Chemical sympathectomy attenuates lipopolysaccharide-induced increase of plasma cytokine levels in rats pretreated by ACTH.

The sympathetic nervous system participates significantly in the regulation of immune functions. In support of this, data indicate that besides vagal afferent and efferent pathway, sympathetic nerves represent crucial component of inflammatory reflex. In addition, it was shown that efferent arm of this reflex might be activated by ACTH. Therefore, we investigated the effect of chemical sympathectomy on lipopolysaccharide (LPS)-induced increases in plasma IL-1ß, IL-6, and TNF-α levels in rats. Plasma IL-10 and corticosterone levels were also evaluated. We also investigated the effect of sympathectomy in rats pretreated with ACTH (1-24). We found that sympathectomy significantly attenuated LPS-induced increases of plasma IL-1ß levels. Administration of ACTH (1-24) reduced LPS-induced increases of plasma IL-1ß and IL-6 and exaggerated the rise of IL-10. In animals treated with ACTH (1-24) sympathectomy attenuated LPS-induced increases of IL-1ß, IL-6, and IL-10 plasma levels. Plasma levels of TNF-α and corticosterone were not affected by any interventions. These data indicate that during acute immune challenge, sympathetic nerves stimulate the immune response. In addition, our data indicate that sympathetic nerves are not significantly involved in the anti-inflammatory effect of ACTH (1-24) and that the anti-inflammatory effect of ACTH (1-24) is independent of plasma corticosterone levels.

Investigation of the origin of prednisolone in cow urine.

After a two-year period of the frequent detection of prednisolone-positive bovine urine samples in the Italian region of Lombardy, studies were initiated to investigate the source. Because the majority of positive samples were detected at the slaughterhouse, researchers hypothesised that, together with increased cortisol and cortisone, a small quantity of prednisolone could be produced by the cows in stressful situations. In the present study, three dairy cows underwent intramuscular treatments with tetracosactide hexaacetate, a synthetic analogue of adrenocorticotropic hormone, to simulate stress. The animals were slaughtered at the end of the study. The results indicated that prednisolone could be detected occasionally in the non-stressful state, but was consistently found in the urine of stressed cows (concentrations ranged from 1.01 to 4.08 ng/mL). To confirm the stress condition, urinary cortisol and cortisone were also detected at high concentrations in the urine, typically at concentrations of hundreds of nanograms per millilitre. The results of this preliminary study did not reveal the metabolic pathway responsible for prednisolone but suggested that this corticosteroid could be produced endogenously.

Immune challenge induces differential corticosterone and interleukin-6 responsiveness in rats bred for extremes in anxiety-related behavior.

Disturbances in mood such as anxiety and depression are often associated with altered hypothalamo-pituitary-adrenal (HPA) axis reactivity, but also with changes in cytokine production, such as interleukin-6 (IL-6), an essential immune factor produced by macrophages and lymphocytes during inflammatory processes. The reciprocal relationship between the HPA axis and the immune system is now well established. In order to understand better the endocrine reactivity of anxious individuals faced with an immune challenge, a model of innate anxiety-related behavior, HAB and LAB rats (HABs, high and LABs, low anxiety-related behavior) was used in this study. We sought to determine whether injection of lipopolysaccharide (LPS) induced a differential HPA axis reactivity and plasma IL-6 release in HABs and LABs. After LPS injection, the plasma adrenal corticotrophic hormone increase did not differ between HABs and LABs, whereas a larger increase in plasma corticosterone levels occurred in HABs than in LABs at 2 h after injection. Moreover, basal IL-6 levels were lower in HABs than in LABs, leading to a higher IL-6 2 h/basal ratio in HABs. In conclusion, we propose for the first time a link between the endocrine and immune systems of HABs and LABs and suggest that IL-6 could be a neuroendocrine correlate of trait anxiety in HABs.

Influence of ACTH-(1-24) and plasma hyperviscosity on free radical production and capillary perfusion after hemorrhagic shock.

OBJECTIVE: The authors investigated the effects of ACTH-(1-24) and a high-viscosity solution in the restoration of microvascular function during resuscitation. They injected NG-monomethyl-L-arginine (L-NMMA) and superoxide dismutase (SOD) before ACTH-(1-24) in hamsters resuscitated with the hyperviscous solution to determine the role of ROS and NO in ACTH-(1-24) protective mechanism in the cheek pouch. Hemorrhagic shock (HS) was induced by withdrawing blood to reduce mean arterial pressure (MAP) to 30 mm Hg for 45 min. METHODS: Animals were injected with ACTH-(1-24) and resuscitated with dextran of low molecular weight (70 kDa) and a small amount (4%) of dextran of high molecular weight (500 kDa) plus ACTH-(1-24), or autologous (shed) blood withdrawn during HS. Microvascular effects were characterized by measuring blood flow, perfused capillary length (PCL), arteriolar diameter, and red blood cell (RBC) velocity. ROS were assayed at the beginning and after 45 min of HS and after 10 and 90 min of resuscitation. RESULTS: Resuscitation with either shed blood or dextrans 70/500 resulted in the restoration of MAP, whereas PCL, RBC velocity, and arterial diameter decreased significantly. ROS increased significantly after HS, 10 and 45 min of resuscitation. ACTH-(1-24) plus dextrans 70/500 increased MAP immediately; it increased vasodilation and PCL, and attenuated significantly ROS production and leukocyte adhesion during resuscitation. L-NMMA injected after 30 min of HS did not change the protection exerted by ACTH-(1-24) and dextrans 70/500, while SOD increased their protective effects. CONCLUSIONS: ACTH-(1-24) appears to enhance the protective effects on the endothelium exerted by increased plasma viscosity by significantly decreasing the oxidative stress and the leukocyte adhesion during resuscitation.

Altered pituitary-adrenal axis responses to provocative challenge tests in adult survivors of childhood abuse.

OBJECTIVE: Early adverse life events may predispose individuals to the development of mood and anxiety disorders in adulthood, perhaps by inducing persistent changes in corticotropin-releasing factor (CRF) neuronal systems. The present study sought to evaluate pituitary-adrenal responses to standard hypothalamic-pituitary-adrenal axis challenge tests in adult female survivors of childhood abuse with and without major depressive disorder. METHOD: Plasma ACTH and cortisol responses to the administration of 1 microg/kg ovine CRF and plasma cortisol responses to the administration of 250 microg ACTH(1-24) were measured in healthy women without early life stress (N=20), women with childhood abuse without major depressive disorder (N=20), women with childhood abuse and major depressive disorder (N=15), and women with major depression but no early life stress (N=11). RESULTS: Abused women without major depressive disorder exhibited greater than usual ACTH responses to CRF administration, whereas abused women with major depressive disorder and depressed women without early life stress demonstrated blunted ACTH responses. In the ACTH(1-24) stimulation test, abused women without major depressive disorder exhibited lower baseline and stimulated plasma cortisol concentrations. Abused women with comorbid depression more often suffered from posttraumatic stress disorder and reported more recent life stress than abused women without major depressive disorder. CONCLUSIONS: These findings suggest sensitization of the anterior pituitary and counterregulative adaptation of the adrenal cortex in abused women without major depressive disorder. On subsequent stress exposure, women with a history of childhood abuse may hypersecrete CRF, resulting in down-regulation of adenohypophyseal CRF receptors and symptoms of depression and anxiety.

Further study of aldosterone secretion-inhibitory factor and brain natriuretic peptide on cortisol production of guinea pig zona fasciculata cells.

The suppressive effect of aldosterone secretion-inhibitory factor (ASIF) and brain natriuretic peptide (BNP-32) on the basal and ACTH-stimulated cortisol production in a primary culture enriched with guinea pig Zona Fasciculata (ZF) cells was further studied. The binding of 125I-labeled ACTH(1-24) and ASIF to ZF cells was found to be displaced by ACTH(1-24), [Phe2, Nle4 and Ala24]-ACTH(1-24), ASIF, and BNP in a concentration-dependent manner. The binding of 125I-labeled [Phe2, Nle4 and Ala24]-ACTH(1-24) to two transformed clones of mammalian cells expressing the guinea pig ACTH receptor was also competitively inhibited by ASIF and BNP. ASIF and BNP significantly suppressed ACTH-stimulated cAMP production in ZF cells. The 10- and 30-min cellular changes in cAMP induced by ASIF and BNP did not correlate in the rank order with the ultimate magnitude of cortisol suppression observed in ZF cells after a 24-hour treatment with these peptides. Nevertheless, the results did conform to the signaling mechanism of their action. Overall, the findings clearly demonstrated that ASIF and BNP suppressed the adrenocortical function and inhibited ACTH for their antagonistic action against ACTH primarily at the ACTH receptor site. These results support the notion that a physiological role of adrenal medulla in regulating the adrenocortical function may be mediated by the neuropeptides through a paracrine pathway.

Adrenocortical ACTH receptors in pigs of differing in vivo response to adrenocorticotropin.

1. Adrenocortical membrane protein was isolated from the adrenal glands of 12 Large White x Landrace male pigs, six with high adrenocortical response to adrenocorticotropic hormone (ACTH) and six with low response. 2. The peptide (Phe2, Nle4) ACTH was iodinated by the chloramine-T method and served as the radioligand in receptor binding studies. 3. Only one class of ACTH receptor was detected, with Kd = 2.57 +/- 0.35 x 10(9) M and Bmax = 1.59 +/- 0.06 pmol/mg protein in high responders and Kd = 1.68 +/- 0.18 x 10(-9) M and Bmax = 1.17 +/- 0.11 pmol/mg protein in low responders. 4. The difference in the Bmax between high and low responders was significant (P < 0.05), the difference in Kd was not statistically significant.

The release of corticosterone and a corticosterone-binding protein by incubated rat adrenal slices.

Stimulation of incubated rat adrenal slices with ACTH(1-24) resulted in an increase in the release of both corticosterone and specific corticosterone-binding protein into the incubation medium. The release of corticosterone and binding protein was dose and calcium dependent with adrenals from animals pretreated with betamethasone. While the secretion of corticosterone was continuous throughout the incubation period, there appeared to be a limit to the increase in binding capacity. The specificity of steroid binding to the adrenal protein showed a similar profile to that of corticosteroid-binding globulin (CBG) in rat serum. A Western blot analysis using anti-rat CBG as the primary antiserum, showed that the adrenal protein was not CBG. [3H]corticosterone binding with disc electrophoresis, run at 2 degrees C, gave a single peak with approximately the same Rf value for rat serum, purified CBG, and adrenal incubate; at 22 degrees C peaks were only seen for rat serum or purified CBG. The data presented provides further evidence for the existence of a specific corticosterone-binding protein of adrenal origin released in conjunction with corticosterone. The adrenal protein would appear to have a lower affinity for corticosterone than does CBG, and to be functionally more labile. It is possible that the adrenal protein may be CBG that has been internalized, modified and released with corticosterone.

A monoclonal anti-peptide antibody mimics adrenocorticotropic hormone activity.

A monoclonal antibody that specifically recognizes the adrenocorticotropic receptor (ACTH) on rat adrenal cells was tested for hormonal activity. The antibody behaved as an agonist based on three different biological activities associated with ACTH. An antibody concentration of 16 micrograms/ml stimulated isolated rat adrenal cells to secrete 800 ng/10(4) cells of corticosterone with a concomitant 10-fold increase of cAMP to 30 pmol/10(5) cells. Antibody concentrations above 16 micrograms/ml inhibited mitotic activity in mouse Y-1 adrenal cells. A radio-immunoassay using an anti-ACTH antibody showed that the monoclonal anti-adrenocorticotropic receptor antibody and ACTH are antigenically related. These findings suggest that the anti-receptor antibody recognizes the ligand binding domain of the ACTH receptor.

Adrenocorticotropin binding sites in rat cardiac tissue.

Using the ACTH analog [125I-Tyr23,Phe2,Nle4] ACTH(1-24), the existence of specific binding sites for ACTH in atrial membrane preparations was demonstrated. The dissociation constants (Kd), determined by Scatchard analysis were not significantly different for membrane preparations of adrenal gland or atrial tissue (being 6.40 x 10(-12)M and 8.86 x 10(-12)M respectively). No binding was observed to membrane preparations from kidney or lung. While the binding of the ACTH(1-24) analog to atrial membranes was inhibited by ACTH(1-24), it was not affected by norepinephrine or epinephrine. It was proposed that the ACTH(1-24) analog may bind to sites located on the adrenergic nerve endings associated with the cardiac tissue, and that such binding would interfere with the neuronal reuptake of the catecholamines.

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain.

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropin receptors in rat adrenal glomerulosa cells.

The results presented here demonstrate, for the first time, the presence of ACTH receptors in the zona glomerulosa of adrenal glands. We obtained the surprising result that the glomerulosa cells carry a higher concentration of ACTH receptors than the fasciculata cells. The analog [Phe2,Nle4]ACTH was iodinated by the iodogen method and separated by HPLC; it was obtained carrier-free and has a specific activity of 600 muCi/micrograms, retaining full biological potency. After 30 min of incubation at 22 C for concentrations of 2 X 10(-11) M [125I]ACTH, specific binding values were 4.85 +/- 0.44% (n = 15) and 1.85 +/- 0.18% (n = 15), respectively, for 50,000 glomerulosa or fasciculata cells. For the glomerulosa, our results indicated a density of 6.5 X 10(4), receptors/cell of the high affinity type (Kd1 = 7.6 X 10(-11) M) and 1.0 X 10(6) receptors of the low affinity type (Kd2 = 1.2 X 10(-9) M). In the zona fasciculata, we found 7.2 X 10(3) receptors of high affinity (Kd1 = 1.1 X 10(-11) M) per cell and 6.3 X 10(5) of low affinity (Kd2 = 2.9 X 10(-9) M). The dissociation constant for the high affinity site of the glomerulosa cells was in excellent correlation with the half-maximal stimulation dose of ACTH for aldosterone and corticosterone (Kd1 = 7.6 X 10(-11) M vs. ED50 of 8 X 10(-11) and 3 X 10(-11) M). Results from primary cultures showed a decrease in binding capacity after 1 day in culture and then an increase to the initial value after 3 days in culture.

[7-Methyltryptophan9]-beta-corticotropin-(1-24): a hyperactive Synacthen analogue.

[7-Methyltryptophan9]-beta-corticotropin-(1-24) has been synthesised. In an isolated adrenal cell bioassay, it has 2.7 times the steroidogenic activity of beta-corticotropin-(1-24) (Synacthen).

[Experience using sinakten-retard in the treatment of muscular sclerosis]. / Opyt primeniia sinakten-retard dlia lecheniia rasseiannogo skleroza.

The experience of the authors demonstrated that the best results in sinakten-retard treatment were attained in patients with mild and moderate degrees of severity of the disease. However, even in patients with a protracted development, treatment by sinakten-retard increased the muscular strength in the paralyzed extremities, diminished the increased muscular tone and increased the volume of movements. All these facts permit the authors to recommend sinakten-retard for the treatment of disseminated sclerosis.

Differences between in-vitro and in-vivo potencies of corticotrophins: an interpretation in terms of metabolic stability.

Relative activities of a series of corticotrophin analogues have been measured by means of five different bioassays using the rat. Similarities in the relative potencies of various ACTH analogues determined using lipolysis or steroidogenesis in vivo and for the lipolytic and steroidogenic responses of fat pads and adrenal slices in vitro emerged and support the concept of a close structural relationship between the ACTH receptors in adipose and adrenal tissues in the rat. Potencies based on the steroidogenic response of isolated adrenal cells, adrenal slices or in-vivo experiments differed markedly from each other. Inactivation of peptides did not occur in the isolated cell assay, so it is likely that this assay estimates potency at the receptor level. A number of arguments suggest that the difference between the isolated cell assay and the other steroidogenic assays lies solely in the effects of peptide inactivation in the latter, and this allows the relative metabolic stabilities for the peptide analogues in these assays to be calculated. In this way it can be shown that: (1) Replacement of L-Ser by D-Ser in amino acid position 1 markedly increases the metabolic stability of the peptide and has only a slight effect on receptor properties. (2) Shortening at the NH2-terminus reduces the activity of peptides at the receptor level by several orders of magnitude, but increases their relative metabolic stability. (3) Introduction of amide groups at the CO2H-terminus markedly increases receptor potency of (1-16), (1-17) and (1-18) ACTH without affecting their metabolic stability in vivo. However, amidation of the CO2H-terminus does have a large effect on metabolic stability in the adrenal slice assay. (4) Replacement of Arg by Lys in positions 17 and 18 of (1-18) ACTH increases potency at the receptor level (adrenal cells) but has little effect on metabolic stability. The comparison of potencies obtained in the various assays, therefore, throws light on the significance of each assay. In addition, the effects of structural modification of analogues can be separately evaluated with respect to the metabolic stability of a peptide and its potency at the receptor level.

Adverse reactions to Zn1-24 ACTH therapy associated with specific cellular immunity.

Lymphocyte transformation to 1-24ACTH, as assessed by the incorporation of tritiated thymidine, has been demonstrated to be associated with severe adverse reactions occurring in patients receiving a Zn-linked 1-24ACTH preparation (Tetra cosactrin depot, 'Synacthen'). Antibodies measured with an isotope-binding assay occurred commonly in all patients receiving therapy and did not correlate with adverse reactions. Lymphocyte transformation with the 1-24ACTH polypeptide, a part of the naturally occurring ACTH molecule, has not been previously recorded. The significance of antibodies and cell-mediated immunity to this polypeptide hormone is discussed.