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Biomedical subjects

J F Tallman

Publications and source records attributed to J F Tallman.

At least 37 records · Page 2Linked to original sources
PubMed1989

Development of beta-adrenergic receptor and G protein messenger RNA in rat brain.

The ontogeny of rat brain beta 1- and beta 2-adrenergic receptor (beta-AR) and G protein messenger RNA (mRNA) was examined by Northern blot analysis using nick-translated cDNA clones for probes. The level of beta 1-AR and beta 2-AR mRNA followed a pattern of development which paralleled that for the receptor binding sites; the levels of mRNA and binding sites were low at day 1 and increased gradually to adult levels by postnatal days 16-25. In contrast, the level of G protein mRNA, including that for Gs alpha, Gi1 alpha, Gi2 alpha, Go alpha and G beta, on postnatal day 1 was equal to or greater than adult levels, increased 40-80% between day 1 and 7 and then decreased to adult values by day 14-25. This developmental time course approximates that reported for the expression of Gs and Gi but not Go and G beta protein levels determined by immunolabeling and toxin catalyzed ADP-ribosylation. The level of beta-actin mRNA was also greater than adult levels on day 1 and then gradually decreased to adult levels by day 14-25. The results indicate that the ontogeny of beta-ARs and G proteins and the relationship between the amount of mRNA and protein are qualitatively different.

Aging↗
PubMed1989

Corticosterone differentially regulates the expression of Gs alpha and Gi alpha messenger RNA and protein in rat cerebral cortex.

The possibility that glucocorticoids regulate specific guanine nucleotide binding regulatory proteins (G proteins) was investigated in rat cerebral cortex. Corticosterone was administered to normal and bilaterally adrenalectomized rats, and hormone regulation of individual G-protein subunits was investigated in cerebral cortex in three ways: (i) immunoblot analysis of subunit protein, (ii) hybridization blot analysis of subunit mRNA, and (iii) ADP-ribosylation analysis of stimulatory G protein (Gs alpha) subunits. Chronic (7 days) corticosterone administration to normal rats increased levels of Gs alpha immunoreactivity, mRNA, and ADP-ribosylation but decreased levels of inhibitory G protein (Gi alpha) mRNA and tended to decrease levels of Gi alpha immunoreactivity. In contrast, levels of Go alpha and G beta immunoreactivity and mRNA were not influenced by corticosterone treatment. In adrenalectomized rats, corticosterone treatment produced a 25-50% increase in the levels of Gs alpha immunoreactivity, mRNA, and ADP-ribosylation, whereas the hormone produced a 20-35% decrease in the levels of Gi alpha immunoreactivity and mRNA. Adrenalectomy, without corticosterone replacement, produced the opposite effects on Gs alpha and Gi alpha compared to sham-operated controls, indicating that these G proteins are regulated by this class of steroid hormone under physiological conditions in vivo. The results indicate that specific G-protein subunits--namely, Gs alpha and Gi alpha--are under the coordinated control of glucocorticoids in rat brain and demonstrate that G proteins are physiological targets of glucocorticoids in vivo. Possible roles played by these G-protein responses in mediating the effects of glucocorticoids on brain function are discussed.

Adenosine Diphosphate Ribose↗
PubMed1988

Isolation of a cDNA clone for the alpha subunit of the human GABA-A receptor.

A cDNA clone of an alpha subunit of the human GABA-A receptor has been isolated. The human clone (pCLL800) contains 1055 nucleotides in an open reading frame and 260 nucleotides in the 5' non-coding region. The 351 amino acid sequence of this human alpha subunit shows 97% homology with its bovine counterpart. Hybridization of pCLL800 to Northern blots shows a 3.9/4.3 Kb RNA doublet in human cortex, rat whole brain, cortex, hippocampus, midbrain, olfactory bulb and cerebellum. Developmental studies show that the levels of the rat alpha mRNA increase between one and three weeks of age in a manner similar to the development of the benzodiazepine binding sites.

Amino Acid Sequence↗
PubMed1988

Phosphorylation of the GABAa/benzodiazepine receptor alpha subunit by a receptor-associated protein kinase.

Partially purified preparations of GABAa/benzodiazepine receptor from rat brain were found to contain high levels of a protein kinase activity that phosphorylated a small number of proteins in the receptor preparations, including a 50-kilodalton (kD) phosphoprotein that comigrated on two-dimensional electrophoresis with purified, immunolabeled, and photolabeled receptor alpha subunit. Further evidence that the comigrating 50-kD phosphoprotein was, in fact, the receptor alpha subunit was obtained by peptide mapping analysis: the 50-kD phosphoprotein yielded one-dimensional peptide maps identical to those obtained from iodinated, purified alpha subunit. Phosphoamino acid analysis revealed that the receptor alpha subunit is phosphorylated on serine residues by the protein kinase activity present in receptor preparations. Preliminary characterization of the receptor-associated protein kinase activity suggested that it may be a second messenger-independent protein kinase. Protein kinase activity was unaltered by cyclic AMP, cyclic GMP, calcium plus calmodulin, calcium plus phosphatidylserine, and various inhibitors of these protein kinases. Examination of the substrate specificity of the receptor-associated protein kinase indicated that the enzyme preferred basic proteins as substrates. Endogenous phosphorylation experiments indicated that the receptor alpha subunit may also be phosphorylated in crude membranes by a protein kinase activity present in those membranes. As with phosphorylation of the receptor in purified preparations, its phosphorylation in crude membranes also appeared to be unaffected by activators and inhibitors of second messenger-dependent protein kinases. These findings raise the possibility that the phosphorylation of the alpha subunit of the GABAa/benzodiazepine receptor by a receptor-associated protein kinase plays a role in modulating the physiological activity of the receptor in vivo.

Animals↗
PubMed1988

Acute and chronic opiate-regulation of adenylate cyclase in brain: specific effects in locus coeruleus.

Acutely, morphine and D-ala2-D-leu-enkephalin (DADLE) inhibited adenylate cyclase in vitro in locus coeruleus (LC), dorsal raphe, frontal cortex and neostriatum and the inhibition by each agonist was blocked by the opiate-receptor antagonist naloxone. Although morphine was equally efficacious in the four brain regions examined (10-15% inhibition), DADLE inhibited cyclic AMP (cAMP) production to a greater extent in cortex and striatum (20-25% inhibition). Pertussis toxin treatment in vitro significantly reduced DADLE-inhibition of adenylate cyclase in all brain areas, indicating that this opiate response is mediated by a pertussis toxin-sensitive G-protein (i.e., Gi and/or Go). Chronic (in vivo) administration of morphine pellets for 5 days, treatment known to induce opiate tolerance and dependence, increased basal, GTP- and forskolin-stimulated adenylate cyclase in the LC, but not in the other three brain regions studied. DADLE was found to inhibit cAMP production in LC in vitro to the same extent in control and morphine-treated rats, suggesting a lack of opiate receptor tolerance. The morphine-induced increase in adenylate cyclase required chronic exposure to the opiate, as shorter treatment times, namely 2 hr and 1 day, failed to produce this effect. In fact, at 2 hr a small decrease in adenylate cyclase in the LC was observed that did not appear to be due to morphine being retained in the membrane fraction. Taken together, the findings of this study provide support for the view that changes in the cAMP system in the LC play a role in mediating acute opiate action as well as in underlying the development of opiate tolerance, dependence and/or withdrawal.

Adenylate Cyclase Toxin↗
PubMed1988

Chronic morphine treatment increases cyclic AMP-dependent protein kinase activity in the rat locus coeruleus.

We have studied a possible role for cyclic AMP-dependent protein kinase in mediating opiate addiction in the central nervous system by focusing on the rat locus coeruleus. This brain region is well suited for these studies because it is relatively homogeneous and because a wealth of electrophysiological and behavioral data indicate that it plays an important role in mediating the chronic effects of opiates in animals, including humans. It was found that chronic, but not acute, in vivo treatment of rats with morphine increased cyclic AMP-dependent protein kinase activity in the locus coeruleus with a time course that closely paralleled the time course by which locus coeruleus neurons become tolerant to and dependent on opiates, based on electrophysiological studies. Concomitant administration of the opiate receptor antagonist naltrexone was found to block the effect of chronic morphine treatment on protein kinase activity, indicating that the effect is mediated via specific activation of opiate receptors. In contrast, chronic morphine treatment did not alter protein kinase activity in several other brain regions studied, including the neostriatum, frontal cortex, and dorsal raphe. We propose that the observed increase in cyclic AMP-dependent protein kinase activity in the locus coeruleus contributes to the biochemical basis of opiate addiction.

Animals↗
PubMed1987

Pertussis toxin or 8-bromo-cAMP block inhibition of the acoustic startle response by the alpha 2-adrenergic agonist ST-91.

Stimulation of supraspinal alpha 2-adrenergic receptors by intraventricular infusion of the alpha 2-adrenergic agonist ST-91 depresses a simple vertebrate behavior, the acoustic startle response. Intraventricular pretreatment with pertussis toxin, an agent known to inactivate the inhibitory guanine nucleotide binding protein (Gi) which can inhibit adenylate cyclase, completely prevented the depressant behavioral effect of ST-91. In contrast, pertussis toxin did not alter the depressant effect of intraventricular infusion of the 5-HT 1B agonist 1-m-chlorophenylpiperazine (mCPP). Intraventricular infusion of the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP also reversed the depressant effect of ST-91 without altering the effect of mCPP. These data suggest that inhibition of adenylate cyclase may be involved in the effect of activation of central alpha 2-adrenergic receptors.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Solubilization of calcium channel antagonist binding sites from rat brain.

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Regional difference in brain benzodiazepine receptor carbohydrates.

The ability to photolabel benzodiazepine receptors from various regions of the rat brain with 3H-flunitrazepam has allowed for the structural examination of these receptors by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Results for all regions studied revealed the labeled receptor to consist of a single major band of radioactivity with the apparent molecular weight of approximately 50,000. Under our conditions of labeling we do not significantly label any higher molecular weight forms of the receptor. Exposure of the benzo-diazepine receptors to either of the glycosidases neuraminidase (N) and endoglycosidase-H (E) results in the specific removal of sialic acids and complete asparagine-linked carbohydrate moieties, respectively. This type of structural modification of the receptor resulted in an apparent decrease in the molecular weight, as determined by increased mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis, for all regions examined (cortex + N + E, 8,000-10,000; hippocampus + N, 7,000, + E, 12,000; cerebellum + N, none, + E, 4,000). These results point to a heterogeneity in the posttranslational glycosylation of the benzodiazepine receptor that may be due to brain region-specific differences in glycosylation. The removal of these carbohydrate moieties alters the binding of agonists and antagonists to the benzodiazepine receptor. Cortical agonist binding following either glycosidase treatment resulted in no apparent shift in the Kd but a significant decrease in the Bmax. The Bmax change may be the result of a large decrease in affinity or denaturation of a subpopulation of benzodiazepine receptors. Antagonist binding also showed no apparent Kd shift but a significant increase in the Bmax. The increase may have resulted from the activation of "hidden" benzodiazepine receptors or a shift of low affinity sites to sites of higher affinity. Cerebellar agonist or antagonist binding was not altered, in terms of either Kd or Bmax, by either enzyme treatment, correlating well with the small amount of carbohydrate removal seen following such treatments. The ability of these enzymes to modify the apparent molecular weight of the benzodiazepine receptors and the strong correlation to altered ligand binding, in a regional specific manner, generally parallel the description given of type 1 and type 2 benzodiazepine receptors.

Acetylglucosaminidase↗
PubMed1986

The role of spinal cord cyclic AMP in the acoustic startle response in rats.

Drugs thought to increase intracellular levels of cAMP were infused intrathecally into the subarachnoid space of the lumbar spinal cord, and the effects on the acoustic startle response in rats were measured. Intrathecal infusions of the cAMP analogs dibutyryl cAMP or 8-bromo cAMP (12.5-100 micrograms) produced marked, dose-dependent increases in startle amplitude compared to the infusion of artificial cerebrospinal fluid (CSF). Local infusions of dibutyryl cAMP at more rostral levels of the spinal cord or brain failed to mimic the excitatory effect seen following lumbar intrathecal infusion. No excitation of startle was seen following intrathecal infusion of cAMP itself, ATP, 5'-AMP, or dibutyryl cGMP. A weak excitation of startle was seen following intrathecal, but not intraventricular, infusion of the water-soluble adenylate cyclase activator forskolin 7-deacetyl-7-O-hemisuccinic acid (forskolin-DHA; 5.0-100 micrograms, in artificial CSF), whereas forskolin itself [0.01-200 micrograms, in dimethyl sulfoxide (DMSO)] was without consistent effect. Finally, intrathecal infusion of the selective phosphodiesterase inhibitor Rolipram (12.5-200 micrograms) produced a marked excitation of startle similar in magnitude to the effects produced by cAMP analogs. The excitatory effects of intrathecally infused dibutyryl cAMP, 8-bromo cAMP, forskolin-DHA, or Rolipram support a functional link between spinal cord cAMP and the acoustic startle reflex. Possible sites of cAMP action on startle are discussed.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Electrophysiological and receptor studies in rat brain: effects of clorgyline.

Acute high doses of clorgyline produce a rapid inhibition of monoamine oxidase type A (MAO A) in the rat brain, together with an increase in norepinephrine and a decrease in the firing rate of locus coeruleus (LC) neurones: this decrease is reversed by piperoxane, an alpha 2 antagonist. In control animals, piperoxane increases LC neuronal firing showing that these noradrenergic neurones are under alpha 2-adrenoceptor-mediated tonic inhibition. Chronic administration of clorgyline, like acute doses of this MAO A inhibitor, significantly decreases cell firing in the LC and the effect is partially reversed by piperoxane. Chronic clorgyline treatment also produces significant decreases in [3H]clonidine and [3H]dihydroalprenolol binding in cerebral cortex, receptor changes which are slightly greater in animals showing greater inhibition of LC neuronal firing: such receptor changes do not occur following a single exposure to clorgyline. Electrophysiological studies in hippocampal pyramidal cells show that the chronic clorgyline treatment does not significantly induced subsensitivity to NE in these adrenoreceptive cells.

Animals↗
PubMed1985

Molecular structure of benzodiazepine receptors.

Benzodiazepines bind to high affinity binding sites in brain and other tissues. One of the high affinity sites in brain possesses properties which make it a likely site of action for these drugs. Binding to the benzodiazepine receptor is saturable and stereospecific; a high degree of correlation can be made between binding to this site and the pharmacological potency of the benzodiazepines. Binding to the site is also enchanced in the presence of GABA, the brain's major inhibitory transmitter. Because of this allosteric activation of benzodiazepine binding and the known electrophysiological interactions of the benzodiazepines with GABA, it appears likely that the site of action of the benzodiazepines in the brain is a GABA receptor complex. Recent evidence indicates that this complex also contains an anion channel which is the site of action of some of the barbiturates and known cage convulsants. The purification of the proteins composing this complex has been carried out and the major proteins appear to be several proteins with molecular weights between 50-60 k daltons; some higher molecular weight subunits may also be present. The benzodiazepine receptor protein can be labeled with 3H-flunitrazepam which labels a protein of molecular weight about 50 k daltons. Proteolytic degradation of the photolabeled benzodiazepine receptor results in the formation of several peptides and one limiting peptide which has recently been purified. Knowledge of its structure should yield interesting information about the origins of the benzodiazepine receptor and provide a useful tool for further molecular studies.

Affinity Labels↗
PubMed1984

Peripheral benzodiazepine binding sites in kidney: modifications by diabetes insipidus.

Using [3H] diazepam as ligand, it is possible to distinguish neuronal binding sites from those present on glial elements and in peripheral tissues (non-neuronal). The function of the "non-neuronal" binding sites is still obscure. Preliminary data showed a distribution of [3H] diazepam binding sites in kidney that could suggest a localization along the renal tubules. This is the site at which a renal peptide, arginine-vasopressin (AVP) is supposed to act. In an attempt to examine the function of these "non-neuronal" sites, we studied the [3H] diazepam binding in kidney of Brattleboro rats which lack AVP and present the symptoms of diabetes insipidus. The homozygous Brattleboro rats showed an increase in the apparent number of benzodiazepine binding sites (Bmax) compared to Long-Evans control rats. Replacement of AVP in these animals results in a reversal of the electrolyte alterations of diabetes insipidus and in an increase of the affinity of the [3H] diazepam binding. These findings may indicate a possible relationship between benzodiazepine binding sites and vasopressin action in kidney and may support receptor function of these "non-neuronal" binding sites.

Animals↗
PubMed1984

Proteolytic degradation of neuronal benzodiazepine binding sites.

The pathway of breakdown of membrane-bound benzodiazepine binding sites has been examined with proteolytic enzymes. Photoaffinity labeled benzodiazepine receptors were degraded for varying amounts of time and at varying enzyme concentrations. The properties of fractions both remaining in the membrane and released into the supernatant were examined for their apparent molecular weight by SDS gel electrophoresis. Trypsin treatment converted the 46K subunits of the GABA/BDZ complex which bind 3H-flunitrazepam into 40K and 27.5K fragments which remained in the membrane and finally a small fragment which was released into the supernatant. An endogenous trypsin-like activity in the membrane fractions has similar proteolytic effects on the membrane bound receptor.

Animals↗
PubMed1983

Consequences of benzodiazepine receptor occupancy.

The pharmacological consequences of the occupancy of benzodiazepine receptors have been a recent area of active research. There is good agreement between the electrophysiological effects of benzodiazepines and their binding to benzodiazepine receptors when both are studied in vitro under identical conditions. Compounds of different structure from the benzodiazepines can occupy the receptor in a way, which produces little overt effect (imidazodiazepines) or actually causes actions opposite to the benzodiazepines (beta-carbolines, inverse agonists). Several biochemical tests (GABA-shift, photo-shift) for distinguishing these different behavioral properties are described. A model is described for the interactions at membranes of agonists, antagonists and inverse agonists with benzodiazepine receptors in the GABA receptor complex.

Animals↗
PubMed1983

Properties of two benzodiazepine binding sites in spinal cord.

Two types of benzodiazepine receptors were demonstrated in spinal cord. Binding to one of these sites (neuronal) was sensitive to the centrally active benzodiazepine, clonazepam, and binding was enhanced both by chloride and GABA. The second site was sensitive to 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl) 2H-1,4-benzodiazepine-2-one (R05-4864) and thus is similar to the site characteristic of non-neuronal tissue. Binding to the neuronal site was inhibited by the putative glycine antagonist, strychnine, both in spinal cord and brain. This inhibition may account for the reported anti-GABAergic properties of strychnine and does not appear to be related to the glycine receptor.

Animals↗