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N Adham

Publications and source records attributed to N Adham.

33 records · Page 2Linked to original sources
PubMed1990

Antagonist characterization of atypical beta adrenoceptors in guinea pig ileum: blockade by alprenolol and dihydroalprenolol.

The present study was undertaken to further characterize the atypical beta adrenoceptor in guinea pig ileum. Tension was developed in isolated segments of ileum using transmural electrical stimulation of enteric cholingeric nerves. The ability of isoproterenol to relax the ileum, via beta-1 adrenoceptor and atypical beta adrenoceptor agonism, was measured. Propranolol (5 x 10(-6) M) and bromoacetylaprenololmetane blocked beta-1 adrenoceptors but, at the concentrations tested, were without affinity at atypical beta adrenoceptors. (-)-Alprenolol and (-)-dihydroalprenolol, however, acted as competitive antagonists at both sites (pA2 values of 8.2 and 8.81 at beta-1 adrenoceptors and 6.47 and 6.43 at atypical beta adrenoceptors, respectively). (-)-Alprenolol also exerted agonistic activity at the atypical beta adrenoceptor. [3H](-)-Dihydroproalprenolol failed to identify beta-1 adrenoceptors or atypical beta adrenoceptors but, instead, bound to a putative lipophilic site unrelated to ileal adrenoceptors. Before this study, nadolol (pA2 = 4.7) was the only documented antagonist at the atypical beta adrenoceptor in guinea pig ileum. Thus, the present results detail two additional pharmacological probes which exhibit about a 100-fold greater affinity than nadolol for the atypical site.

Adrenergic beta-Antagonists↗
PubMed1990

Cloning, expression, and pharmacological characterization of a human alpha 2B-adrenergic receptor.

An alpha 2-adrenergic receptor subtype has been isolated from a human genomic spleen library using the human 5-hydroxytryptamine1A receptor gene (also known as G-21) as a probe. This adrenergic receptor gene encodes a protein of 450 amino acids and does not contain any consensus sequences for N-linked glycosylation in its amino terminus or extracellular loops. This receptor is also distinguished by the presence of 12 consecutive glutamic acid residues in the region of its third intracellular loop. The deduced amino acid sequence shows greatest homology to previously cloned human alpha 2-adrenergic receptors and has structural similarities to other guanine nucleotide-binding protein-coupled receptors. The DNA encoding the human alpha 2 receptor was stably transfected into mouse fibroblast Ltk- cells and radioligand binding studies were performed using the alpha 2 antagonist [3H]rauwolscine. [3H]Rauwolscine bound with high affinity (Kd = 0.33 nM) and in a saturable manner (Bmax = 1.4 pmol/mg of protein). Pharmacological characterization of this receptor indicated a rank order of potency of yohimbine greater than prazosin greater than oxymetazoline. Additionally, 100 microM 5'-guanylylimidodiphosphate, produced a rightward shift in the epinephrine competition curve, with resultant increases in both the Ki value and Hill coefficient, suggestive of a functional interaction of the cloned receptor with native guanine nucleotide-binding protein(s) of Ltk- membranes. The data presented here are consistent with previous biochemical and pharmacological studies on alpha 2 receptors and are supportive of the designation of this receptor as an alpha 2B subtype.

Amino Acid Sequence↗
PubMed1990

[3H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H] ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor.

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to [3H]ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding the serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both [3H]DOB and [3H]ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] to this system caused a rightward shift and steepening of agonist competition curves for [3H] ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity [3H]DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that [3H]DOB and [3H]ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein. These observations are consistent with the classical view of interconvertible agonist affinity states of GTP-binding protein-coupled receptors and strongly support the "two state" over the "two receptor" model for DOB binding to the 5-HT2 receptor.

DOM 2,5-Dimethoxy-4-Methylamphetamine↗
PubMed1987

Exposure of receptor sites on rat muscle membranes by a neurally released factor.

Isolated phrenic nerve-diaphragm preparations of the rat were stimulated electrically via the nerve. The medium bathing the preparation contained a factor which increased the specific binding (Bmax) of [125I]alpha-bungarotoxin to isolated sarcolemmal membranes. Medium from control preparations, left unstimulated, had no significant effect on the binding. The nerve stumps in the stimulated preparations were relatively depleted of the factor which increased the binding. The possibility that latent acetylcholine receptors are exposed by a factor released from the nerve during impulse activity is suggested.

Animals↗
PubMed1987

Effect of parasympathetic denervation on K+ release by rat parotid slices.

1. By measuring the release of 86Rb, potassium movement was compared in parasympathectomized (Px) and contralateral control parotid gland slices three weeks following surgical denervation. 2. Carbachol and phenylephrine elicited a biphasic increase in 86Rb release that was dose related and could be blocked by atropine and phentolamine respectively. The transient phase was of 2-4 min duration and Ca independent whereas the sustained phase of 86Rb release was greatly reduced by the omission of the external Ca. 3. Denervation caused a shift to the left of the 86Rb efflux dose-response curve to carbachol and phenylephrine (3.75- and 3.37-fold respectively). Pilocarpine had similar action to carbachol but it behaved as a partial agonist. 4. Parasympathectomy (Px) increased the intrinsic activity of the partial agonist pilocarpine and converted it to a full agonist. Results of the present study indicate the possibility of an altered receptor-signal transduction mechanism between the receptor and phospholipid turnover/Ca mobilization in the denervated rat parotid gland.

Animals↗
PubMed1986

Effect of phospholipase C on the binding of alpha-bungarotoxin to isolated plasma membranes of rat skeletal muscle.

Sarcolemmal membranes were isolated from hindlimb muscles of the rat and the specific binding of [125I]alpha-bungarotoxin to the membranes was determined. Incubation of the membranes with a purified preparation of phospholipase C (ex Clostridium perfringens) increased the specific binding of the toxin (Bmax). The apparent dissociation constant (Kd) for the binding was unchanged by treatment with the enzyme. The possibility that latent acetylcholine receptors exist in muscle sarcolemma, and that these receptors can be activated by the enzyme, is discussed.

Acetylcholine↗
PubMed1983

Beneficial effect of pancreatic polypeptide in experimental pancreatitis.

Two animal models have been employed to examine the role of pancreatic polypeptide, a potent and selective inhibitor of pancreatic exocrine secretion, in the treatment of acute pancreatitis. In one model pancreatitis was induced by feeding young female Swiss Webster mice an ethionine-supplemented, choline-deficient diet for 48 hr. Animals (N = 30 per group) were injected subcutaneously every 8 hr for 7 days with pancreatic polypeptide (0, 2, 20, and 200 micrograms/kg/day). Treatment with 20 and 200 micrograms/kg/day pancreatic polypeptide significantly (P less than 0.05) reduced mortality from a control rate of 70% to 42% and 33%, respectively. Treated animals also exhibited significant (P less than 0.05) decreases in pancreatic content of activated chymotrypsin and an improvement in pancreatic histology. Pancreatic polypeptide was effective whether treatment was started before or at the same time the test diet was introduced. In contrast, pancreatic polypeptide failed to protect dogs with acute pancreatitis induced by retrograde injection of the pancreas with bile, which may reflect the rapid and mechanical nature of pancreatic damage in this animal model.

Animals↗
PubMed1983

Effects of prostaglandin and indomethacin on diet-induced acute pancreatitis in mice.

This study was performed to determine the effects of exogenous prostaglandin and a prostaglandin synthetase inhibitor on experimental pancreatitis in mice. An ethionine-supplemented choline-deficient diet was used to induce pancreatitis in 4-6-wk-old Swiss Webster mice. Mice were injected subcutaneously with 16,16-dimethyl prostaglandin E2 (0.1, 1.0, 10 micrograms X kg-1 X day-1), indomethacin (0.05, 0.5, 5 mg X kg-1 X day-1), or saline for 7 days. The ethionine-supplemented choline-deficient diet was introduced 24 h after the first injection, and animals ate the test diet for 48 h. A 55% mortality was observed in control animals (n = 100) treated with carrier alone. Treatment with 10 micrograms X kg-1 X day-1 of 16,16-dimethyl prostaglandin E2 significantly decreased (p less than 0.01) mortality to 12% (n = 100). Improved survival was accompanied by a significant (p less than 0.05) decrease in the pancreatic content of free chymotrypsin and a decrease in histologic damage. Treatment with 5 mg X kg-1 X day-1 of indomethacin (n = 30) significantly (p less than 0.01) increased mortality in diet-treated rats from a control rate of 55% to 100%. These studies demonstrate a protective effect of prostaglandin on the pancreas and suggest a role for endogenous prostaglandins in the pathophysiology of pancreatitis.

16,16-Dimethylprostaglandin E2↗
PubMedDate not supplied

Pharmacological characterizations of recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT1D subtypes.

Recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1D alpha cell line) and Y-1 adrenocortical tumor cells (1D beta cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated cAMP accumulation (FSCA). [3H]LSD saturation studies indicated that 5-HT1D receptor expression levels were slightly higher in the 1D beta cell line (Bmax = 1334 +/- 134 fmol/mg protein) than in the 1D alpha cell line (Bmax = 900 +/- 218 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC50 approximately 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine > or = 5-carboxamidotryptamine (5-CT) > LSD > or = 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), with Ki/EC50 ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT1D alpha and 5-HT1D beta receptors with apparent dissociation constants (Kb values) of 12 +/- 1 nM and 3 +/- 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1D alpha cell line, these compounds behaved as partial agonists in the 1D beta cell line. To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1D beta cell line whereas nearly linear relationships were observed for these two compounds in the 1D alpha cell line. Moreover, KA/EC50 ratios of these compounds were significantly larger in the 1D beta (10-32) as compared to the 1D alpha (0.9-2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger-receptor reserve in the 1D beta cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists. Although the potencies (EC50 values) of compounds matched their respective affinity constants (Ki values) for the closely-related 5-HT1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT1D alpha receptor relative to the human 5-HT1D beta subtype may be responsible for the higher intrinsic activities observed in the 1D alpha cell line, even though receptor reserve is apparently lower in this system.

Adenylyl Cyclase Inhibitors↗
PubMedDate not supplied

A single point mutation increases the affinity of serotonin 5-HT1D alpha, 5-HT1D beta, 5-HT1E and 5-HT1F receptors for beta-adrenergic antagonists.

The serotonin 5-HT1B and 5-HT1A receptors bind certain beta-adrenergic antagonists, such as propranolol and pindolol, with high affinity. Other 5-HT1 receptors that display very low affinity for beta-adrenergic antagonists, have either a threonine (T) (5-HT1D alpha, 5-HT1D beta and 5-HT1E) or an alanine (A) (5-HT1F) residue in the homologous position in the seventh transmembrane domain. In the case of the human 5-HT1D beta receptor, replacement of this T with asparagine (N), dramatically increases its ability to bind beta-adrenergic antagonists. To assess whether other 5-HT1 receptors would behave similarly, we have used site-directed mutagenesis to replace the T or A in 5-HT1D alpha, 5-HT1E and 5-HT1F receptors with N. Both the wild-type and mutant genes were expressed transiently in COS-7 cells and radioligand binding studies were performed by using [3H]5-HT and [125I]iodocyanopindolol. Using [3H]5-HT, we found that the affinities of all the mutant receptors for propranolol and pindolol were significantly increased by 100-1000 fold, 5-HT1D alpha and 5-HT1F receptors showing the highest and the 5-HT1E receptor displaying the lowest affinity. On the other hand, the affinities for 5-HT were essentially unchanged as compared to the wild-type receptors. All mutant receptors bound [125I]iodocyanopindolol with high affinity, KD values ranging between 0.04 nM (mutant 5-HT1D alpha) and 0.57 nM (mutant 5-HT1E), whereas the wild-type receptors failed to show any specific binding with this radioligand in the same concentration range used for the mutant receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMedDate not supplied

The cloned human 5-HT1E receptor couples to inhibition and activation of adenylyl cyclase via two distinct pathways in transfected BS-C-1 cells.

The pharmacological profile of coupling of the cloned human serotonin [5-hydroxytryptamine] (5-HT)1E receptors to second messengers was studied in African green monkey kidney cells (BS-C-1). At low concentrations (0.1-100 nM), 5-HT inhibited forskolin-stimulated cAMP accumulation (FSCA) by up to 90% whereas at higher concentrations it potentiated FSCA; potentiation was dependent on receptor density. Pretreatment of cells with pertussis toxin (PTx) or cholera toxin (CTx) eliminated agonist-induced inhibition and potentiation of FSCA, respectively. The potentiation of FSCA was not due to activation of phospholipase C and/or phospholipase A2 since 5-HT had no effect on inositol phosphate release, intracellular Ca2+ mobilization or arachidonic acid mobilization; neither was it affected by pretreatment with the nonselective phospholipase A2 inhibitor, quinacrine, or by the removal of extracellular Ca2+. The pharmacological profiles of the 5-HT1E receptor-mediated inhibition and potentiation of FSCA were very similar, although agonists displayed higher affinity for the former. These results indicate that the human 5-HT1E receptors can potentially couple, with similar pharmacological profiles, to multiple effector pathways. However, the potency and intrinsic activity of the compounds eliciting these responses can differ significantly, depending on the receptor density and the effector pathway studied.

Adenylate Cyclase Toxin↗
PubMedDate not supplied

Cloning and characterization of the guinea pig 5-HT1F receptor subtype: a comparison of the pharmacological profile to the human species homolog.

The anti-migraine compound, sumatriptan, has been shown to have substantial affinity for the cloned human 5-HT1F receptor suggesting that, in addition to 5-HT1B/5-HT1D receptor subtypes, the 5-HT1F receptor may be a therapeutic target for the treatment of migraine. Several investigators have used the guinea pig plasma extravasation model to evaluate potential anti-migraine drugs. Since species differences in the pharmacology of serotonin receptors are well known, we compared the pharmacological profiles of the cloned human and guinea pig 5-HT1F receptors in order to validate the usefulness of the in vivo model in predicting anti-migraine activity of compounds targeted for humans. We have cloned the guinea pig 5-HT1F by homology to the human 5-HT1F receptor and evaluated its pharmacological profile using radioligand binding assays. The cloned guinea pig 5-HT1F gene exhibited 94% amino acid identity to the corresponding human homolog. High affinity (Kd approximately 10 nM) [3H]5-HT binding was detected to membranes obtained from Cos-7 cells transiently expressing the guinea pig 5-HT1F receptor. The cloned guinea pig receptor displayed typical 5-HT1F receptor pharmacology with the following rank order of binding affinities: 5-HT > sumatriptan > 1-NP = DHE > alpha-methyl 5-HT > metergoline > methiothepin > 5-CT. The pharmacological profiles of the cloned guinea pig and human 5-HT1F receptors were very similar as reflected by the high correlation (r2 = 0.72, slope = 0.76) observed between the binding affinities of compounds for these two species homologs. In situ hybridization studies in guinea pig tissue revealed 5-HT1F receptor mRNA expression in the neurons of the trigeminal ganglion, suggesting that the 5-HT1F receptor may play a role in the presynaptic inhibition of neuropeptide release at the level of the intracranial vasculature, thereby blocking the development of neurogenic inflammation. Dorsal root ganglion cells also moderately expressed the 5-HT1F transcripts. The localization of the 5-HT1F receptor to areas involved in the mediation and transfer of nociceptive information implies a role for this receptor in pain processing. These findings indicate that a selective 5-HT1F agonist may be a novel approach to treat migraine.

Amino Acid Sequence↗
PubMedDate not supplied

[3H]RS 57639, a high affinity, selective 5-HT4 receptor partial agonist, specifically labels guinea-pig striatal and rat cloned (5-HT4S and 5-HT4L) receptors.

RS 57639, by being a partial agonist in rat esophagus but a competitive antagonist in guinea-pig ileum, is one of several ligands which operationally discriminate among 5-HT4 receptors in different tissues. The discovery of splice variants of the 5-HT4 receptor, 5-HT4S and 5-HT4L, raises the possibility that this functional heterogeneity among 5-HT4 receptors may be due to differences in the interaction of ligands with different isoforms of the receptor. To test this idea, the functional and binding interactions of RS 57639 with rat 5-HT4S and 5-HT4L receptors were characterized. RS 57639 stimulated adenylate cyclase in cells expressing 5-HT4S or 5-HT4L receptors with similar potency (pEC50 = 7.9 +/- 0.1 and 7.6 +/- 0.1) and efficacy (71 +/- 3 and 59 +/- 4% of 5-HT). [3H]RS 57639 also bound to 5-HT4S and 5-HT4L receptors with similar affinity (Kd = 0.09 +/- 0.01 and 0.11 +/- 0.01 nM) and specificity (SB204070 > GR113808 > SDZ 205557 > cisapride > renzapride > alpha me-5-HT > 5-CT). Therefore, the operational differences among 5-HT4 receptors, detected with RS 57639, are not explained by differences in the interaction of the ligand with 5-HT4S and 5-HT4L receptors. [3H]RS 57639 binding to guinea-pig striatal membranes was also characterized. [3H]RS 57639 bound with high affinity (Kd = 0.25 +/- 0.07 nM) and a specificity similar to that of the 5-HT4 receptor antagonist, [3H]GR113808. Therefore, while the mechanism by which RS 57639 operationally distinguishes among 5-HT4 receptors was not determined, [3H]RS 57639 was shown to specifically label native and cloned 5-HT4 receptors. As the first selective agonist radioligand to be described for this receptor, [3H]RS 57639 may prove useful in further studies of receptor coupling and ligand interactions.

Aminobenzoates↗
PubMedDate not supplied

The molecular biology of serotonin receptors. An overview.

Recently, the family of G protein-coupled serotonin (5-hydroxytryptamine[5-HT]) receptors has begun to yield to molecular analysis. The cloning of the 5-HT1C and 5-HT2 receptors has provided a structural basis for the similarities observed in their pharmacologic properties. Furthermore, pharmacologic characterization of the transfected human 5-HT2 receptor has answered two outstanding questions regarding this receptor. First, the few amino acid differences that exist between the human and the rat genes are sufficient to account for the species differences seen in their pharmacologic properties. Second, the single protein encoded by the human 5-HT2 receptor gene is capable of binding both [3H]DOB and [3H]ketanserin. Analysis of the effects of guanine nucleotides provides further evidence that this single protein binds both ligands, that this receptor has high- and low-affinity states, and that these states are partially interconvertible. Furthermore, the close relationship between the adrenergic receptors and the 5-HT1A receptor has been reaffirmed by the recent cloning of a new adrenergic receptor subtype, alpha 2B, by use of the 5-HT1A receptor sequence. Finally, the detailed level of structural information now available on serotonin receptors has yielded valuable information about the ligand binding site and about the possible functional significance of differing rates of evolutionary change in various parts of the gene.

Animals↗