Search PubMed⌕ Search

Biomedical subjects

E Heldman

Publications and source records attributed to E Heldman.

At least 37 records · Page 2Linked to original sources
PubMed1994

Agonist-stimulated release of von Willebrand factor and procoagulant factor VIII in rats with and without risk factors for stroke.

Lipopolysaccharide (LPS)-induced (i.v. or i.c.v., 1.8 mg/kg) release of von Willebrand factor (vWF) was examined in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. SHR rats released significantly (P < 0.05) more vWF than WKY rats in response to LPS. LPS also inhibited factor VIII procoagulant activity (FVIII:c) which may indicate an increase in thrombin activity. Cultured cerebrovascular endothelial cells (EC) derived from both SHR and WKY rats, as well as human umbilical vein EC (HUVEC) cultures constitutively released vWF. Treatment with agonists including LPS, thrombin and tumor necrosis factor-alpha (TNF alpha) did not affect the in vitro secretion of vWF by cerebrovascular EC cultures but significantly upregulated vWF release by HUVEC cultures. Preincubation of cerebrovascular EC cultures with interleukin-1 (IL-1) +/- TNF alpha or co-culturing in the presence of LPS-activated syngeneic monocytes had no effect on vWF secretion. The findings demonstrate that conditions of hypertension may affect endothelial cells and make them more responsive to agonist stimulation and thereby increase secretion of vWF, an important factor in hemostasis as well as thrombosis. The capacity of LPS to significantly affect the in vivo secretion of vWF in SHR and WKY rats but not cultured cerebrovascular EC indicates that observed elevations in plasma vWF were not derived from cerebrovascular EC. It is suggested that hypertension may function as a risk factor for thrombotic stroke by influencing factors involved in coagulation processes, such as vWF and factor VIII:c.

Animals↗
PubMed1994

Discrete activation of transduction pathways associated with acetylcholine m1 receptor by several muscarinic ligands.

Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC50 values of 2.6, 3.8 and 76 microM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.

Adenylyl Cyclases↗
PubMed1993

Selective signaling via unique M1 muscarinic agonists.

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AF102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the m1 AChR subtype. Notably, the selectivity of the new m1 agonists is reflected also by activation of select second messenger systems via distinct G-proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand-selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimer's disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.

Acetylcholine↗
PubMed1993

Anandamide, a brain endogenous compound, interacts specifically with cannabinoid receptors and inhibits adenylate cyclase.

A putative endogenous cannabinoid ligand, arachidonylethanolamide (termed "anandamide"), was isolated recently from porcine brain. Here we demonstrate that this compound is a specific cannabinoid agonist and exerts its action directly via the cannabinoid receptors. Anandamide specifically binds to membranes from cells transiently (COS) or stably (Chinese hamster ovary) transfected with an expression plasmid carrying the cannabinoid receptor DNA but not to membranes from control nontransfected cells. Moreover, anandamide inhibited the forskolin-stimulated adenylate cyclase in the transfected cells and in cells that naturally express cannabinoid receptors (N18TG2 neuroblastoma) but not in control nontransfected cells. As with exogenous cannabinoids, the inhibition by anandamide of the forskolin-stimulated adenylate cyclase was blocked by treatment with pertussis toxin. These data indicate that anandamide is an endogenous agonist that may serve as a genuine neurotransmitter for the cannabinoid receptor.

Adenylyl Cyclase Inhibitors↗
PubMed1993

Progress in medicinal chemistry of novel selective muscarinic agonists.

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor subtypes. AF102B, AF125, AF150 and AF151 are such rigid analogs of ACh. Whilst AF125 is an M2 > M1 agonist, AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia, only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AF102B for M1 AChRs in rat cortex and in CHO cells stably transfected with the m1 AChR subtype. In various animal models for Alzheimer's disease (AD) all three agonists (AF102B, AF150 and AF151), and in particular AF102B, exhibited positive effects on mnemonic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy in AD. Here we review some current features of these compounds, which may be relevant to a rational treatment strategy in AD. Comparison is made, whenever possible, with some new and old muscarinic agonists.

Acetylcholine↗
PubMed1992

Comparison of stimulated tissue factor expression by brain microvascular endothelial cells from normotensive (WKY) and hypertensive (SHR) rats.

The amounts of tissue factor (TF) expressed by brain microvascular endothelial cells (BMECs) from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were compared after stimulating the cells with different doses of lipopolysaccharide (LPS), thrombin, phorbol myristic acid (PMA), Ca(2+)-ionophore (A23187), or tumor necrosis factor (TNF) and interleukin-1 (IL-1). Treatment of cultured BMECs from WKY and SHR with all of these factors dose-dependently increased their total amount of TF; no substantive differences in the levels of enhanced TF expression were observed between WKY and SHR BMECs. We conclude that stimulated endothelium from rats with hypertension, a major stroke risk factor, is not hyperresponsive with respect to TF expression when compared to normotensive controls.

Animals↗
PubMed1992

Differential long-term effect of AF64A on [3H]ACh synthesis and release in rat hippocampal synaptosomes.

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 microliters/side) or vehicle (saline). Synaptosomes were preloaded with [3H]choline ([3H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [3H]Ch and [3H]acetylcholine ([3H]ACh) and for their ability to synthesize and release [3H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [3H]Ch was reduced to 60% of control; (ii) residing [3H]ACh levels were 43% of control while residing [3H]Ch levels were 72% of control; (iii) basal and K(+)-induced [3H]ACh release were 77% and 73% of control, respectively; (iv) high K(+)-induced synthesis of [3H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.

Acetylcholine↗
PubMed1992

Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1992

Release of proinflammatory and prothrombotic mediators in the brain and peripheral circulation in spontaneously hypertensive and normotensive Wistar-Kyoto rats.

BACKGROUND AND PURPOSE: We reported previously that stroke risk factors prepared the brain stem for the development of ischemia and hemorrhage and induced the production of tumor necrosis factor following an intrathecal injection of lipopolysaccharide, a prototypic monocyte-activating stimulus. This study evaluates whether blood or brain cells of hypertensive rats produce more proinflammatory and prothrombotic mediators than do blood or brain cells of normotensive rats. METHODS: Levels of tumor necrosis factor, platelet-activating factor, 6-ketoprostaglandin F1 alpha, and thromboxane B2 in the cerebrospinal fluid and blood of spontaneously hypertensive and normotensive Wistar-Kyoto rats were monitored before and after a challenge with lipopolysaccharide. RESULTS: Little or no activity from these mediators was found in the cerebrospinal fluid or blood of saline-injected control animals. Intravenous administration of lipopolysaccharide (0.001, 0.1, and 1.8 mg/kg) produced dose-dependent increases in blood levels of all mediators in hypertensive rats. In normotensive rats the levels were less than in hypertensive rats and were not clearly dose-related. When lipopolysaccharide was injected intracerebroventricularly, more tumor necrosis factor was measured in the cerebrospinal fluid than in the blood, suggesting local synthesis of this cytokine. Levels of tumor necrosis factor and platelet-activating factor in the cerebrospinal fluid were higher in hypertensive than in normotensive rats. The thromboxane A2/prostacyclin ratio was not altered significantly between the two rat strains. CONCLUSIONS: It is suggested that the higher incidence of brain stem ischemia and hemorrhage after the intrathecal injection of lipopolysaccharide in hypertensive rats than in normotensive rats might be related to the higher levels of the two cytotoxic factors tumor necrosis factor and platelet-activating factor produced in response to such challenge.

Animals↗
PubMed1991

Mixed nicotinic and muscarinic features of cholinergic receptor coupled to secretion in bovine chromaffin cells.

Acetylcholine evokes release from cultured bovine chromaffin cells by a mechanism that is believed to be classically nicotinic. However, we found that the full muscarinic agonist oxotremorine-M (Oxo-M) induced a robust catecholamine (CA) secretion. By contrast, muscarine, pilocarpine, bethanechol, and McN-A-343 did not elicit any secretory response. Desensitization of the response to nicotine by Oxo-M and desensitization of the response to Oxo-M by nicotine suggest that both nicotine and Oxo-M were acting at the same receptor. Additional experiments supporting this conclusion show that nicotine-induced secretion and Oxo-M-induced secretion were similarly blocked by various muscarinic and nicotinic antagonists. Moreover, secretion induced by nicotine and Oxo-M were Ca2+ dependent, and both agonists induced 45Ca2+ uptake. Equilibrium binding studies showed that [3H]Oxo-M bound to chromaffin cell membranes with a Kd value of 3.08 x 10(-8) M and a Hill coefficient of 1.00, suggesting one binding site for this ligand. Nicotine inhibited Oxo-M binding in a noncompetitive manner, suggesting that both ligands bind at two different sites on the same receptor. We propose that the receptor on bovine chromaffin cells that is coupled to secretion represents an unusual cholinergic receptor that has both nicotinic and muscarinic features.

Acetylcholine↗
PubMed1991

Osmotic strength differentiates between two types of calcium transport pathways regulating catecholamine secretion from cultured bovine chromaffin cells.

Calcium transport and catecholamine secretion was measured in cultured bovine chromaffin cells. Calcium ions which entered the cells following stimulation with either nicotine or 50 mM KCl (high potassium) triggered catecholamine release, but then inactivated the secretory process. The nicotine and the high potassium-induced calcium transport mechanisms were mechanistically distinct, but functionally dependent on each other. The specific evidence is that whereas the high potassium-induced Ca2+ influx was found to be inhibited by hyperosmotic medium, the nicotine-stimulated calcium influx was unaffected under these conditions. High potassium and nicotine-stimulated catecholamine release were also differently affected by hyperosmotic medium. While potassium-stimulated catecholamine release was profoundly inhibited by hyperosmolarity, nicotine-stimulated release was only moderately inhibited. Sequential treatments of cells with nicotine and high potassium, under isotonic physiological conditions, indicate that there is a functional, biochemical communication between the otherwise mechanistically distinct calcium channels. Calcium ions which were found to inactivate these channels may be the basis for such communication.

Adrenal Medulla↗
PubMed1991

Lipopolysaccharide-induced production of tumor necrosis factor activity in rats with and without risk factors for stroke.

Rats produced more TNF activity in cerebrospinal fluid (CSF) than in blood after intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS). After intravenous (i.v.) LPS, blood TNF levels exceeded CSF levels. Thus, brain cells appear to produce TNF in response to LPS. Rats with the stroke-risk factors hypertension or combined hypertension and genetic stroke-proneness produce more TNF in response to a provocative dose of LPS i.v. than control animals free of these risk factors. The possible relevance to stroke vulnerability is discussed.

Animals↗
PubMed1991

Extracellular matrix permits the expression of von Willebrand's factor, uptake of di-I-acetylated low density lipoprotein and secretion of prostacyclin in cultures of endothelial cells from rat brain microvessels.

Microvascular endothelial cells from the adult rat brain were cultured on Matrigel and found to express many differentiated properties including secretion of prostacyclin (PGI2) and von Willebrand's factor (vWF). Brain microvascular endothelial cells (BMECs) were purified by dextran and percoll gradients after enzymatic treatment and cultured under various conditions. BMECs that were plated on Matrigel stained positively for factor VIII-related antigen and incorporated Di-I-acetylated low density lipoprotein, whereas BMEC plated on fibronectin, gelatin, or uncoated dishes did not express any of the above properties which are characteristic of endothelial cells. vWF was measured by a sensitive ELISA in the culture media of BMECs plated on different types of matrices. Specificity of the anti-human vWF antibodies for the rat vWF was verified by immunoabsorption on a solid phase, sodium dodecyl sulfate, and Western blot analysis. BMECs also secreted vWF into the culture media only when the cells were plated on Matrigel, and this secretion was augmented after a 6 h incubation with an interleukin-1 tumor necrosis factor-alpha mixture, but not by lipopolysaccharide. From different matrices tested, only Matrigel permitted the secretion of PGI2 by BMECs. Cells also proved to be sensitive to mechanical stimulation and became refractory to secretagogue if the mechanical stimulation was serially repeated. Under the best conditions, stimulation of the cells with bradykinin (1 microM) substantially increased PGI2 secretion. These data indicate that growth of BMECs on Matrigel in vitro permits the expression of classical endothelial cell markers in a manner similar to the behavior of these cells in situ.

Acetylation↗
PubMed1991

(+-)-cis-2-methyl-spiro(1,3-oxathiolane-5,3')quinuclidine, an M1 selective cholinergic agonist, attenuates cognitive dysfunctions in an animal model of Alzheimer's disease.

AF102B [(+-)-cis-2-methyl-spiro(1,3-oxathiolane-5,3')quinuclidine], a structurally rigid analog of acetylcholine, was investigated in a number of neurochemical, pharmacological and behavioral tests related to cholinergic functions. AF102B induced atropine-sensitive contractions of isolated guinea pig ilea and trachea preparations with EC50 values of 3.5 and 3 microM being 87- and 1.3-fold less potent than acetylcholine, respectively. Binding studies using the radioligands pirenzepine, cis-dioxolane and quinuclidinyl benzilate in rat cerebral cortex and quinuclidinyl benzilate in cerebellar homogenates indicated that AF102B was a potent and highly selective M1-type muscarinic probe, being more selective for M1 receptors than oxotremorine, carbachol and AF102A (the trans-isomer of AF102B). AF102B had a 3-fold higher apparent affinity for M1 receptors than the prototype M1 agonist, McN-A-343, cis- and trans-AF30 (other rigid analogs of acetylcholine). Treatment of rat cortical homogenates with Cu++ ions did not modify the affinity observed for the muscarinic antagonists atropine, scopolamine and pirenzepine, whereas increasing the proportion of high affinity sites for the agonists oxotremorine-M, carbachol and McN-A-343. The apparent affinity of AF102B also increased by Cu++ treatment suggesting that this compound interacts with rat cerebral cortex muscarinic receptors as an agonist. AF102B did not affect high affinity choline transport, choline acetyltransferase and acetylcholinesterase activities in rat brain preparations. In rats treated with AF64A (the cholinotoxin ethylcholine aziridinium ion; 3 nmol/2 microliters/side i.c.v.), AF102B (1 mg/kg p.o. or i.p.), AF102A (1 mg/kg i.p.), cis-AF30 (1 mg/kg, i.p.) and physostigmine (0.06 mg/kg i.p.), each reversed cognitive impairments in a step-through passive avoidance task. Both AF102B and AF102A (1 mg/kg i.p.), but not physostigmine (0.1 mg/kg i.p.), were effective also in reversing reference memory impairments in a Morris water maze test. Repetitive administrations of AF102B (0.2 mg/kg/day i.p.) improved AF64A-induced working memory deficits in the Morris water maze test, but did not affect open field behavior. The data show that the selective M1 agonist AF102B can restore AF64A-induced cognitive impairments, without producing adverse central and peripheral side effects at the effective doses and this can indicate its potential use for the treatment of Alzheimer's disease.

Alzheimer Disease↗
PubMed1990

Distinct muscarinic receptor subtypes differentially modulate acetylcholine release from corticocerebral synaptosomes.

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1989

(+-)-cis-2-methyl-spiro(1,3-oxathiolane-5,3') quinuclidine (AF102B): a new M1 agonist attenuates cognitive dysfunctions in AF64A-treated rats.

(+-)-cis-2-Methyl-spiro(1,3-oxathiolane-5,3')quinuclidine (AF102B), a new muscarinic agonist of utmost rigidity, exhibits a high selectivity for M1 muscarinic receptors. In rats having a cholinergic hypofunction induced by the intracerebroventricular administration of ethylcholine aziridinium (AF64A), AF102B reversed cognitive impairments in a step-through passive avoidance task and in an 8-arm radial maze. AF102B reversed cognitive impairments at significantly lower doses than those needed to induce side-effects. In addition, AF102B exhibited low toxicity. The results suggest that AF102B may prove useful for treatments of cholinergic deficiencies and cognitive impairments, like those reported in Alzheimer's disease.

Alzheimer Disease↗
PubMed1989

Barium ions enter chromaffin cells via voltage-dependent calcium channels and induce secretion by a mechanism independent of calcium.

Barium ions enter chromaffin cells via voltage-sensitive calcium channels, although the intracellular site of barium action is distinct from that of calcium. The entry of barium primarily through voltage-sensitive channels was indicated by experiments showing inhibition of 133Ba2+ uptake by blockers of voltage-dependent calcium channels. In addition, 133Ba2+ uptake was stimulated by 50 mM KCl but not by nicotine. Furthermore, 133Ba2+ uptake was inhibited by hyperosmolarity, which specifically blocks the voltage-sensitive calcium channel but not the receptor-associated calcium channel. These conclusions from studies on barium uptake were also borne out by experiments measuring catecholamine secretion. Thus, blockers of voltage-dependent calcium channels which inhibited barium uptake also inhibited barium-induced catecholamine secretion. In other experiments, simultaneous stimulation with nicotine and barium in the presence of calcium evoked coincident and additive catecholamine secretion. By contrast, when 50 mM KCl was substituted for nicotine in the same experimental design, barium ions inhibited potassium-induced catecholamine secretion at low calcium concentrations. Only at high calcium concentrations were barium-induced and potassium-induced secretion additive. These data also indicate that barium and calcium compete at the voltage-sensitive pathway. Furthermore, these additivity data suggest that once inside the cell, barium and calcium have two distinct mechanisms of action. As predicted by this hypothesis, in digitonin-permeabilized chromaffin cells either calcium or barium stimulated catecholamine release, and in the presence of both cations catecholamine secretion was equivalent to the sum of secretion with either cation alone. Additional support of this concept comes from experiments showing that while calcium-mediated catecholamine secretion is sensitive to trifluoperazine and imipramine, barium-mediated secretion is not. Taken together, all these data indicate that there are two distinct intracellular sites of action for barium and calcium. In contrast to catecholamine secretion, non-exocytotic ascorbic acid secretion was induced by nicotine and potassium in the presence of calcium, but not by barium alone. These data provide additional evidence that barium acts by a different mechanism than calcium, in still another secretory system in chromaffin cells.

Animals↗