Peripheral myelin formation is initiated by axonal cues that trigger a differentiation program in associated Schwann cells. Here, we define one essential differentiation signal: activation of the transcription factor NF-kappaB. In rat sciatic nerves, NF-kappaB was highly upregulated in pre-myelinating Schwann cells, and then its expression progressively declined until it was nearly absent in adults. Similarly, in co-cultures of Schwann cells and sensory neurons, NF-kappaB activation paralleled myelination, and blocking its activity or using cells from mice lacking the NF-kappaB subunit p65 markedly attenuated myelination. Inhibiting NF-kappaB also prevented activation of Oct-6, a transcription factor induced by axonal contact and required for proper myelin formation. These results show that the activation of NF-kappaB is an essential signal for the progression of axon-associated Schwann cells into a myelinating phenotype.

Sympathetic neurons depend on NGF binding to TrkA for their survival during vertebrate development. NGF deprivation initiates a transcription-dependent apoptotic response, which is suggested to require activation of the transcription factor c-Jun. Similarly, apoptosis can also be induced by selective activation of the p75 neurotrophin receptor. The transcriptional dependency of p75-mediated cell death has not been determined; however, c-Jun NH2-terminal kinase has been implicated as an essential component. Because the c-jun–null mutation is early embryonic lethal, thereby hindering a genetic analysis, we used the Cre-lox system to conditionally delete this gene. Sympathetic neurons isolated from postnatal day 1 c-jun–floxed mice were infected with an adenovirus expressing Cre recombinase or GFP and analyzed for their dependence on NGF for survival. Cre immunopositive neurons survived NGF withdrawal, whereas those expressing GFP or those uninfected underwent apoptosis within 48 h, as determined by DAPI staining. In contrast, brain-derived neurotrophic factor (BDNF) binding to p75 resulted in an equivalent level of apoptosis in neurons expressing Cre, GFP, and uninfected cells. Nevertheless, cycloheximide treatment prevented BDNF-mediated apoptosis. These results indicate that whereas c-jun is required for apoptosis in sympathetic neurons on NGF withdrawal, an alternate signaling pathway must be induced on p75 activation.

Activation of ␣ 1 -adrenergic receptors influences both the contractile activity and the growth potential of cardiac myocytes. However, the signaling pathways linking activation of specific ␣ 1 -adrenergic receptor (AR) subtypes to these physiological responses remain controversial. In the present study, a molecular approach was used to identify conclusively the signaling pathways activated in response to the individual ␣ 1A -and ␣ 1B -AR subtypes in cardiac myocytes. For this purpose, a mutant ␣ 1a -AR subtype (␣ 1a -S 290/293 -AR) was constructed based on analogy to the previously described constitutively active mutant ␣ 1b -AR subtype (␣ 1b -S 288 -294 -AR). The mutant ␣ 1a -S 290/293 -AR subtype displayed constitutive activity based on four criteria. To introduce the constitutively active ␣ 1 -AR subtypes into cardiac myocytes, recombinant Sindbis viruses encoding either the ␣ 1a -S 290/293 -AR or ␣ 1b -S 288 -294 -AR subtype were used to infect the whole cell population with >90% efficiency, thereby allowing the biochemical activities of the various signaling pathways to be measured. When expressed at comparable levels, the ␣ 1a -S 290/293 -AR subtype exhibited a significantly elevated basal level as well as agonist-stimulated level of inositol phosphate accumulation, coincident with activation of atrial natriuretic factor-luciferase gene expression. By contrast, the ␣ 1b -S 288 -294 -AR subtype displayed a markedly increased serum response element-luciferase gene expression but no activation of atrial natriuretic factor-luciferase gene expression. Taken together, this study provides the first molecular evidence for coupling of the ␣ 1a -AR and the ␣ 1b -AR subtypes to different signaling pathways in cardiac myocytes.Activation of ␣ 1 -adrenergic receptors (AR) 1 influences both the contractile activity and the growth potential of cardiac myocytes. However, despite intense investigation, the signaling pathways linking activation of specific ␣ 1 -AR subtypes to these particular physiological responses remain controversial (1). The situation is complicated by the diversity of ␣ 1 -AR subtypes. Three distinct ␣ 1 -AR subtypes have been identified by molecular cloning (2-4). Recently, the relationships between the cloned and native ␣ 1 -AR subtypes have been established by comparison of their affinity constants for a wide variety of ␣ 1 -AR subtype-selective antagonists (5, 6). From this comparison, it has been suggested that the cloned ␣ 1b -AR represents the native ␣ 1B -AR subtype; the cloned ␣ 1a/c -AR 2 corresponds to the native ␣ 1A -AR subtype; and the cloned ␣ 1d -AR is considered to represent a novel ␣ 1D -AR subtype. With the recognition that multiple ␣ 1 -AR subtypes exist, the roles of the individual ␣ 1 -AR subtypes in mediating specific physiological effects need to be investigated further.The assignment of particular physiological responses and signaling pathways first requires the elucidation of the specific ␣ 1 -ARs subtypes present in cardiac myocytes. In a previous study, we showed that all t...

A recently discovered endogenous autacoid, C-type natriuretic peptide, was tested in a pheochromocytoma (PC12) cell line for effects on 1) catecholamine release induced by a depolarizing stimulus, 2) guanylyl and adenylyl cyclase activities, and 3) specific 125I-labeled atrial natriuretic peptide (ANP) binding. C-type natriuretic peptide suppressed evoked neurotransmitter release in the absence of guanylyl cyclase activation or adenylyl cyclase inhibition; however, both a "clearance" (ANP-C) receptor binding agent, des-[Gln18Ser19Gly20Leu21Gly22]-ANF-(4-23)-NH2 (cANF), and pertussis toxin prevented this neuromodulatory effect. The C-type natriuretic peptide preferentially bound to receptors that also bound cANF. The results suggest that C-type natriuretic peptide suppressed evoked neurotransmitter efflux by binding to ANP-C receptors coupled to a pertussis toxin-sensitive process; furthermore, the neuromodulatory effect of C-type natriuretic peptide occurred independently of guanylyl cyclase activation or adenylyl cyclase inhibition. The novel aspects of these findings are 1) neuromodulatory effects of C-type natriuretic peptide, 2) guanylyl cyclase-independent actions of C-type natriuretic peptide, and 3) ANP-C receptors mediating C-type natriuretic peptide actions.

Natriuretic peptides suppress adrenergic neurotransmission by a mechanism apparently involving the natriuretic peptide receptor-C (NPR-C) rather than particulate guanylyl cyclase receptors. The bulk of evidence implicating the NPR-C in neuromodulatory effects relies on the pharmacological specificity of peptides believed to be specific for the NPR-C. This study tests for NPR-C effects on neurotransmitter release by examining fragments of the receptor for biological activity in pheochromocytoma (PC12) cells permeabilized with digitonin. A pentadecapeptide segment of the cytoplasmic portion of the NPR-C mimicked the effect of natriuretic peptides to suppress dopamine efflux evoked by calcium approximately 40%. Furthermore, an antibody generated against the pentadecapeptide fragment abolished the neuromodulatory effect of C-type natriuretic peptide in permeabilized cells. In contrast, the carboxy terminal nonadecapeptide portion of the NPR-C failed to attenuate dopamine efflux. These data are consistent with the proposed role of the NPR-C in transducing the biological activity of natriuretic peptides in adrenergic tissue. The most novel aspect of these observations involves the potency of the small cytosolic region of the NPR-C with the region closest to the membrane accounting for neuromodulatory effects.