PubMed Health⌕ Search

Biomedical subjects

J Lai

Publications and source records attributed to J Lai.

At least 19 recordsLinked to original sources
PubMed1992

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.

Animals↗
PubMed1992

Sclerosing peritonitis with gross peritoneal calcification: a case report.

We report the case of a patient on dialysis for 13 years, including continuous ambulatory peritoneal dialysis (CAPD) for 11 years, who developed sclerosing peritonitis with gross peritoneal calcification. The patient first presented with abdominal pain in January 1990, when peritoneal calcification was detected for the first time. Her symptoms settled spontaneously and 1 year later she presented with acute peritonitis and adynamic ileus. The peritonitis settled with antibiotics and Tenchkoff catheter removal, but the ileus persisted. She was commenced on long-term parenteral nutrition, but never recovered useful bowel function. After 8 weeks of hemodialysis and total parenteral nutrition, a further laparotomy for an acute abdomen showed what appeared to be extensive bowel infarction and peritoneal calcification. She died several days later. Of significance, peritoneal calcification was first noted on x-ray and computed tomography (CT) scan while the patient was still largely asymptomatic and before peritoneal ultrafiltration capacity was significantly impaired. Unlike other reported cases of calcifying peritonitis, sclerosing peritonitis was present and calcification was far more extensive. It was not associated with factors such as frequent infective peritonitis or acetate dialysate. Calciphylaxis was not present nor was there any abnormality of calcium-phosphate metabolism. The outcome of this case suggests that patients with recurrent or persistent bowel symptoms on long-term CAPD should have early abdominal x-ray or CT scanning to exclude sclerosing peritonitis or bowel calcification. If present, consideration should be given to transferring the patient to another therapeutic dialysis modality if possible.

Adult↗
PubMed1992

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

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

Amino Acid Sequence↗
PubMed1991

Characterization of a tachykinin peptide NK2 receptor transfected into murine fibroblast B82 cells.

Membranes isolated from a murine fibroblast B82 cell line (SKLKB82#3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of [His(125I)1]neurokinin A (125I-NKA) to a single population of sites with a Bmax of 147 fmol/mg of protein and a Kd of 0.59 nM. Control cell lines had little or no specific binding. The ligand binding in SKLKB82#3 cells was reversible and was inhibited by peptides in the potency rank of neuropeptide gamma greater than neuropeptide K greater than neurokinin A greater than [10-norleucine]neurokinin A-(4-10) greater than substance P much greater than senktide (succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH2). Specific binding was enhanced by Mn2+, Mg2+, and Ca2+ and was inhibited by guanine nucleotide analogues. Thus, SKLKB82#3 cells have been transfected with NK2 receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK2 receptors, NK2 receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of 125I-NKA binding by nucleotide analogues was markedly different in SKLKB82#3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK2 receptors.

Animals↗
PubMed1991

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

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

Adenylyl Cyclase Inhibitors↗
PubMed1991

Pharmacologic comparison of selected agonists for the M1 muscarinic receptor in transfected murine fibroblast cells (B82).

The radioligand binding and functional properties of 10 muscarinic agonists for the M1 muscarinic receptors were characterized on the murine fibroblast B82 cells, which have been transfected with the m1 gene. All of the muscarinic agonists completely inhibited [3H](-)methyl-3-quinuclidinyl benzilate binding to the M1 muscarinic receptor in the transfected B82 cells. Their apparent inhibition constant values for agonist/[3H](-)methyl-3-quinuclidinyl benzilate inhibition experiments correlate well with their EC50 values in stimulating phosphatidylinositide hydrolysis. Based on the maximal functional effects: (+)-cismethyl-dioxolane, oxotremorine-M, acetylcholine, carbachol and methacholine are most efficacious, McN-A-343 and arecoline are least efficacious, whereas the efficacies of oxotremorine and pilocarpine are intermediate. In addition, McN-A-343 inhibited carbachol-stimulated phosphatidylinositide hydrolysis. Spare receptors were detected for oxotremorine-M, methacholine and carbachol, but not the rest of the agonists, by comparing the receptor-occupancy curves with the concentration-response curves. These results suggest that the presence of a quaternary nitrogen (trimethylammonium group) within the structure of the agonist may be important for the expression of full agonist activity.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1990

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

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

Alkaloids↗
PubMed1990

K(+)-stimulation of the phosphoinositide pathway in guinea-pig ileum longitudinal smooth muscle is predominantly neuronal in origin and mediated by the entry of extracellular Ca2+.

1. K+ and scorpion toxin stimulate formation of inositol phosphates in guinea-pig ileum longitudinal smooth muscle slices. The response to these two agents is not additive. 2. The response to K+ is inhibited partially by nifedipine and partially by omega-conotoxin. When given together the effect of these two Ca2+ channel blockers is additive and the response to K+ is reduced by more than 80%. 3. The response to scorpion toxin is inhibited completely by tetrodotoxin, partially by omega-conotoxin but not by atropine or nifedipine. Scorpion toxin induces a similar formation of inositol phosphates in collagenase-dispersed cells to that seen in cross-chopped slices. 4. The responses to scorpion toxin and K+ are inhibited completely when the extracellular Ca2+ concentration is reduced to below cytosolic levels (less than 100 nM). 5. Neither nifedipine nor omega-conotoxin, either alone or in combination, inhibited formation of inositol phosphates by substance P or carbachol. Both of these agonists induced a significant formation of inositol phosphates even when the extracellular Ca2+ concentration was reduced to 10 nM. 6. These results indicate that K+ and scorpion toxin induce formation of inositol phosphates through the mobilisation of extracellular Ca2+. The response to K+ appears to occur predominantly in neuronal cells.

Animals↗
PubMed1989

Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells.

The muscarinic receptors in a B82 cell line which were transfected with the rat m1 muscarinic receptor gene (cTB10 cells) were studied by using radioligand binding assays. Their possible coupling to the hydrolysis of inositol lipids and cyclic AMP formation were also investigated. [(-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] binding to the intact cTB10 cells was saturable and displaceable by 1 microM atropine sulfate. The Kd and maximum binding values of (-)-[3H]QNB from saturation studies were 12 pM and 17 fmol/10(6) cells, respectively. Inhibition studies of (-)-[3H]QNB binding to intact cTB10 cells suggested that these muscarinic receptors are of the M1 type defined by their high affinity for pirenzepine and low affinity for AF-DX 116 [11-[2-diethylamino methyl-1-piperidinylacetyl]-5,11-dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one]. The muscarinic agonist carbachol stimulated [3H]inositol monophosphate accumulation in the cTB10 cells, which could be reversed by the muscarinic antagonists atropine, pirenzepine or AF-DX 116. The rank order of potency of the muscarinic antagonists in inhibiting carbachol-stimulated [3H]inositol monophosphate accumulation was atropine greater than pirenzepine greater than AF-DX 116, in agreement with that from ligand/(-)-[3H]QNB competition experiments. Pertussis toxin and 4 beta-phorbol, 12-beta-myristate, 13-alpha-acetate reduced carbachol-stimulated [3H]inositol monophosphate accumulation. Prostaglandin E1 stimulated cyclic AMP formation in the cTB10 cells. Carbachol at the concentration of 10 mM exhibited no stimulatory or inhibitor effect on the basal or prostaglandin E1-stimulated cyclic AMP formation. These results suggest that the muscarinic receptors encoded by the transfected m1 gene in the cTB10 cells are of the M1 type and are coupled to the hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein.

Alprostadil↗
PubMed1989

The relationship between agonist states of the M1 muscarinic receptor and the hydrolysis of inositol lipids in transfected murine fibroblast cells (B82) expressing different receptor densities.

We studied the relationship between the M1 muscarinic receptor density and the receptor-mediated hydrolysis of inositol lipids in cloned murine fibroblast B82 cells which were transfected with the m1 muscarinic receptor gene. Of the seven clones examined, the M1 muscarinic receptor densities in these cells characterized by (-)[3H]methyl-3-quinuclidinyl benzilate ([-)-[3H]MQNB binding ranged from 12 fmol/10(6) cells in LK3-1 cells to 260 fmol/10(6) cells in the LK3-8 cells. Carbachol/(-)[3H]MQNB competition curves for the LK3-1 cells (with low receptor density) had a Hill coefficient close to unity. The competition curves for carbachol in the clones with higher receptor densities had Hill coefficients less than 1 and were best fitted by a computerized nonlinear least-squares regression program for the two-site model. The percentage of the M1 muscarinic receptors which had high affinity for carbachol decreased as the receptor density increased, suggesting that the presence of endogenous factors in these cells may be important for the agonist affinity state of the receptor. Concentration-response curves for carbachol-stimulated [3H]inositol monophosphate [( 3H]IP1) accumulation were also obtained. A significant correlation was observed between the density of M1 muscarinic receptor with high affinity for carbachol and the maximum [3H]IP1 accumulation in these cells. There is no significant difference among the EC50 values and the dissociation constant of high-affinity state values of the carbachol/(-)[3H] MQNB competition curves. These results suggest that the high-affinity state for carbachol may be the functional state of the M1 muscarinic receptors in these transfected B82 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

The cloned murine M1 muscarinic receptor is associated with the hydrolysis of phosphatidylinositols in transfected murine B82 cells.

A rat genomic DNA clone was isolated by its homology with a conserved primary sequence among the mammalian and avian beta adrenergic and porcine muscarinic receptors. A gene identified in this clone was highly homologous to the rat M1 muscarinic receptor. Stable expression of this gene was achieved in an established murine fibroblast cell line, B82. The gene product exhibits M1 type muscarinic receptor characteristics, as it has high affinity for PZ but low affinity for AF-DX 116. Carbachol stimulated the hydrolysis of phosphatidylinositols in the transfected cells. Pirenzepine had a more potent inhibitory effect on this response than AF-DX 116 since their functional inhibition constants were 13 nM and 480 nM, respectively, which is consistent with an M1 pharmacological profile. These data suggest that the M1 muscarinic receptor encoded by the gene is coupled to the hydrolysis of phosphatidylinositols after transfecting this gene into the B82 cells.

Animals↗
PubMed1988

Evolution of neurotransmitter receptor systems.

The presence of hormones, neurotransmitters, their receptors and biosynthetic and degradative enzymes is clearly not only associated with the present and the recent past but with the past several hundred million years. Evidence is mounting which indicates substantial conservation of protein structure and function of these receptors and enzymes over these tremendous periods of time. These findings indicate that the evolution and development of the nervous system was not dependent upon the formation of new or better transmitter substances, receptor proteins, transducers and effector proteins but involved better utilization of these highly developed elements in creating advanced and refined circuitry. This is not a new concept; it is one that is now substantiated by increasingly sophisticated studies. In a 1953 article discussing chemical aspects of evolution (Danielli, 1953) Danielli quotes Medawar, "... endocrine evolution is not an evolution of hormones but an evolution of the uses to which they are put; an evolution not, to put it crudely, of chemical formulae but of reactivities, reaction patterns and tissue competences." To also quote Danielli, "In terms of comparative biochemistry, one must ask to what extent the evolution of these reactivities, reaction patterns and competences is conditional upon the evolution of methods of synthesis of new proteins, etc., and to what extent the proteins, etc., are always within the synthetic competence of an organism. In the latter case evolution is the history of changing uses of molecules, and not of changing synthetic abilities." (Danielli, 1953). Figure 4 outlines a phylogenetic tree together with an indication of where evidence exists for both the enzymes that determine the biosynthesis and metabolism of the cholinergic and adrenergic transmitters and their specific cholinergic and adrenergic receptors. This figure illustrates a number of important points. For example, the evidence appears to show that the transmitters and their associated enzymes existed for a substantial period before their respective receptor proteins. While the transmitters and enzymes appear to exist in single cellular organisms, there is no solid evidence for the presence of adrenergic or cholinergic receptors until multicellular organisms where the receptors appear to be clearly associated with specific cellular and neuronal communication (Fig. 4). One can only speculate as to the possible role for acetylcholine and the catecholamine in single cell organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Monoclonal antibodies against chicken brain acetylcholinesterase. Their use in immunopurification and immunochemistry to demonstrate allelic variants of the enzyme.

Acetylcholinesterase (AChE) from 1-day chicken brain was enriched over 2000-fold by affinity chromatography using N-methylacridinium-Sepharose. This preparation was used to prepare monoclonal antibodies (mAb) directed against AChE, of which two were extensively characterised for further application. Both mAbs bound to the enzyme from the chicken with high affinity (Kd approximately 8 X 10(-10) M) and one mAb, in addition, recognised AChE from quail brain and muscle. Neither mAb cross-reacted with mammalian or fish AChE. Both mAbs recognised AChE in the endplate region of adult chicken skeletal muscle and bound with equal affinity to the three major oligomeric forms found in early ambryonic muscle. One mAb was used to immunopurify chicken brain AChE to homogeneity (over 12000-fold enrichment), with nearly complete recovery of the enzyme and without detectable proteolytic breakdown. The other mAb recognised AChE after immunoblotting and was used to screen crude brain extracts from individual chickens for allelic variations. Evidence is presented to show that two allelic forms occur, represented in SDS-PAGE by a doublet polypeptide of Mr approximately 110,000, this pattern is maintained after deglycosylation of the N-linked oligosaccharides. This variation was found throughout development and in both the brain and the muscle of individuals. We conclude that the gene encoding the catalytic subunit of chicken AChE is polymorphic with either one or two equally active alleles being expressed.

Acetylcholinesterase↗
PubMed1987

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.

Amino Acid Sequence↗
PubMed1986

Low frequency chronic electrical stimulation of normal and dystrophic chicken muscle.

The fast-twitch posterior latissimus dorsi muscle of normal and genetically dystrophic chickens was subjected to continuous indirect electrical stimulation at 10 Hz for periods of 4-8 weeks. To sustain this in vivo nerve stimulation an internally implantable miniature stimulator device was designed. This regime of stimulation caused complete fatigue of the normal muscle within 5 min of its initiation. The dystrophic muscles maintained a very small degree of contractile activity during this initial phase. Tangible twitching of the muscle returned in 5 week birds between 3 and 5 days and in 10 week birds between 11 and 16 days after implantation. After 4 weeks of stimulation, no significant change was measured in the time-to-peak of the isometric twitch response, nor in the half-relaxation time. The resistance to fatigue was significantly increased in the stimulated muscles when tested with a series of tetani at 40 Hz. The mean fibre area was decreased, in all muscles stimulated for longer than 3 weeks, in comparison to their contralateral controls, except where fibre splitting in dystrophic birds abnormally reduced the control value. The majority fibre type of the muscle was changed from type IIB to IIA. The histochemical reactions for both NADH-linked oxidation and phosphorylase were distinctly increased in the stimulated muscles. In normal muscle, stimulation increased somewhat the number of nuclei per unit area and changed their intracellular distribution, so that a greater proportion was found adjacent to the sarcolemma. The normal posterior latissimus dorsi muscle responded to chronic stimulation with increases of 3-6-fold in its acetylcholinesterase (AChE) activity. The maximum change in AChE occurred after 2 weeks stimulation; a steady level, 3 times that of the control unstimulated muscle, persisted at later times. Chronic stimulation suppressed the over-production of AChE that is characteristic of dystrophic chicken fast-twitch muscle, to attain a level comparable to the AChE activity in a stimulated normal muscle. Stimulation exerted a strong normalizing influence on dystrophic muscle, as assessed morphologically. The characteristic fibre rounding, fibre hypertrophy and myonuclear proliferation were reduced. This influence was most marked where the stimulation was initiated before the major pathological changes had occurred, but was also significant when commenced in strongly affected birds of 10-11 weeks.

Acetylcholinesterase↗