Delineating the pharmacophoric elements of huperzine A: importance of the unsaturated three-carbon bridge to its AChE inhibitory activity.
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Alkaloids↗
Biomedical subjects
M McKinney
Publications and source records attributed to M McKinney.
Pharmacological profiles for rat cortical M1 and M2 muscarinic receptors using selective antagonists: comparison with N1E-115 muscarinic receptors.
We previously showed that M1 and M2 muscarinic receptors in dissociated cells of the adult rat cortex couple to phosphoinositide (Pl) and cyclic AMP (cAMP) metabolism, respectively. To further classify these receptors according to probable subtype, we have employed a group of selective muscarinic antagonists to obtain pharmacological profiles of the cortical M1 and M2 receptors, and to compare them with the muscarinic receptors in N1E-115 cells, which contain M1 receptors mediating cyclic GMP elevation and M4 receptors inhibiting cAMP levels. The M2-mediated inhibition of cAMP levels in cortex was blocked by 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) with higher potency (0.29 nM) than for reported potency in cardiac tissue (approximately 10 nM), indicating that this cortical response is probably not mediated by the m2 gene product. Similarly, the potency of hexahydrosiladiphenidol (HSD) at the cortical M2 receptor (159 nM) was somewhat greater than the reported potency in cardiac tissue (295 nM). The cardioselective drugs AF-DX 116 and methoctramine blocked the cortical M2 response less potently (135 nM and 229 nM, respectively) than would be expected for involvement of the m2 gene product. Thus, the potencies of AF-DX 116, methoctramine, 4-DAMP and HSD suggest that the cortical M2 response, like the striatal M2 receptor, is mediated by a noncardiac M2 receptor, perhaps by the m4 gene product. This postulate was supported by the significant correlations between cortical and striatal M2 receptors as compared to the M4 receptor in N1E-115 cells (r = 0.92 and 0.99, respectively, P less than .025).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Interactions of agonists with M2 and M4 muscarinic receptor subtypes mediating cyclic AMP inhibition.
In this study the similarities and differences between the M2 and M4 subtypes in their recognition of agonists were explored. A CHO-K1 cell line transfected with the human m2 receptor was used as a homogeneous M2 tissue for comparison with two putative M4 systems (rat striatum and the N1E-115 mouse neuroblastoma cell line). The equilibrium binding dissociation constants and intrinsic efficacies for seven muscarinic agonists were determined for their stimulation of cyclic AMP inhibition via the M2 and M4 receptors. Partial receptor occlusion with propylbenzilylcholine mustard was used to determine binding constants for the more efficacious drugs and the reference agonist oxotremorine-M. The binding dissociation constants and relative efficacies for other agonists were then determined in reference to oxotremorine-M by a null method. For the M2 receptor the agonist binding dissociation constants ranged in potency from oxotremorine (1.5 microM) to bethanechol (171 microM), whereas relative efficacies varied from that of muscarine (relative efficacy = 0.9) to the value for McN-A343 (relative efficacy = 0.04). In general, most agonists bound with similar potencies to M2 and M4 receptors (Kd values within a factor of 2-3). However, oxotremorine bound to the N1E-115 and striatal M4 receptors about 3-fold and 10-fold less potently, respectively, than it did to the M2 receptor. Another exception was pilocarpine, which bound to the N1E-115 receptor (1.9 microM) with 8-fold and 12-fold higher potency than to the CHO-K1 M2 receptor and the striatal M4 receptor, respectively. Despite the low affinity of bethanechol for the M2 receptor, it was an efficacious agonist (maximal response equivalent to that of oxotremorine-M; relative efficacy = 0.6) at this subtype, whereas it was a partial agonist (60%) with lesser efficacy in the clonal M4 system. In contrast, McN-A343 and arecoline were significantly more efficacious at the two M4 receptors than they were at the M2 receptor. The M4 system in the rat striatum displayed some similarity to the N1E-115 M4 system, with regard to the efficacy ranking for certain agonists (arecoline greater than bethanechol greater than McN-A343 greater than or equal to pilocarpine). This rank order was different from the ranking of these four agonists in the M2 system, indicating that these two M4 receptors are more similar to each other in efficacy ranking than they are to the M2 receptor. However, the rat striatal and N1E-115 M4 receptor differed in their binding of oxotremorine and pilocarpine, indicating that these two M4 systems were not pharmacologically identical.(ABSTRACT TRUNCATED AT 400 WORDS)
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
Binding of [3H]AF-DX 384 to cloned and native muscarinic receptors.
The binding selectivity of [3H]AF-DX 384 [(+-)-5,11-dihydro-11- ([(2-(2-[(dipropylamino)methyl]-1- piperidinyl)ethyl)amino]carbonyl)-H-pyrido(2,3-b)(1,4)benzodiazepine-6-o ne] was evaluated with cloned human muscarinic receptors (M1-M4) in Chinese hamster ovary (CHO-K1) cell lines as well as in rat heart and brain. There were uniform classes of sites for the radioligand in the M2-rich tissues, heart (Kd = 2.3 nM) and brainstem (Kd = 2.4 nM). However, [3H]AF-DX 384 bound to all four cloned receptor subtypes. Using kinetic methods, the calculated Kd values were M2 (1 nm) greater than M4 (2.2 nM) greater than M3 (15 nM) greater than M1 (55 nM). Scatchard analysis with the CHO cells confirmed the high affinity of this radioligand for the M2 (1.8 nM) and M4 (2.5 nM) receptors. To evaluate the potential for selectively binding to M2 and M4 receptors in cortex and striatum, low concentrations (0.5-0.8 nM) of the radioligand were used and a two-site competition model was used to derive the binding constants for pirenzepine and AF-DX 116 [(+-)-11-2((-((diethylamino) methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H- pyrido(2,3-b)(1,4)-benzodiazepine-6-one] and to compare them with values obtained with cloned M2 and M4 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Desensitization of muscarinic M1 receptors of murine neuroblastoma cells (clone N1E-115) without receptor down-regulation and protein kinase C activity.
Acute desensitization of M1 muscarinic receptor-mediated responses (cyclic GMP formation and inositol phosphate release) was studied in murine neuroblastoma cells (N1E-115 clone). After a 45-min incubation at 37 degrees of N1E-115 cells either in monolayer or in suspension, with the muscarinic agonist carbachol (1 mM), the receptor-mediated cyclic GMP response to carbachol was nearly completely lost. This loss was associated with greater than 80% loss of carbachol-mediated inositol phosphate release. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) inhibited both responses with similar potencies. Carbachol or PMA reduced by 30-40% the number of muscarinic receptor sites for antagonist and agonist on intact cells (determined in binding assays using [3H]N-methylscopolamine) only for cells in monolayer and not for those in suspension. PMA but not carbachol pretreatment of cells in monolayer or in suspension caused a translocation of [3H]phorbol 12,13-dibutyrate binding and protein kinase C activity. In addition, desensitization to carbachol occurred in cells largely depleted of protein kinase C by chronic exposure to PMA. Thus, agonist-mediated down-regulation is not needed for muscarinic M1 receptor desensitization, which may be a result of the activation of a receptor-activated kinase different from protein kinase C.
Animals↗
Selective blockade of receptor-mediated cyclic GMP formation in N1E-115 neuroblastoma cells by an inhibitor of nitric oxide synthesis.
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Animals↗
Manifestations of esophageal disease on plain chest radiographs.
The chest film appearances of various esophageal abnormalities are illustrated. Inability to recognize these abnormalities on chest radiographs can result in confusion and misinterpretation. This pictorial essay should assist the radiologist in recognizing esophageal abnormalities on chest films and in recognizing their place in the spectrum of chest film abnormalities.
Esophageal Diseases↗
Biochemical evidence for somatostatin receptors in murine neuroblastoma clone N1E-115.
Radioligand binding and functional assays were employed to demonstrate the existence of somatostatin receptors in the murine neuroblastoma clone N1E-115. Saturation experiments with [125I][Tyr11]somatostatin-14 indicated the presence of a single class of binding sites in membranes prepared from N1E-115 cells (Kd = 83 pM; Bmax = 21,000 receptors/cell). Somatostatin-14, somatostatin-28 and L363586 (cyclo(N-Me-ALA-TYR-D-TRP-LYS-VAL-PHE] all displaced the 125I-ligand monophasically in N1E-115 cells (Ki values were 28, 82 and 34 pM, respectively), which contrasted with the binding heterogeneity apparent with L363586 in rat brain membranes. The binding of [125I][Tyr11]somatostatin-14 was reduced by GppNHp, indicating that N1E-115 somatostatin receptors interacted with guanine nucleotide binding protein(s). Somatostatin agonists decreased by 30-50% the levels of [3H]cyclic AMP induced in intact cells by forskolin, prostaglandin E1, or vasoactive intestinal polypeptide. The EC50 values for inhibition of the [3H]cyclic AMP response to PGE1 by L363586, somatostatin-14, and somatostatin-28 were 0.24, 0.63 and 1.0 nM, respectively. Pertussis toxin treatment of N1E-115 cells reduced both binding to the receptor and the functional response to somatostatin-14. These data suggest that a single class of somatostatin receptors in N1E-115 cells are linked to the inhibition of adenylate cyclase through a Gi protein.
Adenylate Cyclase Toxin↗
Mood disorders, psychiatric symptoms, and distress in women presenting for infertility evaluation.
Women who undergo treatment for infertility frequently report depression, but it is crucial to distinguish between subjective distress, symptoms, and clinical depressive disorders. In the initial assessment of a prospective, longitudinal study, 59 women presenting for infertility treatment were compared with 35 women presenting for routine gynecological care. Infertility patients and controls were not significantly different on self-report measures of partner satisfaction, sexual functioning, or self-esteem. There was also no difference in psychiatric symptomatology, or in the percentage of subjects who were currently experiencing or had ever experienced a major depressive episode. However, the infertility patients perceived themselves to have been already quite affected by their inability to conceive. For instance, 49.2% reported changes in their sexual functioning and 74.6% reported changes in their mood.
Adolescent↗
Diagnosis of disseminated intravascular coagulation. Role of D-dimer.
Detection of the cross-linked fibrin degradation fragment, D-dimer, in patients at risk for disseminated intravascular coagulation (DIC) is strong evidence for the diagnosis. D-dimer confirms that both thrombin generation and plasmin generation have occurred. Patients at risk for DIC (58) and normal controls (7) were studied. Thirty-three patients had DIC--with fragment D-dimer identified in their serum by immunoblotting. Latex agglutination measurements of fibrin(ogen) degradation products (FDPs) and D-dimer were compared with immunoblotting in the detection of D-dimer. FDP measurement was extremely sensitive but not specific. D-dimer measurement was less sensitive but highly specific. Used in tandem, screening with FDP and confirming with D-dimer, sensitivity and specificity were maximized, rendering a predictive value of a confirmed FDP of 100% in this cohort. D-dimer is a valuable adjunct for the laboratory diagnosis of DIC but is most appropriately used as a confirmatory test for the very sensitive FDP test.
Adult↗
Different agonist-receptor active conformations for rat brain M1 and M2 muscarinic receptors that are separately coupled to two biochemical effector systems.
In dissociated cellular preparations of adult rat cortex, M1 and M2 muscarinic receptors were shown to mediate phosphoinositide metabolism and cAMP inhibition, respectively. Additionally, in dissociated striatum, an M2 receptor was shown to inhibit the level of cAMP. The components of "receptor reserve" in these three receptor-effector systems were evaluated by the method of partial receptor inactivation and the dissociation constants for the full agonist carbachol were determined. In dissociated cellular preparations of cortex, and in the presence of 10 mM lithium ion, carbachol (EC50 = 116 microM) activated an M1 receptor subtype (atropine Ki = 0.9 nM; pirenzepine Ki = 9.3 nM) to elicit up to a 7-fold release over the basal level of [3H]inositol 1-phosphate. Carbachol was 100-fold more potent (EC50 approximately 1 microM) in the inhibition of forskolin-elevated [3H]cAMP levels in both the cortex (maximally 28%) and striatum (maximally 49%). Pirenzepine blocked the [3H]cAMP inhibition responses to carbachol in cortex and striatum with Ki values of 334 nM and 313 nM, respectively, which indicated that cortical and striatal M2 receptors mediate [3H]cAMP inhibition. The equilibrium dissociation constants for the full agonist carbachol in mediating these two biochemical responses were determined after partial receptor inactivation with propylbenzilylcholine mustard. The results indicate that cortical M1 receptor-mediated phosphoinositide metabolism is elicited by carbachol through a low affinity agonist-receptor complex (carbachol Kd = 90 microM). However, the cortical and striatal M2 receptor-mediated inhibition of [3H]cAMP is mediated by a high affinity agonist-receptor complex (carbachol Kd = 8.5 microM and 2.9 microM, respectively). Thus, the agonist is bound in a low affinity active conformation of the M1 receptor but the agonist is bound in a high affinity active conformation of the M2 receptor. In contrast to the cortical M1-phosphoinositide system, the central M2 receptors exhibited a significant receptor reserve in their mediation of [3H]cAMP inhibition, as elicited by the full agonist carbachol. Whereas the ratio of Kd/EC50 for carbachol was 0.9 at the cortical M1 receptor, this ratio was 9.4 and 3.2 at the cortical M2 and striatal M2 receptors, respectively.
Animals↗
Characterization of the striatal M2 muscarinic receptor mediating inhibition of cyclic AMP using selective antagonists: a comparison with the brainstem M2 receptor.
Pharmacological profiles of the striatal and brainstem M2 receptors were developed with a group of selective muscarinic antagonists. The striatal M2 muscarinic receptor was identified by its inhibition of [3H]cyclic AMP levels, whereas the brainstem M2 receptor was characterized using competition with [3H]quinuclidinyl benzilate binding. The potency of pirenzepine does not differentiate clearly between the striatal M2 receptor (Ki approximately 300 nM) and the brainstem M2 receptor (Ki = 219 nM) or peripheral M2 receptors. In the present study, we used 4-diphenylacetoxy-N-methylpiperidine methbromide, hexahydrosiladifenidol, AF-DX 116 and methoctramine to characterize the striatal and brainstem M2 receptors in more pharmacological detail. For comparison, the potencies of these antagonists were also measured at cortical M2 receptors (using competition with [3H]pirenzepine binding). The potencies of 4-diphenylacetoxy-N-methylpiperidine methbromide (KB = 0.19 nM) and hexahydrosiladifenidol (KB = 14 nM) in blocking the striatal M2 receptor suggested similarity to those M2 receptors localized in certain smooth muscles or in glands. However, AF-DX 116 (KB = 155 nM) and methoctramine (KB = 47 nM) were considerably more potent in blocking the striatal M2 receptor than as reported in functional studies in smooth muscle or glands. Thus, the profile of the striatal M2 receptor obtained with these antagonists did not match in all respects with either glandular (probable M4 gene product) or cardiac (probable M2 gene product) muscarinic receptors. In contrast, our data with the brainstem M2 receptor was highly correlated (r = 0.93) with literature data regarding the cardiac muscarinic system.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effect of reduced training volume on cardiac function, VO2 max, and running performance.
This study examined the physiological effects of reducing training mileage in a veteran long distance runner while increasing exercise intensity. Variables measured included stroke volume, cardiac output, maximum oxygen uptake, ventilation threshold and performance time in a 10,000 m run. For 8 weeks, training mileage was reduced from 75.8 miles per week to 42.5 miles per week including interval training twice weekly. Following the specialized training, performance time was 10 seconds faster although VO2max and heart contractility had decreased. It was concluded that distance running performance can be maintained while considerably reducing training mileage and increasing exercise intensity twice a week.
Adult↗
Right to day in court imperils citizens' access to medical care.
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Health Services Accessibility↗
Muscarinic M2 receptor-mediated cyclic AMP reduction in mechanically dissociated rat cortex.
The coupling of the muscarinic receptor to the inhibition of the adenylate cyclase system was studied in adult rat cortical tissue dissociated by teasing tissue minces through finely meshed Nitex filters. The intracellular ATP stores in the final preparation were metabolically prelabeled with [3H]adenine and the [3H]cAMP formed in the tissue was isolated by ion exchange chromatography. Forskolin (3-30 microM) elevated [3H]cAMP levels 5- to 9-fold over basal in the preparation, with maximum stimulation achieved by 10-15 min. In the dissociated cortex, carbachol inhibited forskolin-elevated [3H]cAMP levels with an EC50 value of 1.4 microM; maximal inhibition was in the range of 20-30%. Atropine completely blocked the response (Ki = 1.8 nM), which showed that carbachol stimulates a muscarinic receptor to inhibit [3H]cAMP levels in this preparation. Pirenzepine, an M1-selective antagonist, blocked the response to carbachol with low potency (Ki = 467 nM), which indicated that an M2 muscarinic receptor subtype mediates [3H]cAMP inhibition in the cortex. The response to 10 microM carbachol was not affected by 10 mM EGTA, 50 microM D-tubocurarine, or 100 nM tetrodotoxin; thus, activation of nicotinic receptors or a neuronal release process was not involved. [3H]cAMP reduction in response to muscarinic stimulation was also observed in dissociated tissue prepared from other brain regions. A robust response was encountered in striatal preparations (maximal inhibition 40%), while hippocampal responses were smaller and less reproducible than in the cortex. The striatal response was shown to be pharmacologically similar to the cortical response.
Animals↗
Non-selectivity of amitriptyline for subtypes of brain muscarinic receptors demonstrated in binding and functional assays.
The characteristics of interaction of amitriptyline, a tricyclic antidepressant, with rat brain muscarinic receptors were assessed using both radioligand binding and functional assays. In competition studies, amitriptyline displaced muscarinic ligand binding from a single high-affinity site in homogenates of various brain regions which have a different distribution of M1 and M2 receptor subtypes. The affinity of amitriptyline for muscarinic receptors was also comparable in all brain regions. Furthermore, amitriptyline identified a single species of muscarinic receptors in intact cells dissociated from the cerebral cortex and in cerebrocortical slices. The non-selectivity of amitriptyline for muscarinic receptor subtypes in these preparations was in contrast to the selectivity exhibited by pirenzepine. This non-selective nature of amitriptyline was also evident in functional assays, since this antidepressant was equipotent at antagonizing M1-mediated increase in phosphoinositide hydrolysis and M2-mediated inhibition of cyclic AMP formation in dissociated cortical cells. Atropine was also equipotent at blocking both responses but was 20- to 30-fold more potent than amitriptyline. These results demonstrate that amitriptyline behaves as a non-selective muscarinic antagonist using both radioligand binding and functional measurements.
Amitriptyline↗
A new technique for sleeve resection and major bronchial resection using twin catheters and high frequency jet ventilation.
A new anaesthetic technique that uses two catheters combined with high frequency jet ventilation for sleeve resection of the major bronchus and carina is described. The advantages and disadvantages are discussed and compared with other techniques.
Aged↗
Pseudo-noncompetitive antagonism of muscarinic receptor-mediated cyclic GMP formation and phosphoinositide hydrolysis by pirenzepine.
Pirenzepine selectively antagonized muscarinic receptor-mediated cyclic GMP formation in a noncompetitive fashion in mouse neuroblastoma cells (clone N1E-115). These effects of pirenzepine were time- and concentration-dependent and they were also reversible. Interestingly, whereas atropine elicited competitive antagonism of the cyclic GMP response at low concentrations, it also behaved like a noncompetitive antagonist at higher concentrations and its effects were partially reversible. Using additional approaches to study the mechanisms underlying this anomalous antagonistic profile of pirenzepine, we investigated whether this deviation from competition could be due to the short time of exposure to muscarinic agonists (30 sec) used in cyclic GMP measurements. Our data indicated that the mode of pirenzepine-induced antagonism of ligand binding to muscarinic receptors was different when assessed using nonequilibrium (30 sec) or equilibrium (1 hr) incubations. Thus, pirenzepine appeared to be noncompetitive and competitive under these two conditions, respectively. Furthermore, although pirenzepine blocked receptor-mediated phosphoinositide hydrolysis competitively when the response was measured at 20 min, it was clearly noncompetitive using 5-min incubations. Therefore, the noncompetitive antagonism by pirenzepine detected in cyclic GMP measurements might be only apparent and might be attributed, at least in part, to a lack of an equilibrium state under the specific conditions of these assays.
Animals↗