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Abstract. Botulinum C3 exoenzyme specifically ADPribosylates a group of ms-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 ADPribosylated in situ in a time-and concentrationdependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-l-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 exoenzymeinduced 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. R as and ras-related genes constitute a gene family encoding a series of closely related proteins with guanine nucleotide-binding activities. To date, ~40 ras and ras-related GTP-binding proteins are known, and they are divided into four subfamilies: ms, rho, tab, and others (Hall, 1990;Bourne et al., 1991). These proteins exist in two interconvertible functional states; one in an inactive GDPbound form and the other in an active GTP-bound form. In resting cells, they are present in an inactive GDP-bound form, and upon cell stimulation, converted to the active GTP-bound form and work as molecular switches linking external stimuli to various cellular responses such as growth, differentiation, and secretion. Among the ras-related GTPbinding proteins, rho proteins are believed to be involved in organization of cytoskeleton and maintenance of cell shape. There are at least three members in this family in human, which are called rhoA, B, and C (Yeramian et al., 1987;Chardin et al., 1988). rho proteins are unique among the small GTP-binding proteins in being substrates for ADP ribosylation by the exoenzyme C3 from Clostridium botulinum (Aktories et al., 1987;Morii et al., 1988;Narumiya et al., 1988;Kikuchi et al., 1988). This enzyme ADPribosylates the proteins at an asparagine residue in the putative effector domain and inhibits their biological activities presumably by interfering with their interaction ...

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.

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.