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 ACTH_1–24 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 ACTH_1–24 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 ACTH_1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.     

GTP-sensitive phosphorylation of proteins in a postmitochondrial supernatant from rat brainstem affected by ACTH1–24

The effect of ACTH_1–24 and cyclic nucleotides on the endogenous phosphorylation of proteins from a postmitochondrial supernatant from rat brainstem was investigated in the presence and absence of GTP. Phosphorylation and its modulation by these compounds were studied in vitro by incorporation of labeled phosphate from [-³²P]ATP added to the incubation mixture. Phosphoproteins were subsequently analyzed by autoradiography after one-and two-dimensional separation. Eight ACTH-sensitive phosphoproteins of molecular weights 75 (IEP 4.0), 67, 64, 50 (IEP 4.7), 47 (IEP 4.8), 38, 34, and 24K were found. The effects of ACTH on phosphorylation were mainly inhibitory, and the effected protein bands did not coincide with the phosphoproteins sensitive to cyclic AMP and cyclic GMP. Phosphorylation of those phosphoprotein bands and its ACTH sensitivity appeared to be highly sensitive to GTP. It is suggested that the activity of protein kinases involved in hormone-sensitive phosphorylation in a postmitochondrial rat brainstem fraction is regulated by GTP-dependent mechanisms.     

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.     

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.     

In vitro mitogenic and steroidogenic effects of ACTH analogues on an adrenal tumor cell line (Y-1)

Native porcine adrenocorticotropin (ACTH_1–39) as well as synthetic adrenocorticotropin (ACTH_1–24) increase cAMP and steroid production and inhibit DNA synthesis in an adrenal cell line. The COOH terminal sequence of both peptides as well as β-endorphin have no effects, while the NH₂ terminal sequence of ACTH as well as α-MSH which have very low stimulatory effect on cAMP production, have a mitogenic effect. These results suggest that ACTH might have in vitro some mitogenic action on adrenal cell, but this effect is blunted by cAMP accumulation during hormonal stimulation. The results can also explain the in vivo and in vitro contradictory effects of the hormone on adrenal cell replication.     

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.     

Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH,spermine, and hemin

The incorporation of [-³²P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca²⁺ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 M). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca²⁺-independent protein kinase.     

Effects of memory-modulating neuropeptides on spontaneous and evoked cortical electrical activity in rabbits

Encephalographic correlates of behavioral activity were demonstrated for oligopeptides belonging to the group of memory neuromodulators and of a similar nature: arginine-vasopressin, corticotrophic fragment ACTH_4–7 and its four analogs, leu-enkephalin, and a melanostatin analog. The compounds activated the EEG, the number of low-frequency components in the spectrum was reduced and the number of high-frequency components increased, and the amplitude of the waves was reduced in most cases, The results of a study of the effect of ACTH_4–7 and vasopressin on the parameters of the visual global evoked potential show that, depending on their dose, neuropeptides can reduce the latent period and increase the amplitude of the global potential or, conversely, in a substantially increasing dose they may inhibit these parameters. Effects of vasopressin are long-term in nature and may continue for weeks, whereas the effect of ACTH fragments and leuenkephalin are limited to a few hours.S. M. Kirov Military Medical Academy, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 578–584, November–December, 1982.     

Superpotency and superaffinity phenomena in the stimulation of steroidogenesis in adrenocortical cells by adrenocorticotropin-tobacco mosaic virus conjugates

Summary Adrenocorticotropin-(1-24)-tetracosapeptide was covalently attached to tobacco mosaic virus in two different manners: (i) through a handle near the C-terminus on tyrosine-(23) and (ii) through a handle at the N-terminus on serine-(1). Compounds of type (i) with their N-terminal message sequence freely exposed on the virion surface were considerably more potent for stimulating steroidogenesis in isolated adrenocortical cells than those of type (ii) with a more congested message. Conjugates with 50 or less hormone molecules per virion were less potent per peptide unit than the free handle-substituted hormones, whereas conjugates with 150 ACTH units exhibited superpotency effects. Superpotency disappeared when the substituted virions were disaggregated into (substituted) capsomers, suggesting influences of hormone clustering and virion geometry on biological activity. Superpotent stimulation was irreversible under conditions that immediately inhibited steroidogenesis by ACTH (dilution, addition of a peptide antagonist). Thus, superpotency might be caused by superaffinity arising from a slow rate of dissociation of the conjugates from the target cell receptors. The reason for the slow dissociation rate is still unclear: possible explanations include cooperative affinity, rapid internalization of the conjugate-receptor complexes, or decreased rates of peptide degradation at the receptor site.Abbreviations ACTH_1–39 adrenocorticotropin-(1–39)-nonatriacontapeptide - ACTH_1–24 adrenocorticotropin-(1–24)-tetracosapeptide - ACTH_6–24 adrenocorticotropin-(6–24)-nonadecapeptide - TMV tobacco mosaic virus wild strain To whom reprints should be addressed.     

Tissue culture of rat adult decapsulated adrenal glands

Summary A method of cultivation involving both repeated trypsinisations (at room temperature) and explantation of the tissue fragments on polythene discs has been shown to be apt to the growth in vitro of rat adult decapsulated adreno-cortical tissue. This is the first time that the successful cultivation of such a tissue is reported. The technique and its applications are discussed.The effects of _1–24 corticotrophin (ACTH_1–24) on the rat adult adrenal cultures have been examined by both electron microscopy and autoradiography. Zona fasciculata and reticularis cells grown in the absence of ACTH for long terms (15–16 days) survive and proliferate as dedifferentiated elements. If ACTH_1–24 is added to the cultures, adrenocortical cells will, within 2 days, simultaneously increase their proliferation rate and differentiate. After 7 days of treatment, cortical cells exhibit not only fully differentiated but even hypertrophic morphologic features. Significant stimulations of adrenal DNA, RNA and gross protein synthesis have been found to take place at different times after the starting of the ACTH_1–24 treatment. These data are discussed in relation to the findings previously reported in literature.Rat adult adrenal gland tissue cultures are proposed as a non-previously available tool for investigations into the physiopathology of the adrenal cells to be carried out in a carefully controlled environment.A Preliminary report on part of this material was given at the annual meeting of the Société Française de Microscopie Elécronique, Nantes, May 1972.Authors wish to thank Drs. P. G. Andreis and A. S. Belloni for their skilful assistance in the autoradiographic experiments. Thanks are also due to Mr. G. Gottardo for his excellent technical help in electron microscopic work. This work was partly supported by a contract with the CNR-Italy (No 69.0172/115.3439).     

Inhibition of ACTH-stimulated steroidogenesis in isolated rat adrenal cells treated with neuraminidase

The possible role of membrane sialic acid in the action of ACTH was investigated in rat adrenal cells. After treatment with neuraminidase, the cells showed a diminished steroidogenic response to ACTH while the response to cyclic AMP and dibutyryl cyclic AMP was unaffected. 11β-hydroxylation of deoxycorticosterone (DOC) was also not impaired. Dose response curves for three ACTH peptides (ACTH_1–39′, ACTH_1–24 and ACTH_1–10) with neuraminidase treated cells suggest that sialic acid residues on the glycoproteins of the plasma membrane may either impart affinity to the plasma membrane for ACTH molecule or facilitate transmission of the signal arising from ACTH-receptor interaction to the catalytic site of adenyl cyclase.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

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.     

Evidence That the Synaptic Phosphoprotein B-50 Is Localized Exclusively in Nerve Tissue

The localization of the phosphoprotein B-50 (molecular weight 48,000 isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B-50 protein. Two-dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and in immunochemical detection method employing an anti B-50 antiserum revealed the presence of B-50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B-50 in nervous tissue. In addition, the regional distribution of endogenous B-50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B-50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1-24 is discussed.     

Paradoxical Results after Inadvertent Use of Cosyntropin [Adrenocorticotropin Hormone (1-24)] Rather than Acthrel (Ovine Corticotropin Releasing Hormone) during Inferior Petrosal Sinus Sampling

ObjectiveThe use of ovine corticotropin releasing hormone (oCRH) maximizes the diagnostic accuracy of inferior petrosal sinus sampling (IPSS) in patients with adrenocorticotropin hormone (ACTH)-dependent Cushing’s syndrome (CS). oCRH is marketed as ACTHrel and, understandably, may be confused with cosyntropin [ACTH ^(1-24)]. The inadvertent substitution of synthetic ACTH^(1-24) for oCRH (ACTHrel) during IPSS may cause unexpected and misleading results. The aim of this report is to raise awareness of the potential confounding results created when synthetic ACTH^(1-24) is mistakenly used during IPSS.MethodsWe present 3 patients treated at 3 different centers with ACTH-dependent CS in whom ACTH^(1-24) was mistakenly substituted for oCRH (ACTHrel) during IPSS.ResultsIn all patients, there was an abrupt and unexpected decrease in plasma ACTH in the inferior petrosal sinus (IPS) samples after presumptive stimulation with oCRH. Re-evaluation of the patients’ pharmacy records confirmed that synthetic ACTH^(1-24) had been used rather than oCRH during each procedure. Because “sandwich” immunometric assays for ACTH measure the entire pool of endogenous ACTH, the administration of synthetic ACTH^(1-24) artifactually decreases the endogenous plasma ACTH^(1-39) measurement by binding only to the N-terminal antibody raised against ACTH^(1-17) and not to the C-terminal antibody raised against ACTH^(34-39). This results in a lack of a detectable sandwich complex and explains the apparent reduction in ACTH concentration.ConclusionAn abrupt decrease in ACTH during IPSS suggests that synthetic ACTH^(1-24) rather than oCRH (ACTHrel) has been administered. The labeling of oCRH as ACTHrel poses a potential patient safety problem about which endocrinologists, interventional radiologists, and pharmacists should be aware. (Endocr Pract. 2014;20: 646-649)     

ACTH1–24 Releases a Protein from Synaptosomal Plasma Membranes

Abstract: Brain membranes contain several protein kinases, all of which appear to play a role in the regulation of neuronal functioning. These membranes also contain numerous (phospho) proteins. It has been proposed that the degree of phosphorylation of some of these proteins may affect neuronal membrane properties. In a series of previous reports we showed that ACTH_1-24 inhibits the endogenous phosphorylation of several synaptosomal plasmamembrane (SPM) proteins including the B-50 protein. Although we have speculated that the degree of phosphorylation of B-50 may be important in regulating the turnover of membrane (poly)-phosphoinositides, the exact nature of the interaction between ACTH_1-24 and B-50/B-50 protein kinase is unknown. The purpose of the present study was to determine whether treatment of SPM with ACTH_1-24 will lead to a specific release of proteins from SPM. We found that ACTH_1-24 specifically releases a 41,000 M_r protein from rat brain SPM. Although we are not certain about the biological significance of the release of this polypeptide, it is of sufficient interest for further research in view of the lack of success of finding binding of labeled ACTH to brain membranes.     

ACTH-dependent stimulation of a specific peptide in adrenocortical cells in culture.

Corticotropin (1–24) tetracosapeptide (ACTH_1–24) induces a small but significant increase in the incorporation of radioactive leucine into trichloracetic insoluble proteins of a mouse adrenal cell line Y₁. Neither cyclic AMP, nor cholera toxin or a nitrophenyl sulfenyl derivative of ACTH_1–24 (NPS-ACTH_1–24) have any effects.After being labelled with radioactive leucine in the presence or absence of ACTH, the cells were solbilized in 1 % sodium dodecylsulfate and subjected to 20 % sodium dodecylsulfate polyacrylamide gels electrophoresis. ACTH_1–24 was found to induce a dramatic increase in the incorporation of radioactive leucine into a small peptide (MW 3500). This effect was mimicked by other steroidogenic compounds such as cholera toxin, cyclic AMP, NPS-ACTH_1–24 but not by ACTH_11–24, a non steroidogenic analogue of ACTH.     

Interaction of Adrenal Cell Membrane Receptors with Dimers of Corticotropin Fragments and with Poly-L-Lysine

Abstract

Polycationic peptides are demonstrated to interact with the membrane receptors of the adrenal cell as judged from their effect on steroidogenesis. The corticotropin fragments ACTH_7-24 and ACTH_11-24, when covalently dimerized at their C-termini, strongly antagonize both corticotropin- and angiotensin II -induced steroidogenesis, while dimerized ACTH_1-24 behaves as a mixed agonist/antagonist. A quantitative analysis of the antagonistic potencies shows that the measured effects are consistent with the prediction that electrostatically controlled accumulation of the charged ligand at the cell surface is an important factor in the overall ligand/receptor interaction. Similar antagonizing effects of poly-L-lysine provide further support for this hypothesis.     

Effect of vanadate on brain protein phosphorylation

The effect of vanadate on the phosphorylation of synaptosomal membrane proteins prepared from rat cerebral cortex was studied. Vanadate concentrations of 10^–6, 10^–5, and 10^–4 M increased the endogenous phosphorylation activity by 25%, 37%, and 75%, respectively. Increasing the ATP concentration in the assay medium from 50 to 500 M did not influence the above effect. A commercial preparation of the purified protein kinase was stimulated 40% by 10^–3 M vanadate. Calcium-calmodulin dependent activity was stimulated only 20% by 10^–5 M vanadate. The effect was not enhanced by further increasing vanadate concentration. Addition of calcium ions (above 50 M) suppressed the vanadate effect, while an inhibition was observed at high Ca²⁺ concentration (2.5 mM). Below 50 M calcium ions stimulated phosphorylation activity in the absence of vanadate and did not affect the stimulatory action of vanadate. Cyclic AMP-dependent endogenous phosphorylation was also stimulated by vanadate. Activation by cAMP could not be observed at vanadate concentrations above 10^–6 M. Possible mechanisms of the vanadate effect are discussed.     

Comparison of structural requirements of α-MSH and ACTH for inducing excessive grooming and pigment dispersion

α-MSH and ACTH-like peptides are known to play an important role in the adaptation of many vertebrates to a new environment. These peptides induce pigment dispersion in amphibian melanophores through a receptor-mediated mechanism. In this study we compared the structural requirements of these peptides for melanotropic activity on Xenopus laevis melanophores with those for inducing excessive grooming in the rat. With the exception of ACTH_1–24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle⁴,-D-Phe⁷]-α-MSH is extremely active in both assays. Weak agonists such as [Leu⁹]-α-MSH did not possess antagonistic properties either in the melanophore assay or in the excessive grooming test. The data suggest that the mechanism of action of α-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores.