemiologic Study of Obstructive Airways Disease were surveyed to determine the prevalence of snoring. Major independent risk factors for snoring were male gender, age between 40 and 64 years, obesity, and current cigarette smoking. Furthermore, greater intensity of cigarette smoking also was associated with higher snoring prevalence rates. Snoring prevalence remained elevated in subjects who recently quit smoking, but declined in ex-smokers to the level of never smokers within four years of smoking cessation. The presence of cough or sputum production was associated with an increase in snoring prevalence especially in ex-smokers. Snoring prevalence was slightly increased in subjects who regularly used alcohol or medications as aids to sleep. We conclude that cigarette smoking, obesity, male gender, age over 40, and use of alcohol or sleep medications are important risk factors for snoring. We propose that the effect of smoking may be related to the production of upper airway inflammation and edema by cigarette smoke, and that smoking cessation may eventually reduce snoring risk.

Adult↗

PubMed1988

Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung.

The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist [3H]pirenzepine [( 3H]PZ) and the classical muscarinic antagonist [3H](-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for [3H](-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of [3H](-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.

Adult↗

PubMed1987

Respiratory effects of non-tobacco cigarettes.

Data from the Tucson epidemiological study of airways obstructive disease on smoking of non-tobacco cigarettes such as marijuana were analysed to determine the effect of such smoking on respiratory symptoms and pulmonary function. Among adults aged under 40, 14% had smoked non-tobacco cigarettes at some time and 9% were current users. The prevalence of respiratory symptoms was increased in smokers of non-tobacco cigarettes. After tobacco smoking had been controlled for men who smoked non-tobacco cigarettes showed significant decreases in expiratory flow rates at low lung volumes and in the ratio of the forced expiratory volume in one second to the vital capacity. This effect on pulmonary function in male non-tobacco cigarette smokers was greater than the effect of tobacco cigarette smoking. These data suggest that non-tobacco cigarette smoking may be an important risk factor in young adults with respiratory symptoms or evidence of airways obstruction.

Adolescent↗

PubMed1987

The course and prognosis of different forms of chronic airways obstruction in a sample from the general population.

We examined the course and prognosis in subjects selected from the general population who had chronic airflow obstruction at the time of their enrollment in a longitudinal epidemiologic study. Mortality and the rate of change in lung function were analyzed in relation to the initial clinical characteristics of the subjects. Twenty-seven subjects with symptoms and signs of asthma (Group I) had a higher survival rate and a much lower rate of decline in pulmonary function than the 45 subjects in Group III, whose clinical characteristics were more compatible with an emphysematous form of chronic obstructive pulmonary disease (COPD). The 10-year mortality among subjects in Group III (non-atopic smokers without a history of asthma) was close to 60 percent, whereas it was only 15 percent in Group I (atopic subjects or nonsmokers with known asthma). The mean overall rate of decline in forced expiratory volume in one second was 70 ml per year in Group III but less than 5 ml per year in Group I. Forty-five patients (Group II) who did not clearly fit into either Group I or III had intermediate values for survival and decline in pulmonary function. Previous data on mortality from COPD and the rate of progression of the condition, although compatible with our findings in patients who had an emphysematous form of disease, are not applicable to those with an asthmatic-bronchitic form. Better control of the progression of asthmatic bronchitis with therapy may explain its more favorable prognosis.

Asthma↗

PubMed1987

The interaction of rDNA factor VIII, factor VIIIdes-797-1562 and factor VIIIdes-797-1562-derived peptides with phospholipid.

The interaction of rDNA factor VIII, factor VIIIdes-797-1562 and factor VIIIdes-797-1562-derived peptides with phospholipid were studied with an ELISA system. Factor VIII was observed to bind to phosphatidylserine but not to phosphatidylcholine or phosphatidylethanolamine. Factor VIIIdes-797-1562 also bound to phosphatidylserine with the same affinity, suggesting that residues 797-1562 of the factor VIII molecule are not required for phospholipid binding. In addition, the binding of the purified factor VIII carboxy-terminal Mr 80,000 and amino-terminal Mr 90,000/115,000 polypeptides to phosphatidylserine was investigated. Only the Mr 80,000 polypeptide was observed to bind, suggesting that the carboxy-terminal of factor VIII contains the lipid binding domain.

Electrophoresis, Polyacrylamide Gel↗

PubMed1987

Heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex demonstrated by the selective antagonist AF-DX 116.

Recent studies have demonstrated that the majority of muscarinic receptors in rabbit peripheral lung homogenates bind pirenzepine with high affinity (putative M1 subtype). In experiments of AF-DX 116 inhibiting [3H](-)quinuclidinyl benzilate or [3H]pirenzepine, we found similar inhibitory constants for AF-DX 116 binding in rat heart and rabbit peripheral lung that were 4-fold smaller (i.e. of higher affinity) than the inhibitory constant for rat cerebral cortex. This result demonstrates heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex.

Animals↗

PubMed1987

A muscarinic receptor with high affinity for pirenzepine mediates vagally induced bronchoconstriction.

The nature of the putative muscarinic receptor subtypes involved in vagally mediated bronchoconstriction was examined in the rabbit model utilizing the classical muscarinic antagonist atropine and the selective antagonist pirenzepine. In vivo electrical stimulation of the cervical vagus nerves in anesthetized rabbits resulted in a reproducible increase in pulmonary resistance indicative of bronchoconstriction and a marked negative chronotropic effect on the heart. Both atropine and pirenzepine produced dose-related inhibition of these two vagal effects. Fifty percent inhibition of the vagally induced increase in pulmonary resistance was achieved with an infusion of pirenzepine that was only 8-fold greater than the equi-effective dose of atropine. In contrast, the dose of pirenzepine required to inhibit the vagally induced decrease in heart rate by 50% was 100-fold greater than the atropine dose. Thus, pirenzepine is markedly more potent in inhibiting vagally mediated bronchoconstriction than bradycardia. In vitro inhibition of methacholine-induced contraction of bronchial rings with atropine and pirenzepine yielded pA2 values of 8.86 and 6.88 respectively (95-fold potency ratio), demonstrating that the muscarinic receptors on airway smooth muscle cells that mediate contraction are not of the pirenzepine-sensitive subtype.

Action Potentials↗

PubMed1987

FES for bladder: direct or indirect means?

Efforts to restore function to the neurologically disabled lower urinary tract by direct electrical stimulation of the bladder wall have met with only very limited success. This has been due to pain and cocontraction of bladder outlet mechanisms caused by presumed spread of the large currents required to effectively directly stimulate the detrusor muscle. Stimulation at the four anatomical sites of the sacral neural outflow on the other hand has been more successful. Conus medullaris stimulation has resulted in "good results" in just over half of the 28 patients so treated. Acceptance of this technique has been limited by the poor selectivity of the intramedullary electrodes in stimulating only the target motor neurons and the resultant clinical problems with the consequent stimulus current spread. Sacral anterior root stimulation has been used in at least 88 patients with generally good results. Cocontraction of the detrusor and external urethral sphincter are circumvented by the use of an intermittent pattern of stimulation. The primary disadvantage of this technique is the obligatory placement of the electrodes within the cerebrospinal fluid compartment. Clinical experience with stimulation of the extradural sacral mixed nerves is limited. Experimental studies indicate that success with this technique requires dorsal rhizotomy and pudendal neurotomy. Preliminary clinical experience suggests that these surgical manipulations may not be necessary for a successful outcome. The literature on clinical application of pelvic nerve stimulation is too limited for detailed comment on this technique. A definitive technique for restoration of bladder function by electrical stimulation remains to be developed.

Electric Stimulation Therapy↗