P. Schotman

ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated32P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10−10 to 10−4 M. In a number of protein bands, a biphasic effect of ACTH1–24 was observed: in concentrations of 10−4–10−5 M, a reduced amount of32P was found; in concentrations of 10−6–10−7 M, hardly any effect could be detected, whereas consistently at concentrations around 10−8 M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.

Role of the growth-associated protein B 50/GAP 43 in neuronal plasticity

The neuronal phosphoprotein B-50/GAP-43 has been implicated in neuritogenesis during developmental stages of the nervous system and in regenerative processes and neuronal plasticity in the adult. The protein appears to be a member of a family of acidic substrates of protein kinase C (PKC) that bind calmodulin at low calcium concentrations. Two of these substrates, B-50 and neurogranin, share the primary sequence coding for the phospho- and calmodulin-binding sites and might exert similar functions in axonal and dendritic processes, respectively. In the adult brain, B-50 is exclusively located at the presynaptic membrane. During neuritogenesis in cell culture, the protein is translocated to the growth cones, i.e., into the filopodia. In view of many positive correlations between B-50 expression and neurite outgrowth and the specific localization of B-50, a role in growth cone function has been proposed. Its phosphorylation state may regulate the local intracellular free calmodulin and calcium concentrations or vice versa. Both views link the B-50 protein to processes of signal transduction and transmitter release.

Phosphoprotein B-50 in nerve growth cones from fetal rat brain

The presynaptic, nervous tissue-specific phosphoprotein B-50 is present in infant and adult rat brain. In the present study we demonstrate that B-50 is a major phosphoprotein in nerve growth cones obtained from fetal rat brain. As this protein is an endogenous substrate for protein kinase C, an enzyme linked to cell growth and proliferation, a role for B-50 in nerve growth cone function is suggested.

ACTH-induced inhibition of endogenous rat brain protein phosphorylation in vitro: Structure activity

ACTH1–24 inhibits the endogenous phosphorylation in vitro of distinct SPM protein bands. Using N-terminal fragments of ACTH, the structure-activity requirements for this effect were studied. A rather complex interaction of the ACTH fragments with endogenous SPM phosphorylation was observed. The effects were not only dependent on the primary structure of the peptide used, but also on the protein band studied and the ATP/SPM ratio used in the incubation system. ACTH1–24 did not interfere with the ATP-hydrolyzing activity of the SPM preparation, nor did it influence the endogenous phosphatase activity. Therefore, a direct interaction of ACTH with SPM protein kinase(s) is likely to be responsible for its effect on phosphorylation.

The Kinase C Substrate Protein B-50 and Axonal Regeneration

As reported previously the prominent protein kinase C substrate protein B-50 is present in growth cones isolated from fetal rat brain and in outgrowing hippocampal neurites. These findings suggest that B-50 plays a role in axonal growth during development of the nervous system. In the present paper the fate of B-50 is investigated in regenerating rat sciatic nerve. Using affinity-purified anti-B-50 antibodies B-50 levels have been compared in crushed and contralateral intact nerves by means of immunoblotting and radioimmunoassay. B-50 levels in the crushed nerve increased 5.3-fold as compared to non-crushed controls. Furthermore, the cellular localization of B-50 has been assessed by immunohistochemistry. Virtually no B-50 immunoreactivity was seen in control nerves, but bright immunofluorescence appeared in regenerating sprouts. Our data are in line with current evidence from several laboratories that B-50 is a member of a small family of growth-associated proteins and support the hypothesis that B-50 is involved in axonal growth.

The enhanced recovery of sensorimotor function in rats is related to the melanotropic moiety of ACTH/MSH neuropeptides.

The recovery of sensorimotor function in female rats was studied using a foot-flick response test after crushing the sciatic nerve. Every other day the animals received a subcutaneous injection of small ACTH/MSH-like peptides. Rats treated with ACTH-(4-10), ACTH-(4-9), ACTH-(4-9) analog ORG 2766, ACTH-(6-10) and alpha-MSH showed a faster recovery of sensorimotor function as compared to vehicle-treated rats. Treatment with ACTH-(4-7) and the tripeptide Phe7-D-Lys8-Phe9 (PDLP, the C-terminal part of the ORG 2766) remained ineffective. The effect of alpha-MSH was even stronger than that of the other peptides. The facilitation of the return of sensorimotor function by the ACTH-like peptides is discussed in relation to the corticotropic and melanotropic properties of these peptides. Furthermore, it was shown that treatment with ORG 2766 was effective not only in young adult animals (2--3 months) but also in one-year-old animals.

ACTH like neurotropic peptides: Possible regulators of rat brain cyclic AMP

The influence of behaviorally active, N-terminal fragments of ACTH on the accumulation of cAMP in rat brain investigated in broken cell preparations of subcortical tissue, in slices of neostriatum and in vivo. ACTH1--24 has a biphasic effect on the activity of adenylate cyclase in broken cell preparations of rat brain subcortical tissue: concentrations below 25 micrometer stimulated, whereas concentrations of 0.1 mM and higher inhibited adenylate cyclase activity. The magnitude of the stimulation was dependent on the concentrations of ATP and Mg2+ in the incubation medium. Structure activity studies revealed that at a concentration of 10(-4) M ACTH1--16-NH2 and ACTH4--7 also inhibited the activity of adenylate cyclase, whereas ACTH11--24, ACTH1--10, ACTH4--10, [D-Phe7]ACTH1--10 and [D-Phe7]ACTH4--10 were inactive in this respect. Addition of 0.8 mM EGTA but not of 0.25 mM Ca2+ prevented the inhibition by 10(-4) M ACTH1--24. GMP-N-P (10(-5) M), naltrexone (10(-3) M) and ergometrine (10(-3) M) did not influence the inhibitory effect. ACTH1--24 enhanced the accumulation of cAMP in slices from rat brain neostriatum in a dose-dependent manner. This effect was already maximal 7.5 min after the addition of the peptide and was potentiated by isobutylmethylxanthine, a potent inhibitor or phosphodiesterase. Intraventricular injection of 1 microgram ACTH1--16-NH2 in rats significantly elevated (+ 27%) the concentration of cAMP in the septal region 60 min after the injection of the peptide. The results are discussed in terms of a possible involvement of cAMP as a second messenger in the central nervous system for N-terminal fragments of ACTH.

ACTH sensitive protein kinase from rat brain membranes

—The protein kinase which in rat brain synaptosomal plasma membranes is responsible for the phosphorylation of a protein band B‐50 (MW 48, 000) was inhibited by the behaviorally active peptide ACTH1–24 and not stimulated by cAMP. Treatment with 0.5% Triton X‐100 in 75 mM‐KCl solubilized 15% of the total B‐50 protein kinase activity and preserved the sensitivity of the enzyme to ACTH1–24. The rate of endogenous phosphorylation of protein band B‐50 was different in intact SPM, solubilized fraction and residue. cAMP stimulated the endogenous phosphorylation of the solubilized fraction in a rather general manner. The solubilized membrane material also phosphorylated B‐50 proteins which were previously extracted from membranes. Column chromatography of the solubilized material over DEAE‐cellulose pointed to the presence of multiple protein kinase activities from rat brain synaptosomal plasma membranes, one of which was the ACTH‐sensitive B‐50 protein kinase.

α-Melanocyte-stimulating hormone stimulates the outgrowth of myelinated nerve fibers after peripheral nerve crush

The effect of alpha-melanocyte-stimulating hormone on peripheral nerve regeneration was studied by monitoring functional recovery and quantifying histologic changes that follow crush lesion of the rat sciatic nerve. The results showed that such treatment of rats with a crushed sciatic nerve resulted in a reduction of the recovery period and in an increase in the number of regenerating nerve fibers.

Immunohistochemical localization of a phosphoprotein (B 50) isolated from rat brain synaptosomal plasma membranes

The endogenous phosphorylation in vitro of at least 5 protein bands of rat brain synaptosomal membranes (SPM) is inhibited by the behaviorally active peptide ACTH1-24. One of these proteins, the phosphoprotein band B-50, was isolated from rat brain SPM by SDS-slab gel electrophoresis. An antiserum to B-50 was raised in rabbits. The presence of antibodies to B-50 in the antiserum was demonstrated by immunoperoxidase staining of cryostat sections of a polyacrylamide gel containing the antigen. The production of antibodies was monitored by an indirect immunofluorescence technique using cryostat sections of quick-frozen rat cerebellum. Immunofluorescent staining of the molecular and granular layers was observed, whereas the white matter and the perikarya and Purkinje cells of the cerebellum were not stained. With the use of the peroxidase-antiperoxidase (PAP) method, the immunohistochemical localization of the antigen in the molecular and granular layer of the cerebellum was confirmed. Regions rich in neuropil, like the CA1, CA2, CA3, CA4 and dentate gyrus showed an intense immunostaining. Thus, in agreement with the synaptic origin of B-50, the antiserum reacted with tissue components present in brian regions in synaptic contacts.