NOS-II is involved in early differentiation of murine cortical, retinal and ES cell-derived neurons-an immunocytochemical and functional approach.

Nitric oxide (NO), a cell-derived highly diffusible and unstable gas is regarded to be involved in inter- and intracellular communication in the nervous system. Based on findings about the expression of the inducible NO synthase (NOS) isoform during development of early mouse olfactory as well as vestibulocochlear receptor neurons, we intended to prove a general role of this isoform for neuronal differentiation. Using immunohistochemical techniques, an exclusive expression of the inducible NOS-II isoform in early post-mitotic neurons of the developing mouse cortex and retina can be detected. In a pharmacological approach using cultures of the mouse cortex as well as embryonic stem cell-derived neural precursor cells, we investigated the functional role of NO on initial neuronal differentiation. Effects of NOS inhibitors and NO donors on the morphological differentiation were correlated with developmentally regulated calcium current densities, focusing on the effects of the specific NOS-II inhibitor GW 274150. Furthermore, involvement of the soluble guanylate cyclase (sGC)/cGMP signaling cascade was pharmacologically investigated. Our data indicate that while a specific block of NOS-II provokes a clear inhibition of neurite outgrowth formation as well as a decrease of calcium current densities, the inverse is true for exogenous NO donation. In line with lacking immunoreactivity for the sGC and cGMP there are only minor effects of compounds manipulating the sGC/cGMP pathway, suggesting the downstream sGC/cGMP pathway not to be essential in these early differentiation steps.

Intracerebral transplantation and successful integration of astrocytes following genetic modification with a high-capacity adenoviral vector.

To investigate the ability of genetically modified astrocytes to integrate into adult rat brain, two spontaneously immortalized cell lines and the allogenic nontumorigenic glioma cell line F98 were transduced with a high-capacity adenoviral vector (HC-Adv) expressing the EGFP gene from the hCMV promoter. In organotypic slice cultures the transduced astrocytes were shown to integrate into the brain tissue. Following transplantation of the transduced astrocytes into the striatum of adult rats, the transplanted cells survived at least for 6 weeks, continuously expressed the EGFP transgene, in close neighborhood with cells of the recipient tissue executing their differentiation capacity along the glial lineage. Thus, HC-Adv transduced astrocytes are promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases.

Nestin-specific green fluorescent protein expression in embryonic stem cell-derived neural precursor cells used for transplantation.

Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long-lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell-derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence-activated cell sorting of EGFP-labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.

Activation of mitogen-activated protein kinase cascade and phosphorylation of cytoskeletal proteins after neurone-specific activation of p21ras. I. Mitogen-activated protein kinase cascade.

Alterations in the phosphorylation state of the microtubule-associated protein tau have been associated with the pathogenesis of neurofibrillary degeneration as well as with a neuroprotective action against apoptotic cell death. Mitogen-activated protein kinases (MAPK) phosphorylate tau protein in vitro but the pathophysiological significance of this tau phosphorylation and its effects on neuronal viability is far from clear. Moreover, an in vivo model of activation of MAPK, a key candidate for in vivo tau phosphorylation, is still lacking. The aim of the present study and the accompanying paper was to establish an animal model of stimulated MAPK and to analyse the consequences on tau phosphorylation and the neuronal cytoskeleton. We took advantage of transgenic mice with neurone-specific expression of activated ras protein (p21H-ras(Val12)). The expression of the transgene in these animals is forced to a subset of neurones by the use of the synapsin I promoter. Activity of B-raf was elevated by 37%, while activity of MAPK (ERK1/ERK2) was increased by 25% associated with a subcellular redistribution from the cytoplasmic to the nuclear compartment. Kinases downstream of MAPK such as p90rsk and glycogen synthase kinase 3beta were only marginally affected. Activity of p70S6 kinase was unaltered. The present model might be useful to study the effects of activation of the MAPK cascade on tau phosphorylation and its cell biological sequelae.

Differentiation of green fluorescent protein-labeled embryonic stem cell-derived neural precursor cells into Thy-1-positive neurons and glia after transplantation into adult rat striatum.

OBJECT: The aim of this investigation was to assess new information concerning the capacity of transplanted embryonic stem cell (ESC)-derived neuronal cells to migrate into host brain and to evaluate these cells as a possible source for cell replacement therapy in neurodegenerative disorders such as Parkinson's disease (PD). METHODS: The authors investigated the ability of ESC-derived neural precursor cells to migrate and differentiate in a host striatum by using a D3-derived ESC clone that was transfected stably with a chicken beta-actin cytomegalovirus enhancer-driven green fluorescent protein (GFP)-labeled construct. This procedure allowed easy monitoring of all transplanted cells because of the green fluorescent labeling of donor cells. This approach also afforded easy estimation of cell integration and simultaneous observation of the entire transplanted cell population in relation to immunocytochemically identified neuronal and glial differentiation. After selection of nestin-positive neural precursor cells in a synthetic medium, they were implanted into the striatum of male adult Wistar rats. Their integration was analyzed on morphological studies performed 3 days to 4 weeks posttransplantation. CONCLUSIONS: The investigators found that after transplantation, a subpopulation of GFP-labeled cells differentiated into various neural morphological types that were positive for the mouse-specific Thy-1 antigen, which is known be expressed on neurons, as well as being positive for the astroglial marker glial fibrillary acidic protein. Moreover, GFP-expressing cells that were negative for either of these markers remained close to the injection site, presumably representing other derivatives of the neural lineage. Together, these findings contribute to basic research regarding future transplantation strategies in neurodegenerative diseases such as PD.

Selective expression of the NOS II isoform during mouse vestibulocochlear receptorgenesis.

Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation-dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor-cell-derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS-II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.

Ectopic expression of a chimeric colony-stimulating factor-1/TrkB-receptor promotes CSF-1-dependent survival of cultured sympathetic neurons.

The regulation of the density of innervation and the promotion of survival of neurons are the original effects depending on neurotrophins. Here we analyse such effects evoked by trkB tyrosine kinase in transfected PC12 cells and transfected sympathetic neurons. In order to exclude the previously described modulation of trk kinase activity by the extracellular activation of the low-affinity p75 neurotrophin receptor, we applied a chimeric receptor approach: The extracellular domain of colony-stimulating factor-1 (CSF-1) receptor was fused to the transmembrane and cytoplasmic domain of the trkB tyrosine kinase receptor, allowing its selective activation by the heterologous ligand. Protein expression and CSF-1-induced tyrosine phosphorylation of the chimeric receptor protein was demonstrated in transfected COS cells. After stable transfection into nerve growth factor (NGF)-responsive PC12 cells, CSF-1 mediated the K252a-sensitive induction of fiber outgrowth. Furthermore, we were able to show by heterologous expression of the chimeric receptor, that activation of trkB tyrosine kinase activity is sufficient to promote survival of neurotrophin deprived sympathetic neurons.

Nerve growth factor-stimulated mitogen-activated protein kinase activity is not necessary for neurite outgrowth of chick dorsal root ganglion sensory and sympathetic neurons.

Nerve growth factor (NGF)-stimulated neurite outgrowth in the rat PC12 tumor cell line recently has been shown to depend on the activation of the mitogen-activated protein (MAP) kinase kinase 1 (MEK1) (Pang et al.: J Biol Chem 270:13585-13588, 1995). In this study we have analyzed whether or not function of the MAP kinase pathway is necessary for NGF-stimulated neurite outgrowth in two subtypes of primary neurons derived from the embryonic chick peripheral nervous system (PNS). Treatment of p21ras-dependent dorsal root ganglion (DRG) sensory neurons (E9) with the MEK1 inhibitor PD98059 at concentrations up to 100 microM did not prevent NGF-stimulated neurite outgrowth. At this concentration NGF-stimulated tyrosine phosphorylation of MAP kinase p42 as well as MAP kinase activity both were decreased by approximately 80%. Essentially the same results were obtained with p21ras-independent sympathetic neurons (E12). We conclude that, in contrast to the PC12 tumor cell line, NGF-stimulated MAP kinase activity is not necessary for neurite outgrowth of DRG sensory and sympathetic neurons derived from the chick PNS.

Time-resolved signaling pathways of nerve growth factor diverge downstream of the p140trk receptor activation between chick sympathetic and dorsal root ganglion sensory neurons.

We have recently shown that the small GTP binding protein p21ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time-resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observed within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a.(ABSTRACT TRUNCATED AT 250 WORDS)

GTP-blot analysis of small GTP-binding proteins. The C-terminus is involved in renaturation of blotted proteins.

Recombinant c-Ha-ras, ralA and rap2, but not rap1A or rap1B proteins retained their ability to bind [alpha-32P]GTP after SDS/PAGE and transfer to nitrocellulose. Recombinant c-Has-ras missing the C-terminal 23 amino acid residues failed to bind [alpha-32P]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [alpha-32P]GTP was decreased many-fold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process. Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of [alpha-32P]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [alpha-32P]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [alpha-32P]GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras. Our results demonstrate that detection and quantification of small GTP-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.

Insulin-like growth factor I modulates voltage-dependent Ca2+ channels in neuronal cells.

Insulin and insulin-like growth factors are neuroactive peptides. We investigated the effect of insulin-like growth factor I (IGF-I) on Ca2+ channel currents in 108CC15 neuroblastoma x glioma (N x G) cells and a possible role of protein kinase C (PKC). Whereas the native IGF-I enhanced the Ca2+ channel current density in N x G cells, the boiled IGF-I had no effect. The effect of IGF-I occurred after 1-2 h incubation and reversed within 24 h. Ca2+ channel currents recorded in control cells were mainly of a low-threshold fast inactivating type and showed a mean density of 5.9 +/- 0.3 pA/pF. Current density in cells incubated with IGF-I (0.2 micrograms/ml) for 2 h increased to 9.2 +/- 0.8 pA/pF. Ca2+ channel currents in cells treated with IGF-I showed an enhanced amount of a high-threshold slowly inactivating Ca2+ current type sensitive to the dihydropyridine isradipine and the snail toxin omega-conotoxin. The effect of IGF-I was suppressed by coincubation with the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and staurosporin which were both without effect on current density in control cells. Whereas the inactive phorbol ester phorbol 12-myristate 13-acetate (PMA) failed to modulate Ca2+ channels in N x G cells, stimulation of PKC by the active phorbol ester PMA mimicked the effect of IGF-I. The effects of IGF-I and phorbol ester were not additive. Our data suggest an intracellular mechanism dependent on PKC and we propose a physiological relevance of the observed Ca2+ channel modulation by IGF-I in the neuroactivity of the peptide.

Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells.

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.

Involvement of pertussis toxin-sensitive G-proteins in the hormonal inhibition of dihydropyridine-sensitive Ca2+ currents in an insulin-secreting cell line (RINm5F).

Adrenaline inhibits insulin secretion via pertussis toxin-sensitive mechanisms. Since voltage-dependent Ca2+ currents play a key role in insulin secretion, we examined whether adrenaline modulates voltage-dependent Ca2+ currents of the rat insulinoma cell line, RINm5F. In the whole-cell configuration of the patch-clamp technique, dihydropyridine- but not omega-conotoxin-sensitive Ca2+ currents were identified. Adrenaline via alpha 2-adrenoceptors inhibited the Ca2+ currents by about 50%. Somatostatin which also inhibits insulin secretion was less efficient (inhibition by 20%). The hormonal inhibition of Ca2+ currents was not affected by intracellularly applied cAMP but blocked by the intracellularly applied GDP analog guanosine 5'-O-(2-thiodiphosphate) and by pretreatment of cells with pertussis toxin. In contrast to adrenaline and somatostatin, galanin, another inhibitor of insulin secretion, reduced Ca2+ currents by about 40% in a pertussis toxin-insensitive manner. Immunoblot experiments performed with antibodies generated against synthetic peptides revealed that membranes of RINm5F cells possess four pertussis toxin-sensitive G-proteins including Gi1, Gi2, Go2, and another Go subtype, most likely representing Go1. In membranes of control but not of pertussis toxin-treated cells, adrenaline via alpha 2-adrenoceptors stimulated incorporation of the photo-reactive GTP analog [alpha-32P]GTP azidoanilide into pertussis toxin substrates comigrating with the alpha-subunits of Gi2, Go2, and the not further identified Go subtype. The present findings indicate that activated alpha 2-adrenoceptors of RINm5F cells interact with multiple G-proteins, i.e. two forms of Go and with Gi2. These G-proteins are likely to be involved in the adrenaline-induced inhibition of dihydropyridine-sensitive Ca2+ currents and in other signal transduction pathways contributing to the adrenaline-induced inhibition of insulin secretion.

Inhibition of Ca2+ channels via alpha 2-adrenergic and muscarinic receptors in pheochromocytoma (PC-12) cells.

Biochemical studies have suggested a voltage-dependent dihydropyridine-sensitive catecholamine release in adrenal chromaffin cells. This release is inhibited by activation of alpha 2-adrenergic and muscarinic receptors; the underlying molecular mechanism is not known. We used undifferentiated PC-12 cells to study the effect of epinephrine and carbachol on transmembranous currents. Applying the patch-clamp technique in the whole cell configuration and using Ba2+ as charge carrier, we identified a high voltage-activated Ca2+ channel current. Both epinephrine (10 microM, in the presence of 1 microM propranolol) and carbachol (10 microM) reversibly inhibited the Ca2+ channel current by 30-40%. Yohimbine abolished and clonidine mimicked the effect of epinephrine. Phenylephrine failed to inhibit the Ca2+ channel current. The effect of carbachol was abolished by atropine. Epinephrine and carbachol did not affect the Ca2+ channel current reduced by the dihydropyridine, PN 200-110 (1 microM), suggesting a selective inhibition of dihydropyridine-sensitive Ca2+ channels. The Ca2+ channel current and its inhibition by receptor agonists were not influenced by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM). Pretreatment of cells with pertussis toxin or intracellular infusion of the GDP analogue guanosine-5'-O-(2-thiodiphosphate) was without effects on the control Ca2+ channel current but abolished its hormonal inhibition. Four pertussis toxin-sensitive G proteins were identified in membranes of PC-12 cells: two members of the Gi family, Gi1 and Gi2, and two members of the Go family, Go2 and another Go subtype (possibly Go1). The present data indicate that activated alpha 2-adrenergic and muscarinic receptors inhibit dihydropyridine-sensitive Ca2+ channels via pertussis toxin-sensitive G proteins without the involvement of a cAMP-dependent intermediate step.

Identification of the G-protein alpha-subunit encoded by alpha o2 cDNA as a 39 kDa pertussis toxin substrate.

A novel form of the Go alpha-subunit (alpha o2) has been identified by molecular cloning (Hsu et al., J. Biol. Chem. 265, 11220-11226, 1990). An antibody was generated against a synthetic peptide corresponding to a region of the protein encoded by alpha o2 cDNA. The antibody reacted with an apparently single 39 kDa protein in membrane preparations of rodent brain and with a 39 kDa pertussis toxin substrate in membranes of rodent neuroendocrine and pituitary cells. A previously produced antibody raised against a region common to proteins encoded by alpha o2 cDNA and the previous cloned alpha o1 cDNA (Itoh et al., Proc. Natl. Acad. Sci. USA 83, 3776-3780, 1986) recognized proteins of 39 and 40 kDa in preparations of bovine, porcine and rodent brain and pertussis toxin substrates of 39 and 40 kDa in membranes of rodent neuroendocrine and pituitary cells. We conclude that the 39 kDa Go alpha subunit is encoded by alpha o2 cDNA.

Ras proteins activate calcium channels in neuronal cells.

Ras (p21) proteins are involved in the control of cell growth and differentiation, but the mechanism by which they exert these effects is not yet known. Here we present evidence that c-Ha-ras (p21(Gly-12)) and its oncogenic mutant T24-ras (p21(Val-12)) selectively induce omega-conotoxin and dihydropyridine-sensitive Ca2+ currents within a few hours after introduction into the cytoplasm of neuroblastoma x glioma hybrid cells. Whereas control cells exhibited a mean Ca2+ current of 250 pA, it amounted to 730 pA in cells pretreated with ras protein. In cells loaded with p21(Gly-12), the effect occurred after 2 hours and was terminated after 8 hours. In contrast, introduction of p21(Val-12) resulted in a prolonged delay (6 hours) of the effect which lasted for more than 24 hours. When ras proteins were preactivated with the non-hydrolysable GTP analog GppNHp, the time courses of both p21(Gly-12) and p21(Val-12) effects were fast and sustained, suggesting that in intact cells (i) the GDP/GTP exchange is faster for p21(Gly-12) compared to p21(Val-12) and (ii) inactivation of p21(Gly-12) is mediated by GAP-induced GTPase activity. T-type Ca2+ currents and K+ currents were unaffected by ras proteins.

Development-dependent expression of low molecular weight GTP-binding proteins in embryonic chicken brain.

Expression of low molecular weight GTP-binding proteins in particulate and soluble fractions of embryonic chicken brain was analysed by SDS-PAGE and incubation of blotted proteins with [alpha-32P]GTP. At least seven GTP-binding proteins with apparent molecular weights between 21 and 29 kDa were demonstrated by this technique in membranes and microsomal fractions, whereas only four species were present in the cytosol. Levels of several small GTP-binding proteins were developmentally regulated in membrane and microsomal fractions, but not in the cytosol of embryonic chicken brain. Major GTP-binding proteins G28 and G26 were strongly increased in microsomal but not in membrane fractions between E6 and hatched chicken brain, whereas the minor protein G24 decreased in both membrane and microsomal fractions over this time. The differential expression of low molecular weight GTP-binding proteins in embryonic chicken brain suggests important roles for these proteins in brain development.

Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells.

Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations. Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.

Cholera toxin ADP-ribosylates the receptor-coupled form of pertussis toxin-sensitive G-proteins.

Cholera toxin catalyzes the ADP-ribosylation of 40 kDa pertussis toxin substrates in membranes from NG108-15 cells, which is increased in the presence of the opioid agonist DADLE. The basal ADP-ribosylation can be abolished by the opioid antagonist ICI 174864, suggesting that unoccupied opioid receptors interact spontaneously with the pertussis toxin substrates Gi/Go in the membrane. Treatment of NG108-15 cells with the opioid agonist DADLE leads to a reduction of agonist-stimulated and basal ADP-ribosylation of 40 kDa substrates catalyzed by cholera toxin. This indicates that the spontaneous interaction between opioid receptors and G-proteins is decreased in membranes of cells in which the receptor was desensitized by prolonged exposure to the agonist.

Ras protein expression is developmentally regulated in embryonic chicken brain.

Immunoblot analysis using a panreactive monoclonal antibody directed against ras p21 proteins detects two differentially regulated ras pools in embryonic chicken brain: a membrane pool that is not changed and a microsomal pool that starts at a low level but is strongly increased from E6 to E16 chicken brain. In order to study the distribution of ras proteins in different cell types of the nervous system, immunoblot analysis was performed on total cell proteins. In contrast to histochemical data showing the absence or low levels of ras proteins in glial cells, comparable amounts of ras proteins were found in cell lysates from purified chicken sympathetic neurons, cultured rat Schwann cells and mouse brain astrocytes.