Involvement of non-NMDA receptors in the rescue of weaver cerebellar granule neurons and sensitivity to ethanol of cerebellar AMPA receptors in oocytes.

The cellular mechanism responsible for the death of cerebellar granule neurons in the weaver mutant mouse is still being intensely investigated. To determine if alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors are involved in producing the weaver phenotype or are altered by the weaver gene, we used (1) reverse transcription and polymerase chain reaction (RT-PCR) to detect transcripts of glutamate receptors (GluR1-4) from wild-type and mutant cerebella; (2) immunocytochemistry to establish the types of glutamate receptors present in granule neurons cultured from normal and homozygous weaver postnatal day 5-6 (P5-6) cerebella; (3) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a blocker of glutamate (AMPA/Kainate/NMDA) receptors, and 6,7-dinitroquinoxaline-2,3-dione (NBQX), a blocker of AMPA and kainate receptors, to assess the number of neurons and the number of neurons with long neurites in cultures of homozygous weaver granule neurons; (4) two-electrode voltage clamp recordings to study AMPA glutamate receptor expression in Xenopus oocytes after injection of mRNA isolated from cerebella of normal and weaver P5-6, postnatal day 10 (P10) and postnatal day 23 (P23) mice; and (5) ethanol, which at low 1-10 mM concentrations had been shown previously to rescue homozygous weaver granule neurons in culture [Liesi et al., J. Neurosci. Res. 48 (1997) 571-579], to examine its effect on modulation of AMPA receptors expressed from mRNA. By RT-PCR, the mRNA coding for AMPA receptor subunits GluR1-4 were detected from +/+ and wv/wv cerebella, and by immunocytochemistry, GluR1, GluR2/3 and GluR4 were observed to be expressed in cultured +/+ and wv/wv granule cells. CNQX at 10 microM or NBQX at 10 microM significantly increased the number of surviving neurons and the number with long neurites as compared to wv/wv controls. In addition, CNQX was significantly more effective than NBQX. In oocytes injected with mRNA from P10 normal or weaver cerebella, the amplitudes of the responses to kainate were about equal. In contrast, the amplitudes of the kainate-activated currents in oocytes injected with weaver P23 mRNA were about twice as large as the currents observed in oocytes injected with mRNA from normal P23 cerebella, and both were larger than kainate-activated currents observed after injection of P10 normal and weaver mRNA. Kainate-activated AMPA receptor currents in oocytes injected with mRNA from P10 and P23 normal and homozygous weaver cerebella were inhibited by ethanol. There were no significant differences in the inhibition produced by ethanol on currents from P10 or P23 normal and wv/wv mRNA. Thus, P23 weaver cerebellar mRNA expressed more kainate-activated current in oocytes than P23 normal cerebellar mRNA; both normal and weaver cerebellar granule neurons express mRNA coding for functional AMPA receptors that are susceptible to ethanol inhibition.

Straight-chain alcohols exhibit a cutoff in potency for the inhibition of recombinant glutamate receptor subunits.

The effects of n-alcohols (methanol to 1-decanol) on kainate-activated AMPA receptor subunit GluR1 and GluR3 ion currents were studied in Xenopus oocytes using the two-electrode voltage-clamp recording technique. For short-chain alcohols from methanol to 1-hexanol, potency for inhibition of GluR1 and GluR3 receptor-mediated current increased in proportion to the chain length or hydrophobicity of the alcohol. The IC(50) values of these alcohols for GluR1 were: methanol, 702 mM; ethanol, 170 mM; 1-propanol, 69 mM; 1-butanol, 20 mM; 1-pentanol, 17 mM; and 1-hexanol, 10 mM. For GluR3, IC(50) values were: methanol, 712 mM; ethanol, 238 mM; 1-propanol, 50 mM; 1-butanol, 32 mM; 1-pentanol, 13 mM; and 1-hexanol, 7 mM. For long-chain alcohols, 1-heptanol was less potent than 1-hexanol (estimated IC(50): 19 mM for GluR1 and 18 mM for GluR3), 1-octanol had little effect only on GluR3, and 1-nonanol and 1-decanol did not significantly inhibit both GluR1 and GluR3 responses. The observations indicate that straight-chain n-alcohols exhibit a cutoff in their potency for inhibition of the function of non-NMDA glutamate receptor subunits, GluR1 and GluR3. The cutoff in potency of n-alcohols for inhibition of non-NMDA glutamate receptor function is consistent with the interpretation that alcohols affect the function of these receptor-channels by interacting with an alcohol binding site of specific dimensions on the receptor protein.

Anandamide inhibition of recombinant AMPA receptor subunits in Xenopus oocytes is increased by forskolin and 8-bromo-cyclic AMP.

Anandamide is an endogenous cannabinoid receptor agonist with similar pharmacological effects as D9-tetrahydrocannabinol, the major psychoactive compound in marijuana. Because anandamide does inhibit long-term potentiation, and cannabinoid abuse is known to affect learning and memory, the effects of anandamide on recombinant AMPA glutamate receptor (GluR) subunit currents were studied in Xenopus oocytes. All subunit currents were not affected by SR-1 41716A (a selective CB1 cannabinoid receptor antagonist), but were blocked by the selective AMPA antagonist CNQX and were sensitive to anandamide. Anandamide directly inhibited kainate (KA) activated homomeric GluR1; GluR3 and heteromeric GluR1/3; GluR2/3 receptor currents with IC50 values of 161+/-19, 143+/-12, 148+/-10 and 241+/-107 microM, respectively. The sensitivity of all the subunits to anandamide was not significantly different. Anandamide inhibition was voltage-independent, specific, and could not be duplicated by arachidonic acid or WIN 55,212-2 mesylate. Furthermore, anandamide effects were potentiated by forskolin (an adenylyl cyclase stimulator) and 8-bromo-cAMP (a cAMP analog), whereas MDL-HCl (an adenylyl cyclase inhibitor) caused a reversal of anandamide inhibition of GluR receptor current. Anandamide inhibition appears to be mediated by cAMP synthesis, and may underlie the involvement of this brain cannabinoid agonist in the modulation of fast synaptic transmission in the CNS.

In-vitro and in-vivo action of cannabinoids.

The discovery of endocannabinoids such as anandamide and the wide spread localization of cannabinoid receptors in the brain and peripheral tissues, suggests that the cannabinoid system represents a previously unrecognized ubiquitous net work in the nervous system, whose physiology and function is unfolding. In this study, we tested the hypothesis that some of the actions of anandamide are independent of a cannabinoid receptor mechanism. This was accomplished by the use of cannabinoid agonist and antagonist interaction in an in-vitro and in-vivo test systems. In-vitro, we used Xenopus laevis oocytes expression system and two-voltage clamp technique in combination with differential display polymerase chain reaction to determine whether the differential display of genes following treatment with anandamide may be linked to AMPA glutamate receptor. The differential expression of genes in vivo after the sub-acute administration of anandamide could not be directly linked with the AMPA glutamate receptor. In the voltage clamp studies we investigated the effects of anandamide on recombinant AMPA GluR3 subunit currents generated by kainic acid in oocytes expressing the AMPA glutamate receptor. In the in-vitro studies, we present evidence that anandamide inhibited the kainate activated currents in oocytes expressing AMPA glutamate receptor involves cAMP transduction via a cannabinoid receptor independent mechanism. In the in-vivo studies, SR141716A, the CB1 antagonist, induced anxiolysis, that was dependent on the mouse strain used in the anxiety model and blocked the anxiogenic effects of anandamide or methanandamide whereas SR141716A had no effect on the anandamide inhibition of kainate activated currents in-vitro.

Weaver cerebellar granule neurons show altered expression of NMDA receptor subunits both in vivo and in vitro.

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.

Effect of in vitro incorporation of prostanoid precursors, superoxide radical and hydrogen peroxide on platelet function.

The level of cyclic adenosine monophosphate (cAMP) in human platelets is known to be an important regulator of platelet function. The polyunsaturated fatty acids (PUFA) dihomo-gamma-linolenic acid (DHLA), and eicosapentaenoic acid (EPA), precursors of the prostaglandin (PG) 1 and 3 series respectively, were studied for their ability to stimulate platelet cAMP and/or PGE1 levels, and to inhibit platelet aggregation (PAg). Incubation of washed platelets (1 x 10(8)/ml) with 125 microM DHLA increased intraplatelet levels of PGE1 from 197 +/- 7 to 1622 +/- 9.7 picograms/10(8), cAMP from 3 +/- 0.8 to 31 +/- 1.9 picomoles/10(8), and inhibited collagen-induced PAg. Addition of 1 mumole of xanthine per unit of xanthine oxidase (a superoxide radical generating system) to the incubating medium potentiated the effects of both fatty acids, whereas 240 microM Hydrogen Peroxide (H2O2) inhibited these effects. These results suggest that: (1) DHLA may be more effective in inhibiting PAg than EPA, which has been reported to reduce the incidence of coronary diseases in some human populations; (2) That superoxide radical may activate the platelet cyclooxygenase system to increase lipid peroxidation of these PUFA prostanoid precursors and may result in the inhibition of PAg, whereas H2O2 may have an opposite effect.

Ethanol increases ADP-ribosylation of histones in rat hepatocyte nuclei.

Ethanol was shown previously to increase ADP-ribosylation of hepatocyte proteins. The purpose of this study was to determine the effect of ethanol on ADP-ribosylation of histones in hepatocyte nuclei. Freshly isolated hepatocytes were exposed to 100 mM ethanol for 2 h and ADP-ribosylated histones were separated from nonribosylated histones by phenylboronate agarose chromatography. Both histone factions were then separated into the individual histones by 12% acetic-urea-triton polyacrylamide gel electrophoresis. Ethanol did not change the amounts of outer histone H1 or amounts of core histones (H2A, H2B, H3.1, and H4) but increased the histone variants H3.2 and H3.3. The principal effect of ethanol was to increase the ADP-ribosylation of all the above histones. Exposure of hepatocytes in culture to 100 mM ethanol for 3 days did not increase the synthesis of histones as determined by the incorporation of 14C-L-lysine, but increased the ADP-ribosylation of histones, principally histone H2A, determined from the incorporation of 2, 8, 3H adenosine. These results show that ethanol increases the ADP-ribosylation of histones. This is a potential mechanism for effects of ethanol on the regulation of gene expression and cell differentiation.

Ethanol enhances ADP-ribosylation of protein in rat hepatocytes.

Decreases in hepatocyte NAD+ produced by ethanol are only partially explained by the increased conversion of NAD+ to NADH and NADP+. The purpose of this study was to determine whether a mechanism for the ethanol-induced decrease in NAD+ is its increased use in ADP-ribosylation. Exposure of hepatocytes in culture for 2 hr to 100 mmol/L ethanol increased the incorporation of 14C-ribose from prelabeled NAD+ into 14C-ribosylated proteins. Poly (ADP-ribose) polymerase activity was increased by exposure of isolated hepatocytes to 100 mmol/L ethanol for 10 min. In hepatocyte culture, increases in poly (ADP-ribose) polymerase were not detected after exposure to 100 mmol/L ethanol for 10 min or 2 hr but rather occurred at 24 hr. Ethanol exposure of hepatocytes in culture for 2 hr, however, decreased the Km for NAD+ of poly (ADP-ribose) polymerase. Both nicotinamide and 5-aminobenzamide, which are inhibitors of poly (ADP-ribose) polymerase, prevented the decrease in NAD+ produced by 2-hr exposure of hepatocytes in culture to 100 mmol/L ethanol. The effect of ethanol in decreasing DNA synthesis on days 3 and 4 of culture was not reversed by the inhibitors of poly (ADP-ribose) polymerase. These results indicate that increased ADP-ribosylation of hepatocyte proteins is a mechanism for the effect of ethanol in decreasing NAD+.

Ethanol increases the formation of NADP+ in rat hepatocytes.

The acute effects of ethanol on total (bound + free) pyridine dinucleotides were determined in freshly isolated rat hepatocytes. Pyridine dinucleotides and adenine nucleotides were determined by high-performance liquid chromatography. Exposure of the hepatocytes to 8 mmol/L ethanol resulted in a decrease in NAD+ and an increase in NADP+ after 2 min incubation. There were no significant changes in NADH and NADPH. Ethanol decreased ATP and increased AMP after 2 min, whereas an increase in ADP was only apparent after 15 min of incubation. Ethanol 8 mmol/L and 100 mmol/L resulted in an increased incorporation of [32P] into NADP+ from [32P]-prelabeled NAD+ and ATP. Ethanol increased hepatocyte NAD+ kinase activity; this effect was blocked by 4-methylpyrazole but reproduced by 10 mumol acetaldehyde. These observations indicate that ethanol increases the synthesis of NADP+ and that this effect is most likely the result of increased NAD+ kinase activity. The ethanol-induced decrease of NAD+ may limit ADP ribosylation of nuclear proteins, whereas increases in NADP+ may stimulate the pentose phosphate cycle.

Effect of epinephrine on ethanol metabolism by isolated rat hepatocytes.

The effect of epinephrine on ethanol metabolism was determined in isolated rat hepatocytes. Epinephrine (10 microM) enhanced an initial rapid rate of ethanol elimination observed in the first 5 min. Thereafter, between 5 and 90 min, the rate of ethanol elimination was slower and not affected by epinephrine. Epinephrine resulted in higher acetaldehyde concentrations at 2 min, but not thereafter. Acetaldehyde production in the presence and absence of epinephrine was inhibited by 4-methylpyrazole, by a low free extracellular calcium concentration, and by the alpha 1-adrenergic blocker prazosin. Ethanol alone and epinephrine alone increased oxygen consumption, but the effects were not additive. The ethanol-induced decreases in the cytosolic NAD-/NADH and NADP++NADPH ratios and in the mitochondrial NAD+/NADH ratio were delayed by the presence of epinephrine. An accelerated initial alcohol dehydrogenase activity sufficient to account for the rapid initial rate of ethanol elimination shown with epinephrine was demonstrated by coupling ethanol oxidation with lactaldehyde reduction, a system which increases the rate of dissociation of NADH from the enzyme and its oxidation back to NAD+. The findings in this study indicate that an increased reoxidation of NADH during ethanol oxidation by alcohol dehydrogenase is the basis for the rapid transient increase in ethanol elimination produced by epinephrine.