HLA-A, HLA-B, and HLA-DRB1 allele distribution in a large Armenian population sample.

Human leukocyte antigen (HLA)-A, HLA-B, and HLA-DRB1 gene frequencies were investigated in 4279 unrelated Armenian bone marrow donors. HLA alleles were defined by using PCR amplification with sequence specific primers (PCR-SSP) high- and low-resolution kits. The aim of this study was to examine the HLA diversity at the high-resolution level in a large Armenian population sample, and to compare HLA allele group distribution in Armenian subpopulations. The most frequently observed alleles in the HLA class I were HLA-A*0201, A*0101, A*2402, A*0301, HLA-B*5101, HLA-B*3501, and B*4901. Among DRB1 alleles, high frequencies of DRB1*1104 and DRB1*1501 were observed, followed by DRB1*1101 and DRB1*1401. The most common three-locus haplotype found in the Armenian population was A*33-B*14-DRB1*01, followed by A*03-B*35-DRB1*01. Our results show a similar distribution of alleles in Armenian subpopulations from different countries, and from different regions of the Republics of Armenia and Karabagh. The low level of genetic distances between subpopulations indicates a high level of population homogeneity, and the genetic distances between Armenians and other populations show Armenians as a distinct ethnic group relative to others, reflecting the fact that Armenians have been an 'isolated population' throughout centuries. This study is the first comprehensive investigation of HLA-allele group distribution in a subset of Armenian populations, and the first to provide HLA-allele and haplotype frequencies at a high-resolution level. It is a valuable reference for organ transplantation and for future studies of HLA-associated diseases in Armenian populations.

Activation of the NPTA element of the CYP2A3 gene by NFI-A2, a nasal mucosa-selective nuclear factor 1 isoform.

The aim of this study was to determine whether the NPTA element of the olfactory mucosa-predominant CYP2A3 gene can be activated by NFI-A2, a recently identified member of the nuclear factor 1 family of transcription factors. Isoform-specific RNA-PCR confirmed that NFI-A2 is mainly expressed in rat olfactory mucosa. A full-length NFI-A2 cDNA was isolated from a cDNA library of rat olfactory mucosa and was used for preparation of a construct encoding a fusion protein of NFI-A2 with the yeast GAL4 activation domain. Expression of the fusion protein in yeast was detected with an antibody to NFI-A. The fusion protein activated the expression of a LacZ reporter gene in yeast one-hybrid assays with a reporter construct containing the NPTA element, but not with other constructs lacking the NPTA element. These findings suggest that NFI-A2 may be involved in the tissue-selective transcriptional activation of the CYP2A3 gene in the olfactory mucosa.

The translocation and involvement of protein kinase C in mossy fiber-CA3 long-term potentiation in hippocampus of the rat brain.

The detailed mechanisms underlying long-term potentiation (LTP) are not known. In hippocampal CA1, translocation of protein kinase C (PKC) activity from cytosol to membrane and subsequent phosphorylation of growth associated protein (GAP)-43 have been demonstrated to be critical events for the maintenance phase of LTP. LTP in mossy fiber (MF)-CA3 pathway and the Schaffer collateral/commissural (SC)-CA1 pathway differ in a number of ways: SC-CA1 LTP depends on NMDA receptors while MF-CA3 LTP does not, and SC-CA1 LTP is primarily postsynaptic while MF-CA3 LTP is primarily presynaptic. The role of PKC in MF-CA3 LTP has not been studied. We investigated the role of PKC in CA3 and show that PKC inhibitors prevent LTP, but that PKC activators produce a reversible synaptic potentiation, indicating that PKC activation is an essential but not sufficient component of LTP in CA3. Then using antibodies against specific PKC isozymes we have determined the membrane vs. cytosolic distribution of various PKC isozymes in slices subjected to low or tetanic stimulation, or perfused with phorbol esters (PDAc). Compared with control, LTP and PDAc slices show greater PKC-alpha and -epsilon immunoreactivity in the membrane fraction, indicating that both LTP and phorbol ester treatment induce translocation of PKC-alpha and -epsilon from cytosol to membrane. However, with PKC-beta and PKC-gamma the only detectable translocation from cytosol to membrane was in the phorbol ester-treated slices. Thus, while phorbol ester treatment causes translocation of PKC-alpha, -beta, -gamma and -epsilon, the only detectable translocation associated with CA3 LTP is that of PKC-alpha and -epsilon.

Rapid regulation of a cyclic AMP-specific phosphodiesterase (PDE IV) by forskolin and isoproterenol in LRM55 astroglial cells.

Elevation of intracellular cyclic AMP (cAMP) levels by incubation of intact LRM55 astroglial cells with 0.1 mM forskolin or 0.1 microM isoproterenol (IPR) caused a rapid increase in soluble cAMP phospho-diesterase (PDE) activity. Activation did not require de novo protein synthesis and reached a maximum of > or = 100% increase over basal PDE activity after 15 min of treatment. The increase in activity was recovered in a single peak (peak 3) following DEAE chromatography; the other two peaks separated by this procedure showed no change. Peak 3 had all the characteristics of PDE IV: it was sensitive to rolipram, was insensitive to CI-930 and cyclic GMP (cGMP), had a high affinity for cAMP (K(m) approximately equal to 4 microM), and had a very low affinity for cGMP (K(m) > 100 microM). Forskolin treatment resulted in an increase of the Vmax of peak 3 without affecting its K(m). In vitro treatment of peak 3 with the catalytic subunit of protein kinase A increased activity, whereas treatment with alkaline phosphatase decreased activity. The rapid activation of this specific PDE in response to forskolin and IPR represents a novel regulation of PDE IV by a mechanism that seems to involve its phosphorylation by a cAMP-dependent protein kinase.

Regulation of receptor-mediated shape change in astroglial cells.

Activation of adenylate cyclase in astroglial cells in culture results in a rapid change in cell shape that appears to occur by the active movement of cytoplasm from peripheral cell regions to the perinuclear space with processes being formed along regions that remain extended. Three series of experiments were designed to determine how shape change occurred. First, the Ca(2+)-dependency of shape change was determined by reducing intracellular Ca2+ concentrations to less than or equal to 50 nM or increasing intracellular Ca2+ concentrations to greater than or equal to 1 microM. Neither of these changes significantly affected the rate of receptor-mediated shape change. Second the role that longer-lived, acetylated microtubules play in receptor-mediated shape change was assessed by visualizing microtubules using a polyclonal antibody to brain 6S tubulin or a monoclonal antibody to oligomers of tubulin to monitor total tubulin distribution and a monoclonal antibody to acetylated tubulin to describe the distribution of these microtubules. Three-dimensional distribution of microtubules was observed by optical sectioning of cultures using a laser scanning confocal imaging system. The distribution of acetylated tubules in control cells was similar to that observed with the antibodies to tubulin. Following treatment with 100 nM isoproterenol to stimulate shape change, there was a dramatic redistribution of microtubules; however, the distribution of acetylated tubules was again similar to the total microtubules. Analysis of the optical sections recorded using the confocal attachment revealed that while control cells were relatively flat (cell height = 4 microns), the perinuclear region of isoproterenol-treated cells extended much higher above the substrate (cell height = 13 microns). Third, the role of microtubule assembly and disassembly were assessed using colchicine and taxol. Results from these experiments suggest that microtubule reassembly is necessary for receptor-mediated shape change. Control experiments indicated that colchicine or taxol treatment did not inhibit either cAMP synthesis or another cAMP-dependent process, receptor-mediated taurine release. Together these results indicate that receptor-mediated shape change in astroglial cells occurs by a Ca(2+)-independent mechanism that results in active movement of cytoplasm to the perinuclear region. This process is dependent on microtubule reassembly suggesting that shape change may occur by active movement of material along microtubules or by microtubule redistribution.

The role of osmotic pressure and membrane potential in K(+)-stimulated taurine release from cultured astrocytes and LRM55 cells.

The effects of [K+]o on taurine release from glial cells were studied with primary cultures of cerebellar astrocytes and with LRM55 cells, a continuous glial cell line. The characteristics of K(+)-stimulated taurine release were virtually identical in the 2 cell types. Both cerebellar astrocytes and LRM55 cells released taurine when stimulated with high-K+ medium prepared by isosmotically substituting KCl for NaCl, but neither cell type released taurine when stimulated with hyperosmotic high-K+ medium prepared by adding solid KCl to control medium. The membrane potential of LRM55 cells was measured by intracellular recording and was insensitive to changes in [K+]o below 20 mM. LRM55 cells released taurine when stimulated with nondepolarizing concentrations of K+ (13-22 mM) if the isosmotically prepared high-K+ medium was used, but the cells did not release taurine when treated with a depolarizing concentration of K+ (50 mM) if hyperosmotic high-K+ medium was used. The time course of K(+)-stimulated taurine release was quite slow, having a time to peak of 10-15 min. Small changes (2.5-10%) in the osmolarity of the medium strongly affected taurine release by cerebellar astrocytes and LRM55 cells. K(+)-stimulated taurine release from both cell types was inhibited when the osmolarity was increased with sucrose or NaCl and was enhanced when the osmolarity was reduced. Similarly, baseline taurine release was suppressed by small elevations in osmolarity and increased by reduced osmolarity.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous and beta-adrenergic receptor-mediated taurine release from astroglial cells are independent of manipulations of intracellular calcium.

Stimulation of beta-adrenergic receptors on LRM55 astroglial cells results in cAMP-dependent release of taurine. We have previously demonstrated that extracellular Ca2+ is not required for either spontaneous or receptor-mediated taurine release (Martin et al., 1988b). In the present series of experiments we investigated the relationship between changes in intracellular free Ca2+ ([Ca2+]i) and taurine release. [Ca2+]i was measured using the fluorescent probe fura-2 and was manipulated by changing the concentration of Ca2+ in the incubation medium and by using the Ca2+ ionophore ionomycin. [Ca2+]i was reduced from 150 +/- 95 nM (n = 46) in control medium (containing 1.1 mM CaCl2) to 46 +/- 10 nM (n = 43) in saline containing no CaCl2 and 10 microM EGTA. [Ca2+]i was rapidly elevated to greater than or equal to 1 microM in medium containing 100 microM CaCl2 and 10 microM ionomycin. Taurine release, either spontaneous or stimulated by isoproterenol, was not significantly affected by these manipulations of [Ca2+]i. [Ca2+]i did not change when cells were stimulated with 100 nM isoproterenol in either control saline containing 1.1 mM CaCl2 or in CaCl2-free saline containing 10 microM EGTA. Other secretogogs (serotonin and ethanol) did not cause changes in [Ca2+]i. These data indicate that neither spontaneous or receptor-mediated taurine release from astroglial cells is Ca2+ dependent. However, when cells were preloaded with Ca2+, allowed to recover briefly, and then stimulated with isoproterenol, it was possible to demonstrate transient increases in Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous and beta-adrenergic receptor-mediated taurine release from astroglial cells do not require extracellular calcium.

Astroglial cells release taurine when stimulated by beta-adrenergic agonists and other neuroactive agents. The Ca2+-dependency of taurine release by an LRM55 astroglial cell line was investigated by removing Ca2+ from the perfusion medium and by using three inorganic and three organic Ca2+-channel blockers (Mn2+, Co2+, Cd2+, verapamil, nifedipine, and diltiazem). Spontaneous release and release stimulated by the beta-adrenergic agonist isoproterenol were not inhibited when cells were perfused with medium containing no added Ca2+ and 10 microM EGTA. Isoproterenol-stimulated taurine release was not blocked when extracellular Ca2+ was completely replaced by Mn2+, Co2+, or Cd2+, nor was it blocked by verapamil, nifedipine, or diltiazem. In fact isoproterenol-stimulated taurine release was increased by 50 microM diltiazem and when Ca2+ was replaced by Co2+. The rate of spontaneous release increased slowly and continually when Co2+ was substituted for Ca2+ but was almost unaffected by substitution of Mn2+ or Cd2+. Application of diltiazem increased spontaneous release significantly, while verapamil and nifedipine appeared to cause small increases. These results indicate that entry of Ca2+ from the extracellular medium is not required for either receptor-mediated or spontaneous taurine release from astroglial cells. Some other changes in the medium did strongly affect release. Both spontaneous and isoproterenol-stimulated release were inhibited by elevated osmotic pressure, and spontaneous release was greatly increased when Ca2+ was completely removed without substituting another divalent cation. Spontaneous release increased when antagonistic metal ions were replaced with Ca2+ and when organic channel blockers were removed.

Inactivation of cyclic AMP-dependent taurine release from astroglia.

When astroglial cells are exposed to beta-adrenergic agonists for long periods of time (greater than 20 min), transient increases in taurine release and intracellular cyclic AMP (cAMP) are observed. Three phases of taurine release can be distinguished: activation, inactivation, and an elevated steady state. In this article, we present data describing the relationship between intracellular cAMP levels and inactivation of taurine release. To do this, we compared the apparent first-order rate constants for the inactivation of taurine release (ktau) with the apparent first-order rate constant for the decline of intracellular cAMP (kcAMP). We also measured ktau under experimental conditions that were chosen to provide a wide range of intracellular cAMP concentrations or to stimulate release without the involvement of the beta-adrenergic receptor and adenylate cyclase. When taurine release was stimulated with a saturating concentration of isoproterenol, the inactivation of release was significantly faster than the decline of intracellular cAMP. Furthermore, there were no significant differences in ktau measured under any of the experimental conditions used. Thus, inactivation of taurine release does not involve changes in the activity of the beta-adrenergic receptor and adenylate cyclase, i.e., desensitization, and appears to be independent of the intracellular concentration of cAMP. These results indicate that cAMP-mediated events can be regulated by mechanism(s) in addition to those that control receptor-adenylate cyclase interactions and the synthesis of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine stimulates cAMP-mediated taurine release from LRM55 glial cells.

The possible role of adenosine as a modulator or transmitter in the central nervous system was tested by measuring its effects on LRM55 astroglial cells. Two related cellular responses were measured--receptor activated increases in intracellular cAMP and cAMP-mediated taurine release. Taurine is a neuroinhibitory amino acid that is taken up, stored, and released from primary cultures of astrocytes and astroglial cells. Three-minute incubations of cells with adenosine caused a dose-dependent accumulation of intracellular cAMP and release of the taurine (EC50 = 5.0 x 10(-5) M and 1.6 x 10(-6) M, respectively). That the cellular responses were mediated through the activation of specific adenosine receptors was indicated by the observations that the adenosine receptor antagonist isobutylmethylxanthine (IBMX) but not the beta-adrenergic receptor antagonist 1-propranolol inhibited responses to adenosine. The study of various adenosine analogs showed a rank order of potency (chloroadenosine = 5'-(N-ethyl)carboxamido-adenosine greater than N6-(L-2-phenylisopropyl)-adenosine greater than cyclohexyladenosine = cyclopentyladenosine) characteristic of the low affinity A2-type adenosine receptors that have been associated with cAMP elevation in several tissues. These results indicate that, in addition to directly affecting neurons, adenosine may have a primary site of action on astroglial cells resulting in taurine release and subsequent inhibition of neuronal activity.

Morphology of astroglial cells is controlled by beta-adrenergic receptors.

Astroglial cells in vivo and in vitro respond to hormones, growth factors, and neurotransmitters by changing from an epithelial-like to stellate morphology. We have studied the temporal relationship between receptor activation, second messenger mobilization, and morphological changes using LRM55 astroglial cells. Maintenance of an altered morphology required continuous beta-adrenergic receptor activation. These changes appeared to be mediated by cAMP since they were elicited by its analogue, dibutyryl cAMP, and by forskolin, a direct activator of adenylate cyclase. Changes in cell morphology may require a relatively small increase in intracellular cAMP, since receptor-stimulated changes in cAMP levels were transient and peaked approximately 5 min after receptor activation while changes in morphology took at least 30 min to reach a new steady state. Time-lapse videomicroscopy and high voltage electron microscopy indicated that receptor activation resulted in a sequence of morphological events. Time-lapse observations revealed the development and enlargement of openings through the cytoplasm associated with cytoplasmic withdrawal to the perinuclear region and process formation. Higher resolution high voltage electron microscopy indicated that the transition to a stellate morphology was preceded by the appearance of two distinct cytoplasmic domains. One contained an open network of filaments and organelles. The other was characterized by short broad cytoplasmic filaments. The first domain was similar to cytoplasm in control cells while the second was associated with the development and enlargement of openings through the cytoplasm and regions of obvious cytoplasmic withdrawal.

Regulation of isoproterenol-induced cyclic AMP accumulation in LRM55 glial cells by phosphodiesterase.

Continuous stimulation of LRM55 glial cells with the beta adrenergic agonist isoproterenol (IRP) produced a transient increase in intracellular cyclic AMP (cAMP). Pretreatment of cells for 1, 5 and 30 min with IPR followed by a 1-min challenge with IPR resulted in 20, 50 and 70% drops in maximum stimulation, respectively, with no significant change in the EC50 value (60 nM). Cells stimulated with IRP in the presence of the phosphodiesterase inhibitor RO 20-1724 reached intracellular cAMP levels 6 to 8 times higher than controls and maintained these levels for at least 60 min of continuous stimulation. Addition of RO 20-1724 to cells showing a reduced response after exposure to IPR resulted in an immediate and sustained increase of cAMP levels. Because RO 20-1724 nearly completely inhibited cAMP degradation, the authors conclude that the apparent inactivation of IPR-stimulated cAMP response in the LMR55 cells is due mainly to cAMP degradation by phosphodiesterase activity.

Activation of beta-adrenergic receptors stimulates release of an inhibitory transmitter from astrocytes.

Activation of beta-adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP-mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2'-dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the beta-adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS-receptor-mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.

Beta-receptor-stimulated and cyclic adenosine 3',5'-monophosphate-mediated taurine release from LRM55 glial cells.

Adrenergic stimulation of LRM55 glial cells results in the release of the neuroactive amino acid taurine. The present study characterizes the receptors involved in taurine release and shows that taurine release is mediated by cyclic adenosine 3',5'-monophosphate (cAMP). beta-Receptors in LRM55 cells were first characterized by [125I]iodohydroxybenzylpindolol binding. Binding was stereospecific and saturable with time and ligand concentration. Kinetic analysis of equilibrium binding at 37 degrees C revealed a single component of high affinity (Km = 113 pm; Bmax = 52.1 +/- 5.0 fmol/mg of protein). The pharmacologies of the stimulation of cAMP accumulation and taurine release were similar. The agonists isoproterenol (IPR), epinephrine (E) and norepinephrine (NE) showed a rank order of potency characteristic of a beta-adrenergic system (IPR greater than E greater than or equal to NE). The beta-antagonists alprenolol and propranolol inhibited the IPR stimulation of both processes; the alpha-antagonist phentolamine did not. The dependence of taurine release on cAMP was further suggested by the similarity of the two time courses and was demonstrated by the stimulation of taurine release by the cAMP analogue dibutyryl cAMP. Thus, one physiological response of glial cells to beta-adrenergic stimulation is the release of taurine. Receptor-activated release of taurine from glia represents a previously undescribed neuronal-glial interaction by which glia may actively regulate neuronal excitability.

Modification of sulfhydryl groups of creatine kinase by urate.

The catalytic activity of rabbit skeletal muscle creatine kinase is diminished in solutions of sodium urate. The rate of inactivation follows first-order kinetics and is dependent upon urate concentrations. During the course of inactivation, only partial reactivation can be obtained by exposure to thiol agents. Reduction in the number of reactive sulfhydryl groups from eight per dimer to four accompanies inactivation of the enzyme, but no alteration in electrophoretic mobility is detectable.

Defluorination of methoxyflurane by glutathione and coenzyme B12.

Fluoride was eliminated from methoxyflurane in solutions which contained glutathione and coenzyme B12. The reaction rate was maximal at pH 10 and was dependent upon the concentration of each reactant under specified conditions. A stoichiometric ratio of 2 was found between the amount of glutathione utilized and the amount of fluoride generated in the reaction. Glutathione disulfide was the only stable ninhydrin-positive reaction product identified.

Defluorination of methoxyflurane by a glutathione-dependent enzyme.

Partial purification from rat liver of an enzyme which catalyzes defluorination of methoxyflurane is described. Fractionation of liver homogenates by protamine sulfate and ammonium sulfate precipitation and by Sephadex G-100 chromatography results in a 10-fold purification with 53% recovery. The enzyme requires glutathione for activity, and other sulfhydrhyl compounds cannot be substituted. The enzyme appears to be a glutathione S-transferase, possibly one of several which have recently been characterized.