Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells.

Exposure of A431 human epidermoid carcinoma cells to epidermal growth factor (EGF), bradykinin, and histamine resulted in a time- and concentration-dependent accumulation of the inositol phosphates (InsP) inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3). Maximal concentrations of EGF (316 ng/ml; approximately 50 nM), bradykinin (1 microM), and histamine (1 mM) resulted in 3-, 6-, and 3-fold increases, respectively, in the amounts of inositol phosphates formed over a 10-min period. The K0.

Regulation of inositol trisphosphate accumulation by muscarinic cholinergic and H1-histamine receptors on human astrocytoma cells. Differential induction of desensitization by agonists.

Although activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells results in a linear accumulation of inositol phosphates for up to 60 min in the presence of LiCl [Masters, Quinn & Brown (1985) Mol. Pharmacol. 27, 325-332], activation of H1-histamine receptors resulted in an increase in total inositol phosphate formation that was maintained for less than 5 min.

Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.

The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757].

Receptor reserve in the calcium-dependent cyclic AMP response of astrocytoma cells to muscarinic receptor stimulation: demonstration by agonist-induced desensitization, receptor inactivation, and phorbol ester treatment.

Activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells results in attenuation of cyclic AMP accumulation, apparently as a consequence of increases in phosphoinositide hydrolysis, Ca2+ mobilization, and the activation of a Ca2+ calmodulin-regulated phosphodiesterase. Preincubation of these cells with carbachol for 30-90 min resulted in a 15-30-fold shift to the right of the concentration effect curves for carbachol or methacholine for attenuation of cyclic AMP accumulation, with no change occurring in the maximal effect observed with either agonist. In contrast, preincubation with carbachol for 30-90 min resulted in an essentially complete loss of effects on cyclic AMP accumulation of the muscarinic receptor agonists oxotremorine, arecoline, and bethanechol.

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.

Adenosine and muscarinic cholinergic receptors attenuate cyclic AMP accumulation by different mechanisms in 1321N1 astrocytoma cells.

An adenosine receptor has been characterized to unambiguously demonstrate that the inhibitory guanine nucleotide regulatory protein, Gi, of 1321N1 human astrocytoma cells is fully capable of functionally coupling to adenylate cyclase. Adenosine receptor agonists attenuated cyclic AMP accumulation by 35 to 75% with the order of potency of N6(R-phenylisopropyl)-adenosine greater than adenosine = 2-chloroadenosine greater than N6-methyladenosine = N6-benzyladenosine. 3-Isobutyl-1-methylxanthine competitively antagonized the effect of adenosine receptor agonists.

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).

Comparison of binding of 125I-iodopindolol to control and desensitized cells at 37 degrees and on ice.

Binding of 125I-iodopindolol (IPIN) to intact 1321N1 human astrocytoma cell B-adrenergic receptors was measured at 37 degrees and on ice. Control cells showed a single component of IPIN binding on ice with the same total number of receptors as measured at 37 degrees. In desensitized cells (pretreated for 20 min with 1 microM isoproterenol) approximately 40% of IPIN binding on ice exhibited kinetics similar to those observed in control cells.

Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Relation to efficacy of agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization.

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were 'full' agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were 'partial' agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested.

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors.

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al.

Rapid down regulation of beta adrenergic receptors by combining antidepressant drugs with forced swim: a model of antidepressant-induced neural adaptation.

The hypothesis that behavioral responses to antidepressant drugs in the forced swim test are related to a rapid neural adaptation produced by the combination of drug treatment and swim stress was explored. As a measure of adaptation, brain beta adrenergic receptors were assayed using [3H]dihydroalprenolol [( 3H]DHA) binding to brain membranes from rats that were processed in the forced swim test. The combination of swim stress and imipramine treatment antagonized immobility induced by forced swimming and resulted in a reduction in [3H] DHA binding to membranes from forebrain preparations which did not include the corpus striatum.

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.

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.

Effects of tunicamycin on the expression of beta-adrenergic receptors in human astrocytoma cells during growth and recovery from agonist-induced down-regulation.

Tunicamycin, which inhibits formation of asparagine-linked glycoproteins, caused a concentration-dependent blockade of beta-adrenergic receptor (beta-AR) accumulation in 1321N1 human astrocytoma cells during growth in culture. A concentration of tunicamycin (0.1 microgram/ml) that inhibited receptor accumulation and [3H]mannose or [3H]glucosamine incorporation into glycoproteins by 90% had only a small effect (10%) on [3H]leucine incorporation into protein, and reduced the rate of cell growth.

Muscarinic cholinergic receptor-mediated attenuation of adenylate cyclase activity in rat heart membranes.

The regulation of adenylate cyclase by muscarinic cholinergic receptors has been studied in rat heart membranes. In the presence of isoproterenol the K0.5 for Mg2+-mediated activation of adenylate cyclase was 0.

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.

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.

Use of a density shift method to assess beta-adrenergic receptor synthesis during recovery from catecholamine-induced down-regulation in human astrocytoma cells.

Exposure of postconfluent 1321N1 human astrocytoma cells to 1.0 microM isoproterenol for 12-24 hr results in a 90% loss of beta-adrenergic receptors. Upon removal of agonist, recovery of beta-receptors to control levels occurs within 72 hr.

Muscarinic cholinergic receptors of two cell lines that regulate cyclic AMP metabolism by different molecular mechanisms.

The attenuation of cyclic AMP accumulation occurs by different mechanisms in 1321N1 astrocytoma cells and NG108-15 neuroblastoma X glioma cells. In 1321N1 cells, cholinergic agonists reduce cyclic AMP accumulation through a Ca2+-dependent activation of phosphodiesterase; in NG108-15 cells, muscarinic receptor-mediated effects on cyclic AMP metabolism occur through inhibition of adenylate cyclase. The goal of the current study was to determine whether different pharmacological specificities were expressed by the muscarinic receptor populations of these two cell lines.

Relationship between an altered membrane form and a low affinity form of the beta-adrenergic receptor occurring during catecholamine-induced desensitization. Evidence for receptor internalization.

We have investigated the relationship between the catecholamine-induced occurrence in 1321N1 human astrocytoma cells of beta-adrenergic receptors that exhibit low apparent affinity for hydrophilic ligands in short-time assays with intact cells and a population of beta-adrenergic receptors that migrate in a light vesicle fraction on sucrose density gradients. Pretreatment of cells with concanavalin A prevents the generation of both of these forms of the receptor during incubation with agonists but does not prevent the agonist-induced decrease in isoproterenol-stimulated cyclic AMP production that also occurs during desensitization. Selective labeling of the low affinity beta-receptors with 125I-pindolol followed by centrifugation on sucrose density gradients revealed that all of the receptors in the light vesicle fraction from desensitized cells were of the low affinity type, but that a portion of the low affinity receptors also migrated in a heavier sucrose fraction together with the plasma membrane.

Histoplasma capsulatum. Isolation from an Australian cave environment and from a patient.

The fungus Histoplasma capsulatum, although commonly found in bat-frequented caves in many countries, has not previously been isolated from that environment in Australia. This report describes the isolation of H. capsulatum from several sources, including cave soil, the organs of mice exposed to the cave environment, and the sputum of a patient clinically diagnosed as having acute pulmonary histoplasmosis after exposure to the cave environment.

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.

Relationships between phosphoinositide and calcium responses to muscarinic agonists in astrocytoma cells.

Activation of muscarinic receptors in human astrocytoma (1321N1) cells stimulates phosphoinositide metabolism and calcium mobilization. The muscarinic effect on phosphoinositide turnover is evidenced by increased formation of [3H]inositol 1-phosphate (Ins1P) and by increased [3H]inositol incorporation into PtdIns. The muscarinic effect on calcium mobilization is seen as a large increase in undirectional 45Ca2+ efflux from cells equilibrated with 45Ca2+ and a small increase in unidirectional 45Ca2+ influx.

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.

Regulation of cyclic AMP accumulation by peptide hormone receptors in immunocytochemically defined astroglial cells.

Primary cultures of neonatal murine brain have been reported to express multiple receptors that regulate adenylate cyclase activity. Since for the most part these results were obtained with mixed cell cultures, it has been difficult to define receptor profiles for specific cell types. With this concern in mind a series of studies has been initiated designed to identify specific receptors present on highly purified, immunocytochemically defined astroglia derived from the cerebral cortices of neonatal rats.