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M R Hanley

Publications and source records attributed to M R Hanley.

At least 37 records · Page 2Linked to original sources
PubMed1994

Expression of the MAS proto-oncogene in the retinal pigment epithelium of the rhesus macaque.

The MAS proto-oncogene codes for a seven transmembrane protein which has been previously localized to specific regions of the hippocampus and cerebral cortex in the rat central nervous system. Because MAS has biological properties related to the growth and differentiation of cells of neuroectodermal origin, we investigated the distribution of MAS expression in the rhesus macaque retina by in situ hybridization. A 330 base pair (bp) segment of the human MAS sequence was subcloned and used to generate single-stranded cRNA probes for these studies. Our results demonstrated little, if any, positive signal over the neurons of the retina. The use of epi-polarization microscopy, however, revealed a distinct positive labelling of retinal pigment epithelial (RPE) cells. These studies suggest the use of MAS as a possible marker for the retinal pigment epithelium.

Animals↗
PubMed1994

Attenuation of agonist-induced desensitization of the rat substance P receptor by microinjection of inositol pentakis-and hexakisphosphates in Xenopus laevis oocytes.

Recently, inositol hexakisphosphate (phytic acid) was shown to bind to photoreceptor arrestin and block its interaction with rhodopsin. Such an interaction might predict that inositol polyphosphates could alter G protein-coupled receptor desensitization. To investigate the possible roles of higher inositol polyphosphates on receptor desensitization, we have expressed the rat substance P receptor in Xenopus laevis oocytes. The functional expression of substance P receptor was monitored by voltage-clamp recording of substance P-induced Ca(2+)-dependent Cl- currents. When control oocytes were stimulated with substance P (30 nM), after 10 min of washing the second responses to substance P were approximately 15% of the first responses. Cytosolic injection of inositol pentakisphosphate (100 microM) or inositol hexakisphosphate (100 microM) inhibited the reduction of the second substance P-induced current responses, maintaining the second responses to 57-58% of the initial responses. The protective effects of inositol pentakisphosphate and inositol hexakisphosphate against agonist-induced desensitization were concentration and time dependent and structurally specific, in that inositol hexasulfate and inositol tris- and tetrakisphosphate isomers were inactive. Microinjection of inositol hexakisphosphate did not (a) change the potency of substance P or the sensitivity of the expressed substance P receptor to substance P, (b) inhibit 12-O-tetradecanoylphorbol-13-acetate-induced loss of substance P-induced current responses, or (c) alter the currents elicited by microinjection of inositol-1,4,5-trisphosphate. These results suggest that inositol pentakisphosphate and inositol hexakisphosphate have specific inhibitory effects on the agonist-induced loss of responsiveness of the rat substance P receptor. Moreover, these protective effects of inositol hexakisphosphate against desensitization were also observed with the endogenous lysophosphatidic acid/phosphatidic acid receptor, indicating that this mechanism is not specific to ectopic receptors. These results suggest that inositol pentakisphosphate and inositol hexakisphosphate may be novel pharmacological tools for the study of agonist-induced desensitization.

Animals↗
PubMed1993

Receptor regulation of phosphoinositide 3-hydroxykinase in the NG115-401L-C3 neuronal cell line: stimulation by insulin-like growth factor-I.

The activation of phosphoinositide 3-hydroxykinase (P13K) is currently believed to represent the critical regulatory event which leads to the production of a novel intracellular signal. We have examined the control of this pathway by a number of cell-surface receptors in NG115-401L-C3 neuronal cells. Insulin-like growth factor-I stimulated the accumulation of 3-phosphorylated inositol lipids in intact cells and the appearance of P13K in antiphosphotyrosine-antibody-directed immunoprecipitates prepared from lysed cells, suggesting that P13K had been activated by a mechanism involving a protein tyrosine kinase. In contrast, P13K in these cells was not regulated by a variety of G-protein-coupled receptors, nerve growth factor acting via a low affinity receptor, or receptors for transforming growth factor-beta and interleukin-1. The receptor-specificity of P13K activation in these cells places significant constraints on the possible physiological function(s) of this pathway.

Animals↗
PubMed1993

Characterization of metal ion-induced [3H]inositol hexakisphosphate binding to rat cerebellar membranes.

The binding of [3H]inositol hexakisphosphate ([3H] InsP6) to rat cerebellar membranes has been characterized with the objective of establishing the role, if any, of a membrane protein receptor. In the presence of EDTA, we have previously identified an InsP6-binding site with a capacity of approximately 20 pmol/mg protein (Hawkins, P. T., Reynolds, D. J. M., Poyner, D. R., and Hanley, M. R. (1990) Biochem. Biophys. Res. Commun. 167, 819-827). However, in the presence of 1 mM Mg2+, the capacity of [3H]InsP6 binding to membranes was increased approximately 9-fold. This enhancing effect of Mg2+ was reversed by addition of 10 microM of several cation chelators, suggesting that the increased binding required trace quantities of other metal cations. This is supported by experiments where it was possible to saturate binding by addition of excess membranes, despite not significantly depleting radioligand, pointing to removal of some other factor. Removal of endogenous cations from the binding assay by pretreatment with chelex resin also prevents the Mg(2+)-induced potentiation. Consideration of the specificity of the chelators able to abolish this potentiation suggested involvement of Fe3+ or Al3+. Both these ions (but not several others) were able to increase [3H]InsP6 binding to chelex-pretreated membranes at concentrations of 1 microM. It is possible to demonstrate synergy between Fe3+ and Mg2+ under these conditions. We propose that [3H]InsP6 may interact with membranes through non-protein recognition, possibly via phospholipids, in a manner dependent upon trace metals. The implications of this for InsP6 biology are considered.

Animals↗
PubMed1992

Phosphatidic acid and lysophosphatidic acid stimulate receptor-regulated membrane currents in the Xenopus laevis oocyte.

External application of dioleoyl-phosphatidic acid and oleoyl-lysophosphatidic acid stimulated Ca(2+)-dependent chloride currents in voltage-clamped Xenopus laevis oocytes. The responses were observed in oocytes from which follicular cells had been removed, indicating they were intrinsic to the oocyte itself. The lipid-induced Ca(2+)-dependent chloride currents were observed in the absence of extracellular calcium, were blocked by intracellular injection of the calcium chelator, bis(O-aminophenoxy)-ethane N,N,N'N'-tetraacetic acid, and could not be elicited by direct intracellular injection of the active lipids. The thresholds for dose-dependent current responses to dioleoyl-phosphatidic acid (100 nM) and for oleoyl-lysophosphatidic acid (10 nM) indicated that the lipid activities on oocytes were potent. With repeated or prolonged administration of either active lipid, responses exhibited desensitization. These results demonstrate that the Xenopus oocyte expresses endogenous functional responses for the mitogenic lipids phosphatidic acid and lysophosphatidic acid and thus provides a powerful model for characterization of the pharmacology and transduction pathways of these responses.

Animals↗
PubMed1992

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes.

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology.

Animals↗
PubMed1992

Stimulation of adenylate cyclase by amylin in CHO-K1 cells.

The CHO-K1 cell line responds to the peptide amylin by a rapid elevation of cAMP. The related peptide calcitonin gene-related peptide (CGRP) is 100 times less potent at stimulating adenylate cyclase than is amylin. The actions of amylin at this receptor are concentration dependent and not antagonized by the CGRP antagonist CGRP-(8-37). Although these cells have receptors for calcitonin, amylin is unable to take part in any high affinity interaction with these receptors, as assessed by radioligand binding. The CHO-K1 cell line has receptors for amylin that are distinct from those for calcitonin and CGRP.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.

The role of ATP-dependent calcium uptake into intracellular storage compartments is an essential feature of hormonally induced calcium signaling. Thapsigargin, a non-phorboid tumor promoter, increasingly is being used to manipulate calcium stores because it induces a hormone-like elevation of cytosolic calcium. It has been suggested that thapsigargin acts through inhibition of the endoplasmic reticulum calcium pump. We have directly tested the specificity of thapsigargin on all of the known intracellular-type calcium pumps (referred to as the sarcoplasmic or endoplasmic reticulum Ca-ATPase family (SERCA]. Full-length cDNA clones encoding SERCA1, SERCA2a, SERCA2b, and SERCA3 enzymes were expressed in COS cells, and both calcium uptake and calcium-dependent ATPase activity were assayed in microsomes isolated from them. Thapsigargin inhibited all of the SERCA isozymes with equal potency. Furthermore, similar doses of thapsigargin abolished the calcium uptake and ATPase activity of sarcoplasmic reticulum isolated from fast twitch and cardiac muscle but had no influence on either the plasma membrane Ca-ATPase or Na,K-ATPase. The interaction of thapsigargin with the SERCA isoforms is rapid, stoichiometric, and essentially irreversible. These properties demonstrate that thapsigargin interacts with a recognition site found in, and only in, all members of the endoplasmic and sarcoplasmic reticulum calcium pump family.

Amino Acid Sequence↗
PubMed1991

Regulation of c-fos and c-jun protooncogene expression by the Ca(2+)-ATPase inhibitor thapsigargin.

Thapsigargin, a non-phorbol-ester-type tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca(2+)-ATPase. We used this drug to analyze the involvement of Ca2+ and Ca(2+)-ATPases in the control of growth- and transformation-related genes. Here we show that treatment of mouse NIH 3T3 fibroblasts with thapsigargin induced rapid expression of the c-fos and c-jun protooncogenes. Inhibition or depletion of protein kinase C partially diminished the c-fos but not the c-jun response. Furthermore, thapsigargin could synergize with the tumor promoter phorbol 12-myristate 13-acetate to induce c-fos but not c-jun. However, thapsigargin had no effect on basal or phorbol ester-induced protein kinase C activity. Our results indicate that Ca2+ is a potent second messenger that controls expression of growth- and transformation-related genes. Since inhibition of the endoplasmic reticulum Ca(2+)-ATPase results in a strong induction of these genes, our data suggest that this Ca2+ pump may act as a negative regulator of cell growth.

Animals↗
PubMed1991

myo-inositol pentakisphosphates. Structure, biological occurrence and phosphorylation to myo-inositol hexakisphosphate.

1. Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate, 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346, 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).

Animals↗
PubMed1991

A monoclonal anti-idiotypic antibody to mu and delta opioid receptors.

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.

Animals↗