Activation of sphingosine kinase in pheochromocytoma PC12 neuronal cells in response to trophic factors.

Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), dibutyryl cAMP and forskolin, known differentiating agents for pheochromocytoma PC12 cells, induced sustained activation of sphingosine kinase, the enzyme responsible for the formation of the sphingolipid second messenger, sphingosine-1-phosphate, which mediates the mitogenic effects of certain growth factors. In contrast, epidermal growth factor and insulin-like growth factor-1, which stimulate proliferation of PC12 cells, induced only small and transient increases in sphingosine kinase activity. Of the growth factors examined, NGF was the most potent activator of sphingosine kinase, inducing a 4-fold increase in Vmax. Sphingosine kinase activity induced by NGF, but not FGF, was blocked by the protein kinase inhibitor K252a when added simultaneously, with minimal effect when added after 60 min. Thus, activation of sphingosine kinase may have an important role in neural differentiation.

Developmental expression of G proteins that differentially modulate adenylyl cyclase activity in mouse brain.

Changes in the relative abundance of the G protein alpha subunits were observed during early mouse development Gs alpha was almost exclusively present as a large form (Gs-1) in prenatal brain. Postnatally with a substantial increase in Gpp[NH]p stimulated adenylyl cyclase activity, the small form (Gs.s) increased in amount while Gs-1 decreased. These results suggest that the Gs-s may be the more effective cyclase activator and that changes in alternative splicing are developmentally regulated. Gi1 and Go appeared before birth whereas Gi2 developed postnatally. Opiate stimulation of GTPase and inhibition of adenylyl cyclase were fully expressed prenatally.

Characterization of Xenopus laevis proenkephalin gene.

Enkephalins are opiate peptides found in a variety of tissues including brain and pituitary. In brain, they function as neurotransmitters, neuromodulators and neurohormones. Recent studies show that proenkephalin mRNA is expressed early in development both in mammals and the amphibian, suggesting that enkephalins may play a unique role in embryogenesis. In order to characterize factors which regulate the onset and patterning of expression of this gene in adult and developing frog embryos, the proenkephalin A gene was cloned from Xenopus laevis. The clones have been characterized by DNA sequencing and restriction endonuclease mapping. The gene is made up of three exons which span approximately 12 kb. Exon I encodes the 5' untranslated region of the mRNA. Exon II contains the signal peptide and the N terminus of the mature protein. Biologically active opioid peptides are generated from exon III. Comparison to mammalian proenkephalin genomic sequence indicated that nucleotide sequences of the 5' flanking region, noncoding exon I and exon II were not well conserved but exon III was highly conserved. Primer extension and RNase protection assay analyses of the RNA transcripts revealed two major 5' ends. The putative TATA box, CAAT box, CRE and Pit 1 elements have been identified on this gene by sequence homology to published consensus sequences. To assay for sequences that could potentially regulate Xenopus proenkephalin expression, we transfected constructs that contained upstream genomic sequences linked to the CAT reporter gene into various eukaryotic cell lines. The expression of the fusion gene constructs were detected and could be induced 10- to 30-fold upon treatment with forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related reduced affinity in [3H]nitrendipine labeling of brain voltage-dependent calcium channels.

Literature data indicate a reduced calcium uptake in synaptosomes prepared from old rat brains. On this line, the present paper investigates the binding of ([3H]NDP) to brain synaptic membranes prepared from rats at different ages from birth up to 24 months of age. The binding is undetectable at birth but reaches within 9-18 day the values observed in adults [3H]NDP binding affinity and sensitivity to calcium were decreased in old rats (24 months). Tritiated dihydropyridines are believed to label voltage-dependent calcium channels (VDC). The observed age-related reduction in binding suggests that the characteristics of VDC in the aged brain may change.

In vivo chronic lead exposure alters [3H]nitrendipine binding in rat striatum.

The effect of lead as a neurotoxic agent has been associated with alterations in calcium metabolism. On this line, the present study shows that lead alters the characteristics of [3H]nitrendipine ([ 3H]NDP) binding to rat striatal membranes. In vitro, lead shares the action of calcium in enhancing [3H]NDP binding although it is more potent on a molar basis. In vivo, lead exposure through drinking water enhances [3H]NDP binding to crude synaptosomal membrane preparations. This effect is lost when membranes are washed with EDTA-EGTA, indicating that the increased binding is due to the persistence of lead in the brain of treated rats.

Chronic ethanol changes opiate receptor function in rat striatum.

Ethanol produces supersensitivity of striatal delta-opiate receptor sites labeled by [3H]DADLE and [3H]etorphine. The impairment may be ascribed to the diminished enkephalin release detected in rat striatum after chronic ethanol consumption. On the contrary, a lower affinity of striatal mu-opiate receptors results after same ethanol exposure. In fact, the Kd values of [3H]Met-enkephalin and [3H]DHM are enhanced when measured in striata of ethanol-dependent rats. The diverse sensitivity of the various classes of opiate receptors to ethanol may be ascribed to different ethanol effects on enkephalinergic transmission.

Cyclic AMP-dependent protein phosphorylation is reduced in rat striatum after chronic ethanol treatment.

Endogenous protein phosphorylation by cyclic AMP-dependent protein kinase was found reduced in striatal membranes obtained from chronic ethanol-treated rats. Experiments using an exogenous substrate show that the decreased response is due to a deficiency in the phosphorylating activity of the cyclic AMP-dependent protein kinase and not to a lack of endogenous substrate for phosphorylation.

Developmental changes in Gs and G(olf) proteins and adenylyl cyclases in mouse brain membranes.

Guanine nucleotide-binding (G) proteins, Gs and G(olf) mediate the increase in cAMP formation through the activation of adenylyl cyclases. The developmental profiles of Gs, G(olf) and adenylyl were determined in mouse striatum and whole brain using immunobloting with specific antisera. Gs and the 115 kDa and 150 kDa adenylyl cyclases were present at the earliest age tested, embryonic day (E) 14.5 G(olf) and the 160 kDa adenylyl cyclase emerged in parallel, postnatally; during this period the increase in the relative abundance of the 150 kDa was observed. Gpp[NH]p activated Gs/G(olf) in a dose dependent manner, with a smaller response observed in embryos compared to adults. Mn2+ and forskolin activated the adenylyl cyclases and this activation increased during development. At E 14.5, maximal activation with Mn2+ and forskolin elicited a similar increase in cAMP levels, but from postnatal day 1, a nearly two fold higher response was obtained with forskolin compared to Mn2+; at the same time the 160 kDa adenylyl cyclase was detected. These data suggest that the appearance of certain forms of stimulatory G proteins was developmentally correlated with the expression of specific adenylyl cyclases.

Acute ethanol effect on calcium antagonist binding in rat brain.

We investigated the effect of acute ethanol administration on voltage-sensitive calcium channels (VSCC) by measuring [3H]nitrendipine ([3H]NTP) binding to crude synaptosomal membrane preparations from different rat brain areas, i.e. cerebral cortex, hippocampus and striatum. Ethanol enhances the number of binding sites shortly after the administration (40 min), then Bmax returns towards control values while the binding affinity increases. Kd decreased peaks 8 h after the oral administration and returns within the range of control values at 36 h. The in vitro addition of ethanol has no effect on [3H]NTP binding at various concentrations up to 600 mM. These results suggest that acute ethanol treatment modifies VSCC supporting the concept that the short-term neurochemical alterations induced by in vivo ethanol administration involve calcium channels.

Prenatal processing of pro-opiomelanocortin in the brain and pituitary of mouse embryos.

The processing of pro-opiomelanocortin (POMC) to ACTH- (adrenocorticotropin), MSH- (melanotropin) and endorphin-related peptides was studied in mouse embryos with the ultimate aim of determining the role of the POMC-related peptides in early development especially in the CNS. Mouse embryos at gestational days 10.5, 11.5, 12.5 and 14.5 were analyzed for POMC-derived peptides by SDS-PAGE, HPLC and radioimmunoassay using antisera specific for various regions of the prohormone. At embryonic day 10.5 (E 10.5) the prohormone was the major product detected. At E 11.5, POMC was processed to ACTH(1-39), des-acetyl alpha-MSH and beta-endorphin(1-31) and beta-endorphin(1-27). The amounts of these peptides increased at E 12.5, and at E 14.5. At E 14.5, there was a major increase in ACTH(1-39) and beta-endorphin(1-31) peptides. This was attributed to the large increase of corticotrophs in anterior pituitary at this stage. Des-acetyl alpha-MSH levels, however, were similar at E 12.5 and E 14.5 and the peptide was confined mainly to the central nervous system. gamma-MSH was not detected until E 16.5 in the brain. No alpha-MSH or acetylated beta-endorphin was detected between E 11.5 and E 14.5. Thus in early embryonic development, POMC is processed to des-acetyl alpha-MSH, beta-endorphin(1-31), beta-endorphin(1-27) and gamma-MSH in the brain, and primarily to ACTH(1-39) and beta-endorphin(1-31) in the anterior pituitary. Some differences exist in the forms of POMC-derived peptides found in embryonic versus adult brain and pituitary. The embryonic forms of the peptides may be significant in playing a role during development.

The prenatal development profile of expression of opioid peptides and receptors in the mouse brain.

Although the postnatal development of opioid systems of mammalian brain has been well studied, little is known about the ontogeny of and relationship between embryonic (E) opioid peptides and their receptors. Moreover, a simultaneous assessment of levels of the 3 classes of opioid peptides and their putative receptors during embryonal development has not been made. To this end, the ontogeny of opioid peptides and receptors in mouse brain were examined during the period E11.5 to postnatal day 1 (P1). Met-enkephalin, dynorphin and beta-endorphin immunoreactivity were detected before their putative opioid receptors. beta-Endorphin can be discerned as early as E11.5, whereas mu binding was first observed at E12.5. Although dynorphin and Met-enkephalin were measurable at the same time as beta-endorphin, kappa-receptors were not detected until E14.5 and delta sites were not found at all prenatally. Differences in immunoreactivity levels of the 3 peptides occur with dynorphin being lower than Met-enkephalin and beta-endorphin, consistent with a low Bmax for kappa binding. Expression of the 3 opioid peptides as well as mu and kappa opioid receptors rapidly increase in parallel from E14.5 to E18.5. Interestingly, levels of beta-endorphin diminish by P1, the stage at which a sharp rise of mu receptors occurs. In a comparative study of the binding of beta-endorphin 1-31, its truncated form (1-27) and their N-acetyl derivatives to E14.5 brain membranes, beta-endorphin 1-31 exhibited the highest affinity.

Differential development of beta-endorphin and mu opioid binding sites in mouse brain.

Mouse brains of various ages from embryonal day 14 (E14) to adult were analyzed for opioid receptor binding using the enkephalin analog Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and the opiate alkaloid dihydromorphine (DHM) as mu-selective radioligands. Binding parameters were estimated from homologous and heterologous competition binding curves. During the postnatal period, Kd values for [3H]DAMGE did not change but Bmax values (fmol/mg protein) increased 2.7 fold from postnatal day 3 (P3) to P7. Minor receptor density fluctuations were evident from P7 to adult. Similar results were obtained with [3H]DHM. In contrast, estimation of total mu binding sites (fmol/brain) revealed a continuous rise from P3 to the adult. The postnatal developmental profile of total mu binding sites was comparable to the weight gain of mouse brain and the increase in protein content. In contrast, during the same period beta-endorphin immunoreactivity (IR) levels undergo an increase that is inversely proportional to mu opioid receptor Bmax values. [3H]DAMGE binding to E14 membrane preparations was inhibited to a greater extent by Gpp(NH)p than that to P1 or adult. Additional characterization of mu receptors was accomplished by heterologous competition binding assays. IC50 values for beta-endorphin in competition with [3H]DHM and [3H]DAMGE were age dependent and differed for the two radioligands. These results suggest that mu receptor selectivity for mu-specific peptide and alkaloid ligands changes as a function of age.

Age-dependent increase in [3H]verapamil binding to rat cortical membranes.

[3H]Nitrendipine bound to cerebral cortex membranes is displaced more efficiently by verapamil in old rats (24 months old) compared to young ones (3 months old). In addition, [3H]verapamil binding was studied in detail in 3-, 12- and 24-month-old rats. Aging increases the Bmax of [3H]verapamil, leaving the affinity unchanged. These observations further indicate that aging may affect calcium channels leading to a derangement of calcium movements which in turn alter neuronal activity.

Regional modification of brain calcium antagonist binding after in vivo chronic lead exposure.

Lead toxicity in the central nervous system seems to be partially related to specific effects of the metal on calcium metabolism and in particular on calcium transport. On this line, the present study investigates the characteristics of [3H]nitrendipine binding to several rat brain regions after in vitro lead addition or after in vivo chronic exposure to this metal. In vivo a lead induced increase in [3H]nitrendipine binding, Bmax, is observed in cerebral cortex and striatum while the binding is unmodified in hippocampus. The in vitro studies are in agreement with in vivo data; lead addition stimulates the binding in synaptic membranes prepared from cortex and striatum but not from hippocampus where the binding is slightly inhibited. The data suggest that lead interferes with neuronal calcium channels in an area-selective manner.

Pituitary adenylate cyclase activating polypeptide (PACAP) potently enhances tyrosine hydroxylase (TH) expression in adrenal chromaffin cells.

In primary cultured bovine adrenal chromaffin cells (BACC), pituitary adenylate cyclase activating polypeptide 1-38 (PACAP) produced a dose related increase in tyrosine hydroxylase (TH) Vmax when measured 48 hours after the beginning of the treatment; a significant increase was observed with 0.5 nM and the maximal induction of close to 2.5-fold was found with 0.1 microM PACAP. The potency of PACAP was nearly 3 orders of magnitude greater than forskolin and VIP in inducing TH activity. These effects were preceded by an increase in TH mRNA levels, that started 2 hours after treatment and peaked 12 hours later. The presence of the phosphodiesterase inhibitor HL 725 further increased the stimulation of TH activity by PACAP, indicating that this activation was mediated via a cascade of events initiated by cAMP. Nicotine (1 microM) failed to increase TH activity significantly, however, when added in association with PACAP, a statistically significant increase of TH was elicited with peptide concentrations 5 times lower (0.1 nM) than the threshold dose of the peptide. The stimulation of nicotinic receptors facilitates the TH induction elicited by PACAP.

Differential sensitivity of [3H]nitrendipine binding to cations of toxicological interest in various rat brain areas.

[3H]Nitrendipine ([3H]NTP) is a radiolabelled calcium antagonist which can be used to study neuronal calcium (Ca2+) channels. The interaction of Mn2+, Zn2+, Pb2+ and La3+ on [3H]NTP binding was studied in 3 brain areas particularly rich in [3H]NTP binding sites. Differences were observed in the brain regional distribution of [3H]NTP binding as well as in their sensitivity to the metal ions Pb, Mn and Zn. The binding data suggest that neuronal Ca2+ channels in different brain areas display distinct sensitivity to selected divalent cations.

Acute ethanol and acetaldehyde administration produce similar effects on L-type calcium channels in rat brain.

The present study investigates the effect of acute ethanol and acetaldehyde administration on neuronal L-type calcium channels by measuring the binding of 3H-nitrendipine (3H-NTP). Acute ethanol (3 g/kg orally) transiently increases (+40% at 40 min) 3H-NTP binding. Acetaldehyde has a similar effect, but the onset of action is shorter; in fact the binding increase peaks 15 min following administration and is completely reversible within 2 hours. Disulfiram pretreatment does not modify the effect produced by acute ethanol on 3H-NTP binding. The results indicate that acetaldehyde may participate in mediating the action of ethanol on voltage sensitive L-type calcium channels with consequent alterations of neuronal excitability.

Chronic ethanol induces changes in opiate receptor function and in met-enkephalin release.

Ethanol induces supersensitivity of striatal delta-opiate receptor sites labelled by 3H-Etorphine. This effect may be ascribed to the diminished enkephalin release detected in striatal slices after chronic ethanol consumption. On the other hand, Kd values for 3H-Met-enkephalin and 3H-DHM (mu-opiate receptors) specific binding are enhanced. The different sensitivity of the two classes of opiate receptors to ethanol may be due to specific effects on enkephalinergic transmission. It has been hypothesized that the decrease of 3H-Met-enkephalin and 3H-DHM affinity for their receptors takes place because endogenous substances from ethanol metabolism (for example salsolinol) behave as mu opioid agonists. This hypothesis is confirmed by "in vitro" studies demonstrating that salsolinol displaces 3H-Met-enkephalin and 3H-DHM but not 3H-DADLE binding. On the contrary, it seems that delta-receptors become supersensitive because of the decreased endogenous peptide release.

Chronic ethanol exposure alters dopaminergic signal transduction processes.

A number of data suggest that the chronic ethanol treatment induces derangements of cell membrane structure leading to modifications of membrane related processes. In particular, alterations have been observed in the mechanisms of neurotransmitter recognition and in the coupling of the receptor with the effector system. Phosphorylation of specific proteins by cyclic AMP stimulated protein kinases represent the final step in the biological response in several distinct functional processes. Ethanol neurotoxic action therefore may affect neurotransmitter availability and release as well as receptors effector systems and protein phosphorylation. In this line, chronic ethanol treatment in rats decreases cyclic AMP dependent protein kinase activity in rat striatal membrane fractions. When lysine rich histone type III was used as exogenous substrate, cyclic AMP stimulated 32P incorporation was still decreased in the ethanol group. These data favor the hypothesis of a decreased capability of the enzyme to phosphorylate in response to cAMP.

Altered calcium signal transduction after chronic ethanol consumption.

Calcium and calcium-calmodulin dependent phosphorylation of several protein bands was found altered in synaptosomal membranes prepared from ethanol treated rats. The ethanol induced effect on Ca++ and Ca++-calmodulin phosphorylation presented regional differences. In particular 32P incorporation was lower in the striatum and cerebellum, higher in the hippocampus and unmodified in the cortex. Part of the phosphorylated bands had an apparent molecular weight similar to that of the phosphoproteins involved in neurotransmission. These results extend previous observations indicating that calcium movement control is modified during chronic ethanol consumption and suggest that ethanol may interfere at various steps in the calcium-promoted events.