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M W Martin

Publications and source records attributed to M W Martin.

At least 37 records · Page 2Linked to original sources
PubMed1987

[3H]propylbenzilylcholine mustard-labeling of muscarinic cholinergic receptors that selectively couple to phospholipase C or adenylate cyclase in two cultured cell lines.

Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma X glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PBCM), and the mobilities of the [3H]PBCM-labeled species of both cells were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1321N1 and NG108-15 cells each primarily expressed a single [3H]PBCM-labeled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. [3H]PBCM labeling was completely inhibited by 1 microM atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the [3H]PBCM-labeled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Since muscarinic receptors are glycoproteins, the contribution of carbohydrate groups to the difference in apparent size of the [3H]PBCM-labeled proteins was determined by treatment of [3H]PBCM-labeled membranes with endoglycosidase F, an enzyme that removes both complex and high mannose type N-linked carbohydrate chains. Endoglycosidase F treatment reduced the apparent size of the [3H]PBCM-labeled species in 1321N1 cells from 92,000 to approximately 77,000 Da and in NG108-15 cells from 66,000 to 45,000 Da. Neuraminidase produced no further reduction of the apparent size of the [3H]PBCM-labeled species from either cell after endoglycosidase F treatment, suggesting the absence of sialic acid containing O-linked carbohydrate chains on the muscarinic receptors of the two cell lines. The results suggest that different muscarinic receptor proteins may be responsible for the two different biochemical responses to muscarinic receptor activation.

Adenylyl Cyclases↗
PubMed1986

H1-histamine receptors on human astrocytoma cells.

The H1-histamine receptor antagonist [3H]mepyramine bound with high affinity (Kd = 3-5 nM) to membranes derived from 1321N1 human astrocytoma cells. The H1-receptor antagonists triprolidine and diphenhydramine inhibited [3H]mepyramine binding with Kj values of 1-5 nM, whereas the Kj of the H2-histamine receptor antagonist cimetidine was greater than 100 microM. Histamine also inhibited [3H]mepyramine binding to 1321N1 cell membranes, and the histamine inhibition curve was shifted to the right and steepened in the presence of 1 microM guanosine 5'-O-(3-thiotriphosphate). Treatment of 1321N1 cells with pertussis toxin had no effect on the capacity of histamine to inhibit [3H]mepyramine binding either in the absence or presence of guanosine 5'-O-(3-thiotriphosphate). Therefore, agonist-occupied histamine receptors in these cells apparently interact with a guanine nucleotide regulatory protein that is not the inhibitory guanine nucleotide regulatory protein of adenylate cyclase. Although adenylate cyclase activity was not affected by histamine in a cell-free preparation, incubation of 1321N1 cells with histamine resulted in an attenuation of cyclic AMP accumulation. Analysis of cyclic AMP degradation in the presence of histamine indicated that the effects of histamine on cyclic AMP accumulation are mediated through activation of phosphodiesterase. This idea was supported by the fact that the phosphodiesterase inhibitor 1-isobutyl 3-methylxanthine blocked attenuation of cyclic AMP accumulation by histamine in a noncompetitive manner. Histamine also markedly increased phosphoinositide breakdown and 45Ca2+ efflux in 1321N1 cells. These histamine-induced effects apparently are mediated through H1-receptors, since triprolidine, but not cimetidine, potently inhibited histamine action. As for histamine interaction with its receptor, pertussis toxin had no effect on histamine-induced phosphoinositide breakdown, 45Ca2+ efflux, or attenuation of cyclic AMP accumulation. Taken together, these data indicate that 1321N1 human astrocytoma cells are a useful model system for the study of H1-histamine receptors and the biochemical responses mediated through these receptors.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Identification of the phosphodiesterase regulated by muscarinic cholinergic receptors of 1321N1 human astrocytoma cells.

Agonist occupation of muscarinic cholinergic receptors of 1321N1 human astrocytoma cells results in an activation of phosphodiesterase and a resultant 50-75% attenuation of isoproterenol-stimulated cyclic AMP accumulation. The effects of a series of phosphodiesterase inhibitors on muscarinic receptor-mediated inhibition of cyclic AMP accumulation and on the activities of partially purified, soluble phosphodiesterase have been compared to determine which form of phosphodiesterase activity is regulated by muscarinic receptors. The phosphodiesterase inhibitors (50 microM) 1-methyl-3-isobutylxanthine (MIX), 1-methyl-3-isobutyl-7-benzylxanthine (7-BzMIX), 1-methyl-3-isobutyl-8-methoxymethylxanthine (8-MeOMeMIX), and 2-O-propoxyphenyl-8-azapurin-6-one (MB 22948) blocked the effect of muscarinic receptor activation. However, 1-isoamyl-3-isobutylxanthine (IIX) and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) did not block muscarinic receptor-mediated effects but enhanced isoproterenol-stimulated cyclic AMP accumulation. Three forms of soluble phosphodiesterase activity were resolved by DEAE-cellulose chromatography and sucrose density gradient centrifugation. A calmodulin-stimulated phosphodiesterase activity was inhibited by MIX, 7-BzMIX, 8-MeOMeMIX, and MB 22948 (IC50 values = 1-10 microM) but was not inhibited by IIX and Ro 20-1724. The similar relative capacities of the phosphodiesterase inhibitors for blocking both the muscarinic receptor-mediated attenuation of cyclic AMP accumulation and the calmodulin-stimulated phosphodiesterase activity in vitro suggest that it is this form of enzyme that is regulated by muscarinic receptor stimulation.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Further evidence that muscarinic cholinergic receptors of 1321N1 astrocytoma cells couple to a guanine nucleotide regulatory protein that is not Ni.

Inhibitory coupling of receptors to adenylate cyclase previously has been shown to be relatively sensitive to inactivation by alkylation with N-ethylmaleimide (NEM). Modification of the inhibitory guanine nucleotide regulatory protein, Ni, has been proposed to be responsible for this effect. The effects of NEM on GTP-sensitive binding of carbachol to muscarinic cholinergic receptors has been compared in a cell line (1321N1 human astrocytoma cells) in which these receptors stimulate phosphoinositide breakdown and in a cell line (NG108-15 neuroblastoma X glioma cells) in which activation of these receptors results in inhibition of adenylate cyclase. Pretreatment of membrane preparations from 1321N1 cells with NEM resulted in a concentration-dependent decrease in the extent of pertussis toxin-catalysed [32P]ADP-ribosylation of a 41 000 Da protein previously proposed to be the alpha subunit of Ni. Under conditions where 32P-labelling of Ni in 1321N1 membranes was reduced by NEM by 90%, no effect was observed on the extent of guanine nucleotide-sensitive high-affinity binding of carbachol to muscarinic cholinergic receptors. In contrast, treatment of NG108-15 membranes with NEM under the same conditions resulted in complete loss of high-affinity guanine nucleotide sensitive binding of carbachol. These results illustrate another difference between the muscarinic receptor population of these two cell lines, and support the previous proposal that muscarinic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is not Ni.

Adenosine Diphosphate↗
PubMed1985

Pertussis toxin does not inhibit muscarinic-receptor-mediated phosphoinositide hydrolysis or calcium mobilization.

Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.

Animals↗
PubMed1985

Guanine nucleotide-sensitive, high affinity binding of carbachol to muscarinic cholinergic receptors of 1321N1 astrocytoma cells is insensitive to pertussis toxin.

Activation of muscarinic cholinergic receptors of 1321N1 human astrocytoma cells attenuates cyclic AMP accumulation. This effect results from an activation of phosphodiesterase with no direct inhibition of adenylate cyclase activity. In spite of this lack of coupling of muscarinic receptors to adenylate cyclase, guanine nucleotides reduce the apparent binding affinity of the agonist carbachol in a washed membrane preparation of 1321N1 cells. The order of potency for this effect is guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl-imidodiphosphate = GTP = GDP; ATP has no effect. The occurrence of a Mr = 41,000 protein labeled in the presence of [32P]NAD and pertussis toxin as well as the occurrence of guanine nucleotide-mediated inhibition of forskolin-stimulated adenylate cyclase activity indicate that the functional inhibitory guanine nucleotide regulatory component of adenylate cyclase (Ni) is present in 1321N1 cells. Pertussis toxin pretreatment of NG108-15 neuroblastoma X glioma cells, which express muscarinic receptors that link through Ni to inhibit adenylate cyclase, blocked the GTP-sensitive, high affinity binding of carbachol. In contrast, pretreatment of 1321N1 cells with a concentration of pertussis toxin that blocked [32P]ADP ribosylation of the Mr = 41,000 substrate and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity had no effect on GTP-sensitive high affinity binding of carbachol. These results suggest that muscarinic cholinergic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is distinct from Ni.

Adenylate Cyclase Toxin↗
PubMed1985

Regulation of cyclic AMP metabolism by muscarinic cholinergic receptors.

The occurrence of muscarinic cholinergic receptor-mediated activation of phosphodiesterase in 1321N1 cells does not represent an isolated phenomenon, since a similar response to cholinergic stimuli is observed in thyroid slices (45) and WI-38 fibroblasts (1,42). Both muscarinic-receptor-mediated inhibition of adenylate cyclase and activation of phosphodiesterase occur in WI-38 fibroblasts (42). Work currently under way in our laboratory is directed toward determining if a guanine nucleotide regulatory protein is involved in the activation of phosphodiesterase in these cells and whether common or separate populations of muscarinic receptors are coupled to these two mechanisms of cyclic AMP metabolism. The analysis of acute hormonal regulation of phosphodiesterase in intact cells is sufficiently complicated to have previously discouraged investigators from pursuing this question in mammalian tissues. The 1321N1 cell line provides a simple model system in which at least one mechanism of hormonal regulation of phosphodiesterase can be examined. In light of the widespread occurrence of muscarinic-receptor-mediated effects on Ca2+ mobilization, it would not be surprising to find that this mechanism represents an important part of cholinergic action in both the peripheral and central nervous systems. Indeed, this system could provide an important regulatory link between Ca2+ -mediated and cyclic-AMP-mediated events in target cells. The potential importance of such a mechanism also need not be restricted to the muscarinic receptor system, since any neurotransmitter or hormone receptor system coupled to events involved in Ca2+ mobilization might produce phenomena similar to that observed for muscarinic receptors in 1321N1 cells. Our studies emphasize that two mechanisms for regulation of cyclic AMP accumulation by muscarinic cholinergic receptors exist. The data to date suggest that separate receptor subtypes are involved in these mechanisms of cholinergic regulation and provide another biochemical basis whereby the well-known interaction of Ca2+ with the cyclic AMP system can be effected. Thus, identification of the molecular events involved in the regulation of PI turnover and its consequences may be crucial in defining the basis of this aspect of cholinergic action. In addition, more extensive analyses of the phosphodiesterase system using cell-free preparations have the potential of providing clues to the molecular basis of this mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1984

Pertussis toxin differentiates between two mechanisms of attenuation of cyclic AMP accumulation by muscarinic cholinergic receptors.

It has been proposed elsewhere [Meeker, R.B. & Harden, T. K. (1982) Mol. Pharmacol. 22, 310-319] that muscarinic cholinergic receptor-mediated attenuation of cAMP accumulation occurs through activation of phosphodiesterase in 1321N1 human astrocytoma cells. Pertussis toxin, which ADP-ribosylates the guanine nucleotide regulatory protein involved in receptor-mediated inhibition of adenylate cyclase (Ni), has been utilized to further differentiate between the mechanism of cholinergic regulation of cAMP metabolism in 1321N1 cells and the mechanism involving inhibition of adenylate cyclase in other tissues. Muscarinic receptor-mediated regulation of cAMP accumulation in NG108-15 neuroblastoma-glioma cells occurs through inhibition of adenylate cyclase. Pretreatment of these cells with pertussis toxin completely blocked the capacity of carbachol to attenuate cAMP accumulation. In contrast, concentrations of pertussis toxin two to three orders of magnitude higher than those effective in NG108-15 cells had no effect on muscarinic receptor-mediated attentuation of cAMP accumulation in 1321N1 cells. In addition, no effect of pertussis toxin was observed either on the control rate or the carbachol-stimulated rate of cAMP degradation measured directly in intact 1321N1 cells. A 41,000 Mr protein previously proposed to be the alpha subunit of Ni was labeled during incubation of a plasma membrane fraction from 1321N1 cells with [32P]NAD and pertussis toxin. Pertussis toxin is apparently active in 1321N1 cells, since this protein substrate was not labeled in plasma membrane preparations from cells previously incubated with toxin. Functional activity of Ni was demonstrated by the observation that guanosine 5'-[gamma-thio]triphosphate- and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity occurred in cell-free preparations from 1321N1 cells. The inhibitory activity of these guanine nucleotides was lost in membrane preparations from pertussis toxin-treated cells. The data suggest that adenylate cyclase is not involved in cholinergic action in 1321N1 cells and, furthermore, Ni is not involved in muscarinic receptor-mediated activation of phosphodiesterase in these cells. Thus, pertussis toxin can be used to differentiate between two mechanisms of cholinergic regulation of cAMP metabolism.

Adenosine Diphosphate Ribose↗
PubMed1984

Modulation of muscarinic cholinergic receptor affinity for antagonists in rat heart.

Modulation of the affinity of agonists and antagonists at muscarinic cholinergic receptors in rat heart membranes was investigated using the radiolabeled antagonist, [3H]quinuclidinyl benzilate ([3H]QNB), and the radiolabeled agonist, [methyl-3H]oxotremorine acetate ([3H]OXO). Receptor affinity for oxotremorine measured in competition binding assays with [3H]QNB or by equilibrium binding of [3H]OXO was increased when the incubation temperature was reduced to 4 degrees C. In contrast, the receptor affinity for [3H]QNB was decreased at lower incubation temperatures and a marked effect of guanine nucleotides on the affinity for [3H]QNB was revealed. Guanine nucleotides increased receptor affinity for [3H]QNB without changing the total number of binding sites. The GTP-induced increase in the affinity for [3H]QNB was reflected by an increase in the rate constant for association of [3H]QNB. At subsaturating ligand concentrations, guanine nucleotides increased [3H]QNB binding and decreased [3H]OXO binding with the same order of potency: GppNHp = GTP gamma S greater than GTP greater than guanosine 5'-diphosphate greater than GMP. Free Mg++ ion was required to observe guanine nucleotide effects on antagonist binding. Pretreatment of heart membranes with N-ethyl-maleimide increased [3H]QNB affinity and blocked the effects of guanine nucleotides. N-Ethylmaleimide also decreased [3H]OXO binding and increased [3H]QNB binding with a similar concentration-effect relationship. Thus, antagonist and agonist binding to muscarinic cholinergic receptors is modulated in a reciprocal manner by a number of factors; this modulation appears to reflect interaction of agonist and antagonist-occupied receptors with a guanine nucleotide regulatory protein, Ni.

Animals↗
PubMed1983

Interaction of a radiolabeled agonist with cardiac muscarinic cholinergic receptors.

The interaction of a radiolabeled muscarinic cholinergic receptor agonist, [methyl-3H]oxotremorine acetate [( 3H]OXO), with a washed membrane preparation derived from rat heart, has been studied. In binding assays at 4 degrees C, the rate constants for association and dissociation of [3H]OXO were 2 X 10(7) M-1 min-1 and 5 X 10(-3) min-1, respectively, Saturation binding isotherms indicated that binding was to a single population of sites with a Kd of approximately 300 pM. The density of [3H]OXO binding sites (90-100 fmol/mg of protein) was approximately 75% of that determined for the radiolabeled receptor antagonist [3H]quinuclidinyl benzilate. Both muscarinic receptor agonists and antagonists inhibited the binding of [3H]OXO with high affinity and Hill slopes of approximately one. Guanine nucleotides completely inhibited the binding of [3H]OXO. This effect was on the maximum binding (Bmax) of [3H]OXO with no change occurring in the Kd; the order of potency for five nucleotides was guanosine 5'-O-(3-thio-triphosphate) greater than 5'-guanylylimidodiphosphate greater than GTP greater than or equal to guanosine/diphosphate greater than GMP. The [3H]OXO-induced interaction of muscarinic receptors with a guanine nucleotide binding protein was stable to solubilization. That is, membrane receptors that were prelabeled with [3H]OXO could be solubilized with digitonin, and the addition of guanine nucleotides to the soluble, [3H]OXO-labeled complex resulted in dissociation of [3H]OXO from the receptor. Pretreatment of membranes with relatively low concentrations of N-ethylmaleimide inhibited [3H]OXO binding by 85% with no change in the Kd of [3H]OXO, and with no effect on [3H]quinuclidinyl benzilate binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

Effect of testosterone propionate on ABP levels in rats hypophysectomised at different ages using individual sampling.

The effect of age at hypophysectomy on the response of the regressed rat testis to testosterone propionate (TP) and FSH with respect to androgen-binding protein (ABP) levels was studied in individual animals. All treatments were begun 30 days after surgery. Treatment of rats 35, 45, 55 and 75 days of age at surgery with TP (1 mg/260 g for 25 days) significantly increased the level of ABP in the testes of animals in all age groups except those hypophysectomized at 35 days of age. TP treatment did not significantly elevate epididymal levels of ABP above those found in untreated rats in any age group. In animals hypophysectomized at 100 days of age, acute treatment (3 days) with FSH (150 and 300 mug/day) significantly increases the ABP levels per testis and per epididymis. Similar treatment with 750 mug TP/day did not result in a statistically significant increase in testicular ABP. No synergism between the two hormones was noted under the conditions described. Significant restoration of testicular ABP levels per mg protein was achieved with 1 mg TP/day by 5 days of treatment. Treatment of hypophysectomized adult rats with FSH raised the epididymal/testicular ratio of ABP to about 40% of that found in intact rats while comparable treatment with TP (750 mug/day for 3 days or 1 mg/day for 10 days) only slightly affected the ratio. It is postulated that FSH may facilitate ABP transport to the epididymis in addition to affecting its production by the testis.

Aging↗