The immunomodulating action of microwaves in the induction of an immune response to Vi antigen

In this work the possibility of using microwaves for immunomodulation in the immunization of animals with thymus-independent antigen was studied. The projection zones of the thymus and adrenal glands of the test animals were subjected to the action of decimeter, or ultrahigh frequency (UHF), waves, while the corresponding zones of the control animals were subjected to imitation UHF irradiation. Vi-antigen was shown to be a thymus-independent antigen for rabbits (according to the results of the evaluation of the functional state of thymocytes, as manifested by the adsorption of acridine orange by the chromatin of thymocyte nuclei and by the content of RNA and DNA in the thymus). The action of UHF waves on the projection zone of the thymus was accompanied by a decrease in the glucocorticoid activity of the adrenal cortex, observed simultaneously with a pronounced immunostimulating effect. The UHF irradiation of the zone of the adrenal glands was accompanied by immunosuppression in combination with enhanced glucocorticoid activity of the adrenal cortex.     

Regulation of aldosterone synthesis

The effects of angiotensin II and ACTH on cyclic AMP and aldosterone synthesis were studied in cells isolated from the bovine adrenal cortex. Angiotensin is a more potent stimulus of aldosterone synthesis than ACTH and the action of ACTH on aldosterone synthesis in cells from the glomerulosa is augmented by the presence of cells from the fasciculata. Angiotensin stimulates aldosterone synthesis in the absence of detectable changes in cyclic AMP, but the cells do respond to dibutyryl cyclic AMP leaving open the possibility that a cyclic nucleotide may play a role in the steroidogenic action of this hormone in the outer zone of the bovine adrenal cortex.     

Extra-adrenal glucocorticoids and mineralocorticoids: evidence for local synthesis, regulation, and function

Glucocorticoids and mineralocorticoids are steroid hormones classically thought to be secreted exclusively by the adrenal glands. However, recent evidence has shown that corticosteroids can also be locally synthesized in various other tissues, including primary lymphoid organs, intestine, skin, brain, and possibly heart. Evidence for local synthesis includes detection of steroidogenic enzymes and high local corticosteroid levels, even after adrenalectomy. Local synthesis creates high corticosteroid concentrations in extra-adrenal organs, sometimes much higher than circulating concentrations. Interestingly, local corticosteroid synthesis can be regulated via locally expressed mediators of the hypothalamic-pituitary-adrenal (HPA) axis or renin-angiotensin system (RAS). In some tissues (e.g., skin), these local control pathways might form miniature analogs of the pathways that regulate adrenal corticosteroid production. Locally synthesized glucocorticoids regulate activation of immune cells, while locally synthesized mineralocorticoids regulate blood volume and pressure. The physiological importance of extra-adrenal glucocorticoids and mineralocorticoids has been shown, because inhibition of local synthesis has major effects even in adrenal-intact subjects. In sum, while adrenal secretion of glucocorticoids and mineralocorticoids into the blood coordinates multiple organ systems, local synthesis of corticosteroids results in high spatial specificity of steroid action. Taken together, studies of these five major organ systems challenge the conventional understanding of corticosteroid biosynthesis and function.     

Effect of corticotropin treatment in vivo on the synthesis of a specific adrenal cytosolic protein. Characterization by dual-labelling technique and polyacrylamide-gel electrophoresis.

The effect of corticotropin in vivo on total and specific protein synthesis in the adrenal was studied. Adrenal slices from control and corticotropin-treated animals were incubated with [14C]- and [3H]-leucine respectively, followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of subcellular components. With this sensitive dual-labelling technique the following results were obtained. There was a general trophic effect on most adrenal proteins, but corticotropin produced a marked stimulation of a specific adrenal cytosolic protein. This protein has mol.wt. approx. 30 000 and pI 5.5. Corticotropin increased the incorporation of labelled leucine into proteins within 4 h, but no effect was observed before 2 h and after 16 h there was no further increase. These data suggest that this protein is not involved in the corticosteroidogenic action of corticotropin, but rather in the trophic action of this hormone.     

Steroid regulation of monoamine oxidase activity in the adrenal medulla

Administration of different steroid hormones in vivo has distinct and specific effects on the MAO activity of the adrenal medulla. In an effort to reconstitute these effects in defined cells, we have isolated endothelial cells and chromaffin cells from the bovine adrenal medulla and tested each cell type for sensitivity to these steroids. As in the intact animal, we found that endothelial cell MAO activity was stimulated 1.5- 2.5-fold by 10 microM progesterone, hydrocortisone, and dexamethasone, inhibited by ca. 50% by 17-alpha-estradiol, but unaffected by testosterone. The type of MAO in the endothelial cells was found to be exclusively of the A type. The chromaffin cells had MAO B exclusively and were inert to treatment with dexamethasone. The mode of action of the various steroids on MAO A activity in endothelial cells seemed to be that of affecting the number of MAO molecules, as binding of [3H]pargyline, an MAO inhibitor, changed in proportion to changes in enzyme activity. Consistently, the kinetic parameters for MAO A showed changes in Vmax but not Km under all conditions. The specificity of steroid action on MAO A activity was also supported by the fact that steroid-induced changes in total cell division ([14C]thymidine incorporation) and total protein synthesis ([14C]leucine incorporation) were seen after changes in MAO A. We conclude that the differential effects of steroids on MAO activity in the intact adrenal medulla can be reproduced in cultured adrenal medullary endothelial cells but not in chromaffin cells. Therefore we suggest that the action of these steroid hormones on the intact adrenal medulla may be restricted to the endothelial cell component of this tissue.     

Mechanism of corticotropin action on adrenal protein synthesis. The effects of inhibitors of protein synthesis and calcium chelators

We have recently shown that beside a general stimulation of most adrenal proteins, corticotropin induces a marked increase in a specific adrenal cytosolic protein, protein E, in intact and hypophysectomized rats. To further clarify the mechanisms by which corticotropin exerts its trophic action we have investigated the effects of cycloheximide, calcium and calcium chelator administration on intact and hypophysectomized animals. These substances were injected in rats with or without corticotropin, and slices of adrenal glands from control and treated animals were removed 5 h later, incubated with [14C]- or [3H]-leucine for 2 h, and cytosolic proteins analyzed by polyacrylamide gel electrophoresis using a dual labelling technique. When high doses of cycloheximide (higher than 500 micrograms) were injected in rats, incorporation of labelled leucine in adrenal slices of control and corticotropin-treated animals was inhibited. With 500 micrograms cycloheximide per rat, incorporation of labelled leucine in adrenal slices of control animals was normal, but the corticotropin stimulation of both protein E and total protein synthesis was inhibited. Lower doses of cycloheximide (100 micrograms per rat) completely inhibited the stimulatory effect of corticotropin on total protein synthesis but did not affect protein E synthesis, while after 50 micrograms per rat both stimulatory effects were preserved. The two higher doses of cycloheximide (500 and 100 micrograms per rat) could not completely block the steroidogenic effect of the hormone. The effects of calcium and calcium chelators were studied in 1-day hypophysectomized rats. Calcium alone or injected simultaneously with corticotropin has no effect. Calcium chelators injected simultaneously with corticotropin partially inhibited the stimulatory effects of corticotropin on steroidogenesis but totally inhibited stimulation of total protein synthesis, while the stimulation of protein E persisted. Our results show that after corticotropin, stimulation of protein E synthesis correlates better with steroidogenesis than with total protein synthesis.     

Lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat.

In the adrenal gland of the rat, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme of cholesterol synthesis, is shown to be regulated by cholesteerol carried in plasma lipoproteins. When plasma cholesterol levels were lowered 90% by administration of the drug 4-aminopyrazolopyrimidine, the cholesteryl ester content of the adrenal gland declined by more than 90% and this was associated with a 150- to 200-fold increase in the activity of adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase and a 30-fold increase in cholesterol synthesis from [14C]acetate. The subsequent intravenous infusion of cholesterol contained in either rat or human high density or low density lipoproteins restored the adrenal content of cholesteryl esters and reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase to basal levels. The depletion of adrenal cholesteryl esters and the enhancement in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that occurred in the 4-aminopyrazolopyrimidine-treated rat required the action of adrenocorticotropic hormone (ACTH) since neither was observed when ACTH secretion was blocked by administration of dexamethasone. The current data indicate that the low rate of cholesterol synthesis normally observed in the rat adrenal gland is due to a suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that is mediated by plasma lipoproteins.     

Effect of ethanol on corticosterone production by dispersed adrenal cells of the rat

The action of ethanol on adrenal steroidogenesis "in vitro" has been studied. It has been found that ethanol did not change the basal production of corticosterone by dispersed adrenal cells, but significantly reduced its response to ACTH stimulation. It is suggested that the inhibitory action of ethanol on steroidogenesis "in vitro" could have a physiological meaning, because the response to ACTH stimulation of adrenal cells from rats treated "in vivo" with ethanol showed a clear dose-related inhibition.     

Sex differences in adrenocortical structure and function. III. The effects of postpubertal gonadectomy and gonadal hormone replacement on adrenal cholesterol sidechain cleavage activity and on steroids biosynthesis by rat adrenal homogenates.

In rats postpubertal orchiectomy results in an increase in the adrenal weight, testosterone replacement restores the adrenal weight to the normal level. Neither ovariectomy (8 weeks of duration) nor estradiol replacement has an effect on adrenal weight in female rats. Pregnenolone synthesis as well as corticosterone and blue tetrazolium-positive steroids secretion is significantly higher in homogenates of adrenals from female rats than from males. Orchiectomy results in a marked increase in pregnenolone biosynthesis, testosterone replacement restores the value to the normal levels. Neither ovariectomy nor estradiol replacement has an effect on pregnenolone synthesis in v i t r o. In both sexes gonadectomy causes a marked decrease in corticosterone output by adrenal homogenates, concomitantly the increase in the adrenal 5alpha-reductase activity is observed. The ratio of secreted corticosterone to pregnenolone is significantly lower in gonadectomized rats of both sexes than in control animals. Estradiol or testosterone replacement inhibits the adrenal 5alpha-reductase activity and restores the corticosterone output as well as corticosterone/pregnenolone ratio to the normal values. The above described findings show that the sex differences in steroids secretion by the rat adrenal are partially conditioned by a cholesterol sidechain cleavage activity. Testosterone inhibits this activity while estradiol under applied experimental conditions has no effect on the cholesterol sidechain cleavage activity.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cortisol treatment on brain and adrenal corticotropin-releasing hormone (CRH) content and other parameters regulated by CRH

Corticotropin-releasing hormone (CRH) has been found in both hypothalamic and extrahypothalamic sites of the brain and also in the adrenal medulla. To study the timing and location of delayed glucocorticoid action in rats, we measured the effects of 2-day and 7-day cortisol treatment on immunoreactive CRH concentrations in hypothalamus, cerebral cortex, hippocampus, cerebellum, and adrenal gland. The activity of the hypothalamo-pituitary-adrenal (HPA) axis and the sympathoadrenal system were also measured. Studies were carried out both in the afternoon and/or in the morning, to get information about possible circadian changes. CRH contents were not changed in any brain areas studied, except there was a trend of decrease in the hypothalamus compared to vehicle in the afternoon due to the lack of circadian increase after 7-day cortisol treatment. Pituitary ACTH content decreased significantly after 7-day treatment, while beta-endorphin did not. Plasma levels of ACTH, corticosterone, norepinephrine and epinephrine and adrenal ACTH and beta-endorphin contents decreased after 2-day, adrenal CRH content after 7-day treatment with cortisol. Our findings suggest, that chronic cortisol treatment inhibits the circadian activation of the HPA axis at all levels but has variable effects on baseline measures because it causes different changes in release and synthesis at different sites.     

Immunoglobulin synthesis by free polysomes of mouse myeloma cells

Free polyribosomes isolated from mouse myeloma cells in tissue culture synthesize immunoglobulin chains. The presence of these peptide chains in the cytoplasm of intact myeloma cells has been investigated. Some immunoglobulin chains were observed, but it could not be ruled out that these were originally inside cisternae of the endoplasmic reticulum, which were broken during hogenization. We have also investigated the transport of the hypothetical cytoplastic immunoglobulins into the cisternae of the endoplasmic reticulum after incubation with radioactive amino acids and subsequent chase in the absence of protein synthesis. A model to account for synthesis of immunoglobulins on free polysomes is presented. This model assigns specificity for translation on membrane-bound polysomes to the N-terminal region of secretory proteins.     

N-acylethanolamine--a new class of natural adrenotropic modulators

N-acyl-ethanolamines (NAE) belong to the new class of naturally occurring biologically active regulators. Anandamide--a well known representative of NAEs--is an agonist of central (CB1) and peripheral (CB2) cannabinoid receptors. Adrenal cortex contains the CB2 receptor. The aim of present work is to investigate the influence of saturated and polyunsaturated NAEs on the corticosteroid synthesis in adrenal gland in vitro. It was shown that the rat adrenal gland is the main target of N-([1-14C]palmitoyl)ethanolamine incorporation. Saturated NAE enhanced the incorporation of [3H]-cholesterol into the aldosterone by 25% (p < 0.06) and corticosterone by 17% (p < 0.05) of rat adrenal slices in vitro. Mixture of polyunsaturated NAEs containing mainly 18:1w9, 18:2w6, 18:3w3, 20:1w9, 22:1w9 increased the labeling of aldosterone by 70% (p < 0.05) and corticosterone by 20% (p < 0.05). Thus, the saturated NAEs as well as polyunsaturated analogs, act by the similar manner on the corticosteroid synthesis. The fact that saturated NAEs possess poor affinity to CB receptors provides us opportunity to suggest the non-receptor mechanism of NAEs adrenotrophic action. Further we used the purified bovine serum albumin to test the binding kinetic of N-([I14C]palmitoyl)ethanolamine with its hydrophobic domains. It was found that NAEs have high affinity to hydrophobic domains of protein with K = 2.5 x 10(8) M-1. This finding could support the idea of the existence of putative allosteric modulation of regulatory protein function. It was concluded that NAEs exert a stimulatory effect on the corticosteroid synthesis in rat adrenal gland in vitro and this action do not mediated trough cannabinoid receptor.     

Effect of interleukin-1 on adrenal function in mice

The influence of macrophage secretion product on the functional activity of the adrenal glands has been studied in mice. It has been shown that intravenously injected interleukin-1 caused dose-dependent increase in corticosterone content in the blood plasma. The hormonal response of the adrenal glands to interleukin-1 injection was abolished by the inhibition of prostaglandin synthesis. The results obtained indicate that interleukin-1 stimulates the functional activity of the adrenal glands, with the effect mediated by prostaglandins.     

Involvement of cycloheximide-sensitive mediators in the steroidogenic action of adrenocorticotropin and angiotensin II

The involvement of short-lived proteins in the steroidogenic action of corticotropic peptides has been investigated in vitro by means of a perifusion technique using frog adrenal glands. Graded concentrations of cycloheximide (10(-7) M to 10(-5) M) led to a dose-related inhibition of corticosterone and aldosterone production. The perifusion model gives detailed information on the kinetics of the inhibitory effect of cycloheximide. This effect was rapidly observed (the lag period was about 15 min), maximum inhibition being obtained 25 min after the end of administration of the protein synthesis inhibitor. Whatever the concentration of cycloheximide, corticosteroid output returned to basal values 2 h after the onset of cycloheximide infusion. Stimulation of steroidogenesis by ACTH and angiotensin II was totally inhibited by cycloheximide (10(-6) M) indicating that the synthesis of a labile protein was required for the adrenal response to corticotropic peptides. In addition, the stimulatory effect of cAMP and PGE1, which are considered to be the second messengers of ACTH and angiotensin II in amphibian interrenal gland, was blocked by cycloheximide. Taken together, these data suggest that a labile protein is involved in an early step of corticosteroid biosynthesis in the frog.     

Effects of adenosine 3':5'-cyclic monophosphate and guanosine 3':5'-cyclic monophosphate on 3H-uridine incorporation into glomerulosal cells of hypophysectomized rat adrenal cortex.

The effects of cyclic AMP and cyclic GMP on the incorporation of 3H-uridine into glomerulosal cells of hypophysectomized rats were investigated by high resolution autoradiography. The quantitative analysis of autoradiographs shows that cyclic nucleotides, like ACTH, enhance the tracer incorporation into both nuclear and mitochondrial compartments. These findings are discussed in relation to previous results indicating that both cyclic nucleotides function as intracellular mediators of the trophic action of ACTH on the rat adrenal zona glomerulosa. The hypothesis that the mechanism of the glomerulotrophic action of ACTH and cyclic nucleotides involves the stimulation of nuclear and mitochondrial RNA synthesis is discussed.     

Induction and suppression of immunoglobulin synthesis in cultures of human lymphocytes: effects of pokeweed mitogen and Staphylococcus aureus Cowan I

The synthesis and secretion of immunoglobulins by human peripheral blood mononuclear cells in cultures stimulated with pokeweed mitogen or Staphylococcus aureus Cowan I were evaluated by enumeration of cells containing cytoplasmic immunoglobulins and cells actively secreting immunoglobulins, and by quantitation of immunoglobulins released into culture supernatants. The two mitogens caused comparable stimulation of immunoglobulin production; however, in contrast to pokeweed mitogen, S. aureus was active in cultures depleted of T lymphocytes, and its stimulatory effects were resistant to the influence of suppressor T cells generated by co-stimulation with concanavalin A or by preincubation without mitogenic stimulus. These results indicate distinct pathways of induction and suppression of immunoglobulin synthesis for these two polyclonal B cell activators, and suggest that stimulation by S. aureus is less thymus dependent than that induced by pokeweed mitogen.     

Influence of alcohol on peripheral neurotransmitter function

Presently available evidence for the action of ethanol on the peripheral noradrenergic system is reviewed. Acute ethanol treatment produces an initial elevation in catecholamine levels in blood, and in the excretion of catecholamines, which may be followed by a decrease in catecholamine release. Correspondingly, a hyposensitivity of peripheral beta receptors has been noted. Other tissues such as vascular smooth muscles show a biphasic response to ethanol: a hypersensitivity to norepinephrine at low concentrations of ethanol, and hyposensitivity to norepinephrine at high ethanol concentrations. In the adrenal medulla the activity of various enzymes involved in catecholamine synthesis is increased by ethanol treatment. Chronic treatment with ethanol does not alter endogenous norepinephrine levels in heart, but does increase its turnover. Chronic treatment also lowers the number of beta-adrenergic receptors in the heart, and decreases the response to peripherally administered isoproterenol, which suggests a hyposensitivity of peripheral beta receptors. Tolerance may develop to the chronic effects of ethanol in the heart and the vas deferens, but in the adrenals the activity of enzymes for catecholamine synthesis remains elevated with chronic treatment. During withdrawal, there is generalized hyperexcitability of the sympathetic nervous system. Norepinephrine and amine turnover in the heart is increased, as is the activity of adrenal medullary enzymes involved in catecholamine synthesis.     

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.