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Biomedical subjects

D Gurwitz

Publications and source records attributed to D Gurwitz.

At least 73 records · Page 4Linked to original sources
PubMed1989

Proteolytic regulation of neurite outgrowth from neuroblastoma cells by thrombin and protease nexin-1.

This review summarizes studies on the reciprocal regulation of neuroblastoma neurite outgrowth by thrombin and protease nexin-1 (PN-1). PN-1 recently was shown to possess the same deduced amino acid sequence as the glial-derived neurite-promoting factor. The neurite outgrowth activity of PN-1 depends on its ability to inhibit thrombin. Thrombin not only blocks the neurite outgrowth activity of PN-1, but it also brings about neurite retraction in the presence of PN-1. Thrombin also produces neurite retraction in the absence of PN-1 and other regulatory factors. This suggests that its activity is due to a direct action on cells. The neurite retraction by thrombin depends on its proteolytic activity. It does not occur with the other serine proteases that have been tested, indicating that it is a specific effect and is not due to a general proteolytic effect that could detach neurites from the culture dish. Serum brings about neurite retraction in certain neuroblastoma cells and primary neuronal cultures; most of this activity is due to residual thrombin in the serum. Together, these results suggest that PN-1 and thrombin (or a thrombin-like protease) play a role in regulation of neurite outgrowth.

Amyloid beta-Protein Precursor↗
PubMed1989

Protease nexin-1, an antithrombin with neurite outgrowth activity, is reduced in Alzheimer disease.

Protease nexin-1 (PN-1) is a cell-secreted protein that inhibits certain proteases, particularly thrombin, by forming SDS-stable complexes with the catalytic site serine of the protease. PN-1 was recently shown to be identical to a glial-derived neurite-promoting factor/glial-derived nexin present in rat brain. Its neurite outgrowth activity depends on inhibition of thrombin, presumably because thrombin brings about neurite retraction. Here we show that human brain contains PN-1 and that PN-1 activity in brains of individuals with Alzheimer disease (AD) was only 14% of control values (total of 14 AD patients and 7 control individuals). PN-1 activity in the hippocampus, a region with marked neuropathology in AD, was 15% of control values (10 AD patients and 4 control individuals). Western blot analysis indicated a large decrease in free PN-1 protein and an increase in PN-1-containing complexes that comigrated with PN-1-thrombin complexes. Northern blot analysis indicated that PN-1 mRNA levels were about equal in brains from AD patients and control individuals. Thus these results suggest that the decreases in PN-1 activity and free PN-1 protein are due to formation of PN-1-protease complexes.

Alzheimer Disease↗
PubMed1988

Thrombin modulates and reverses neuroblastoma neurite outgrowth.

Previous studies have shown that neuroblastoma cells and several types of primary neuronal cells in culture rapidly extend neurites when switched from serum-containing to serum-free medium. The present studies on cloned neuroblastoma cells show that thrombin blocked this spontaneous differentiation at 2 nM with a half-maximal potency of 50 pM. This required the catalytic activity of thrombin and was reversed upon thrombin removal. Thrombin also caused cells in serum-free medium to retract their neurites at equally low concentrations. Two other serine proteases, urokinase and plasmin, did not block or reverse neurite extension even at 100-fold higher concentrations. A specific assay for thrombin indicated that thrombin detected in serum-containing medium from neuroblastoma cultures was derived from serum and that it was likely responsible for much of the known capacity of serum to maintain neuroblastoma cells in a nondifferentiated state. This was supported by the finding that heparin addition reduced the thrombin concentration in serum-containing medium and stimulated neurite outgrowth from neuroblastoma cells in serum-containing medium. Studies on the ability of thrombin to modulate neurite outgrowth by other agents showed that it blocked and reversed the neurite outgrowth activity of two thrombin inhibitors: protease nexin-1 (which is identical to glial-derived neurite-promoting factor) and hirudin. Thrombin, however, did not block the neurite-promoting activity of dibutyryl cAMP or prostaglandin E1. These results suggest a specific role for thrombin in control of neurite outgrowth.

Alprostadil↗
PubMed1987

Dual pathways in muscarinic receptor stimulation of phosphoinositide hydrolysis.

The relationships between phosphoinositide hydrolysis induced by various muscarinic agonists and by membrane depolarization agents were investigated in rat cerebral cortex and heart atrium slices. In both preparations, phosphoinositide hydrolysis was stimulated by a combination of carbamylcholine and membrane depolarization with 40 mM K+ in a synergistic fashion. The synergism was more pronounced at lower external calcium ion concentrations and was sensitive to verapamil. Lower external calcium ion concentrations were required for demonstration of the synergism in heart atrium slices than in cerebral cortex slices. The carbamylcholine-induced stimulation was only partially additive with membrane depolarization via Na+ channel gating by batrachotoxin. In addition, K+ depolarization eliminated the sensitivity of carbamylcholine-stimulated phosphoinositide hydrolysis to the sodium channel blocker tetrodotoxin. Our results suggest that muscarinically stimulated phosphoinositide hydrolysis in rat cerebral cortex and heart atrium slices may occur by dual pathways which interact synergistically and that only one of the pathways is depolarization-dependent. Different muscarinic agonists could preferentially utilize these pathways, thus perhaps explaining their different potencies in stimulating phosphoinositide hydrolysis.

Animals↗
PubMed1985

Increased agonist affinity is induced in tetranitromethane-modified muscarinic receptors.

Tetranitromethane (TNM) modifies the muscarinic receptors from rat cerebral cortex. The modified receptor possesses an increased binding affinity (6-9-fold) toward several agonists such as acetylcholine, carbamoylcholine, arecoline, etc. The binding of antagonists (Bmax and Kd) is only slightly altered. The effects of TNM treatment can be prevented by atropine, thus indicating that TNM modifies residue(s) at the binding site. We carried out a series of successive chemical modifications which indicated that the modified residue(s) is (are) most probably a tyrosyl and not a cysteinyl residue. This conclusion gains support from the pH profile of agonist binding, which suggests the involvement of a residue with an apparent pK comparable to that of the phenolic hydroxyl of a nitrotyrosyl residue. The binding properties of the modified receptor, when compared to those of the native one, clearly indicate that the response to TNM modification with respect to the binding of agonists such as acetylcholine and carbamoylcholine is different from that when oxotremorine and its analogue are employed. This is interpreted as being the result of different binding modes exhibited by the various agonists. Nitration of the receptors can be prevented by the presence of an antagonist but not by an agonist. We propose that this differential response is due to the formation of ligand-receptor complexes that differ with respect to the microenvironment of the modified tyrosyl residue.

Acetylcholine↗
PubMed1985

Rat brain and heart muscarinic receptors: modification with tetranitromethane.

Tetranitromethane at a concentration of 50 microM modifies the muscarinic receptors in membrane preparations from rat striatum, hippocampus and heart atrium, but not from the rat brain stem. While the binding of antagonists is only slightly altered, the modified receptor possesses an increased affinity of up to 8-fold for [3H]-acetylcholine binding to the high affinity state. This effect is absent if the nitration is carried out in the presence of an antagonist, but not in the presence of an agonist. The affinity for carbamylcholine is increased for both the high and the low affinity state of the receptor, as is evident from its ability to compete with a labeled antagonist. In addition, the proportion of binding sites (alpha) exhibiting the high affinity state for [3H]-acetylcholine or for carbamylcholine is increased upon nitration. This increase cannot be protected against by an antagonist, and is enhanced when nitration takes place in the presence of an agonist. With the agonists oxotremorine and [3H]-oxotremorine-M only the latter effect (i.e., increase in alpha) is observed following nitration, while their dissociation constants for the receptor are unchanged. Data are discussed with respect to the proposed existence of subtypes of muscarinic receptors, as well as the importance of the agonist chosen for studies of ligand-receptor interactions.

Acetylcholine↗
PubMed1985

High affinity binding of [3H]acetylcholine to muscarinic receptors. Regional distribution and modulation by guanine nucleotides.

The interaction of [3H]acetylcholine ([3H]AcCh) with the muscarinic receptor was studied in seven distinct rat brain regions and in heart atrium by employing 10 microM atropine to define specific binding. The specific binding exhibited by the labeled neurotransmitter was found to be sensitive to muscarinic but not to nicotinic drugs. The muscarinic high affinity agonist-binding sites were characterized with respect to their binding properties, regional distribution, pharmacology, and modulation by guanyl nucleotides and by transition metal ions. In all tissues examined, specific binding of [3H]AcCh was saturable over the range of 4-200 nM and occurred in a receptor population that was apparently homogeneous and had a dissociation constant of approximately 19-39 nM in most of the regions. The ratio of muscarinic receptors labeled by [3H]AcCh to those labeled by the potent antagonist [3H]N-methyl-4-piperidylbenzilate varied markedly among tissues, from 0.15 in the hippocampus to 0.71 in the atrium. This ratio was lower in brain regions rich in muscarinic receptors, where smaller sensitivity of [3H]AcCh binding to guanyl nucleotides was also observed. In the presence of the latter [3H]AcCh binding was decreased by 25 to 90% in different tissues, with the greatest decreases occurring in the atrium and brainstem. In the latter preparations, transition metal ions do not affect [3H] AcCh binding, while in the other preparations studied they induce an increase in the binding capacity for the labeled neurotransmitter, which is sensitive to guanine nucleotides.

Acetylcholine↗
PubMed1984

Copper ions and diamide induce a high affinity guanine-nucleotide-insensitive state for muscarinic agonists.

The binding capacity of [3H]-acetylcholine for muscarinic receptors of rat cerebral cortex membranes is increased in the presence of Cu2+ ions from 690 to 1320 fmol/mg protein with no significant change in affinity. Membranes treated with 50 microM Cu2+ and washed retain the increased binding capacity. Agonist binding in copper-treated membranes is insensitive to guanylylimidodiphosphate even at high concentrations (greater than 200 microM). Similar results were obtained when the sulfhydryl oxidizing agent, diamide (2 mM) was substituted for Cu2+ in the treatment of membranes. These data suggest the involvement of inter- or intra-molecular SH/S-S transitions in the interaction between the muscarinic receptor and a guanine nucleotide binding regulatory protein.

Animals↗
PubMed1984

Expression of muscarinic binding sites in primary human brain tumors.

The expression of muscarinic binding sites was examined in a collection of primary brain tumors of different cellular origins and various degrees of dedifferentiation, as compared to control specimens. Eleven gliogenous tumors were examined, all of which contained substantial amounts of muscarinic binding sites. Most of the other tumor types examined did not display detectable binding of [3H]N-methyl-4-piperidyl benzilate ([3H]4NMPB). Scatchard analysis indicated the existence of homogeneous antagonist sites in both normal forebrain and glioblastoma multiforme, with Kd values of 1.2 nM and 0.9 nM, respectively. The density of muscarinic binding sites varied between tumors from different patients, and also between specimens prelevated from different areas of the same tumor. This variability, as well as the average density of binding sites, appeared to be larger in highly malignant tumors than in less malignant ones. In contrast, the density of muscarinic receptors from control specimens was invariably high, but within the same order of magnitude. To test whether the muscarinic binding activity in the brain tumors is correlated to other cholinoceptive properties, cholinesterase activity was also examined. Individual data for density of [3H]4NMPB binding sites were then plotted against corresponding values of cholinesterase activity. The pattern of distribution of these values was clearly different in tumor specimens, when compared to that observed in samples derived from non-malignant brain. Our observations indicate that human brain cells of gliogenous origin are capable of expressing muscarinic binding sites, and that, if a correlation exists between muscarinic receptors and cholinesterase levels in gliogenous tumors, it differs from that of non-malignant brain tissue.

Adult↗
PubMed1984

Recognition of the muscarinic receptor by its endogenous neurotransmitter: binding of [3H]acetylcholine and its modulation by transition metal ions and guanine nucleotides.

Agonist binding to the muscarinic receptor in rat cerebral cortex membranes was studied by using the neurotransmitter itself, [3H]acetylcholine [( 3H]AcCho). By using 10 microM atropine or oxotremorine to define specific binding, it was possible to demonstrate specific binding of [3H]AcCho that was sensitive to muscarinic but not to nicotinic ligands. Equilibrium binding experiments with 5-240 nM [3H]AcCho indicated specific binding of the ligand to a saturable population of muscarinic receptors (361 +/- 29 fmol/mg of protein; Kd = 76 +/- 17 nM). This value represented 25% of the available binding sites for a labeled antagonist in the same preparation and corresponds to the proportion of high-affinity agonist binding sites observed previously in competition experiments with labeled antagonists. Inclusion of transition metal ions (e.g., 2 mM Ni2+) in the assay increased the equilibrium binding of [3H]AcCho (628 +/- 38 fmol/mg of protein, Kd = 86 +/- 21 nM) but did not affect equilibrium binding of 3H-labeled antagonists, indicating conversion of low- into high-affinity muscarinic agonist binding sites. The increase developed slowly over 30 min of incubation at 25 degrees C but could be reversed rapidly (approximately equal to 2 min) by the chelating agent EDTA or by guanine nucleotides. These data directly reveal a slow though quickly reversible interconversion of low- into high-affinity muscarinic agonist binding sites.

Acetylcholine↗
PubMed1983

Long-term evaluation of fenoterol by two different modes of administration (oral versus metered aerosol).

Thirteen asthmatic children were treated double-blind for 6 weeks each with either inhaled or oral fenoterol (a beta-2-selective adrenergic bronchodilator) three times a day. The oral dose regimen resulted in superior bronchodilation on the basis of peak expiratory flow rates, although clinical symptom scores did not differ with the route of administration. We conclude that oral fenoterol can be used on a chronic basis for the treatment of moderate asthmatics. Doses of inhaled fenoterol higher than 0.4 mg three times per day used in this study may be required to produce a similar effect to 0.8 mg/kg of oral fenoterol in three divided doses.

Administration, Oral↗
PubMed1983

Assessment of a new device (aerochamber) for use with aerosol drugs in asthmatic children.

We compared the pulmonary response to fenoterol delivered by a conventional MDI with the response after MDI plus the aerochamber (AC). Twelve children with moderate to severe asthma (mean age 10.8 years) participated in this double-blind crossover study. On two consecutive days, in random order, subjects received one puff of fenoterol hydrobromide (200 mcg) either by MDI or MDI plus AC. Pulmonary function, cardiovascular response and tremor were assessed over a period of four hours. In a similar fashion, the children received one puff of fenoterol three times a day for six weeks by either method in a randomized open study. Daily symptoms and peak flows before and after drug were recorded in daily diaries. The patients returned to the pulmonary function laboratory at 6 and 12 weeks for four hour pulmonary function tests. In the short-term study, the magnitude and time course of pulmonary response was similar for both treatment regimens (p greater than 0.4). No significant effects on blood pressure or pulse or differences in tremor assessment were seen. Similarly, in the long term study the baseline pulmonary function and pulmonary response were markedly consistent between the two treatment regimens (p greater than 0.05). Peak flows were significantly higher in the evening than in the morning (p less than 0.001) and the magnitude of the response to drug was higher in the morning than the evening (p less than 0.001). There were no differences between MDI and MDI plus AC in daily symptom scores or use of concomitant medication. We conclude that acute or chronic administration of fenoterol aerosol to asthmatic children by MDI with or without the AC produces similar benefits in pulmonary function, symptomatology and concomitant medication. The AC device would be a useful adjunct for a child who has difficulty in coordinating the metered dose inhaler.

Aerosols↗
PubMed1982

Improved respiratory prognosis in patients with cystic fibrosis with normal fat absorption.

The clinical presentation, courses, and sweat chloride values of 72 CF patients with normal fat absorption are described. In general, these patients had milder clinical symptoms and a lower mean sweat chloride value than their counterparts with steatorrhoea. Pulmonary function tests, including FEV1, FVC, FEF25%-75%, PaO2, and RV/TLC%, were significantly better in patients with normal fat absorption compared with both male and female patients who had steatorrhoea. The maintenance of better pulmonary function, coupled with the low mortality, suggests that patients without steatorrhoea have a better prognosis. This difference remains unexplained, but may be contributed to by nutritional, genetic, or pancreatic factors.

Adolescent↗
PubMed1981

Increased incidence of bronchial reactivity in children with a history of bronchiolitis.

To assess bronchial reactivity in children who have had bronchiolitis, we studied 48 children by challenging them with methacholine nine or ten years after admission to hospital with bronchiolitis. Pulmonary function was also evaluated. Fifty-seven percent of children studied had bronchial hyperreactivity. Thirty-three percent of first-degree relatives of those with a positive MCH challenge had a positive response. There was a significant correlation between the occurrence of a positive MCH challenge and a history of recurrent bronchiolitis. Pulmonary function tests demonstrated lower flow rates in the positive responders. Fourteen children had a history of asthma or wheezing, but this did not appear to be severe or frequent, and few required long-term therapy. There appears to be a strong genetic component in the prevalence of bronchial reactivity in these children. Bronchial hyperreactivity may be a risk factor in the development of COPD.

Bronchi↗