ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00754.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractRecently, we developed a panel of monoclonal antibodies (MoAbs) to rat IL‐1β and found that MoAbs binding to the aminoacid sequences 66–85 and 123–143 of mature rIL‐1β inhibited the binding of rIL‐1β to murine EL4 cells. Here we study whether MoAbs to these and other domains of IL‐1 interfere with the biological effects of rIL‐1β in adult male ratsin vivo.Administration of rIL‐1β (1 or 5 μg/kg i.v.) enhanced the plasma concentrations of ACTH, corticosterone (CORT) and of IL‐6 in a time‐ (0.5–4 h) and dose‐dependent manner. Because 2 h after 5μg/kg i.v., all three parameters were consistently elevated, this dose and time interval was used for further studies. Prior to injection, rIL‐1β was incubated alone or in the presence of a MoAb (10 mg/kg) for 30 min at 37°C or at 4°C. Plasma ACTH, CORT and IL‐6 responses to these mixtures are compared to those obtained after preincubation of rIL‐1β with a non‐IL‐1 binding MoAb (PEN7). SILK 3, a MoAb that binds to the 66–85 domain of rIL‐1β, reduced the ACTH and IL‐6 responses by 48 and 45% respectively. In contrast, a MoAb to the 123–143 domain (SILK 5) and SILK 16, which binds to the 106–124 domain did not affect any of the IL‐1 induced responses, whereas a MoAb directed to domain 78–97 of rIL‐1β (SILK 20) enhanced the ACTH and CORT responses by 51 and 41% respectively, but not the IL‐6 response.These observations lead us to conclude that the domain 66–85, harbours sequences that are important for receptor binding and for the biological actions of rIL‐1β in the rat and that this biologically active domain is located at the ‘closed side’ rather than at the ‘open side’ of the IL‐1β molecule where domains i

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00755.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractCorticotrophin‐releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre‐proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within thetrans‐Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO‐K1 cells expressing the rat pre‐proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR‐CRH displayed an apparent molecular weight of approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule.In this paper, we further characterized the bitopological, i.e. nuclear and cytoplasmic localization of proCRH within transfected CHO‐K1 cells. Immunoreactive nuclear CRH was not extractable using detergents (Triton X‐100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digestion with DNase I. These results therefore suggest that nuclear proCRH is in close association with DNA/chromatin. Treatment of transfected cells with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very stable with a long half life. Brefeldin A treatment had no effect upon the nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reticulum compartments) of the transfected cells do not play a role in proCRH nuclear transport. We also demonstrate that proCRH synthesized within stably transfected CHO‐K1 cells is capable of stimulating ACTH release from primary cultures of anterior pituitary cells, therefore showing for the first time that the intact precursor is also biologically active and could act as an ACTH se

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00756.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractNeuropeptide Y (NPY) is a powerful stimulus to food intake in the rat. Exogenous NPY given into the third ventricle or into the paraventricular nucleus (PVN) of the hypothalamus stimulates both food consumption as well as the hypothalamus‐pituitary‐adrenal (HPA) axis. Presumably NPY activates the adrenocortical system through direct stimulation of CRF containing cells in the PVN. Food intake is also a major regulator of adrenocortical activation. Rhythms in HPA axis activity follow rhythms in food consumption, and rats that have been food deprived overnight have inhibited HPA axis responses to restraint stress and corticosteroid feedback the following morning. To investigate the interaction of NPY with both feeding and HPA axis activation three sets of experiments were performed: Animals fed ad lib were injected icv with NPY (2.5 μg) and allowed access to food or not post injection; animals were fasted overnight prior to NPY injection; finally, dose response experiments were performed to examine the relative sensitivities of feeding and HPA axis activation to exogenous NPY. Ad lib fed animals allowed access to food after NPY injection had slightly greater ACTH responses to NPY while glucocorticoid and insulin responses were not significantly different from ad lib fed animals not allowed access to food post injection. Animals allowed to eat post injection had significantly decreased food consumption the night following injection, however, total 24 h food consumption was not different between these animals and those given food 8 h post NPY injection. In overnight fasted animals NPY injections produced ACTH responses of equal magnitude to those in ad lib fed animals. Insulin responses to NPY were significantly elevated compared to CSF controls in overnight fasted animals. Dose response studies revealed that the adrenocortical system responds to icv NPY with at least as great sensitivity as feeding systems. NPY is discussed as a potential integrator of feeding and responsiveness in the HPA

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00757.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00758.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractThe localization at the cellular level and the regulation by progesterone of the estrogen‐sensitive oxytocin binding sites was studied in the rat telencephalon and the hypothalamus by using quantitative film‐autoradiography and histoautoradiography. Male rats (castrated or not) and ovariectomized females (estradiol supplemented or not) were used to characterize these sites and to precise their localization. They were detected in the striatal cell bridges, the olfactory tubercle, the principal nucleus of the bed nucleus of the stria terminalis and the medial nucleus of the amygdala of the telencephalon and in the medial preoptic, the ventromedial and the ventral premammillary nuclei of the hypothalamus. Estrogen administration in addition induced expression of oxytocin binding sites in the major island of Calleja, the anterior hypothalamic area and the terete nucleus. The density of the estrogen‐sensitive oxytocin binding sites varied during the estrous cycle, but differently in the telencephalon and the hypothalamus. in the telencephalon it peaked at proestrus 9 h and was already decreased at proestrus 21 h, whereas in the hypothalamus it was similarly high at proestrus 9h and proestrus 21 h, suggesting the intervention of progesterone in the regulation of the hypothalamic estrogen‐sensitive oxytocin bindin

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00759.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractNeuropeptide Y (NPY), a 36 amino acid residue peptide, is involved in stimulation of LHRH and LH surges on proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. Recently, we observed that NPY gene expression in the medial basal hypothalamus (MBH) was increased before the onset of the LH surge in the ovarian steroid‐primed ovx rats. Since the ovarian steroidal milieu during the estrous cycle is markedly different from that prevailing after ovarian steroid injections in ovx rats, we evaluated in cycling rats the temporal relationship between MBH preproNPY mRNA levels and the preovulatory LH surge on the day of proestrus and compared that with diestrus II, concomitant with basal LH levels. PreproNPY mRNA levels in the MBH were measured by solution hybridization/RNAse protection assay, using a cRNA probe. On the day of diestrus II, preproNPY mRNA levels changed little between 1000 and 1800 h. Quite unexpectedly, preproNPY mRNA levels at 1000 h on proestrus were similar to diestrus II levels, despite additional exposure to ovarian steroids during this interval. However, from these low levels at 1000 h, the preproNPY mRNA profile displayed a biphasic rise. During the first phase, preproNPY mRNA rose significantly at 1200 h and remained elevated at 1300 and 1400 h concomitant with basal serum LH levels. Thereafter, a second rise in preproNPY mRNA began at 1500 h, peaked rapidly at 1600 h and declined significantly at 1800 h. This secondary activation of NPY gene expression occurred with a slow, two‐fold increase in serum LH at 1500 h, followed by a rapid ascension to peak levels at 1800 h and was associated with an increase at 1400 h of serum progesterone levels which reached their peak at 1800 h. These results demonstrate that a dynamic, biphasic augmentation in hypothalamic NPY gene expression occurs selectively on proestrus, and that the first incremental response is observed some time before the onset of preovulatory LH hypersecretion. Because preproNPY mRNA levels at 1000 h on proestrus were similar to the low levels seen on the preceding diestrous II phase, a neural timing mechanism, and not changes in ovarian hormone levels during this phase may be responsible for the increase in NPY gene expression after 1000 h of proestrus. Because of our previous observations that progesterone can rapidly augment preproNPY mRNA in the MBH and because a rise in serum progesterone occurs hours before the onset of the LH surge, we suggest that the secondary rise in preproNPY mRNA is facilitated by this antecedent increase in serum progesterone. Cumulatively, these results are in accord with the thesis that activation of hypothalamic NPY gene expression is one of the key early neural events initiated by the neural clock that times the preovulatory LHRH and LH sur

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00760.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractThe physiological role of melanin‐concentrating hormone (MCH) in mammals is still very elusive, but this peptide might participate in the central control of the hypothalamopituitary adrenal (HPA) axis during adaptation to stress. Cloning and sequencing of the rat MCH (rMCH) cDNA revealed the existence of additional peptides encoded into the MCH precursor. Among these peptides, neuropeptide (N) glutamic acid (E) isoleucine (I) arnide (NEI) is co‐processed and secreted with MCH in rat hypothalamus. In the present work we examined: (1) The pattern of rMCH mRNA expression during the light and dark conditions in the rat hypothalamus and (2) The effect of intracerebroventricular (ICV) injections of rMCH and NEI in the control of basal or ether stress‐modified release of corticotropin (ACTH), prolactin (PRL) and growth hormone (GH) secretionin vivoin light‐on and light‐off conditions. Our data indicate that rMCH mRNA levels do not change during the light‐on period, but increase after the onset of darkness. Either alone or co‐administered, rMCH and NEI do not modify basal secretion of GH and PRL at any time tested nor do they alter ether stress‐induced changes in these two hormonal secretions. At the end of the light on period corresponding to the peak of the circadian rhythm in ACTH, administration of rMCH but not NEI leads to a decrease in ACTH levels while MCH is not effective during the light off period of the cycle (i.e. when basal ACTH levels are already low). Using a moderate ether induced stress, ACTH levels are only stimulated during the dark phase of the cycle. rMCH (63 or 210 nmoles) prevents the rise in ACTH release while NEI alone does not modify the stress response. Co‐administration of both peptides before stress results in an abolition of the rMCH induced inhibition of ACTH plasma levels. Taken together, these data indicate that rMCH may act as a central corticotropin inhibitory factor involved in the circadian rhythmicity of plasma ACTH levels and that NEI antag

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00761.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractThe natriuretic peptide receptors (NPR) are membrane‐bound guanylate cyclases with extracellular binding domains specific for particular members of the natriuretic peptide family. NPR‐A binds atrial natriuretic peptide (ANP) with high affinity, whereas the NPR‐B appears to be specific for C‐type natriuretic peptide (CNP). Previous data indicating extensive overlap between localization of ANP and CNP in hypothalamic neuroendocrine circuits suggest the importance of determining whether specificity of natriuretic peptide action may be conferred via receptor type present on target cells. To address this issue, we usedin situhybridization histochemistry to localize NPR‐A and NPR‐B mRNA in the hypothalamus. NPR‐A mRNA was not found in substantial abundance in any hypothalamic nucleus; however, detectable NPR‐A signal was observed in other brain regions, including the subfornical organ and medial habenula. In contrast, NPR‐B mRNA was expressed throughout the hypothalamus, including neurons of the magnocellular and parvocellular paraventricular, the arcuate, and the supraoptic nuclei. Expression was also seen in other nuclei essential to neuroendocrine control, including the median preoptic, anteroventral periventricular, tuberomammillary, ventromedial and suprachiasmatic nuclei. NPR‐B mRNA was also observed in the neural lobe of the pituitary gland, suggesting expression by pituicytes. The results suggest that NPR‐B is the primary natriuretic peptide receptor in hypothalamus, and by inference indicate that CNP is the primary active natriuretic peptide in neur

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00762.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractThe Goto‐Kakizaki (GK) rat is a new model of diabetes mellitus and in this study we have characterized the diabetic and growth hormone (GH) secretory status of male GK rats at 6 and 16 weeks of age. We have also investigated the role of endogenous somatostatin (SS) and cholinergic manipulation on the GH responses to GH‐releasing hormone (GHRH). GK rats were non‐obese with significant fasting hyperglycaemia, hyperinsulinaemia and absent insulin responses to IV glucose. The GH response to GHRH was reduced at 16 weeks compared with normal, age‐matched Wistar rats but no differences were observed at 6 weeks. Pretreatment of older rats (16 weeks) with anti‐somatostatin antibodies (SS‐Ab) significantly increased GH responses to GHRH in both normal and GK groups. Cholinergic augmentation with pyridostigmine (PD) reversed the blunted GH responses to GHRH in older GK rats but had no effect in the normal or young (6 weeks) GK rats. These results indicate that SS release mediates the blunted GH response to GHRH in GK rats and that reduced hypothalamic cholinergic signalling to the somatostatinergic neurone may mediate the increase in SS release. This view is supported by the results fromin vitrostudies in which cholinergic muscarinic blockade with pirenzepine (PIR) caused dose‐related stimulation of SS release from normal rat hypothalami but was without effect on GK rat hypothalami. The cause of this alteration in hypothalamic function is, at pre

ISSN:0953-8194    

DOI:10.1111/j.1365-2826.1995.tb00763.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY