Inhibition of PMA-induced, LFA-1-dependent lymphocyte aggregation by ADP ribosylation of the small molecular weight GTP binding protein, rho.

Botulinum C3 exoenzyme specifically ADP-ribosylates a group of ras-related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32P]ADP-ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADP-ribosylated in situ in a time- and concentration-dependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-1-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzyme-induced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells.

Regulation of human corticotropin-releasing hormone gene expression by 3',5'-cyclic adenosine monophosphate in a transformed mouse corticotroph cell line.

In order to characterize potential mechanisms regulating the expression of the human CRH (hCRH) gene, an intact genomic fragment including 5'-flanking sequence of the hCRH gene was stably transfected into the mouse corticotroph AtT20 cell line. The exogenous hCRH gene was expressed at a high frequency with accurate and efficient transcription in transformed cells. Northern blot analysis revealed a single species of CRH mRNA of 1.6 kilobases which was identical in size to human placental CRH mRNA. S1 analysis demonstrated a single cap site in both placenta and transformed AtT20 cells, corresponding to a site 23 base pairs downstream of the TATA box. Treatment with 8-bromo cAMP and phorbol ester resulted in a dose-dependent increase in CRH secretion during a 1-h incubation. Treatment with forskolin or 8-bromo-cAMP also produced a dose-dependent 4- to 10-fold increase in CRH mRNA levels, which was rapid (1 h) and sustained (6, 12, 24, and 48 h). These effects in a well characterized continuous cell culture system demonstrate pretranslational regulation of CRH expression by a cAMP-dependent pathway.

Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man.

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.

Influence of sex steroid hormones on rat growth hormone-releasing factor and somatostatin in dispersed pituitary cells.

The modulatory effects of glucocorticoid and sex steroid hormones on the effects of rat GH-releasing factor (GRF) and somatostatin (SRIF) on GH release and biosynthesis were studied in monolayer cultures of rat anterior pituitary cells with RIA and quantitative immunoprecipitation methods. Dexamethasone (10(-7) M), a potent synthetic glucocorticoid, increased both the sensitivity and maximum response of GH release stimulated by GRF. Progesterone (10(-7) M) also enhanced GH release stimulated by GRF. The stimulatory effects of dexamethasone and progesterone were dose dependent and required a latent period of at least 24 h to be evident. Testosterone, dihydrotestosterone, and 17 beta-estradiol showed no apparent influence on GRF-induced GH release under the same conditions. None of the hormones studied showed significant influences on basal or SRIF-suppressed GH release. Progesterone added to the maximally effective concentrations of dexamethasone had no additional effects on GRF-induced GH release. The effect of progesterone was attenuated by both 5 alpha-dihydronorethindrone, a progesterone antagonist and 17 alpha-methyltestosterone, a glucocorticoid antagonist. In terms of GH synthesis, stimulatory effects of GRF on GH synthesis were apparent only when pituitary cells were pretreated with dexamethasone. These results indicate that: pretreatment with glucocorticoid or progesterone enhances the effects of GRF on GH release and/or synthesis; these two steroids share at least one common step to enhance GRF effects; and steroid hormones have little influence on basal or SRIF-suppressed GH release.

Effects of rat growth hormone (rGH)-releasing factor and somatostatin on the release and synthesis of rGH in dispersed pituitary cells.

The effects of rat hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) on the release and biosynthesis of rat GH were studied by RIA and quantitative immunoprecipitation using monolayer cultures of rat anterior pituitary cells. In kinetic studies, GRF stimulation of GH release appeared at the first sampling time (20-min incubation) and the effect began to diminish after 2-h incubation with GRF. On the other hand, total (cell plus medium) content of GH significantly increased only after 24-h incubation. To examine the GH-synthesizing effect of GRF more directly, newly synthesized GH labeled by [35S]methionine during incubation with GRF was quantified by immunoprecipitation. The amount of immunoprecipitable GH increased significantly and specifically (compared with the total amount of labeled proteins) also only after 24-h incubation. When GH pools were labeled with [35S]methionine under different schedules, the basal release of newly synthesized GH, which was labeled for 1 h immediately before chase incubation was lower during the first 15 min than stored GH which had been labeled earlier. Basal newly synthesized GH secretion exceeded stored GH secretion after 30 min. GRF stimulated the release of GH from both pools but the stimulation of stored GH was greater. In this system, SRIF suppressed both the basal and stimulated release of GH but did not modify GH biosynthesis under either condition. Newly synthesized GH showed significant degradation during 24-h incubation; neither GRF nor SRIF affected the rate of GH degradation during the same incubation period. These results indicate that 1) GRF stimulates both release and synthesis of GH; 2) these two effects have different kinetics and different sensitivities to SRIF; and 3) GRF stimulates the release of GH from heterogeneous pools disproportionally.

Distribution and characterization of immunoreactive corticostatin in the hypothalamic-pituitary-adrenal axis.

Using an antiserum against synthetic rabbit corticostatin-1 (CS-1), we established a specific RIA for CS-1 and examined its distribution in various tissues, including the hypothalamic-pituitary-adrenal axis. Among the tissues examined, the highest levels of CS-1-like immunoreactivity (-LI) were found in the lung and spleen. CS-1-LI was also detected at relatively high levels in the pituitary, adrenal medulla, and small intestine, while it was barely detectable in the hypothalamus. Immunocytochemical studies revealed the widespread distribution of CS-1 in these tissues. Plasma CS-1 levels averaged 7.8 ng/ml and increased to 185.4 ng/ml in the presence of infection. CS-1-LI in the adrenal gland, small intestine, and hypothalamus also increased in rabbits with active inflammation. These data suggest that CS-1 may modify the hypothalamic-pituitary-adrenal axis in an endocrine or paracrine manner in response to infection.

Roles of interleukin-1 alpha and -1 beta in endotoxin-induced suppression of plasma gonadotropin levels in rats.

Using specific antagonists to rat interleukin (IL)-1 alpha and IL-1 beta, the roles of these IL-1s in endotoxin-induced suppression of plasma gonadotropin levels in freely-moving rats were studied. In orchiectomized rats, recombinant rat IL-1 alpha and IL-1 beta administered into the lateral ventricles almost equipotently suppressed plasma LH levels. Twenty five micrograms of bacterial endotoxin or lipopolysaccharide (LPS) administered similarly showed a comparable effect as that of 1 microgram IL-1 alpha or IL-1 beta, and completely lowered plasma LH levels by 60 min after the injection. To examine the roles of endogenous IL-1 alpha and IL-1 beta, anti-rat IL-1 alpha antiserum (anti-IL-1 alpha) and a recombinant human IL-1 receptor antagonist (IL-1ra) were used as specific blockers for IL-1 alpha and IL-1 beta, respectively. Anti-IL-1 alpha (10 microliters) or IL-1ra (10 micrograms) administered intracerebroventricularly (icv) with 25 micrograms LPS, significantly attenuated the LPS-induced effect on plasma LH levels during the first 60 min after LPS infusion, but not during the second 60 min. LPS at a dose of 5 micrograms induced smaller but still significant changes in plasma LH levels compared with 25 micrograms LPS or 1 microgram IL-1 beta. IL-1ra (10 micrograms) completely blocked LH suppression induced by 1 microgram IL-1 beta, but did not completely reverse the changes of LH induced by 5 micrograms LPS. IL-1ra injected iv also significantly attenuated the early suppressive effect of iv administered LPS, but not its late effect on plasma LH levels. However, iv administered IL-1ra had no influence on the effects of icv administered LPS. These data indicate that at least a part of plasma LH suppression caused by icv administered LPS is mediated via IL-1 alpha and IL-1 beta synthesized within the brain, while factor(s) other than IL-1 also participate in the LPS-induced change, particularly during the later period. A similar mechanism may also work peripherally in the case of iv administered LPS-induced plasma LH suppression.

Bacterial lipopolysaccharide-induced expression of interleukin-6 messenger ribonucleic acid in the rat hypothalamus, pituitary, adrenal gland, and spleen.

Whereas the stimulatory effect of interleukin-6 (IL-6) on the hypothalamic-pituitary-adrenal (HPA) axis is well established, its mode of action in this axis has yet to be fully elucidated. To further study the role of IL-6 in the HPA axis, we compared the expression of IL-6 messenger RNA (mRNA) in the rat hypothalamus, pituitary, and adrenal gland with that in the spleen after ip or intracerebroventricular (icv) administration of bacterial lipopolysaccharide (LPS). After either ip or icv administration, LPS induced the expression of IL-6 mRNA, which consists of 1.2 kilobases (kb) and 2.4 kb subclasses, in all these tissues of the HPA axis as well as in the spleen. Although we used 100 times less amount of LPS for the icv administration than that used for ip LPS, plasma ACTH levels in both the conditions rapidly reached comparable levels. This icv dose induced IL-6 mRNA expression in the hypothalamus faster than ip dose but also stimulated IL-6 mRNA expression in the hypothalamus, pituitary, and adrenal gland more effectively and smoothly than the ip LPS dose did. Northern blot analysis revealed that in the hypothalamus, pituitary, and adrenals, the predominant subclass of IL-6 mRNA was not 1.2 kb but 2.4 kb. In contrast, this subclass was the minor component in the spleen induced under the same circumstances. These findings indicate that IL-6-synthesizing cells in the HPA axis differ in character from those in the spleen, and that LPS applied in vivo may modulate IL-6 expression in these cells directly and/or indirectly through secondarily activated functions in the neuronal or endocrine systems.

Prostaglandin-dependent in vitro stimulation of adrenocortical steroidogenesis by interleukins.

The effects of interleukins on adrenal steroidogenesis and their mode of action were studied using cultured rat adrenal cells. The addition of rat interleukin-1 alpha (IL-1 alpha) or rat IL-2 increased corticosterone levels in the medium in a concentration-dependent manner during 24 h of incubation. The minimum, half-maximum, and maximum effective concentrations of both rat IL-1 alpha and rat IL-2 were almost same (approximately 3, 10, and 100 U/ml, respectively). After a latent period, the effect became apparent after 12 h of incubation. Human IL-1 beta and human IL-6 also showed a stimulatory effect on corticosterone production, whereas human IL-2 was inactive in this system. To clarify the cellular mechanism of these stimulatory effects, we measured the levels of prostaglandin E2 (PGE2) and cAMP in the cells and media as well as the corticosterone levels. Corticosterone production stimulated by IL-1 alpha or IL-2 was accompanied by intracellular and extracellular cAMP and PGE2 accumulation. Although the stimulation of both cAMP and corticosterone was observed only after 12 h of incubation, PGE2 levels increased during the first 4 h of incubation. Corticosterone, cAMP, and PGE2 production stimulated by ILs was almost completely blocked by the addition of 0.1 mM aspirin, a cyclooxygenase inhibitor. Lipoxygenase inhibitors, i.e. AA-861, nordihydroguaiaretic acid, and 5,8,11,14-eicosatetrynoic acid, did not abolish corticosterone production stimulated by ILs. Submaximal doses of IL-1 alpha and IL-2 synergistically stimulated PGE2 production, but did not have even additional effects on cAMP and corticosterone levels. On the other hand, submaximal doses of ACTH, which did not significantly affect PGE2 levels, acted synergistically with IL to increase cAMP and corticosterone levels in these cells. These results indicate that 1) IL-1 alpha and IL-2 directly stimulate glucocorticoid synthesis in a dose- and time-dependent manner; 2) a half-maximum effective concentration of ACTH acts synergistically with IL in stimulating glucocorticoidogenesis; 3) the stimulatory process initially requires PGs, followed by the activation of the adenylate cyclase system; 4) although the profiles of steroidogenic action of IL-1 alpha and IL-2 are quite similar, they may exert their effects through different mechanisms in their early steps of PGE2 production; and 5) the low effective concentrations of both cytokines suggest possible physiological or pathophysiological roles of circulating cytokines in the glucocorticoidogenesis under certain conditions.

Substantial rise of plasma beta-endorphin levels after insulin-induced hypoglycemia in human subjects.

To elucidate whether insulin-induced hypoglycemia enhances the release of beta-endorphin in man, plasma extracts obtained from healthy subjects and patients with Graves' disease before and 45 min after insulin injection were subjected to gel chromatography, and the fractions obtained were measured by RIA for beta-endorphin. In four healthy subjects, basal plasma beta-endorphin levels were less than 3 to 3.1 pg/ml, and the levels rose substantially to 47.5 +/- 12.4 pg/ml (mean +/- SE) 45 min after insulin injection. Basal plasma beta-endorphin levels in three hyperthyroid patinets (less than 3 to 3.8 pg/ml) did not seem to be different from those in healthy subjects; however, the rise after insulin injection tended to be higher in cases of hyperthyroidism, with a peak value of 68.5 +/- 9.7 pg/ml. Plasma beta-lipotropin and ACTH levels also rose in parallel with beta-endorphin in response to insulin-induced hypoglycemia in both healthy subjects and hyperthyroid patients. It would thus appear that beta-endorphin, like ACTH or beta-lipotropin, is released in human subjects by hypoglycemic stress.

Biphasic changes in hypothalamo-pituitary-adrenal function during the early recovery period after major abdominal surgery.

Regulatory mechanisms of the hypothalamo-pituitary-adrenal (H-P-A) axis during and after major abdominal surgery were studied in a group of patients who underwent upper abdominal surgery. We first examined the general profile of the changes of the H-P-A axis from the day before surgery to the seventh day after surgery. On the day of surgery, plasma levels of CRH, ACTH, and cortisol were all significantly elevated after skin incision (phase I). During the next 2 days, plasma cortisol levels remained significantly elevated, and the both plasma CRH and ACTH levels were suppressed below the control levels obtained on the day before surgery (phase II). Several additional studies, carried out to analyze the mechanism that maintains the high plasma cortisol levels, revealed the following features of the H-P-A axis during phase II. Plasma free cortisol levels in this phase were higher than those during the preoperative period. The exogenously administered hydrocortisone clearance rate in phase II did not differ from that observed on the day before surgery. Dexamethasone administration resulted in a decrease in plasma cortisol levels similar to that observed preoperatively. Conversely, the ACTH-stimulated cortisol increase was significantly greater in phase II than that observed preoperatively. These results suggest that during and after major surgical stress, the H-P-A axis undergoes a biphasic change in the pattern of the stress response and during the second phase, not the continuous hypothalamo-pituitary drive but the increased adrenal responsiveness to ACTH is responsible at least in part for maintaining the elevated plasma cortisol level.

Evidence for gamma-MSH-like immunoreactivity in ectopic ACTH-producing tumors.

Using a specific radioimmunoassay for gamma-MSH, a predicted peptide in the cryptic N-terminal portion of the adrenocorticotropin-beta-lipotropin precursor, gamma-MSH-like immunoreactivity (gamma-MLI) was detected in two ectopic ACTH producing tumors. Gel chromatographic studies on Bio-Gel P-60 revealed one or two peaks of gamma-MLI; one was eluted near th elution position of beta-LPH, compatible with gamma-MLI in human pituitary and the other emerged near the position of beta-endorphin. These results indicate that ectopic ACTH-producing tumors eleborate not only ACTH, beta-endorphin but also gamma-MLI.

Effects of repetitive administration of corticotropin-releasing hormone combined with lysine vasopressin on plasma adrenocorticotropin and cortisol levels in secondary adrenocortical insufficiency.

To examine the functioning of the hypothalamo-pituitary-adrenocortical axis in secondary adrenocortical insufficiency, we administered 100 micrograms synthetic human CRH, iv, plus 10 U lysine-8-vasopressin (LVP), im, three times daily for 3 consecutive days. The changes in plasma ACTH and cortisol levels during the administration and the response to an insulin tolerance test (ITT) conducted before and after the administration were determined. In three patients with isolated ACTH deficiency, basal plasma ACTH and cortisol levels were undetectablly low, and there was no response noted in the ITT or during CRH-LVP administration throughout the observation period. In four patients with adrenocortical insufficiency who had undergone successful transsphenoidal microadenomectomy for Cushing's disease and in six patients who had undergone curative unilateral adrenalectomy for Cushing's syndrome, basal plasma ACTH levels were low, but responded considerably to both stimulation tests. Along with the 3 days of CRH-LVP stimulation, however, neither the peak nor the time-integrated ACTH response was significantly enhanced, because of the variability of the responses among the patients. Compared with the ACTH response on the last day of CRH-LVP stimulation, the subsequent ITT tended to induce a lower ACTH response in the post-Cushing's disease patients and a higher response in the post-Cushing's syndrome patients. Regarding the plasma cortisol levels, the basal, peak, and integrated responses tended to increase daily during CRH-LVP administration. Conversely, the ITT after repetitive CRH-LVP administration induced a higher cortisol response than the test before CRH-LVP administration in the post-Cushing's disease patients. No serious complications were noted in any of the patients during or after the treatment. The present findings indicate that 1) repetitive administration of CRH in combination with LVP is a safe and valuable provocation test to examine the pituitary ACTH reserve and the integrity of the pituitary-adrenocortical axis; 2) isolated ACTH deficiency is usually due to a defect at the pituitary level; 3) with respect to adrenocortical responsiveness, post-Cushing's disease patients show a better accumulation of the provocative effect than do post-Cushing's syndrome patients; and 4) both hypothalamic and pituitary dysfunction are responsible for adrenal hypofunction in patients after hypercortisolemia, but post-Cushing's syndrome patients (especially those with a short period of hypercortisolemia) appeared to have less impairment of hypothalamic ACTH-releasing activity than post-Cushing's disease patients.

A patient with hypocortisolism and Cushing's syndrome-like manifestations: cortisol hyperreactive syndrome.

One patient is reported who has the manifestations of Cushing's syndrome in spite of persistent hypocortisolemia. His serum levels of cortisol and free cortisol were below normal, and 24-h urinary excretion of 17-hydroxycorticosteroids and cortisol were decreased. There was a rapid and substantial increase in serum cortisol in response to synthetic ACTH-(1-24). Plasma levels of ACTH were marginally increased by successive administration of CRH and vasopressin, which were followed by substantial increases in serum cortisol. Glucocorticoid activity of the patient's serum, as measured by a RRA was low. There were no responses of urinary 17-hydroxycorticosteroids after metyrapone treatment. These laboratory examinations ruled out any known clinical conditions resulting in hypocortisolemia. The clinical condition could also be explained by cortisol hyperreactivity of the patient's cells. In vitro hyperreactivity to glucocorticoids was demonstrated in cultured skin fibroblasts whose aromatase activity was increased 1.5- to 1.8-fold above that of normal cells, and [3H]thymidine incorporation was inhibited more effectively by the addition of cortisol or dexamethasone. The mechanism by which the patient is hyperreactive to glucocorticoids remains unexplained.

Regulation of interleukin-1 receptors on AtT-20 mouse pituitary tumour cells.

To study the cellular mechanisms of interleukin-1 (IL-1) in the pituitary corticotroph, we studied the properties of IL-1 receptors on a mouse pituitary ACTH-producing cell line, AtT-20. Scatchard plot analysis revealed a single type of receptor with a Kd (dissociation constant) of 93 pM, and 482 binding sites/cell. [125I]IL-1 alpha binding competed with IL-1 alpha and IL-1 beta in an equimolar fashion. A 24 h pre-incubation with either CRH, epinephrine or nor-epinephrine increased the [125I]IL-1 alpha binding sites in the AtT-20 cells and conversely, a similar pre-incubation with either dexamethasone or tumour necrosis factor-alpha (TNF alpha) decreased them without affecting the affinity of the receptors in either case.

Expression of the human pro-opiomelanocortin gene introduced into a rat glial cell line.

A fragment of human genomic DNA containing the entire pro-opiomelanocortin (POMC) gene was introduced by transfection into the rat glial cell line C6. Blot analysis using poly(A)-rich RNA from the transformed C6 cells showed several hybridization bands. One band was similar in size (1.2 kb) to the POMC mRNA of human pituitary, while two were larger (2.6 and 2.2 kb) and the fourth smaller (800 bp). S1 nuclease mapping revealed that the POMC transcripts in transformed C6 cells were similar to those in non-pituitary tissues. Immunoreactive ACTH (ir-ACTH) was measurable in both the culture medium and cells. Gel chromatography showed that ir-ACTH in the medium eluted at a position identical to that of so-called big ACTH (approximately 40 kDa) which is found in the plasma of patients with ectopic ACTH syndrome. The human POMC gene could thus be expressed in the non-pituitary rat glial cell line C6, although the transcripts and translation products in C6 cells differ from those in the human pituitary. These results suggest that the transformed C6 cell may be a useful tool for studying the regulation of human POMC gene expression in non-pituitary cells.

Effects of recombinant human interleukin-1 alpha, -1 beta, 2 and 6 on ACTH synthesis and release in the mouse pituitary tumour cell line AtT-20.

The effects of recombinant human interleukin (rhIL)-1 alpha, -1 beta, 2 and 6 on the release of ACTH from the ACTH-producing tumour cell line AtT-20 of the mouse were studied during relatively long periods of incubation. Levels of ACTH in the media, measured by radioimmunoassay, were increased by the addition of rhIL-1 alpha or -1 beta after latent periods of more than 4 h. RhIL-1 alpha and -1 beta were almost equally potent in this experiment and the minimum, half-maximum and maximum effective concentrations of both rhIL-1 alpha and -1 beta were about 0.1 pmol/l, 1-3 pmol/l and 10-100 pmol/l respectively. During incubation with rhIL-1 beta, immunoreactive ACTH levels and mRNA levels of the ACTH precursor pro-opiomelanocortin in cells also increased without apparent changes in the growth rate of the cells. Although the AtT-20 cells used in this study were quite insensitive to human/rat corticotrophin-releasing hormone (CRH), the cells showed a significant response to CRH after incubation with rhIL-1 beta. RhIL-6 showed similar effects to those of rhIL-1 beta on ACTH synthesis and release; increasing ACTH in cells and media after a certain latent period. On the other hand, rhIL-2 did not change ACTH levels in the AtT-20 cells in this study. These observations indicate that rhIL-1 alpha, -1 beta and rhIL-6 have direct effects on ACTH-producing cells to stimulate the release and synthesis of ACTH after a latent period.

Effects of corticostatin-I on rat adrenal cells in vitro.

We have examined the mechanism by which corticostatin-I (CS-I) acts to attenuate ACTH-induced steroidogenesis in rat adrenal cells. CS-I inhibited ACTH-induced corticosterone production in a dose-dependent manner, without any effects on the basal corticosterone level in adrenal cells. When the cells were stimulated by 100 pg ACTH/ml, the minimum effective concentration of CS-I was 100 ng/ml, and 0.3-1.0 micrograms CS-I/ml produced a 50% reduction of the stimulated corticosterone production. The inhibitory effect of CS-I on ACTH-stimulated corticosterone production became apparent within 15 min of incubation, and the effect was reversed quickly by the removal of CS-I from the media. CS-I had no effect on angiotensin II-stimulated aldosterone production by adrenal zona glomerulosa cells. CS-I also did not affect cyclic AMP- or forskolin-stimulated corticosterone production. In an in-vitro binding study using 125I-labelled CS-I, CS-I showed considerable specific binding to rat adrenal cells, and the binding competed with ACTH in a dose-dependent manner. These experiments suggest that CS-I competes with ACTH on their binding sites and exerts an inhibitory effect on the adrenal cells.

Cyclic AMP-responsive region of the human proopiomelanocortin (POMC) gene.

Transcription of the human proopiomelanocortin (POMC) gene is regulated by cAMP. To identify the region in the human POMC gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5'-flanking region of the human POMC gene fused to the structural sequence encoding the bacterial reporter enzyme chloramphenicol acetyltransferase (CAT). The transcriptional activity of the fusion genes introduced into the rat glial cell line C6 was assayed by measuring CAT activity in the cell lysate. Forskolin, an adenylate cyclase-activating agent, stimulated the expression of POMC-CAT fusion genes. Deletion analysis demonstrated that the region between -417 and -97 bp from the transcriptional origin of the human POMC gene was responsible for regulation by cyclic AMP.

Endocrine-paracrine interaction in communication between the immune and endocrine systems. Activation of the hypothalamic-pituitary-adrenal axis in inflammation.

There are bidirectional communications between the immune and endocrine systems. Cytokines produced in inflammatory foci cause changes in the endocrine system, including activation of the hypothalamic-pituitary-adrenal (HPA) axis. Hormones produced in the endocrine system, especially glucocorticoids, affect the immune system to modulate its function. This is an important endocrine system for the defence mechanism. In addition, bacterial lipopolysaccharide produces cytokines in the brain and endocrine organs which are considered to act through the paracrine mechanism to regulate the HPA axis. Endocrine-paracrine interaction is important for the defence mechanism of the organism.