Journal of Neurochemistry

Ferris, Christopher D.; Hirsch, David J.; Brooks, Brian P.; Snyder, Solomon H.

doi: 10.1111/j.1471-4159.1991.tb08213.xpmid: 1650393

Santiago, M.; Westerink, B. H. C.

doi: 10.1111/j.1471-4159.1991.tb08214.xpmid: 1677674

The extracellular concentration of dopamine (DA) and 3,4‐dihydroxyphenylacetic acid in the substantia nigra (SN) and striatum was estimated by microdialysis. The dialysate content of DA from the SN was recorded during infusion of a DA uptake blocker (nomifensine; 5 μmol/L) dissolved in the perfusion fluid. Perfusion of tetrodotoxin (1 μmol/L) produced a virtually complete disappearance of nigral and striatal DA release. Dendritic as well as terminal release of DA was inhibited for several hours when the nerve impulse flow in dopaminergic neurons was blocked by systemic administration of γ‐butyrolactonc (750 mg/kg, i.p.). The systemic administration (0.3 mg/kg, i.p.) as well as infusion (1 μmol/L) of the D2 agonist (‐)‐N‐0437 (2‐(n‐propyl‐N‐2‐thienylethylamino)‐5‐hydroxytetralin) produced a significant decrease in the release of DA in both the striatum and the SN. DA levels were recorded in the striatum both with and without addition of nomifensine to the perfusion fluid. The decrease in the striatum after (‐)‐N‐0437 was suppressed in the presence of nomifensine. Infusion (1 μmol/L) as well as systemic administration (40 mg/kg) of sulphide caused a similar increase in the release of striatal DA; this increase was, in both experiments, potentiated by nomifensine coinfusion. Sulpiride administration induced a small increase in the release of nigral DA. Infusion of (‐)‐N‐0437 or (‐)‐sulpiride into the nigra caused a moderate decrease and increase, respectively, of striatal DA level. It was possible to estimate the relative contribution of nigral and striatal au‐toreceptors to the typical changes in striatal DA release seen after peripheral administration of D2 agonists or antagonists. These results suggest that the dendritic release of DA fulfills classical release criteria.

Palestini, P.; Masserini, M.; Fiorilli, A.; Calappi, E.; Tettamanti, G.

doi: 10.1111/j.1471-4159.1991.tb08215.xpmid: 1861146

GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long‐ chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a(∼9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long‐ chain‐base analysis showed that newly‐formed GM1 contained ∼68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing ∼ 10% hydrolysis. Long‐ chain‐base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipal‐mitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long‐chain base are more molecularly dispersed than those containing C18:1 long‐chain base.

Giorgi, O.; Pibiri, M. G.; Biggio, G.

doi: 10.1111/j.1471-4159.1991.tb08216.xpmid: 1677675

The present study was designed to examine the steady‐state density and the turnover rates of D1 dopamine (DA) receptors in the striatum, nucleus accumbens, substantia nigra, and retina of the rat. The turnover rates were measured by monitoring the repopulation kinetics of D1 DA receptors labelled with (3H)SCH 23390 after the irreversible inactivation induced by a single dose of N‐ethoxycarbonyl‐2‐ethoxy‐ 1, 2‐dihydroquinoline (10 mg/kg s.c.). The repopulation of D1 DA receptors could be described adequately in all the neural tissues investigated by a theoretical model that assumes a constant rate of receptor production (i.e., zero order) and a rate of degradation that is dependent on the receptor density at any time (i.e., first order). The quantitative analysis of the experimental data using this theoretical model revealed significant regional differences in the rates of receptor production and degradation. Thus, the receptor production rates determined in the nucleus accumbens and striatum (8.03 and 9.96 fmol/mg of protein/h, respectively) were four‐ to sixfold larger than those measured in the substantia nigra (1.80 fmol/mg of protein/h) and retina (1.50 fmol/mg of protein/h). On the other hand, the receptor degradation rates in the striatum, nucleus accumbens, and retina (0.0093 h−1, 0.0110 h−1, and 0.0123 h−1, respectively) were 2.6–3.5‐fold larger than the receptor degradation rate in the substantia nigra (0.0035 h−1).

Owen, P. Jane; Boarder, Michael R.

doi: 10.1111/j.1471-4159.1991.tb08217.xpmid: 1861147

Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine‐insensitive channels, and also that bradykinin stimulated (poly)phos‐ phoinositide hydrolysis. To investigate membrane‐bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus‐secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with (3H)arachidonic acid or (3H)glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with 32Pi for 2.5 h we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of (32P)PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin‐stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate (32P)PA substantially, whereas long‐term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of (32P)PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C. A preliminary indication that G proteins may be involved was suggested by the observation that AIF4 stimulates (32P)PA accumulation whereas N‐ethylmaleimide inhibits bradykinin‐stimulated (32P)PA accumulation.

Owen, P. Jane; Jones, J. Alison; Boarder, Michael R.

doi: 10.1111/j.1471-4159.1991.tb08218.xpmid: 1861148

Using primary cultures of bovine adrenal chromaffin cells labelled with 32Pi, we show that stimulation with bradykinin, nicotine, or a depolarising concentration of potassium stimulates the accumulation of (32P)phosphatidic acid. The effects of nicotine and potassium are smaller than the effect of bradykinin, and are dependent entirely on extracellular calcium. The diacylglycerol kinase inhibitor R 59 022 attenuates the formation of phosphatidic acid by nicotine and depolarising concentrations of potassium. This inhibitor also blocks the nicotine and potassium stimulation of nor‐ adrenaline release from Chromaffin cells. Using 45Ca2+ influx studies, we show that the nicotine‐evoked calcium influx is also attenuated by R 59 022. These observations contrast with those in another report in which we showed that bradykinin stimulation of either (32P)phosphatidic acid accumulation or noradrenaline release is not affected by R 59 022. It is likely that the calcium influx produced by nicotine and depolarising potassium is blocked by R 59 022 by a mechanism that is independent of its ability to block diacylglycerol kinase. The nicotine‐ and potassium‐stimulated (32P)phosphatidic acid accumulation is a consequence of this calcium influx and presumably reflects calcium activation of either phospholipase C or phospholipase D.

Millington, William R.; Smith, Debra L.

doi: 10.1111/j.1471-4159.1991.tb08219.xpmid: 1861149

β‐Endorphin is posttranslationally processed to six derivatives, which, although structurally similar, produce distinctly different biological effects. β‐Endorphin 1–31 is a potent opioid receptor agonist, but β‐endorphin 1–27 exhibits antagonist properties, and β‐endorphin 1–26 and the α‐N‐ acetyl derivatives of all three peptides lack opioid receptor activity. In the present study, we identified the β‐endorphin peptides synthesized in human hypothalamus using cation exchange HPLC. First, we tested whether postmortem changes occur by storing rat hypothalami at 4°C. This demonstrated that relative amounts of the six β‐endorphin forms did not change for up to 24 h, although total β‐endorphin immunoreactivity significantly declined after 6 h. HPLC analysis of human hypothalami revealed that β‐endorphin 1–31 was the principal form, constituting 58.4 ± 5.4% of total immunoreactivity. Substantial amounts of β‐endorphin 1–27 (13.4 ± 1.2%) and β‐endorphin 1–26 (13.1 ± 1.6%) were also present, but α‐N‐acetylated forms were quantitatively minor, each comprising ∼5% of total β‐endorphin. A similar processing pattern occurred in preoptic and supra‐ chiasmatic areas of the hypothalamus. These results show that, despite differences in primary sequence, β‐endorphin is processed similarly in both rat and human hypothalamus. Opiate‐active β‐endorphin 1–31 is the principal form in both species.

Levison, Steven W.; McCarthy, Ken D.

doi: 10.1111/j.1471-4159.1991.tb08220.xpmid: 1861150

Studies on glial cultures have demonstrated that fetal bovine serum contains a factor that induces bipotential glial precursors known as oligodendrocyte‐type 2 astrocyte (O‐2A) progenitors to become type 2 astroglia rather than oligodendroglia. The goal of this research project was to characterize and purify this factor, which we refer to as the astroglia‐inducing molecule (AIM). Using cultures enriched in O‐2A progenitors, we determined that AIM is present in human and bovine sera and that fetal bovine serum qualified as the best serum for purifying AIM. AIM is heat and trypsin labile and may be a plasma glycoprotein. A 240‐fold enriched AIM preparation was produced by applying an ammonium sulfate precipitate of fetal bovine serum to heparin and then lentil lectin‐agarose, followed by gel nitration chromatog‐ raphy. In crude preparations, AIM activity migrated at 50 kDa by gel nitration. With enrichment, activity was seen at several molecular masses, all of which were approximate multiples of 50 kDa. Treatment with 6 M guanidine hydrochloride generated an AIM with a molecular mass between 12 and 18 kDa, a result suggesting that AIM aggregates. On a preparative isoelectric focusing gel, AIM activity most frequently migrated between pH values of 3 and 4; however, proteins with isoelectric points of >9 or at 6 also had activity in several experiments. These data suggest that either multiple AIMs exist or that a single AIM exists that associates with other proteins. Immunofluorescence for ganglioside GD3 and glial fibrillary acidic protein confirmed that AIM preparations induce type 2 astroglia from O‐2A progenitors and suggests that AIM has little effect on type 1 astroglia. Because none of the known growth factors that have been tested to date mimics its effects, AIM may be a novel differentiation factor.

Mack, Kenneth J.; Todd, Richard D.; O'Malley, Karen L.

doi: 10.1111/j.1471-4159.1991.tb08221.xpmid: 1861151

To understand the possible involvement of dopamine receptors in the pathogenesis of various neurological disorders, we have cloned and sequenced a dopamine D2A receptor gene from the mouse. A mouse genomic library was screened with probes derived from the published sequence of a rat D2A receptor cDNA. Using restriction endonuclease mapping, Southern blotting, and DNA sequencing, we have determined the cDNA sequence and genomic organization of the mouse D2A receptor gene. Unlike other guanine nucleotide‐binding protein‐coupled receptors, but similar to its rat and human counterparts, the mouse D2A receptor gene has seven introns and spans at least 30 kb of genomic DNA. The mouse D2A sequence shows 99% amino acid homology with the rat and 95% amino acid homology with the human sequence. As would be predicted, sequence differences are significantly more frequent outside of the hypothesized transmembrane spanning domain regions of the protein. Using the polymerase chain reaction with primers made from neighboring exons, we have identified two alternatively spliced D2A transcripts in the mouse. However, in contrast to the other species studied, the mouse expresses primarily the mRNA representing the larger, 444‐amino‐acid form of the receptor. Mouse pituitary expresses only the mRNA of the 444‐amino‐acid form of the D2A receptor. Hence, the mouse may offer the best model to study the in vivo physiology of the long form of the D2A receptor.

Clark, Edwin A.; Lee, Virginia M.‐Y.

doi: 10.1111/j.1471-4159.1991.tb08222.xpmid: 1861152

We examined the short‐term regulation of the phosphorylation of the mid‐sized neurofilament subunit (NF‐M) by kinases which were activated in rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) and/or 12‐O‐tet‐radecanoylphorbol 13‐acetate (TPA). We found that NGF and TPA, alone or in combination, increased (a) the incorporation of (32P)Pi into NF‐M and (b) the rate of conversion of NF‐M from a poorly phosphorylated to a more highly phosphorylated form. This was not due to increased synthesis of NF‐M, because NGF alone did not increase NF‐M synthesis and TPA alone or TPA and NGF together inhibited the synthesis of NF‐M. Further, an increase in calcium/phospholipid‐dependent kinase (PKC) activity resulting from the treatment of PC12 cells with NGF and TPA was observed concomitant with the increased phosphorylation of NF‐M. This PKC activity was determined to be derived from the PKCα and PKCβ isozyme. Finally, when PC12 cells were rendered PKC‐deficient by treatment with 1 μM TPA for 24 h, NGF maintained the ability to induce an increase in NF‐M phosphorylation, though not to the level attained in cells which were not PKC‐deficient. These data suggest (hat NGF with or without TPA stimulates NF‐M phosphorylation as a result of a complex series of events which include PKC‐in‐dependent and PKC‐dependent pathways.