Physiological role of corticotropin-releasing factor in the control of adrenocorticotropin-mediated corticosterone release from the rat adrenal gland.

Marked fluctuations in adrenal sensitivity to ACTH have been reported under both physiological (e.g. diurnal) and experimental conditions. Recently, we reported that immunoneutralization of CRF reduces resting corticosterone (cort) levels in rats without inducing concomitant reductions in plasma ACTH. We postulated an endogenous CRF mechanism that controls the adrenal sensitivity to ACTH. In the present study, this hypothesis was tested by iv infusion of human ACTH (0, 1, 3, and 10 ng/kg.min for 60 min) into dexamethasone-treated anaesthetized male Wistar rats. Serial blood samples were taken for the determination of ACTH and corticosterone by RIA (ACTHi, corti). Infusion of ACTH resulted in dose-dependent steady state plasma ACTHi levels, ranging from 50-600 pg/ml, which were not affected by prior administration of a rat monoclonal antibody to rat CRF (PFU 83). As expected, infusion of ACTH resulted in a dose-dependent increase in plasma corti. In PFU 83-treated rats, preinfusion plasma corti levels were reduced compared to those of rat immunoglobulin G-treated controls (7.8 +/- 1.2 vs 25.3 +/- 3.2 ng/ml). In addition, the corti responses to infusion of 1 and 3 ng/kg.min ACTH were suppressed by PFU 83. However, at a (near) maximally effective dose of ACTH (10 ng/kg.min), no differences in plasma corti were found between PFU 83 and immunoglobulin G-treated rats. These findings suggest that immunoneutralization of endogenous CRF results in a 3-fold reduction of the adrenal sensitivity to ACTH. Subsequently, we studied the possible effects of exogenous CRF on the isolated perfused adrenal gland in situ. In this preparation, CRF alone (1-100 pmol) or ACTH alone (5 fmol) did not affect the corti secretion rate or the flow rate of the perfusion medium through the gland. However, when given together a marked (up to 3.2 times) CRF dose-dependent stimulation of corti secretion and an increase (up to 1.7 times) in adrenal flow rate were obtained. In experiments with freshly dispersed adrenal cells in vitro, PFU 83 (1 microM) or CRF (0.1-10 nM) did not influence corti secretion when given alone and did not affect ACTH-induced corti secretion. It is unlikely, therefore, that CRF acts directly on the steroid-producing cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies in passive immunization studies: characteristics and consequences.

Antibodies to neuropeptides or hormones are frequently used in passive immunization studies to unravel their physiological role in signal transfer. In such in vivo experiments antibodies are considered to bind and thereby to biologically inactivate the endogenous substance during its journey from its site of secretion to its site of action (signalling time). However, little is known about the mechanism of action and characteristics of antibodies that determine such biological activity. Since the signalling time in neuronal and hormonal communication is short, the kinetics of antibody binding is an important feature. Here, we present a theoretical framework to describe antibody binding kinetics which can contribute to the design of passive immunization protocols. The specific effects of variation in antibody concentration, dissociation constant, and on-rate constant on these binding kinetics are demonstrated. Simple methods are described to determine these parameters, which may guide the selection of antibodies for passive immunization studies. When time is limited, the on-rate constant and the local antibody concentration are the most important determinants. Several points are illustrated for CRF signal transfer in the rat. CRF signalling time in the hypothalamo-pituitary complex, as established from dye transport experiments, was 3-7 sec. Based on parameters measured for a rat monoclonal antibody to CRF (PFU 83), we computed that half-maximal and full blockades of ether-induced ACTH secretion were associated with approximately 85% and more than 99% binding of CRF, respectively. From the theoretical framework presented in this study we conclude that, in general, the kinetics of antigen binding are sufficiently fast for antibodies to interfere with hormonal and probably nonsynaptic neuronal signal transfer. However, interference with fast signalling processes (less than 10 msec), which may occur in the brain, is unlikely.

Uptake of a monoclonal antibody to corticotropin-releasing factor (CRF) into rat hypothalamic neurons.

Previous immunocytochemical studies reported that when specific monoclonal antibody directed against vasopressin (VP) (VP-MAb) was injected in vivo above the rat hypothalamic nuclei, it penetrated and was specifically transported by VP-producing neurons. In this study, using the same methodological approach, the fate of monoclonal antibody directed against corticotropin-releasing factor (CRF) (CRF-MAb) injected in vivo above the paraventricular nucleus (PVN) of the rat brain was investigated by immunocytochemistry in male Zucker rats and adrenalectomized or colchicine-pretreated male Long-Evans rats. The simultaneous immunocytochemical localization of the injected CRF-MAb and endogenous peptides and enzyme synthesized by the neurons penetrated by the antibody, demonstrated that CRF-MAb was mainly detected in CRF neurons. But the CRF-MAb was also detected in VP, oxytocin, neuropeptide Y and tyrosine hydroxylase-producing neurons of the PVN. CRF-MAb was therefore localized in PVN neurons which synthesize CRF and in PVN neurons with physiological and morphological relationships with the CRF peptidergic system. Before obtaining biological effects of injected CRF-MAb, the results described here suggest that specific monoclonal antibodies provide a useful specific tool for elucidating the functional relationships between neuronal systems.

Central activation of thermogenesis and fever by interleukin-1 beta and interleukin-1 alpha involves different mechanisms.

Interleukin-1 exists in two forms (alpha and beta) which are assumed to act on the same receptor. Both forms of the molecule stimulated fever and thermogenesis in the rat when injected into the brain, but interleukin-1 beta was more effective, and combined injection of alpha and beta elicited additive responses. The actions of interleukin-1 beta were inhibited by pretreatment of the animals with either a receptor antagonist or monoclonal antibody to corticotrophin releasing factor. The effects of interleukin-1 alpha were unaltered by these treatments. The results indicate that brain corticotrophin releasing factor mediates thermogenesis and fever induced by interleukin-1 beta but not by interleukin-1 alpha.

Characterization and biological activity of a rat monoclonal antibody to rat/human corticotropin-releasing factor.

To produce a rat monoclonal antibody directed to rat CRF (rCRF), female Wistar rats were actively immunized with a rCRF-bovine thyroglobulin conjugate. Immunization resulted in the formation of CRF antibodies, as indicated by binding of [125I]iodo-rCRF and attenuation of the plasma corticosterone responses to ether stress. Rat spleen cells were fused with mouse myeloma P3 cells, and a hybridoma clone (PFU 83) was selected according to its capacity to bind [125I]iodo-rCRF and inhibit rCRF-induced ACTH release from cultured rat pituitary cells. PFU 83 antibodies (IgG2a subclass) are directed to the extreme C-terminal part (amino acids 38-39) of rCRF and bind with an affinity constant of 21 nM. In vitro, PFU 83 causes a parallel shift to the right of the rCRF dose-ACTH response curve, with an apparent affinity of 10 nM. PFU 83 neither binds nor blocks the ACTH-releasing activity of oCRF. Intravenous administration of PFU 83 ascites (generated in nude mice) to Wistar rats caused a dose-dependent inhibition of ether-induced ACTH secretion. Full blockade of the ACTH response to ether was found at a dose of 10 nmol PFU 83/rat. Based on the dynamics of rCRF binding and its in vitro and in vivo effects, we conclude that the rCRF-blocking bioactivity of PFU 83 is due to binding of PFU 83 to native rCRF and formation of a biologically inactive complex. Finally, we found that PFU 83 did not affect resting or ether-induced alpha MSH secretion, indicating that CRF does not play a major role in the control of alpha MSH secretion.

Stage specific embryonic carbohydrate surface antigens of primordial germ cells in mouse embryos: FAL (S.S.E.A.-1) and globoside (S.S.E.A.-3).

Immunodetections of carbohydrate surface antigens were carried out for SSEA-1 and SSEA-3. Using alkaline phosphatase for the detection of primordial germ cells these surface antigens were detected at the cell membrane and the cytoplasm of the germ cells at E 10.

Ferritin in bean leaves with constant and changing iron status.

Normal green leaves contain low levels of ferritin which stores 5 to 10% of the total iron (approximately 350 iron atoms/molecule). Chlorotic leaves do not have measurable amounts of ferritin, whereas iron-loaded leaves contain high levels of well-filled ferritin (1,500 to 2,500 iron atoms/molecule). The role of ferritin during a transient iron surplus in leaves was investigated. It is suggested that a short-term overdose of iron transported into the leaf is largely stored in or near the vessels in such a form that it can be quickly mobilized for export. Iron that reaches the mesophyll cells in an overdose situation is stored in ferritin and, when released, is most likely used for the leaf cells themselves and not for export.