Adolescent alcohol exposure alters the central brain circuits known to regulate the stress response.

Adolescent alcohol exposure (AAE) may exert long-term effects on the adult brain. Here, we tested the hypothesis that the brain regions affected include the rat hypothalamic-pituitary-adrenal (HPA) axis. Specifically, we examined the consequences of AAE [postnatal days (PND) 28-42] on the HPA axis-related brain circuitry of male rats challenged with an intragastric (ig) administration of alcohol in young adulthood (PND 61-62). Adolescent rats were exposed to alcohol vapors, while controls did not receive the drug. The mean blood alcohol level in adolescence on PND 40 was 212.8±5.7 mg %. Using immunohistochemistry and in situ hybridization procedures, we measured signals for c-fos and corticotropin releasing factor (CRF) in the paraventricular nucleus (PVN) of the hypothalamus, as well as signals for c-fos and phenylethanolamine N-methyltransferase (PNMT) in the adrenergic brain stem regions (C1 and C2). PVN CRF mRNA expression was significantly blunted in AAE rats tested at PND 61-62, compared to their controls. These animals also displayed a significant increase in the mean number of PNMT-ir cells/brain stem section in the C2 area. Collectively, these results suggest that exposure to alcohol vapors during adolescence exerts long-term effects on the ability of the PVN to mount a response to an acute alcohol administration in young adulthood, possibly mediated by medullary catecholamine input to the PVN.

Synthesis and biological activity of GnRH antagonists modified at position 3 with 3-(2-methoxy-5-pyridyl)-alanine.

Degarelix is a potent very long-acting GnRH antagonist after subcutaneous administration. In this paper, we describe the synthesis of two analogs of degarelix incorporating racemic 3-(2-methoxy-5-pyridyl)-alanine (2-OMe-5Pal, 5) at position 3. The two diastereomers were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and the absolute stereochemistry at position 3 in the peptides was determined by enzymatic digestion with proteinase K. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analog 7 with D2-OMe-5Pal was potent in vitro (IC50 = 5.22 nM); however, analog 8 with L2-OMe-5Pal at position 3 in degarelix lost potency as an antagonist of the human GnRH receptor (IC50 = 36.95 nM). Both the analogs were found to be short-acting in vivo.

Nitric oxide and carbon monoxide have a stimulatory role in the hypothalamic-pituitary-adrenal response to physico-emotional stressors in rats.

We tested the hypothesis that nitric oxide and carbon monoxide, which are produced in the brain by nitric oxide synthase (NOS) and heme oxygenase (HO), modulate the hypothalamic-pituitary-adrenal response to physico-emotional stressors by acting at the hypothalamus. Accordingly, we determined 1) whether the intracerebroventricular (icv) injection of NOS or HO inhibitors at doses that were confined to the brain attenuated electroshock-induced ACTH release; and 2) whether the decreases in this ACTH response were concurrent with decreases in NOS or HO activity levels at the hypothalamus. Icv injection of the NOS inhibitor Nomega-nitro-L-arginine-methylester (L-NAME; 50 microg) or the HO inhibitor tin protoporphyrin (SnPP; 20-25 microg) significantly blunted the plasma ACTH response to a 45-min session of intermittent electroshocks. Importantly, in these same animals there were concurrent decreases in hypothalamic NOS or HO activities, respectively. There were little or no effects of these inhibitors on anterior pituitary NOS or HO activities, indicating that there was only minimal leakage of the drug from the brain after icv administration. The specificity of action of these inhibitors was confirmed by the fact that SnPP did not affect NOS activity, and L-NAME did not affect HO activity. Finally, L-NAME produced no effect, whereas SnPP produced only transient increases in blood pressure, suggesting that these inhibitors do not affect activity indirectly through alterations in blood pressure. These data support the hypothesis that in the whole animal, both NO and CO exert a stimulatory influence on the acute ACTH response to physico-emotional stressors, and that the hypothalamus is the critical site of their actions.

Design of potent dicyclic (4-10/5-8) gonadotropin releasing hormone (GnRH) antagonists.

With the ultimate goal of identifying a consensus bioactive conformation of GnRH antagonists, the compatibility of a number of side chain to side chain bridges in bioactive analogues was systematically explored. In an earlier publication, cyclo[Asp(4)-Dpr(10)]GnRH antagonists with high potencies in vitro and in vivo had been identified.(1) Independently from Dutta et al. (2) and based on structural considerations, the cyclic [Glu(5)-Lys(8)] constraint was also found to be tolerated in GnRH antagonists. We describe here a large number of cyclic (4-10) and (5-8) and dicyclic (4-10/5-8) GnRH antagonists optimized for affinity to the rat GnRH receptor and in vivo antiovulatory potency. The most potent monocyclic analogues were cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Asp(4),DArg(6),Xaa(10)]GnRH with Xaa = D/LAgl (1, K(i) = 1.3 nM) or Dpr (2, K(i) = 0.36 nM), which completely blocked ovulation in cycling rats after sc administration of 2.5 microgram at noon of proestrus. Much less potent were the closely related analogues with Xaa = Dbu (3, K(i) = 10 nM) or cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Glu(4),DArg(6),D/LAgl(10)]GnRH (4, K(i) = 1.3 nM). Cyclo(5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Glu(5),DArg++ +(6),Lys(8), DAla(10)]GnRH (13), although at least 20 times less potent in the AOA than 1 or 2 with similar GnRHR affinity (K(i) = 0.84 nM), was found to be one of the most potent in a series of closely related cyclo(5-8) analogues with different bridge lengths and bridgehead chirality. The very high affinity of cyclo(5,5'-8)[Ac-DNal(1), DCpa(2),DPal(3),Glu(5)(betaAla),DArg(6),(D or L)Agl,(8)DAla(10)]GnRH 14 (K(i) = 0.15 nM) correlates well with its high potency in vivo (full inhibition of ovulation at 25 microgram/rat). Dicyclo(4-10/5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Asp (4),Glu(5),DArg(6), Lys(8),Dpr(10)]GnRH (24, K(i) = 0.32 nM) is one-fourth as potent as 1 or 2, in the AOA; this suggests that the introduction of the (4-10) bridge in 13, while having little effect on affinity, restores functional/conformational features favorable for stability and distribution. To further increase potency of dicyclic antagonists, the size and composition of the (5-8) bridge was varied. For example, the substitution of Xbb(5') by Gly (30, K(i) = 0.16 nM), Sar (31, K(i) = 0.20 nM), Phe (32, K(i) = 0.23 nM), DPhe (33, K(i) = 120 nM), Arg (36, K(i) = 0.20 nM), Nal (37, K(i) = 4.2 nM), His (38, K(i) = 0.10 nM), and Cpa (39, K(i) = 0.23 nM) in cyclo(4-10/5,5'-8)[Ac-DNal(1),DCpa(2),DPal(3),Asp(4),G lu(5)(Xbb(5')), DArg(6),Dbu,(8)Dpr(10)]GnRH yielded several very high affinity analogues that were 10, ca. 10, 4, >200, 1, ca. 4, >2, and 2 times less potent than 1 or 2, respectively. Other scaffolds constrained by disulfide (7, K(i) = 2.4 nM; and 8, K(i) = 450 nM), cyclo[Glu(5)-Aph(8)] (16, K(i) = 20 nM; and 17, K(i) = 0.28 nM), or cyclo[Asp(5)-/Glu(5)-/Asp(5)(Gly(5'))-Amp(8)] (19, K(i) = 1.3 nM; 22, K(i) = 3.3 nM; and 23, K(i) = 3.6 nM) bridges yielded analogues that were less potent in vivo and had a wide range of affinities. The effects on biological activity of substituting DCpa or DFpa at position 2, DPal or DTrp at position 3, and DArg, DNal, or DCit at position 6 are also discussed. Interestingly, monocyclo(5-8)[Glu(5), DNal(6),Lys(8)]GnRH (18, K(i) = 1. (ABSTRACT TRUNCATED)

Design of monocyclic (1-3) and dicyclic (1-3/4-10) gonadotropin releasing hormone (GnRH) antagonists.

Careful analysis of the NMR structures of cyclo(4-10)[Ac-Delta(3)Pro(1),DFpa(2),DTrp(3),Asp(4),DNal (6), Dpr(10)]GnRH, dicyclo(4-10/5-8)[Ac-DNal(1),DCpa(2),DTrp(3), Asp(4), Glu(5),DArg(6),Lys(8),Dpr(10)]GnRH, and dicyclo(4-10/5, 5'-8)[Ac-DNal(1),DCpa(2),DPal(3),Asp(4), Glu(5)(Gly),DArg(6),Dbu(8), Dpr(10)]GnRH showed that, in the N-terminal tripeptide, a type II beta-turn around residues 1 and 2 was probable along with a gamma-turn around DTrp(3)/DPal(3). This suggested the possibility of constraining the N-terminus by the introduction of a cyclo(1-3) scaffold. Optimization of ring size and composition led to the discovery of cyclo(1-3)[Ac-DAsp(1),DCpa(2),DLys(3),DNal(6), DAla(10)]GnRH (5, K(i) = 0.82 nM), cyclo(1,1'-3)[Ac-DAsp(1)(Gly), DCpa(2),DOrn(3),DNal(6),DAla(10)]GnRH (13, K(i) = 0.34 nM), cyclo(1, 1'-3)[Ac-DAsp(1)(Gly),DCpa(2),DLys(3),DNal(6),DA la(10)]GnRH (20, K(i) = 0.14 nM), and cyclo(1,1'-3)[Ac-DAsp(1)(betaAla), DCpa(2), DOrn(3),DNal(6),DAla(10)]GnRH (21, K(i) = 0.17 nM), which inhibited ovulation significantly at doses equal to or lower than 25 microgram/rat. These results were particularly unexpected in view of the critical role(s) originally ascribed to the side chains of residues 1 and 3.(1) Other closely related analogues, such as those where the [DAsp(1)(betaAla), DOrn(3)] cycle of 21 was changed to [DOrn(1)(betaAla), DAsp(3)] of cyclo(1,1'-3)[Ac-DOrn(1)(betaAla), DCpa(2),DAsp(3),DNal(6),DAla(10)]GnRH (22, K(i) = 2.2 nM) or where the size of the cycle was conserved and [DAsp(1)(betaAla), DOrn(3)] was replaced by [DGlu(1)(Gly), DOrn(3)] as in cyclo(1, 1'-3)[Ac-DGlu(1)(Gly),DCpa(2),DOrn(3),DNal(6),DA la(10)]GnRH (23, K(i) = 4.2 nM), were approximately 100 and 25 times less potent in vivo, respectively. Analogues with ring sizes of 18 ¿cyclo(1, 1'-3)[Ac-DGlu(1)(Gly),DCpa(2),DLys(3),DNal(6),DA la(10)]GnRH (24)¿ and 19 ¿cyclo(1,1'-3)[Ac-DGlu(1)(betaAla),DCpa(2),DLys( 3),DNal(6), DAla(10)]GnRH (25)¿ atoms were also less potent than 21 with slightly higher K(i) values (1.5 and 2.2 nM, respectively). These results suggested that the N-terminal tripeptide was likely to assume a folded conformation favoring the close proximity of the side chains of residues 1 and 3. The dicyclic analogue dicyclo(1-3/4-10)[Ac-DAsp(1),DCpa(2),DLys(3),Asp (4),DNal(6), Dpr(10)]GnRH (26) was fully active at 500 microgram, with a K(i) value of 1 nM. The in vivo potency of 26 was at least 10-fold less than that of monocyclic cyclo(1-3)[Ac-DAsp(1),DCpa(2),DLys(3),DNal(6), DAla(10)]GnRH (5); this suggested the existence of unfavorable interactions between the now optimized and constrained (1-3) and (4-10) cyclic moieties that must interact as originally hypothesized. Tricyclo(1-3/4-10/5-8)[Ac-DGlu(1),DCpa(2), DLys(3),Asp(4),Glu(5), DNal(6),Lys(8),Dpr(10)] GnRH (27) was inactive at 500 microgram/rat with a corresponding low affinity (K(i) = 4.6 nM) when compared to those of the most potent analogues (K(i) < 0.5 nM).

Design of potent dicyclic (1-5/4-10) gonadotropin releasing hormone (GnRH) antagonists.

In three earlier papers, the structures and biological potencies of numerous mono- and dicyclic antagonists of GnRH were reported. Among these, two families, each containing two to four members were identified that had very high antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K(i) < 0.5 nM) for the rat GnRH receptor (rGnRHR). The most favored cycles bridged the side chains of residues (4-10),(1,2) (5-8),(2) (4-10/5-8),(2) (1-3),(3) and (1-3/4-10).(3) Our goal was to identify a consensus model of bioactive conformations of GnRH antagonists, yet these biocompatible constraints did not sufficiently restrain the spatial location of the N-terminal tripeptide with respect to the C-terminal heptapeptide, due largely to the rotational freedom about the bonds connecting these regions. Examination of models derived from NMR studies of cyclo(4-10) analogues suggested a large number of possible cyclic constraints such as cyclo (0-8), (1-8), or (2-8). All analogues tested with these substitutions were inactive as antiovulatory agents at 1 mg/rat (5-9) and had low affinity for rGnRHR. On the other hand, bridging positions 3 and 8 with a [DAsp(3)] to [Dbu(8)] (12, K(i) = 13 nM) or [Orn(8)] (13, K(i) = 14 nM) in the parent compound cyclo(3-8)[Ac-DNal(1),DCpa(2),DXaa(3), Arg(5),DNal(6),Xbb(8),DAla(10)]GnRH yielded analogues that blocked ovulation at 250 microgram/rat. Analogue 14 (K(i) = 2.3 nM), with a [DAsp(3), Lys(8)] bridge, was fully active at 50 microgram/rat. Loss of potency (>20-fold) was observed with the substitution of [DAsp(3)] in 14 by [DGlu(3)] in 15 (K(i) = 23 nM). Dicyclic analogues possessing the (4-10) cycle and selected (1-6), (2-6), and (2-8) cycles led to analogues that were inactive at doses of 500 microgram/rat or larger. Two analogues with (1-8/4-10) cycles (16, K(i) = 1.1 nM) or (3-8/4-10) cycles (22, K(i) = 17 nM) showed full antiovulatory potency at 250 microgram/rat. None of these substitutions yielded analogues potent enough (>80% inhibition of ovulation at 5 microgram/rat or less and K(i) < 0.5 nM) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1, 1'-5/4-10) analogues met this criterion: dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (32, K(i) = 0.22 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DNal(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (34, K(i) = 0.38 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(betaAla),DCpa(2), DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (40, K(i) = 0.15 nM), and dicyclo(1, 1'-5/4-10)[Ac-Glu(1)(Gly), DCpa(2),DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (41, K(i) = 0.24 nM). Since they differed slightly in terms of the (1,1'-5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive conformations that satisfy a very discriminating receptor, since many other closely related analogues were significantly less potent.

Sprague-Dawley rats obtained from different vendors exhibit distinct adrenocorticotropin responses to inflammatory stimuli.

The purpose of this work was to compare the plasma adrenocorticotropin (ACTH), corticosterone and interleukin-6 (IL-6) responses that rats of the outbred Sprague-Dawley strain obtained from two different vendors: Charles River (CR) and Harlan (HSD). Basal plasma ACTH and IL-6 concentrations were similar in rats from either vendor (HSD or CR), while CR animals exhibited slightly elevated corticosterone levels in late afternoon. Inflammatory stimuli such as lipopolysaccharide (LPS) (1 microgram/kg, i.v.) or turpentine (50 microliter/100 g, i.m.) which induce the production of endogenous cytokines, produced a significantly larger ACTH response in CR, compared to HSD rats, while the overall corticosterone responses were comparable in both rat groups. This could probably not be accounted for by a greater ACTH responsiveness in CR rats per se because CR and HSD rats showed similar peak ACTH responses to electrofootshock. Furthermore, in contrast to when the stimulus was one that induced endogenous cytokine production, the administration of exogenous interleukin-1beta (IL-1beta, 200 ng/kg, i.v.) produced a 2-fold greater rise in plasma ACTH concentrations in HSD rats compared to CR rats. The plasma IL-6 responses to the inflammatory stimuli showed a similar pattern to ACTH, with LPS and turpentine tending to pruduce greater IL-6 responses in CR rats, though these differences were not statistically significant. In contrast HSD rats had a significantly greater IL-6 response to IL-1beta than did CR rats. Collectively, these results show that Sprague-Dawley rats obtained from different commercial sources can differ in immune-neuroendocrine responses to inflammatory stimuli.

Constrained corticotropin releasing factor antagonists (astressin analogues) with long duration of action in the rat.

In an earlier report we identified specific modifications and substitutions of corticotropin releasing factor (CRF) that led to the discovery of antagonists with extended duration of action as compared to that of astressin {cyclo(30-33)[DPhe(12),Nle(21),Glu(30), Lys(33),Nle(38)]hCRF((12)(-)(41))}. These additional modifications included elongation of the peptide chain by three residues at the N-terminus, its acetylation, and the [CalphaMeLeu(27)] substitution to yield cyclo(30-33)[DPhe(12), Nle(21),CalphaMeLeu(27),Glu(30), Lys(33),Nle(38)]Ac-hCRF((9)(-)(41)), which was found to be longer acting than astressin (Rivier, J.; et al. J. Med. Chem. 1998, 41, 5012-5019). To further increase the efficiency (potency, duration of action, and bioavailability) of this family of antagonists, we introduced two or more CalphaMe-leucine residues at positions shown in earlier studies to be favorable (Hernandez, J.-F.; et al. J. Med. Chem. 1993, 36, 2860-2867). Whereas the introduction of CalphaMe-leucine residues at positions 27 and either 18 (11), 37 (17), or 40 (19) resulted in dramatic increases in duration of inhibitory action in the adrenalectomized (adx) rat after intravenous injection, the same substitution at positions 27 and either 15 (7, 8), 17 (9), 19 (12, 13), or 41 (20) led to short acting analogues. Other substitutions by CalphaMeLeu at positions 27 and either 10 (4), 13 (5), 14 (6), 21 (14), 24 (15), 36 (16), or 38 (18) yielded analogues with duration of action intermediate between those mentioned above. Cyclo(30-33)[DPhe(12), Nle(21), CalphaMeLeu(27),Glu(30),Lys(33),Nle(38), CalphaMeLeu(40)]Ac-hCRF((9)(-)(41)) (astressin B, 19) was one of the most efficacious analogues of this series (>4 h inhibition of ACTH secretion at 25 microgram/adx rat). It was found to be even longer acting via subcutaneous administration in either an aqueous (>24 h inhibition of ACTH secretion at 100 microgram/adx rat) or lipid milieu (DMSO/peanut oil, >24 h inhibition of ACTH secretion at 30 microgram/adx rat) than after intravenous administration (<12 h inhibition of ACTH secretion at 100 microgram/adx rat). We concluded that Calpha-methylation at some positions may favor a bioactive conformation while also preventing degradation and/or elimination, resulting in significant extension of duration of action.

Effects of prenatal exposure to alcohol on the release of adenocorticotropic hormone, corticosterone, and proinflammatory cytokines.

Prenatal alcohol exposure has been shown to produce hyperresponsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to immune challenges. Because cytokines, which are released in response to immune challenges, are known to activate the HPA axis, this study determined whether altered release of cytokines contribute to the HPA hyperresponsiveness to immune challenges observed after prenatal alcohol exposure. Pregnant dams were exposed to alcohol vapors (6-7 hr daily) between days 7 and 18 of gestation. At postnatal days 45 and 60, control (C) and prenatal alcohol-exposed (E) offspring were subjected to three different types of immune challenges: injections of interleukin-1beta or endotoxin (lipopolysaccharide), or turpentine-induced tissue injury. We observed the expected higher plasma adrenocorticotropic hormone and corticosterone levels in E compared with C rats, and this HPA hyperresponsiveness was greater in E females compared with E males. Plasma tumor necrosis factor-alpha or interleukin-6 responses were comparable in the C and E groups. Females exhibited significantly higher corticosterone, tumor necrosis factor-alpha, and interleukin-6 responses than males. These results indicate that (1) prenatal alcohol exposure produces HPA hyperresponsiveness to immune challenges; (2) prenatal alcohol treatment does not influence the release of cytokines to immune challenges; and (3) there are gender differences in the secretory pattern of corticosterone and cytokines to immune challenges. Therefore, these data do not support the hypothesis that cytokines play a role in the hyperresponsiveness of the HPA axis to immune challenges observed after prenatal alcohol exposure.

Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action.

Glucocorticoids are hormone products of the adrenal gland, which have long been recognized to have a profound impact on immunologic processes. The communication between immune and neuroendocrine systems is, however, bidirectional. The endocrine and immune systems share a common "chemical language," with both systems possessing ligands and receptors of "classical" hormones and immunoregulatory mediators. Studies in the early to mid 1980s demonstrated that monocyte-derived or recombinant interleukin-1 (IL-1) causes secretion of hormones of the hypothalamic-pituitary-adrenal (HPA) axis, establishing that immunoregulators, known as cytokines, play a pivotal role in this bidirectional communication between the immune and neuroendocrine systems. The subsequent 10-15 years have witnessed demonstrations that numerous members of several cytokine families increase the secretory activity of the HPA axis. Because this neuroendocrine action of cytokines is mediated primarily at the level of the central nervous system, studies investigating the mechanisms of HPA activation produced by cytokines take on a more broad significance, with findings relevant to the more fundamental question of how cytokines signal the brain. This article reviews published findings that have documented which cytokines have been shown to influence hormone secretion from the HPA axis, determined under what physiological/pathophysiological circumstances endogenous cytokines regulate HPA axis activity, established the possible sites of cytokine action on HPA axis hormone secretion, and identified the potential neuroanatomic and pharmacological mechanisms by which cytokines signal the neuroendocrine hypothalamus.

Influence of carbon monoxide, and its interaction with nitric oxide, on the adrenocorticotropin hormone response of the normal rat to a physico-emotional stress.

We determined whether the gas carbon monoxide (CO) altered the adrenocorticotropin hormone (ACTH) response to mild inescapable electrofootshocks, and whether it interacted with nitric oxide (NO). Peripheral injection of the NO synthase (NOS) inhibitor Nwnitro-L-arginine-methylester (L-NAME), a compound which readily crosses the blood-brain barrier, produced the expected blunting of the ACTH response to the shocks. This effect was mimicked by other arginine analogues such as L-nitroarginine (L-NNA) and NG-methyl-L-arginine (NMMA). The subcutaneous (s.c.) administration of the heme oxygenase (HO) blockers tin mesoporphyrin (SnMP) or tin protoporphyrin (SnPP) significantly decreased brain HO levels, indicating that both compounds had penetrated the brain. Blood pressure showed a modest increase in response to SnMP, and no change after SnPP. SnMP and SnPP both decreased shock-induced ACTH release, though the magnitude of this effect was slightly less than that of L-NAME. The influence of SnPP was further augmented in rats with concomitant blockade of NO formation, which suggests that both NO and CO are necessary for the full response of this axis to electrofootshocks. Finally, the ability of SnPP to significantly blunt the expression of the mRNA for the immediate early gene NGFI-B in the paraventricular nucleus (PVN) of rats exposed to shocks, indicates that the influence of CO was exerted on hypothalamic neuronal activity. Collectively, our results show that NO and CO exert a stimulatory effect on the HPA axis response to mild electrofootshocks, and that at least part of this influence takes place on hypothalamic neurons and/or their afferents.

Interleukin-1beta regulates pituitary follistatin and inhibin/activin betaB mRNA levels and attenuates FSH secretion in response to activin-A.

Activins and follistatins regulate all levels of the reproductive axis, including the pituitary where they stimulate and inhibit FSH production, respectively. Gonadotropes are known to express inhibin/activin betaB and activin-B (betaBbetaB) functions as an autocrine modulator of FSH production. By contrast, the mRNA for the activin-binding protein, follistatin, is present in most pituitary cells and folliculo-stellate cells may be the major source of the protein secreted by the anterior pituitary. Interleukin-1beta (IL-1beta) is one of several cytokines known to also influence the reproductive axis. IL-1beta inhibits the hypothalamo-pituitary-gonadal (HPG) axis by suppressing GnRH and gonadal steroid production. Because several pituitary cell types, including follistatin-producing folliculo-stellate cells, are targets of IL-1beta, cytokine effects on gonadotrope function were evaluated using cultured rat anterior pituitary cells. Activin-A (0.01 to 1 nM; 24h) increased basal FSH secretion approximately 2-fold. IL-1beta (0.005 to 0.5 nM) by itself had no effect on basal FSH secretion. However, IL-1beta attenuated FSH secretion in response to all concentrations of activin-A. These results suggest that the cytokine might stimulate the local production of a factor, such as follistatin, that antagonizes the action of activin-A. RNase protection analysis indicated that IL-1beta (0.005 to 5 nM) stimulated follistatin and inhibin/activin betaB mRNA accumulation in a time-dependent manner. These in vitro effects of IL-1beta were blocked by the specific IL-1 receptor antagonist (IL-lra) and were not mimicked by either rhIL-6 or lipopolysaccharide (LPS). Treatment of intact male rats with LPS (50 microg, i.v.), which increases plasma IL-1beta and induces IL-1beta expression in many tissues, including the pituitary, produced similar time-dependent increases in pituitary follistatin and inhibin/activin subunit mRNA levels. These results suggest that IL-1beta can modulate gonadotrope responses to activins by influencing the local balance of activin-B and follistatin within the pituitary.

Intracerebroventricular passive immunization. I. The effect of intracerebroventricular administration of an antiserum to tumor necrosis factor-alpha on the plasma adrenocorticotropin response to lipopolysaccharide in rats.

The present study tested the hypothesis that the cytokine tumor necrosis factor-alpha (TNF-alpha) is an important central nervous system mediator of the rat hypothalamo-pituitary-adrenal (HPA) axis response to the i.v. administration of lipopolysaccharide (LPS; 5 microg/kg). LPS produced a rapid (within 30 min) rise in plasma TNF-alpha levels, which preceded elevations in plasma ACTH (commencing at 45 min). Despite a lack of detectable TNF-alpha biological activity in the brain 30 min to 2 h after LPS administration, intracerebroventricular (i.c.v.) pretreatment (-20 h) with 5 microl anti-TNF-alpha antiserum significantly delayed the onset of the plasma ACTH response to LPS, suggesting that TNF-alpha acts within the brain. However, we also noted that the i.c.v. infusion of anti-TNF-alpha 20 h earlier produced experimentally significant concentrations of the same anti-TNF-alpha antibodies in systemic blood. This suggested the possibility that the effect of this antiserum was due to its leakage to the periphery. Indeed, 5 microl anti-TNF-alpha administered i.v. at -20 h produced an inhibition of the ACTH response to LPS that was temporally and quantitatively similar to that produced by i.c.v. anti-TNF-alpha. Intracerebroventricular administration of anti-TNF-alpha immediately before LPS produced only low systemic blood levels of corresponding anti-TNF-alpha antibodies and did not significantly alter the plasma ACTH response, whereas i.v. administration of anti-TNF-alpha immediately before LPS was clearly effective. Collectively, these results show that 1) biologically active levels of TNF-alpha in systemic plasma and the ensuing ACTH responses to LPS were always temporally and qualitatively related; and 2) even though i.c.v. administration of anti-TNF-alpha could inhibit the HPA axis response to LPS, this was apparent only when substantial amounts of anti-TNF-alpha antibodies had reached systemic blood. We, therefore, conclude that at the dose of LPS used in this study (5 microg/kg), TNF-alpha is an important mediator of the HPA axis response to LPS by an action within the periphery, but probably not within the brain.

Intracerebroventricular passive immunization. II. Intracerebroventricular infusion of neuropeptide antisera can inhibit neuropeptide signaling in peripheral tissues.

The findings of the preceding article suggest that intracerebroventricular (i.c.v.) administration of small amounts (5 microl) of antisera to rats may produce effectual immunoneutralization of peptides in blood/tissues outside of the central nervous system (CNS). In the present work we sought to test this hypothesis by determining the titers of corresponding antibodies in jugular venous plasma after i.c.v. infusion of three different antisera: a sheep anti-CRF, a rabbit anti-CRF, and a rabbit anti-GnRH. For all antisera tested, corresponding antibodies were detected in systemic plasma within 30 min of i.c.v. infusion of 5 microl antiserum. By 8 h, blood levels of the corresponding antibodies were similar whether the antisera had been infused i.c.v. or i.v. When the dilutions of antibodies equivalent to those in systemic blood 1-24 h after i.c.v. infusion of 5 microl antiserum were employed in rat anterior pituitary cell culture assays, they proved effective at inhibiting CRF- or GnRH-induced hormone secretion. Furthermore, in rats pretreated i.c.v. with 5 microl anti-CRF (at -4 h), pituitary ACTH secretion induced by i.v. CRF (0.3 nmol/kg) was reduced by 88%. Collectively, these data demonstrate that shortly after i.c.v. infusion of neuropeptide antisera, the levels of corresponding antibodies found in systemic blood are sufficient to inhibit neuropeptide signaling within peripheral tissues. As i.c.v. passive immunization procedures have been used extensively in the investigation of the biological roles of neuropeptides within the CNS, these findings indicate a critical reevaluation of the peripheral vs. CNS functions of neuropeptides.

Betidamino acid scan of the GnRH antagonist acyline.

Strong clinical evidence suggests that GnRH antagonists will replace GnRH agonists in a number of indications because of their ability to inhibit gonadotropin secretion as long as an adequate concentration of the analogue is present in the circulation whereas superagonists will take approximately 2 weeks to desensitize the gonadotrophs. Until recently, antagonists were either too weak and/or would release histamine. Azaline B {[Ac-D2Nal1,D4Cpa2,D3Pal3, 4Aph5(atz),D4Aph6(atz),ILys8,DAla10] GnRH} and long-acting members of the azaline family {Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(X)-D4Aph(Y) -Leu-ILys-Pro-DAla-NH2}, however, appear to be promising drug candidates. Because these antagonists tend to form gels (due to the formation of beta-sheet structures) and, as a result, are not readily amenable to formulation for long-term delivery, we have investigated ways of increasing hydrophilicity while retaining high potency and lack of histamine releasing activity. Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'-acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. We have used unresolved N alpha-Boc,N'alpha-Fmoc-aminoglycine, and N alpha-Boc,N'alpha-(CH3)Fmoc-aminoglycine as templates for the introduction of betidamino acids in acyline (Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2), a long acting member of the azaline B family, to test biocompatibility of these betide derivatives. Diastereomeric peptides could be separated using RP-HPLC in most cases. Biological results obtained in vitro (binding affinity to rat pituitary gland membranes) and in vivo (rat antiovulatory assay, AOA) indicate in most cases small differences in relative potencies (< 5-fold) between the D- and L-nonalkylated betidamino acid-containing acylines. Importantly, most betide diastereomers have high affinity for the GnRH receptor and were equipotent with acyline in the AOA. Greater differences in affinity and potency between diastereomers were observed after introduction of a methyl group on the side chain nitrogen ("beta" position) of the same analogues, with one of the diastereomer having an affinity and a potency in the AOA equivalent to that of acyline. These results suggest that chirality at the alpha-carbon coupled to side chain orientation is important for receptor recognition. The duration of action of some of the most potent analogues was also determined in the castrated male rat in order to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone release. Data suggest that introduction of a betidamino acid results in reduction of duration of action. Also, introduction of betidamino acids results in peptides with increased hydrophilicity (as determined by elution times on C18 silicas at pH 7.3) compared to that of the parent compound. N'-Methyl substitution results in parallel increase in retention times on C18 silicas as expected.

Gonadotropin-releasing hormone antagonists: novel members of the azaline B family.

A series of antagonists of gonadotropin-releasing hormone (GnRH) homologous to azaline B ([Ac-DNal1,DCpa2,DPal3,Aph5(Atz),DAph6+ ++(Atz),ILys8,DAla10]GnRH) was synthesized, characterized, and tested in a rat antiovulatory assay (AOA). Selected analogues were also tested in both an in vitro dispersed rat pituitary cell culture assay for inhibition of GnRH-stimulated luteinizing hormone release and an in vitro histamine release assay. The duration of action of some of the most potent and safest analogues in those assays was also determined in the castrated male rat in order to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone release. Structurally, this series of analogues has novel substitutions (X and Y) in the structure of the azaline B precursor: [Ac-DNal1,DCpa2,DPal3,-Aph5(X),DAph6(Y),++ +ILys8,DAla10]GnRH. These substitutions were designed to confer increased hydrophilicity as compared to that of azaline B (determined by relative retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.3) or to make them more easily accessible synthetically. Some bulky substituents were introduced in order to probe the spatial limitations of the receptor's cavity. These substitutions include acylated 4-aminophenylalanine at positions 5 and/or 6 (29 analogues), N alpha-methylated backbone substitutions (six analogues), N omega-isopropylaminophenylalanine at position 8, and hydrophilic amino acids at position 1. Out of 20 novel analogues tested for long duration of action in this series, only seven ([Ac-DNal1,DCpa2,DPal3,Aph5,DAph6,ILys8 ,DAla10]GnRH, [Ac-DNal1,DCpa2,DPal3,Aph5(For),DAph6(For) ,ILys8,DAla10]GnRH, [Ac-DNal1,DCpa2,DPal3,Aph5(Ac),DAph6(Ac),- ILys8,DAla10]GnRH (acyline), [Ac-DNal1,DCpa2,DPal3,Aph5(Pio),DAph6++ +(Pio),ILys8,DAla10]GnRH, [Ac-DNal1,DCpa2,DPal3,Aph5(Atz),DAph6++ +(Ac),ILys8,DAla10]GnRH, [Ac-DNalDCpa2,DPal3,Aph5(Atz-beta Ala),DAph6(Atz-beta Ala),ILys8, DAla10]GnRH, [Ac-DNal1,DCpa2,DPal3,Aph5(Atz-Gab), DAph6(Atz-Gab),ILys8,DAla10]GnRH) had relative potencies and/or duration of action comparable to those of azaline B. The others were one-half to one-tenth as effective as azaline B. N alpha-Methylated backbone substitutions at position 5 yielded analogues that were significantly more hydrophilic presumably because of the breakage of the NH alpha-Tyr5 to Arg8-CO hydrogen bond reported to stabilize a beta-turn encompassing residues 5-8 and which favored beta-sheet formation as shown earlier by Haviv et al. This substitution resulted, however, in an increased potency in the histamine release assay and in significantly shorter duration of action. Similarly, attempts at replacing isopropyllysine in position 8 by either isopropyl-4-aminophenylalanine or isopropyl-4-(aminomethyl)phenylalanine resulted in loss of potency in the AOA. Changes in chirality at position 1 or 10 resulted in analogues that were one-tenth and one-half as potent, respectively, as acyline.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat corticostatin R4: synthesis, disulfide bridge assignment, and in vivo activity.

We have synthesized significant amounts of the most potent member of the rat corticostatins that inhibits ACTH-induced corticosteroid and compared its structure to that of the natural hormone. The cystine bridging arrangement that corresponds to that reported for a human defensin (3-31, 5-20, 10-30) was determined. The in vitro corticostatic activity of the synthetic rat corticostatin R4 paralleled that of the natural R4. Biological studies in vivo showed that doses of 8 or 12 mg corticostatin/kg effectively interfered with corticosterone release in stressed rats. We conclude that in the assays that were used, the biological activity of the synthetic and natural molecules was identical. The availability of significant amounts of synthetic material will make possible studies investigating the physiological role played by corticostatins in modulating the activity of the hypothalamic-pituitary-adrenal axis.

Synthesis and theoretical conformational studies of gonadotropin-releasing hormone analogs containing tetrahydroisoquinoline carboxylic acid.

A series of analogs of gonadotropin-releasing hormone (GnRH) containing the conformationally restrictive residue tetrahydroisoquinoline carboxylic acid (Tic) or its non-restricted parent phenylalanine were synthesized and evaluated for anti-ovulatory activity in the rat. The series, based on the potent linear parent compound Ac-DNal1-DCpa2-DPal3- Ser4-Tyr5-DPal6-Leu7-Arg8-Pro9-DAla10-NH2, included L-Tic in positions 1, 2, 3, 7 and 9, D-Tic in positions 1, 2, 3, 6 and 9, or D-Phe in positions 2 and 3 for comparison. The most potent analog in this series, with D-Tic in position 6, fully inhibited ovulation at 2.5 micrograms compared to near complete inhibition at 0.5 microgram for the parent compound. A theoretical analysis of the conformational restrictions imposed on mainchain and sidechain torsional angles by the incorporation of Tic was conducted in vacuo using molecular mechanics techniques. Using cyclo(4-10)-[Ac-delta 3Pro1,DCpa2,DTrp3,Asp4,DNal6,Dpr10]- GnRH as a template conformer for which NMR conformational data is available, it was found that the potency of the different analogs correlated with the strain energy required to deform the mainchain and backbone angles of residues to values which would be expected if Tic were present and if the analog assumed the same solution structure. In particular, the effect of DTic at position 6 is to maintain the type II' beta-turn involving residues 5-8 found in active GnRH analogs.

New effective gonadotropin releasing hormone antagonists with minimal potency for histamine release in vitro.

In order to minimize the adverse effect of histamine release in the rat of some gonadotropin releasing hormone (GnRH) antagonists, such as [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH, new structures with modifications at positions 1, 2, 3, 5, 6, 7, and 10 were synthesized and tested in several biological systems. In vitro: the affinity for the pituitary GnRH receptor was measured as was the ability of the analogues to inhibit GnRH-stimulated release of luteinizing hormone (LH) by dispersed anterior pituitary cells in culture and to release histamine from rat mast cells. In vivo: inhibition of ovulation in the cycling rat was determined after subcutaneous (sc) injection of the peptides at noon on the day of proestrus; the duration of action of the peptides was evaluated by measuring LH levels in the castrated male rat after sc injection of some selected analogues. [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,D-4-p-methoxy benzoyl-2-aminobutyric acid6,DAla10]-GnRH was found to be one of the most potent analogues of this series, causing a 100% inhibition of ovulation at 5 micrograms/kg or less. Release of histamine was observed at doses 10-25 times that required for [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH. Thus, introduction of arginine in position 5 with a hydrophobic amino acid in position 6 is compatible with high potency in several biological systems and results in compounds with lowered potency to release histamine compared to homologous peptides with tyrosine in position 5 and D-arginine in position 6.

Increased pituitary sensitivity to glucocorticoid feedback during the stress nonresponsive period in the neonatal rat.

Neonatal rats show a diminished response to stress [the stress-nonresponsive period (SNRP)] from day 2-3 until day 14 of age; the physiological bases for the SNRP are unknown. We examined whether enhanced sensitivity of the brain or pituitary to the inhibitory feedback effects of circulating glucocorticoids (GC) contributes to the SNRP. Age-related changes in the ability of corticosterone (CORT) and dexamethasone (DEX) to inhibit the ACTH secretion induced by urethane or CRF were studied. We also examined the ACTH response to ether stress or CRF in intact or 24 h-adrenalectomized 5-day-old rats. Plasma ACTH did not increase in intact rats after ether stress (basal: 64.6 +/- 9.1 pg/ml vs. stressed: 66.8 +/- 8.9 pg/ml; P greater than 0.05), whereas small elevations occurred after CRF challenge (184.6 +/- 40 pg/ml; P less than or equal to 0.01). Five-day-old adrenalectomized rats, which had elevated basal ACTH concentrations, increased ACTH secretion after exposure to ether or CRF. Thus, negative feedback appears to mediate critically the SNRP. Furthermore, sensitivity to such feedback was enhanced during the SNRP since the capacity of CORT to inhibit urethane-induced ACTH secretion in vivo declined with age; 1 mg/kg BW was the minimal dose that inhibited ACTH secretion at day 10, whereas at day 18, the threshold for a similar inhibition was 5 mg/kg BW. In contrast, at both ages, a dose of 10 micrograms/kg BW DEX inhibited ACTH release. In vitro dose response studies in whole pituitaries further demonstrated the enhanced pituitary sensitivity to GC feedback during the SNRP since the IC50 for CORT inhibition of CRF-induced ACTH release increased from days 3-5 to days 22-23. A similar, although not statistically significant trend was observed for DEX inhibition. Thus, neonatal rats exhibit an enhanced pituitary sensitivity to GC during the SNRP and removal of this inhibition allows ACTH secretion in response to ether stress.