A defect in the structure of the obese gene is responsible for development of obesity in the ob͞ob mouse. The product of expression of the gene is the protein hormone leptin. Leptin causes weight loss in ob͞ob and normal mice, it is secreted by adipocytes, and it is an important controller of the size of fat stores by inhibiting appetite. The ob͞ob mouse is infertile and has a pattern of gonadotropin secretion similar to that of prepubertal animals. Consequently, we hypothesized that leptin might play a role in the control of gonadotropin secretion and initiated studies on its possible acute effects on hypothalamic-pituitary function. After a preincubation period, hemi-anterior pituitaries of adult male rats were incubated with leptin for 3 hr. Leptin produced a dose-related increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release, which reached peaks with 10 ؊9 and 10 ؊11 M leptin, respectively. Gonadotropin release decreased at higher concentrations of leptin to values indistinguishable from that of control pituitaries. On the other hand, prolactin secretion was greatly increased in a dose-related manner but only with leptin concentrations (10 ؊7 -10 ؊5 M). Incubation with leptin of median eminence-arcuate nuclear explants from the same animals produced significant increases in LH-releasing hormone (LHRH) release only at the lowest concentrations tested (10 ؊12 -10 ؊10 M). As the leptin concentration was increased, LHRH release decreased and was significantly less than control release at the highest concentration tested (10 ؊6 M). To determine if leptin can also release gonadotropins in vivo, ovariectomized females bearing implanted third ventricle cannulae were injected with 10 g of estradiol benzoate s.c., followed 72 hr later by microinjection into the third ventricle of leptin (0.6 nmol in 5 l) or an equal volume of diluent. There was a highly significant increase in plasma LH, which peaked 10-50 min after injection of leptin. Leptin had no effect on plasma FSH concentrations, and the diluent had no effect on either plasma FSH or LH. Thus, leptin at very low concentrations stimulated LHRH release from hypothalamic explants and FSH and LH release from anterior pituitaries of adult male rats in vitro and released LH, but not FSH, in vivo. The results indicate that leptin plays an important role in controlling gonadotropin secretion by stimulatory hypothalamic and pituitary actions.

A BSTRACT : During infection, bacterial and viral products, such as bacterial lipopolysaccharide (LPS), cause the release of cytokines from immune cells. These cytokines can reach the brain by several routes. Furthermore, cytokines, such as interleukin-1 (IL-1), are induced in neurons within the brain by systemic injection of LPS. These cytokines determine the pattern of hypothalamic-pituitary secretion that characterizes infection. IL-2, by stimulation of cholinergic neurons, activates neural nitric oxide synthase (nNOS). The nitric oxide (NO) released diffuses into corticotropin-releasing hormone (CRH)secreting neurons and releases CRH. IL-2 also acts in the pituitary to stimulate adrenocorticotropic hormone (ACTH) secretion. On the other hand, IL-1 ␣ blocks the NO-induced release of luteinizing hormone-releasing hormone (LHRH) from LHRH neurons, thereby blocking pulsatile LH but not folliclestimulating hormone (FSH) release and also inhibiting sex behavior that is induced by LHRH. IL-1 ␣ and granulocyte macrophage colony-stimulating factor (GMCSF) block the response of the LHRH terminals to NO. The mechanism of action of GMCSF to inhibit LHRH release is as follows. It acts on its receptors on ␥ -aminobutyric acid (GABA)ergic neurons to stimulate GABA release. GABA acts on GABAa receptors on the LHRH neuronal terminal to block NOergic stimulation of LHRH release. IL-1 ␣ inhibits growth hormone (GH) release by inhibiting GH-releasing hormone (GHRH) release, which is mediated by NO, and stimulating somatostatin release, also mediated by NO. IL-1 ␣ -induced stimulation of PRL release is also mediated by intrahypothlamic action of NO, which inhibits release of the PRL-inhibiting hormone dopamine. The actions of NO are brought about by its combined activation of guanylate cyclase-liberating cyclic guanosine monophosphate (cGMP) and activation of cyclooxygenase (COX) and lipoxygenase (LOX) with liberation of prostaglandin E2 and leukotrienes, respectively. Thus, NO plays a key role in inducing the changes in release of hypothalamic peptides induced in infection by cytokines. Cytokines, such as IL-1 ␤ , also act in the anterior pituitary 5 M C CANN et al. : MECHANISM OF ACTION OF CYTOKINES IN INFECTION gland, at least in part via induction of inducible NOS. The NO produced inhibits release of ACTH. The adipocyte hormone leptin, a member of the cytokine family, has largely opposite actions to those of the proinflammatory cytokines, stimulating the release of FSHRF and LHRH from the hypothalamus and FSH and LH from the pituitary directly by NO.

Previous studies indicated that there is a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH). An FSH-releasing factor (FSHRF) was purified from rat and sheep hypothalami, but has not been isolated. We hypothesized that FSHRF might be an analogue of mammalian luteinizing hormone-releasing hormone (m-LHRH) and evaluated the activity of many analogues of m-LHRH and of the known LHRHs found in lower forms. Here we demonstrate that lamprey (l) LHRH-III has a potent, dose-related FSH-but not LH-releasing action on incubated hemipituitaries of male rats. l-LHRH-I on the other hand, had little activity to release either FSH or LH. m-LHRH was equipotent to l-LHRH-III to release FSH, but also had a high potency to release LH in contrast to l-LHRH-III that selectively released FSH. Chicken LHRH-II had considerable potency to release both LH and FSH, but no selectivity in its action. Salmon LHRH had much less potency than the others tested, except for l-LHRH-I, and no selectivity in its action. Because ovariectomized, estrogen, progesterone-treated rats are a sensitive in vivo assay for FSHand LH-releasing activity, we evaluated l-LHRH-III in this assay and found that it had a completely selective stimulatory effect on FSH release at the two doses tested (10 and 100 pmols). Therefore, l-LHRH-III is a highly potent and specific FSH-releasing peptide that may enhance fertility in animals and humans. It may be the long sought after m-FSHRF.Although the evidence from physiological studies for a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH) is compelling (1-5), proof of the separate hypothalamic control of FSH rests on the identification of an FSH-releasing factor (FSHRF). Crude extracts of the organum vasculosum lamina terminalis contained much more FSH-releasing activity than accounted for by the content of LH-releasing hormone (LHRH), and the slope of the dose-response curve in the in vitro bioassay for FSH release was much steeper than that obtained with LHRH (6). Extracts of the posterior median eminence similarly contained more FSH-releasing activity than could be accounted for by the content of LHRH (7).FSHRF was purified originally from sheep hypothalami by gel filtration on Sephadex G-25 followed by carboxymethyl cellulose chromatography (2, 3). The release of FSH was measured by bioassay using either in vivo or in vitro assays of FSH-releasing activity (2, 3). Because LHRH has intrinsic FSH-releasing activity (8) and because Schally et al. could not separate FSH-from LH-releasing activity by fractionation on Sephadex G-25 using in vitro assays and measurement of FSH by immunoassay, they concluded that there was only one gonadotropin-releasing hormone (9).Lumpkin et al. (10) separated the FSH-from the LHreleasing activity on the same column of Sephadex G-25 used originally (2) with bioassay of the activity in vivo in the ovariectomized (OVX), estrogen-progesterone-...

Stimulation of corticotropin-releasing factor (CRF) release from the hypothalamus by interleukin 2 (IL-2) was recently demonstrated. Cytokines induce nitric oxide synthase (NOS), an enzyme that converts L-arginine into L-citrulline and nitric oxide (NO). NO is believed to be responsible for the cytotoxic action of these agents. The constitutive form of NOS occurs in neurons in the central nervous system and NO appears to play a neurotransmitter role in cerebeilar and hippocampal function. We explored the probability that IL-2 and synaptic transmitters might release CRF via NO. The effects of L-arginine, the substrate for NOS, and NG_ monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, on IL-2-induced CRF release were studied using mediobasal hypothalami (MBHs) incubated in vitro in KrebsRinger bicarbonate buffer. L-Argoinue did not alter basal and IL-2-induced CRF release after 30 min of incubation but significantly elevated both basal and IL-2-induced CRF release when MBHs were incubated 30 min longer, presumably because the endogenous substrate had been depleted after the initial 30-min incubation period. In 30-min incubations, both carbachol, an acetylcholineomimetic drug, and norepinephrine stimulated CRF release. There was an additive effect of incubation of the MBHs in the presence of carbachol (10-7 M) and IL-2 (10-13 M). On the other hand, coincubation of MBHs with norepinephrine (10-6 M) and IL-2 (10-13 M) did not produce any additive effect. Addition of NMMA, an inhibitor of NOS, at 1 or 3 x 10-4 M completely suppressed IL-2-induced release of CRF as well as that caused by IL-2 plus carbachol. In contrast, the release of CRF induced by norepinephrine was not blocked by 3 x 10-4 M NMMA. The data indicate that IL-2 can activate constitutive NOS leading to increased NO release, which activates CRF release. It appears that NO is also involved in the release of CRF induced by carbachol but not by norepinephrine.Recent reports have demonstrated a stimulatory action of interleukin 2 (IL-2) on corticotropin-releasing factor (CRF) release using~ptoerfused hypothalami (1) or after static incubation with mediobasal hypothalami (MBHs) (unpublished data). In peripheral tissues, cytokines have been found to induce nitric oxide synthase (NOS), an enzyme that converts L-arginine (L-Arg) into L-citrulline and a reactive gas, nitric oxide (NO) (3, 4). The NO can induce cell death. It takes a number of hours for a cytokine to induce NOS. Consequently, this form of the enzyme has been termed the inducible NOS. A second type of NOS exists in a number of cells throughout the body, for example, in vascular endothelium, and it is termed the constitutive form of the enzyme (3, 4). In contrast to the inducible form, the constitutive form requires activation by an increase in intracellular calcium and its interaction with calmodulin. Both forms of NOS require L-Arg as the substrate (3, 4) and are inhibited by L-Arg analogues, such as NG-monomethyl-L-arginine (NMMA) (3, 4).The constitutive form of the enzyme ...

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Several monokines, proteins secreted by monocytes and macrophages, alter release of hormones from the anterior pituitary. We report here the ability offemtomolar concentrations of interleukin 2 (IL-2), a lymphokine released from T lymphocytes, to alter directly pituitary hormone release. The effects of concentrations of IL-2 ranging from 10-i7 to 10-9 M on anterior pituitary hormone release were evaluated in vitro. Hemipituitaries were preincubated in 1 ml of KrebsRinger bicarbonate buffer (KRB) followed by incubation for 1 or 2 hr with KRB or KRB containing different concentrations of IL-2. This was followed by incubation for 30 min in 56 mM potassium medium to study the effect ofpretreatment with IL-2 on subsequent depolarization-induced hormone release. Prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), corticotropin (ACTH), growth hormone (GH), and thyrotropic hormone (TSH) released into the incubation medium were measured by radioimmunoassay. IL-2 stimulated the basal release of PRL at 1 or 2 hr but suppressed the subsequent depolarization-induced PRL release, perhaps because the readily releasable pool of PRL was exhausted. The minimal effective dose (MED) was 10-15 M. Conversely, IL-2 significantly suppressed the basal release of LH and FSH at 1 or 2 hr, with a MED of 10-16 M, thus demonstrating a reciprocal action of the cytokine on lactotrophs and gonadotrophs. The subsequent depolarization-induced release of LH and FSH was suppressed, indicative of a persistent inhibitory action of IL-2. IL-2 stimulated ACTH and TSH release at 1 hr and the MEDs were 10-12 and 10-1' M, respectively. Conversely, IL-2 significantly lowered the basal release of GH at 1 hr, with a MED of 10-'s M. The release of GH was not altered at 2 hr. The high potassium-induced release of ACTH, TSH, and GH was not affected. The results demonstrate that IL-2 at picomolar concentrations affects the release of anterior pituitary hormones. This cytokine may serve as an important messenger from lymphocytes exerting a direct paracrine action on the pituitary by its release from lymphocytes in the gland or concentrations in the blood that reach the gland may be sufficient to activate it.Bidirectional intercellular communication between the neuroendocrine and immune systems is firmly established (1, 2). The interplay between these systems is believed to be mediated by proteins termed cytokines (3). Interleukin 2 (IL-2) is a lymphokine, synthesized and secreted by T lymphocytes activated by antigen or mitogen (4,5). It is a glycoprotein (15.5 kDa) that stimulates the proliferation of T cells, B cells, natural killer cells, and lymphocyte-activated killer cells (3). IL-2 has been reported to enhance the expression of the proopiomelanocortin (POMC) gene by cultured normal and pituitary tumor (AT-20) cells (6). A similar activation of POMC mRNA has already been demonstrated for IL-1 (7). IL-2 increased plasma corticotropin (ACTH) and glucocorticoid levels when injected into patients with cancer or acquired immuno...

Because ascorbic acid (AA) is concentrated in synaptic vesicles containing glutamic acid, we hypothesized that AA might act as a neurotransmitter. Because AA is an antioxidant, it might therefore inhibit nitric oxidergic (NOergic) activation of luteinizing hormonereleasing hormone (LH-RH) release from medial basal hypothalamic explants by chemically reducing NO. Cell membrane depolarization induced by increased potassium concentration [K ؉ ] increased medium concentrations of both AA and LH-RH. An inhibitor of NO synthase (NOS), N G -monomethyl-L-arginine (NMMA), prevented the increase in medium concentrations of AA and LH-RH induced by high [K ؉ ], suggesting that NO mediates release of both AA and LH-RH. Calcium-free medium blocked not only the increase in AA in the medium but also the release of LH-RH. Sodium nitroprusside, which releases NO, stimulated LH-RH release and decreased the concentration of AA in the incubation medium, presumably because the NO released oxidized AA to dehydro-AA. AA (10 ؊5 to 10 ؊3 M) had no effect on basal LH-RH release but completely blocked high [K ؉ ]-and nitroprusside-induced LH-RH release. N-Methyl-D-aspartic acid (NMDA), which mimics the action of the excitatory amino acid neurotransmitter glutamic acid, releases LH-RH by releasing NO. AA (10 ؊5 to 10 ؊3 M) inhibited the LH-RH-releasing action of NMDA. AA may be an inhibitory neurotransmitter that blocks NOergic stimulation of LH-RH release by chemically reducing the NO released by the NOergic neurons.is present in high concentrations in the hypothalamus (1-3) but its role in the modulation of hypothalamic hormone release is obscure. The brain accumulates AA from the blood and maintains a relatively high concentration (1.1-1.7 mM), independent of AA ingestion (4-7). Neuronal intracellular AA concentrations are 10 times greater than extracellular AA concentration (6, 7). Neuronal activity was shown to increase extracellular AA in vitro (6,(8)(9)(10)(11)(12) and in vivo as determined by voltametry (13). AA is further concentrated in isolated nerve terminals (14). Synaptic vesicles concentrate AA by active transport (15). AA is present in high concentrations in synaptic vesicles of glutamergic neurons (13) and in adrenal medullary catecholamine secretory granules (16,17).The presence of high concentrations of AA in synaptic vesicles and the evidence that it could be released into the extracellular space by neuronal activity suggested to us that AA might be a neurotransmitter. Since high concentrations of AA are already known to be present in the hypothalamus, we hypothesized that AA might control luteinizing hormone-releasing hormone (LH-RH) release, possibly by its antioxidant properties. LH-RH release is controlled by release of nitric oxide (NO) from NOergic interneurons that release the soluble gas in juxtaposition to the LH-RH terminals (18,19). In the terminals, NO activates guanylyl cyclase, leading to an increase in cyclic guanosine monophosphate (cGMP), cyclooxygenase, resulting in increased concentrations of prostagla...

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