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Biomedical subjects

M D Browning

Publications and source records attributed to M D Browning.

At least 37 records · Page 2Linked to original sources
PubMed1996

Regional and subunit specific changes in NMDA receptor mRNA and immunoreactivity in mouse brain following chronic ethanol ingestion.

Chronic ethanol treatment of mice has been shown to result in increased binding of dizocilpine and glutamate to hippocampal NMDA receptors. These changes were suggested to reflect an increase in NMDA receptor number that may underlie certain signs of the ethanol withdrawal syndrome. However, there was no change in binding of a competitive NMDA receptor antagonist, or of ligand binding to the glycine co-agonist site on the receptor after chronic ethanol treatment. Differential changes in the binding of particular ligands at the NMDA receptor suggested the possibility that chronic ethanol ingestion might selectively affect the expression of particular NMDA receptor subunits. Our current work demonstrates that chronic ethanol ingestion by mice, which results in the generation of physical dependence, also produces increases in the NMDA receptor NR1 subunit protein in the hippocampus and cerebellum (approximately 50% and 95%, respectively), and produces increases in the NR2A subunit protein in the hippocampus and cortex (approximately 25% and 40%, respectively). However, the mRNA levels for these subunits were not increased in the respective brain areas by the same ethanol treatment. The changes in NMDA receptor subunit expression in discrete areas of the brain may contribute to the previously observed changes in ligand binding and, possibly, signs of ethanol withdrawal.

Alcoholism↗
PubMed1996

Enhancement of recombinant alpha 1 beta 1 gamma 2L gamma-aminobutyric acidA receptor whole-cell currents by protein kinase C is mediated through phosphorylation of both beta 1 and gamma 2L subunits.

The gamma-aminobutyric acidA (GABA)A receptor (GABAR) beta 1 and gamma 2L subtypes have been shown to be phosphorylated in vitro by protein kinase C (PKC) [J. Biol. Chem. 267:14470-14476 (1992); Neuron 12:1081-1095 (1994)]. To determine the physiological consequences of phosphorylation of GABAR isoforms containing the beta 1 and gamma 2L subtypes, the specific serine residues phosphorylated by PKC (beta 1 S409, gamma 2L S327 and S343) were changed to alanines through site-directed mutagenesis. Wild-type (alpha 1 beta 1 gamma 2L GABARs) and three mutant GABAR isoforms [alpha 1 beta 1 gamma 2L(S327A, S343A), alpha 1 beta 1(S409A) gamma 2L, and alpha 1 beta 1(S409A) gamma 2L(S327A, S343A) GABARs) were expressed in mouse L929 fibroblasts through transient cotransfection. Recordings were obtained from each cell with the use of the whole-cell patch-clamp technique. The initial recording was made with the use of control intrapipette solution, and a second recording from the same cell was obtained with pipettes containing either constitutively active PKC [protein kinase M (PKM)] or control solution to obtain paired GABA concentration-response relationships. All GABAR isoforms studied had equivalent maximal GABA currents and similar GABA concentration-response profiles under the control condition. Intracellular PKM treatment increased the maximal current and EC50 value in cells expressing wild-type GABARs. However, PKM reimpalement did not significantly change these parameters in cells expressing any of the mutant GABAR isoforms, indicating that the mutation of either the beta 1 or gamma 2L subtype alone was sufficient to prevent enhancement of GABAR current by PKM. No significant changes were obtained during control reimpalement recordings of wild-type or mutant receptors. Furthermore, PKM treatment did not after the time constants of GABA current desensitization kinetics measured from cells expressing wild-type or mutant receptors. These data thus suggest that PKC phosphorylation of the beta 1 and gamma 2L subtypes enhances GABAR current and that both subtypes are required for complete PKC-mediated enhancement of alpha 1 beta 1 gamma 2L GABAR current.

Alanine↗
PubMed1995

Enhancement of high threshold calcium currents in rat primary afferent neurons by constitutively active protein kinase C.

Protein kinase C has been implicated in the modulation of calcium channel function. However, controversy exists concerning the actions of agents such as phorbol esters or diacylglycerol (DAG) that activate endogenous PKC, with both enhancement and inhibition of Ca2+ currents described. In this article we report the effects of direct intracellular application of a constitutively active form of PKC (PKM) on whole cell calcium currents in acutely dissociated rat dorsal root ganglion neurons. PKM application significantly enhanced high threshold voltage-activated calcium currents elicited from holding potentials of -80 mV and -40 mV. The rate of current rundown in PKM-treated cells was not significantly different from controls. The enhancement observed with PKM was not due to a shift in the voltage dependence of the peak current. Synthetic PKC inhibitor peptide (PKC-I) added to recording solutions containing PKM (PKM+PKC-I) abolished the PKM-associated enhancement. The rate of current rundown was significantly increased in the presence of PKM+PKC-I, and PKC-I alone, suggesting that substantial enhancement of voltage-activated calcium currents by endogenous PKC occurred in this preparation of rat dorsal root ganglion neurons. The portions of current attributable to N-, L-, and non-N,L-type currents [determined by applying the N- and L-type calcium antagonists omega-conotoxin GVIA and nifedipine (3-10 microM)] were not affected by PKM, suggesting that both N and L current components were enhanced by PKM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

ABT-200, a norepinephrine uptake inhibitor, blocks Ca2+ signals in chromaffin cells.

We previously described inhibition by racemic (+/-)-(1'R*,3R*)-3-phenyl-1- [1',2',3',4'-tetrahydro-5',6'-methylenedioxy-1'- napthalenyl-methyl]-pyrrolidine methanesulfonate (ABT-200), and its two constituent enantiomers, SS,ABT-200 and RR,ABT-200, of nicotine-stimulated but not histamine-stimulated catecholamine release from bovine adrenal chromaffin cells. To test the hypothesis that this inhibition reflects a blockade of Ca2+ influx, we used fura-2 loaded chromaffin cells to investigate cytosolic Ca2+ signals. We found that SS,ABT-200 inhibited nicotine- and K(+)-stimulated Ca2+ signals, both of which depend on Ca2+ influx. However, the early phase of the histamine-stimulated Ca2+ signals, which depends on Ca2+ mobilization from intracellular stores, was unaffected. We also examined ion flux through the nicotinic receptor by measuring 86rubidium+ (86Rb+) efflux from preloaded mouse midbrain synaptosomes. We found that SS,ABT-200 partially inhibited nicotine-stimulated 86Rb+ efflux, suggesting that it blocks ion flux through the nicotinic receptor directly. These data support a model in which ABT-200 blocks nicotine-stimulated catecholamine release by inhibiting cation flux through multiple channels.

Adrenal Glands↗
PubMed1994

Regulation of mitogen-activated protein kinase activation by protein kinases A and C in a cell-free system.

Previously pp60v-src, cyclin A, p39mos, and maturation-promoting factor (composed of Cdc2 and cyclin B) have been shown to activate mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) in cell-free extracts of Xenopus oocytes. The pp60v-src pathway is dependent on a functional Ras signal whereas the cyclin/maturation-promoting factor pathway is not. Here we show that protein kinase C (PKC) is also able to stimulate MAPK in a Ras-dependent manner, but PKC is not necessary for signaling by pp60v-src. In addition, preincubation of extracts with cAMP-dependent protein kinase (PKA) blocks stimulation of MAPK by cyclin, p21V12ras, PKC, or pp60v-src, by at least 50%, but stimulation by c-Mos is unaffected. Furthermore, inhibition of endogenous PKA by the heat-stable PKA inhibitor is sufficient to stimulate MAPK activity in these extracts in the absence of protein synthesis and without dependence on a functional Ras protein. These results suggest that independent pp60v-src and PKC pathways converge at Ras and that PKA acts to block MAPK activation by both Ras-dependent and -independent signals.

Amino Acid Sequence↗
PubMed1994

Modulation of AMPA/kainate receptors in cultured murine hippocampal neurones by protein kinase C.

1. The patch clamp technique, together with intracellular perfusion of the catalytic fragment of protein kinase C (PKCM), was employed to investigate the role of this enzyme in the intracellular regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors in cultured hippocampal neurones. 2. The responses evoked by near-maximal concentrations of kainate (250 microM) and AMPA (100 microM) were potentiated by the introduction of PKCM, whilst co-application of the inhibitory peptide fragment PKCI(19-36) prevented this action. 3. Modulation of kainate responses by PKCM was dependent upon the concentration of agonist applied. Currents evoked by kainate were potentiated at concentrations above those which caused 50% of the maximal response (EC50) and depressed at lower concentrations. Furthermore, okadaic acid, a specific inhibitor of phosphatases 1 and 2A, had a similar effect upon concentration-response relationships when currents activated by kainate were recorded using the perforated patch technique. 4. In addition, the mean amplitude and/or time constant of decay of miniature excitatory synaptic currents (mediated by AMPA/kainate receptors) was increased by the intracellular injection of PKCM. 5. These observations suggest that the function of postsynaptic excitatory amino acid receptors can be modulated by the activity of PKC as well as by endogenous phosphatases. This regulation may contribute to some forms of synaptic plasticity within the central nervous system.

Animals↗
PubMed1994

Calcium signalling in bovine adrenal chromaffin cells: additive effects of histamine and nicotine.

In a previous report, we described the ability of two secretogogues, histamine and nicotine, to stimulate additive effects on catecholamine (CA) release and synapsin II phosphorylation in bovine adrenal chromaffin cells (BACC) [Firestone and Browning (1992), J. Neurochem., 58:441-447]. We hypothesized that these results were due to the combined effects on cytosolic Ca++ of the two distinct signalling pathways. We therefore examined the intracellular Ca++ signals stimulated by histamine and nicotine, alone and together. In Ca(++)-deficient medium, nicotine-stimulated signals were abolished, whereas histamine-stimulated signals were maintained, demonstrating that nicotine depended entirely on Ca++ influx for its effects. Indeed, the nicotine-stimulated signal could also be prevented using a Ca++ channel blocker, nicardipine. Further, the observation that exposure of BACC to thapsigargin reduced histamine-stimulated Ca++ signals verified that histamine mobilizes Ca++ from intracellular stores. Thus, the two secretogogues mobilize Ca++ from distinct pools. When BACC were stimulated with the two secretogogues together, the resulting Ca++ signal was greater than that from either alone. These data are consistent with a model in which two distinct sources of Ca++ can summate within the cell, producing a greater Ca++ signal and, hence, a greater effect on neurotransmitter release.

Adrenal Glands↗
PubMed1994

Protein kinase C enhances recombinant bovine alpha 1 beta 1 gamma 2L GABAA receptor whole-cell currents expressed in L929 fibroblasts.

The beta 1 and gamma 2L subunits of the gamma-aminobutyric acid type A receptor (GABAR) contain phosphorylation sites for PKC. To determine the effect of PKC on GABAR function, whole-cell recordings were obtained from mouse fibroblasts expressing recombinant alpha 1 beta 1 gamma 2L receptors, and catalytically active PKC (PKM) was applied via the recording pipette. The first experiment was a population study. Intracellular application of PKM increased GABAR currents, and the enhancement was antagonized by coapplication of the PKC inhibitory peptide. No acceleration or deceleration of GABAR desensitization was observed. The second experiment was a reimpalement study in which paired recordings were made successively from individual cells. Enhancement of GABAR currents by PKM was again obtained. PKM increased GABAR currents at high (> 10 microM) but not at low (< 10 microM) GABA concentrations, resulting in increases in both EC50 and maximal GABAR current. Thus, PKC phosphorylation enhanced recombinant alpha 1 beta 1 gamma 2L GABAR current by increasing maximal current without increasing the affinity of GABA for the GABARs.

Animals↗
PubMed1994

Differential distribution of the synapsins in the rat olfactory bulb.

The distribution of the different forms of synapsin in the rat olfactory bulb was investigated by biochemical and immunocytochemical methods. Western blots of tissue derived from microdissection of the surface and core regions of the olfactory bulb were performed using antibodies to synapsin I and synapsin II. The relative levels of the synapsins in the core region of the olfactory bulb were similar to the cerebral cortex. In contrast, the surface region of the olfactory bulb had significantly higher levels of synapsin IIa and significantly lower levels of synapsin I, relative to the cortex. Immunocytochemical localization of synapsin I and synapsin II revealed that synapsin I immunoreactivity was the most dense in the external plexiform layer and in the glomeruli; immunoreactivity was also present in the granule cell layer and the periglomerular regions. Synapsin II immunoreactivity was the most dense in the glomeruli. The external plexiform layer, internal plexiform layer, and granule cell layer exhibited much lower immunoreactivity. To determine the source of synapsin II immunoreactivity in the glomeruli, the olfactory epithelium was damaged to decrease the primary afferent input to the bulb. Three to four days later, olfactory bulb sections were double labeled with anti-olfactory marker protein (OMP) antibodies and anti-synapsin II antibodies. Following denervation, both OMP and synapsin II immunoreactivities were diminished, and continued to colocalize in regions retaining immunoreactivity. Individual puncta were immunoreactive for both OMP and synapsin II. Occasional puncta contained only synapsin II immunoreactivity. These results indicate that the distribution of the synapsins in the olfactory bulb differs from most other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Significant reductions in synapsin but not synaptophysin specific activity in the brains of some schizophrenics.

The levels of the synaptic vesicle-associated proteins, synapsin and synaptophysin, were examined in human postmortem hippocampus from the brains of schizophrenics and age-matched controls using a quantitative western blot analysis. The schizophrenic samples had significantly lower levels of synapsin I than controls. In individual data, five of the seven schizophrenic samples had extremely low levels of synapsin, whereas two of the schizophrenic samples had normal levels of synapsin. This deficit in synapsin does not appear to be due to some non-specific neuronal loss as the levels of the other synaptic vesicle marker, synaptophysin, were near normal in all seven schizophrenics. Given that synapsin is thought to regulate neurotransmitter release, it is possible that this deficit in synapsin could result in abnormal processing of neuronal information as is seen in various sensory processing abnormalities associated with schizophrenia.

Adolescent↗
PubMed1993

Hippocampal plasticity induced by primed burst, but not long-term potentiation, stimulation is impaired in area CA1 of aged Fischer 344 rats.

The effect of two types of electrical stimulation designed to induce long-lasting plasticity of the Schaffer/commissural inputs to CA1 pyramidal neurons was investigated using in vitro hippocampal slices made from young (3-6 month) and old (24-27 month) Fischer 344 rats. The first stimulation paradigm, primed burst (PB) stimulation, consisted of a total of five physiologically patterned stimuli: a single priming pulse followed 170 ms later by a burst of four pulses at 200 Hz. The second stimulation paradigm, long-term potentiation (LTP) stimulation, consisted of a 200 Hz/1 second train (a total of 200 stimuli). Primed burst and LTP stimulation were equally effective at inducing a lasting increase in the population spike recorded from slices made from young rats. However, the enhancement of population spike amplitude produced by PB, but not LTP, stimulation was significantly less in slices made from old rats. These results suggest that the capacity of the hippocampus to demonstrate long-lasting synaptic plasticity is not altered with age, but that engaging plasticity-inducing mechanisms becomes more difficult. Furthermore, these data suggest that physiologically patterned paradigms for inducing long-lasting synaptic plasticity may more accurately assess the functional status of hippocampal memory encoding mechanisms than does conventional LTP stimulation.

Action Potentials↗
PubMed1993

Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: analysis of the substrate domain.

Previous work has shown that the GABAA-receptor (GABAA-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit/subtypes of the GABAA-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the beta 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the beta 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315-1317) that both PKA and PKC phosphorylate the beta-subunit of the GABAA-R.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of a regulated form of actin depolymerizing factor.

Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.

Actin Depolymerizing Factors↗
PubMed1993

Ca2+/calmodulin-dependent protein kinase II and protein kinase C phosphorylate a synthetic peptide corresponding to a sequence that is specific for the gamma 2L subunit of the GABAA receptor.

The gamma 2 subunit of the GABA receptor (GABAA-R) is alternatively spliced. The long variant (gamma 2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336-351 of the intracellular loop of the gamma 2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The Km values for PKC- and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 microM, respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the gamma 2L subunit has been shown to be necessary for ethanol potentiation of the GABAA-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.

Amino Acid Sequence↗
PubMed1993

Immunochemical characterization of the beta 2 subunit of the GABAA receptor.

To date three beta subunits of the GABAA receptor have been identified in rat brain as a result of cDNA library screening. The beta 2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying beta 2 mRNA. To study the beta 2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315-334 of the intracellular loop of the beta 2 subunit. The antibody, which had been affinity-purified, recognized the beta 2 peptide but did not immunolabel homologous beta 1 and beta 3 subunit peptides, indicating that this antibody is specific for the beta 2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54-58 kDa and exhibited minor labeling of lower-molecular-mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system-specific labeling of a 55-kDa band that comigrated with the 55-kDa band of the purified receptor. Quantitative immunolabeling of this 55-kDa polypeptide permitted direct determination of the relative amounts of the beta 2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the beta 2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of cytochrome P4502E1 (CYP2E1) by calmodulin dependent protein kinase, protein kinase C and cAMP dependent protein kinase.

Phosphorylation of pure cytochrome P4502E1 (CYP2E1) was achieved in vitro using Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). The stoichiometry and time-course of phosphorylation were determined. CaM kinase II was the most efficient enzyme capable of catalyzing this phosphorylation reaction: the maximum incorporation of 32PO4 was 0.8 mol/mol CYP2E1 in 20 min. PKA phosphorylated a maximum of 0.7 mol of 32PO4/mol of cytochrome within 60 min. The phosphorylation by PKC reached a maximum of 0.19 mol of 32PO4/mol of cytochrome and this occurred within a few minutes of incubation. Limited digestion by S. aureus V8 protease (SAP) of CYP2E1, which had been phosphorylated by either PKA and PKC, yielded a single major phosphopeptide with an M(r) of approximately 18,000. Limited digestion of CYP2E1, that had been phosphorylated by CaM kinase II, yielded phosphorylated polypeptides with M(r) of approximately 18,000 and 15,000. These results raise the possibility that these three kinases may be involved in the regulation of CYP2E1.

Animals↗
PubMed1993

Actions of A-75200, a novel catecholamine uptake inhibitor, on norepinephrine uptake and release from bovine adrenal chromaffin cells.

The balance between catecholamine (CA) release and reuptake is closely regulated and determines the effective level of transmitter at the synaptic cleft. Drugs that block CA uptake have potential utility as antidepressant medications. One such drug is racemic (+/-)-(1' R*,3R*)-3-phenyl-1-[1',2',3',4'-tetrahydro-5',6'- methylenedioxy-1'-naphthalenyl-methyl]-pyrrolidine methanesulfonate (A-7500), a novel polycyclic compound developed at Abbott Laboratories. This compound is known to bind to CA transporters in the central nervous system, however, its effects on an intact neurosecretory system have not been studied. In this regard, norepinephrine (NE) release from bovine adrenal chromaffin cells (BACC) is a classic model system for CA release and is an excellent system in which to examine the effects of drugs which modulate neurotransmitter release. We compared the effects of A-75200 and its two constituent enantiomers, A-74111 and A-74112, to the effects of three well-characterized uptake inhibitors, desipramine (DMI), nomifensine and cocaine. We found that the Abbott compounds inhibit [3H]norepinephrine ([3H]NE) uptake with an EC50 comparable to cocaine. In addition, unlike nomifensine and cocaine, these compounds inhibited nicotine- and K(+)-stimulated NE release, whereas histamine-stimulated release was preserved. Thus, the Abbott compounds block the effects on secretion of two agonists (nicotine and K+) which depend on a depolarization-dependent influx of extracellular calcium. We conclude that in addition to blocking NE uptake by inhibiting the NE transporter, the Abbott compounds may modulate peripheral NE release by inhibiting calcium flux through voltage-gated channels. This study demonstrates the utility of bovine adrenal chromaffin cells for preclinical trials of drugs that affect catecholaminergic neurotransmission.

Adrenal Medulla↗