Phosphorylation of the neuronal protein kinase C substrate B-50: In vitro assay conditions alter sensitivity to ACTH

We have explored the hypothesis that changes in the in vitro assay conditions alter both the extent of endogenous phosphorylation of B-50 protein in synaptosomal plasma membrane (SPM) and also the ability of the neuropeptide, ACTH-(1–24) to inhibit the phosphorylation of this protein. B-50 phosphorylation is influenced by preincubation, pH and ionic strength. ACTH-(1–24)-induced inhibition of B-50 phosphorylation varies with ionic strength and SPM protein concentration. Reduction of the buffer ionic strength and the SPM protein concentration enhances the ability of ACTH-(1–24) to inhibit B-50 phosphorylation. Furthermore, loss of ACTH-(1–24) by adsorption to plastic pipettes and test tubes reduces the peptide concentration in the assay. Addition of a low concentration of bovine serum albumin (BSA) essentially eliminates this loss without affecting the extent of phosphate incorporation into B-50. These data provide an explanation for the relatively high (and variable) IC₅₀ values for ACTH-(1–24)-induced inhibition of B-50 phosphorylation reported in the literature. Further, these data suggest that in vitro assay conditions must be carefully investigated before modulation of protein phosphorylation can adequately be studied.A preliminary report of these findings was presented at the 1986 Society for Neuroscience Annual Meeting in Washington, D.C.     

Dynorphin-(1–13) induces spontaneous feeding in rats

Dynorphin-(1–13), a recently isolated opioid peptide stimulates food ingestion in rats after intracerebroventricular administration at doses of 1 and 10 μg. The latency until food ingestion is 22.4 ± 1.9 min. The ability of dynorphin-(1–13) to initiate food ingestion is antagonized by the opiate antagonist, naloxone. The food ingestion is accompanied by excessive grooming behavior.     

Comparison between excessive grooming induced by bombesin or by ACTH: the differential elements of grooming and development of tolerance

Bombesin and ACTH-(1-24) induce a dose dependent increase in grooming behavior. Lower doses of bombesin induce a more general type of compulsive grooming in which most elements are involved, whereas higher amounts of bombesin induce a shift towards the element scratching at the cost of bodily grooming and sexual grooming. In contrast ACTH-(1-24) induces a dose dependent increase of all elements of grooming. It is concluded that the grooming displayed by animals treated with ACTH-(1-24) or with bombesin is of a completely different nature. In addition it is observed that under the conditions used tolerance occurs for the grooming inducing effect of ACTH-(1-24), but not for that of bombesin. Moreover, it appears that no cross tolerance exists between bombesin and ACTH-(1-24).     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.     

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 ACTH_1–24 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 ACTH_1–24. 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.     

Intracerebroventricular ACTH activates the pituitary-adrenal system:dissociation from a behavioral response.

In the rat, intracerebroventricular injection of synthetic ACTH (ACTH_1–24, ACTH_1–16) elevated plasma corticosterone levels and induced the display of excessive grooming behavior. The grooming response could be elicited in hypophysectomized rats without concommittant elevation of plasma corticosterone. In intact rats subcutaneous injection of ACTH_1–24 and not of ACTH_1–16-NH₂ stimulated the release of adrenal corticosteroids, whereas no excessive grooming was observed. In contrast to the reduced effectiveness of a second icv injection of ACTH in inducing the behavioral response, no single-dose tolerance was observed for the effect of icv ACTH on the pituitary-adrenal system. Therefore it was concluded that two different central mechanisms underly the observed responses to the icv applied ACTH.     

Studies on bombesin-induced grooming in rats

The gross behavior induced by centrally administered bombesin in rats was compared to that elicited by ACTH-(1–24) and the somatostatin analog, des AA^{1,2,4,5,12,13}[D-Trp⁸]-somatostatin (ODT8-SS). Bombesin (0.001–1 μg, ICV) caused dose-related excessive scratching which was qualitatively different from that associated with the other two groom-inducing agents. Bombesin-induced grooming was not markedly affected by behaviorally nondepressant doses of haloperidol, morphine, naloxone or neurotensin. Bombesin was active in genetically hypotrichotic (essentially furless) rats; and, again in such animals, even after numbing the area caudal to the shoulders with lidocaine. Tolerance and cross-tolerance studies with bombesin and ODT8-SS indicated that they produce scratching through different mechanisms. Bombesin caused scratching when injected directly into the periaqueductal gray, but not when administered intravenously. Neither hypophysectomy nor adrenalectomy markedly affected bombesin-induced grooming. This behavior appears to be initiated in the central nervous system and is produced independently of the pituitary-adrenal axis.     

Peptide-induced excessive grooming in the rat: The role of opiate receptors

We have investigated the possibility that opiate peptides induce excessive grooming behavior in the rat via a direct action on an opiate receptor by comparing the opiate agonist dynorphin(1–13) with its non-opioid fragment des-tyrosine¹-dynorphin(1–13) (dT-Dyn). We have shown that both peptides are capable of inducing grooming and that this behavior can be suppressed by pretreatment with naloxone. Analysis of the grooming pattern revealed that the response induced by dT-Dyn is qualitatively similar to that induced by ACTH(1–24) and dynorphin(1–13). Cross-tolerance was demonstrated among the various peptides. We conclude that peptide-opiate receptor interaction is not the primary event in the induction of grooming and that the opiate receptor(s) involved are located at another site underlying peptide-induced grooming.     

Evidence for the occurrence of the opioid octapeptide dynorphin-(1–8) in the neurointermediate pituitary of rats

Evidence is provided for the existence of the opioid peptide dynorphin-(1–8) in the neurointermediate pituitary of rats. The octapeptide was isolated by immunoadsorption to antibodies directed against porcine dynorphin-(1–13) followed by a variety of chromatographic separation procedures. The identity of the purified material with dynorphin-(1–8) was indicated by the following criteria: comigration with synthetic dynorphin-(1–8) on gelfiltration chromatography and high-performance liquid chromatography systems and liberation of a peptide with the same chromatographic behavior as leucine-enkephalin after sequential cleavage with trypsin and carboxypeptidase B.Radioimmunological estimations revealed that dynorphin-(1–8) is a major dynorphin-related opioid peptide in the pituitary of rats.     

Purification and Some Characteristics of an ACTH-Sensitive Protein Kinase and Its Substrate Protein in Rat Brain Membranes

Abstract: ACTH inhibits the phosphorylation of a rat brain membrane-bound protein (B-50). Both the protein kinase and the substrate protein could be extracted from the membranes by means of treatment with Triton X-100 in 75 mM-KCl. Using column chromatography over DEAE-cellulose and ammonium sulphate precipitation a protein fraction (ASP 55–80) enriched in endogenous B-50 phosphorylating activity was obtained. The time course of the endogenous phosphorylation of B-50 in this fraction showed a linear incorporation with time for at least 10 min and reached an estimated maximal incorporation of 0.65 mol P/mol B-50 after 60 min. The inhibition by ACTH_{1_24} of the B-50 protein kinase in ASP 55–80 was dose-dependent; the half-maximal effective concentration was 5 × 10⁻⁶ M, being 10 to 50 times lower as compared with intact synaptic plasma membranes (SPM). cAMP, cGMP and various endor-phins had no effect on the B-50 protein kinase. The B-50 protein kinase required both magnesium and calcium for optimal activity. Using two-dimensional electrophoresis on polyacrylamide slab gels the B-50 protein kinase and the B-50 protein could be identified and purified. The isoelectric point (IEP) of the kinase is 5.5 and the apparent molecular weight 70,000, whereas the IEP of the substrate protein B-50 is 4.5 and the apparent molecular weight 48,000. Amino acid analysis on microgram quantities of purified kinase and B-50 protein revealed basic/acidic amino acid ratios in agreement with the respective lEP's. It is speculated that the inhibition of B-50 protein kinase may be related to known modulatory effects of ACTH and related peptides on certain types of neurotransmission and behaviour.     

Detergents and Peptides Alter Proteolysis and Calmodulin Binding of B-50/GAP-43 In Vitro

Abstract: The neuronal growth-associated protein B-50/GAP-43 is a substrate for protein kinase C, binds to calmodulin in a calcium-independent manner, and in vitro is subject to an endogenous and chymotrypsin-mediated hydrolysis in the vicinity of the single kinase C phosphorylation site. All of these processes can be influenced by corticotrophin (ACTH). In the present study we have investigated whether these biochemical interactions involving B-50 could have common structural determinants. Chymotryptic digestion of B-50 in the presence or absence of a nonionic detergent and ACTH demonstrated that hydrolysis is potentiated by a lipid-like environment that primarily affects the protein rather than the protease or the peptide. Furthermore, this lipid dependency appears to extend to the binding of dephosphorylated B-50 to calmodulin, which appears to occur only in the presence of a nonionic detergent or lipid and the absence of calcium. A structure-activity study for ACTH-mediated inhibition of B-50 proteolysis by an endogenous protease that copurifies with B-50 in a detergent extract of synaptosomal plasma membranes showed that ACTH_1–24, ACTH_5–24, ACTH_5–16, dynorphin, and corticostatin inhibited the conversion of rat B-50 to B-50_41–226. In contrast, ACTH_7–16, Org₂₇₆₆, and neurotensin had no detectable effect on B-50 proteolysis at concentrations of 10 and 50 µM. The results indicate that in common with effects in other B-50-containing systems, inhibition of proteolysis is related to the presence of a basic amphiphilic helix in those ACTH fragments and analogues that were inhibitory and, moreover, the presence of this motif in other peptides appears to confer inhibitory activity. The results are discussed with reference to the putative secondary structure of B-50 and changes that may take place in the presence of membrane lipids or nonionic detergents. The conclusions of this study suggest that in vitro B-50 is subject to regulation by posttranslational enzymes and binding proteins as a consequence of its ability to adapt an amphiphilic helix conformation.     

Limbic-midbrain lesions and ACTH-induced excessive grooming.

The induction of excessive grooming by intraventricular administration of ACTH_1–24 was studied in rats with lesions in midbrain-limbic structures. Such areas have been reported to be implicated in mediating ACTH-induced effects on avoidance behavior, sexual excitement or stretching and yawning. Electrolytic lesions in the septal complex, the anterior hypothalamic/preoptic area, the mammillary bodies, the amygdala, the posterior thalamus and dorsal or ventral hippocampus did not interfere with ACTH-induced excessive grooming. Lesioning of the hippocampal complex by aspiration led to an inhibition of excessive grooming depending on the degree of hippocampal damage. Amygdala and hippocampal lesions enhanced the display of stretching and yawning activity after treatment with the peptide. The data indicate differences in the neural substrates mediating the effect of ACTH on extinction of conditioned avoidance behavior, excessive grooming, sexual excitement and stretching and yawning.     

Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain

Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent M_r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH_1-24) (10⁻⁵ M and 10⁻⁴ M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of M_r 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the M_r of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M _r of the "B-50 protein" varies from species to species.     

Adrenocorticotropin regulates angiotensin II receptors in bovine adrenal cells in vitro

The role of ACTH-(1–24) on angiotensin II receptors has been studied in bovine adrenal glomerulosa cells in primary culture. Angiotensin II receptors were measured in cells pretreated or not by ACTH-(1–24) on day 4 of culture. ACTH-(1–24) decreased angiotensin II binding sites in a time and a dose-dependent manner. After 24 hours of treatment the minimal effective dose of ACTH-(1–24) was 10^?11M and the maximal effect was obtained with 10^?8M. Moreover, ACTH-(1–24) 10^?8M decreased significantly angiotensin II receptors after 6 hours of treatment. Scatchard plot analysis showed that ACTH-(1–24) treatment did not modify the affinity of angiotensin II receptors (Ka = 0.42 and 0.44 × 10⁹M^?1 in control and treated cells respectively) but reduced by about half the number of angiotensin II sites per cell. Like ACTH-(1–24), 8-Bromo-cAMP, forskolin and cholera toxin decreased angiotensin II receptors. Factors such as prolactin, somatostatin, ACTH-(11–24) and dopamine which are bound to adrenal membranes without increasing cAMP production had no effect. In conclusion, these studies $\text{in vitro}$ demonstrate for the first time that ACTH decreases angiotensin II receptors by a direct mechanism acting on glomerulosa cells, and they also suggest that this effect could be mediated by cAMP.     

A specific radioimmunoassay for the novel opioid peptide dynorphin

Dynorphin was recently isolated from porcine pituitary extracts and shown to be the most potent known opioid peptide. Antisera were prepared to synthetic dynorphin-(1–13), the biologically active NH₂-terminal fragment of the peptide. A high-titer, sensitive antiserum was characterized with fragments from dynorphin-(1–13). Leucine-enkephalin, which is contained in dynorphin, is not recognized at all by the antiserum. To study distribution in tissue, a procedure using hot acidified methanol extraction of rat pituitary neurointermediate lobe preparations was developed and validated. ¹²⁵I-labelled dynorphin-(1–13), when added to tissue, remained intact throughout this extraction procedure, and added dynorphin-(1–13) was almost completely recovered. There was no destruction of radiolabelled peptide during incubation in the radioimmunoassay. Serial dilutions of pituitary extracts yielded curves that were parallel to the dynorphin-(1–13) standard curve. The immunoreactivity from tissue was completely destroyed by papain treatment.     

Dynorphin in bovine adrenal medulla. II. Isolation, partial characterization and biological activity of two distinct molecules

Two highly potent dynorphin-like peptides were isolated from bovine adrenal medulla by successive chromatography of an acid (HCl) extract on Sephadex G-10, carboxymethylcellulose, Sephadex G-50 and partition chromatography on Sephadex G-50. Amino acid analysis of both peptides revealed the presence of 24 amino acids including the composition of dynorphin-(1-13) and differing from each other only by a few residues. Both peptides were shown to have the same activity as dynorphin-(1-13) in the guniea pig ileum assay and reacted as well as dynorphin-(1-13) with a specific antibody (R-31) directed against the synthetic material.     

The colliculus superior modulates ACTH-induced excessive grooming

In previous studies the involvement of nigrostriatal dopaminergic activity in ACTH(1-24)-induced grooming has been established. It was suggested that the dopaminergic modulation of ACTH(1-24)-induced excessive grooming is exerted through the striato-nigro-collicular pathway. To obtain further evidence it was investigated, whether local application of GABAergic agents into the colliculus superior modulates excessive grooming occurring after an intraventricular injection with ACTH(1-24). It appeared that intra-collicular picrotoxin (a GABAergic antagonist) suppressed ACTH-induced grooming, whereas muscimol (a GABAergic agonist) enhanced the grooming response. The picrotoxin-induced R(unning) F(it) B(ehavior), elicited from the colliculus superior was also seen after intraventricular administration of picrotoxin. A detailed comparison of this behavioral response seen after both routes of administration of picrotoxin suggests that intraventricularly injected picrotoxin may well induce the RFB via a direct effect on the colliculus superior. Lesions placed in the colliculus superior completely abolished picrotoxin-induced RFB, exploration and orientation behavior. Yet, these lesions did not reduce excessive grooming suggesting that although this region may be involved in the modulation of ACTH-induced grooming it is not the primary site of peptide action.     

Corticotropin (ACTH) inhibits the specific proteolysis of the neuronal phosphoprotein B-50/GAP-43

The modulatory action of corticotropin (ACTH) on the specific proteolysis of B-50 to B-60 [B-50(41-226)] was investigated using a previously characterized synaptosomal membrane extract [ASP(57-82)] that was highly enriched in B-50, B-50 kinase activity and a B-50 protease. In common with the previously described inhibitory action on B-50 phosphorylation at Ser41, ACTH(1-24) inhibited B-50 proteolysis in a concentration and structurally dependent manner, while ACTH(1-10) and ACTH(11-24) or a combination of both peptides were ineffective. Structural similarities between segments of ACTH(1-24) and B-50(40-55) may explain the inhibitory action of the peptide on B-50 phosphorylation and proteolysis. In addition, calmodulin, which binds to the N-terminus of the dephosphorylated form of the protein, also protected B-50 from degradation.     

ACTH-(1-24) and alpha-MSH antagonize dopamine receptor-mediated inhibition of striatal dopamine and acetylcholine release

The effects of ACTH-(1-24), alpha-MSH and ACTH-(4-10) were studied on the electrically evoked release of 3H-dopamine and 14C-acetylcholine from striatal slices in the absence and presence of the dopamine receptor agonist TL-99. None of the peptides affected transmitter release when TL-99 was not present. ACTH-(1-24) and alpha-MSH concentration-dependently antagonized the inhibition of striatal transmitter release induced by dopamine receptor stimulation due to the presence of TL-99. ACTH-(1-24), 10(-7)M, reduced the TL-99-induced inhibition of the release of both dopamine and acetylcholine by approximately 50%, and 5 X 10(-6) M ACTH-(1-24) restored the release fully to control values. alpha-MSH was less effective by a factor 20-30 in counteracting the release-inhibiting effect of TL-99. ACTH-(4-10) had no effect at any of the concentrations tested. These results show that ACTH/MSH-like neuropeptides may act by modulating dopamine receptor functions in rat striatum.     

ACTH-induced excessive grooming in the rat: the influence of environmental and motivational factors.

Intraventricularly administered ACTHt_1–24 in rats initiated excessive grooming followed by stretching and yawning syndrome. The present study provides evidence that novelty is not an essential prerequisite for its expression and that a variety of environmental variables is not able to influence the peptide-induced behavior. Only very strong motivational variables as severe hunger/thirst and anxiety are able to modulate the ACTH-initiated excessive grooming: This response is significantly depressed in water-deprived rats bar pressing for water in a Skinner box, as well as in rats receiving unavoidable electric foot shock. The results are indicative of the strength of the ACTH-initiated motivation to groom, and it is suggested that excessive grooming is a secondary response serving to dearouse the organism after activation by ACTH.