Discovery of a functionally selective ghrelin receptor (GHSR1a) ligand for modulating brain dopamine.

SignificanceThe modulation of growth hormone secretagogue receptor-1a (GHSR1a) signaling is a promising strategy for treating brain conditions of metabolism, aging, and addiction. GHSR1a activation results in pleiotropic physiological outcomes through distinct and pharmacologically separable G protein- and ß-arrestin (ßarr)-dependent signaling pathways. Thus, pathway-selective modulation can enable improved pharmacotherapeutics that can promote therapeutic efficacy while mitigating side effects. Here, we describe the discovery of a brain-penetrant small molecule, N8279 (NCATS-SM8864), that biases GHSR1a conformations toward Gαq activation and reduces aberrant dopaminergic behavior in mice. N8279 represents a promising chemical scaffold to advance the development of better treatments for GHSR1a-related brain disorders involving the pathological dysregulation of dopamine.

Neurotrophic factor-α1 prevents stress-induced depression through enhancement of neurogenesis and is activated by rosiglitazone.

Major depressive disorder is often linked to stress. Although short-term stress is without effect in mice, prolonged stress leads to depressive-like behavior, indicating that an allostatic mechanism exists in this difference. Here we demonstrate that mice after short-term (1 h per day for 7 days) chronic restraint stress (CRS), do not display depressive-like behavior. Analysis of the hippocampus of these mice showed increased levels of neurotrophic factor-α1 (NF-α1; also known as carboxypeptidase E, CPE), concomitant with enhanced fibroblast growth factor 2 (FGF2) expression, and an increase in neurogenesis in the dentate gyrus. In contrast, after prolonged (6 h per day for 21 days) CRS, mice show decreased hippocampal NF-α1 and FGF2 levels and depressive-like responses. In NF-α1-knockout mice, hippocampal FGF2 levels and neurogenesis are reduced. These mice exhibit depressive-like behavior that is reversed by FGF2 administration. Indeed, studies in cultured hippocampal neurons reveal that NF-α1 treatment directly upregulates FGF2 expression through extracellular signal-regulated kinase-Sp1 signaling. Thus, during short-term CRS, hippocampal NF-α1 expression is upregulated and has a key role in preventing the onset of depressive-like behavior through enhanced FGF2-mediated neurogenesis. To evaluate the therapeutic potential of this pathway, we examined, rosiglitazone (Rosi), a PPARγ agonist, which has been shown to have antidepressant activity in rodents and humans. Rosi upregulates FGF2 expression in a NF-α1-dependent manner in hippocampal neurons. Mice fed Rosi show increased hippocampal NF-α1 levels and neurogenesis compared with controls, thereby indicating the antidepressant action of this drug. Development of drugs that activate the NF-α1/FGF2/neurogenesis pathway can offer a new approach to depression therapy.

In vivo and in vitro analyses of amygdalar function reveal a role for copper.

Mice with a single copy of the peptide amidating monooxygenase (Pam) gene (PAM(+/-)) are impaired in contextual and cued fear conditioning. These abnormalities coincide with deficient long-term potentiation (LTP) at excitatory thalamic afferent synapses onto pyramidal neurons in the lateral amygdala. Slice recordings from PAM(+/-) mice identified an increase in GABAergic tone (Gaier ED, Rodriguiz RM, Ma XM, Sivaramakrishnan S, Bousquet-Moore D, Wetsel WC, Eipper BA, Mains RE. J Neurosci 30: 13656-13669, 2010). Biochemical data indicate a tissue-specific deficit in Cu content in the amygdala; amygdalar expression of Atox-1 and Atp7a, essential for transport of Cu into the secretory pathway, is reduced in PAM(+/-) mice. When PAM(+/-) mice were fed a diet supplemented with Cu, the impairments in fear conditioning were reversed, and LTP was normalized in amygdala slice recordings. A role for endogenous Cu in amygdalar LTP was established by the inhibitory effect of a brief incubation of wild-type slices with bathocuproine disulfonate, a highly selective, cell-impermeant Cu chelator. Interestingly, bath-applied CuSO4 had no effect on excitatory currents but reversibly potentiated the disynaptic inhibitory current. Bath-applied CuSO4 was sufficient to potentiate wild-type amygdala afferent synapses. The ability of dietary Cu to affect signaling in pathways that govern fear-based behaviors supports an essential physiological role for Cu in amygdalar function at both the synaptic and behavioral levels. This work is relevant to neurological and psychiatric disorders in which disturbed Cu homeostasis could contribute to altered synaptic transmission, including Wilson's, Menkes, Alzheimer's, and prion-related diseases.

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes.

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

Role of serotonin in the paradoxical calming effect of psychostimulants on hyperactivity.

The mechanism by which psychostimulants act as calming agents in humans with attention-deficit hyperactivity disorder (ADHD) or hyperkinetic disorder is currently unknown. Mice lacking the gene encoding the plasma membrane dopamine transporter (DAT) have elevated dopaminergic tone and are hyperactive. This activity was exacerbated by exposure to a novel environment. Additionally, these mice were impaired in spatial cognitive function, and they showed a decrease in locomotion in response to psychostimulants. This paradoxical calming effect of psychostimulants depended on serotonergic neurotransmission. The parallels between the DAT knockout mice and individuals with ADHD suggest that common mechanisms may underlie some of their behaviors and responses to psychostimulants.

Anterior pituitary hypoplasia and dwarfism in mice lacking the dopamine transporter.

Deletion of the dopamine transporter (DAT) results in increased dopaminergic tone, anterior pituitary hypoplasia, dwarfism, and an inability to lactate. DAT elimination alters the spatial distribution and dramatically reduces the numbers of lactotrophs and somatotrophs in the pituitary. Despite having normal circulating levels of growth hormone and prolactin in blood, hypoplastic glands from DAT-/- mice fail to respond to secretagog stimulation. The effects of DAT deletion on pituitary function result from elevated DA levels that down-regulate the lactotroph D2 DA receptors and depress hypothalamic growth hormone-releasing hormone content. These results reveal an unexpected and important role or DA in the control of developmental events in the pituitary gland and assign a critical role for hypothalamic DA reuptake in regulating these events.

Mice lacking the norepinephrine transporter are supersensitive to psychostimulants.

The action of norepinephrine (NE) is terminated, in part, by its uptake into presynaptic noradrenergic neurons by the plasma-membrane NE transporter (NET), which is a target for antidepressants and psychostimulants. Disruption of the NET gene in mice prolonged the clearance of NE and elevated extracellular levels of this catecholamine. In a classical test for antidepressant drugs, the NET-deficient (NET-/-) animals behaved like antidepressant-treated wild-type mice. Mutants were hyper-responsive to locomotor stimulation by cocaine or amphetamine. These responses were accompanied by dopamine D2/D3 receptor supersensitivity. Thus altering NET expression significantly modulates midbrain dopaminergic function, an effect that may be an important component of the actions of antidepressants and psychostimulants.

Targeted deletion of GD3 synthase protects against MPTP-induced neurodegeneration.

Parkinson's disease is a debilitating neurodegenerative condition for which there is no cure. Converging evidence implicates gangliosides in the pathogenesis of several neurodegenerative diseases, suggesting a potential new class of therapeutic targets. We have shown that interventions that simultaneously increase the neuroprotective GM1 ganglioside and decrease the pro-apoptotic GD3 ganglioside - such as inhibition of GD3 synthase (GD3S) or administration of sialidase - are neuroprotective in vitro and in a number of preclinical models. In this study, we investigated the effects of GD3S deletion on parkinsonism induced by 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP was administered to GD3S-/- mice or controls using a subchronic regimen consisting of three series of low-dose injections (11 mg/kg/day × 5 days each, 3 weeks apart), and motor function was assessed after each. The typical battery of tests used to assess parkinsonism failed to detect deficits in MPTP-treated mice. More sensitive measures - such as the force-plate actimeter and treadmill gait parameters - detected subtle effects of MPTP, some of which were absent in mice lacking GD3S. In wild-type mice, MPTP destroyed 53% of the tyrosine-hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNc) and reduced striatal dopamine 60.7%. In contrast, lesion size was only 22.5% in GD3S-/- mice and striatal dopamine was reduced by 37.2%. Stereological counts of Nissl-positive SNc neurons that did not express TH suggest that neuroprotection was complete but TH expression was suppressed in some cells. These results show that inhibition of GD3S has neuroprotective properties in the MPTP model and may warrant further investigation as a therapeutic target.

Hepatic prolactin binding in female Sprague-Dawley rats fed a diet high in corn oil.

The relationship between dietary fat content and hepatic prolactin (PRL) binding during the four stages of the estrous cycle was examined. Serum 17 beta-estradiol, PRL, and progesterone were also monitored in the same animals. Female SD rats were fed 5 or 24% corn oil in nutritionally balanced diets, given 2.5 mg 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6] or vehicle by gavage at 8 weeks of age, and decapitated at noon on 1 of the 4 days of the estrous cycle 2-5 weeks later. Analysis of hepatic PRL binding data by Scatchard plots revealed no effect of diet or DMBA. PRL binding was lowest at proestrus, increased at estrus, remained elevated at metestrus, and declined at diestrus. Diet also had no effect on serum hormone concentrations, which showed the expected pattern during the estrous cycle in all treatment groups. Results reported by others of an effect of dietary corn oil content on hepatic PRL binding may have been due to use of control diets low in essential fatty acids.

Absence of an effect of dietary corn oil content on plasma prolactin, progesterone, and 17 beta-estradiol in female Sprague-Dawley rats.

Enhancement of mammary tumorigenesis in rats by high-fat diets has been postulated to be due to altered hormonal status. Elevated serum prolactin and, in some cases, estrogen have been reported in rats fed diets high in corn oil or lard that increase 7,12-dimethylbenz(a)anthracene (DMBA) tumorigenesis. However, we have found no difference in plasma prolactin during the proestrus surge or at the other stages of the estrous cycle in rats fed a diet high (24%) in corn oil that augments DMBA-induced tumorigenesis or a control diet containing 5% corn oil. There was no effect of the same dietary treatment on plasma progesterone or serum estradiol in the same experiments. In addition, we found that DMBA administration did not change the blood concentration of any of the three hormones. Female Sprague-Dawley rats, bearing atrial cannulae and given a carcinogenic dose of DMBA at 8 weeks of age, were studied 2 to 5 weeks or 10 to 13 weeks after DMBA administration. Rats given only the oil vehicle for DMBA were studied at the same ages. Blood samples were taken from rats with 4-day estrous cycles at 3-hr intervals on the day of proestrus and at 6- to 12-hr intervals on the other days of the cycle. No effect of dietary corn oil content or of DMBA administration on plasma prolactin and progesterone was detected in either age group. On the afternoon of proestrus, rats were sampled more frequently to examine the hormonal patterns in detail; again, no dietary effect was detected. Serum estradiol was measured in rats that were fed the control or high-corn-oil diets, treated with DMBA or vehicle, and decapitated on one of the 4 days of the estrous cycle. There was no detectable effect of dietary corn oil content or DMBA treatment during any stage of the cycle. All three hormones showed qualitative and quantitative patterns identical to normal cycling rats at both ages studied. Our results indicate that earlier reports of an effect of dietary fat on blood prolactin and estrogen content may have been due either to diets low in essential fatty acids or to anesthesia-induced hormonal responses.

Cerebellar defects in Ca2+/calmodulin kinase IV-deficient mice.

The Ca(2+)/calmodulin-dependent protein kinase CaMKIV was first identified in the cerebellum and has been implicated in nuclear signaling events that control neuronal growth, differentiation, and plasticity. To understand the physiological importance of CaMKIV, we disrupted the mouse Camk4 gene. The CaMKIV null mice displayed locomotor defects consistent with altered cerebellar function. Although the overall cytoarchitecture of the cerebellum appeared normal in the Camk4(-/-) mice, we observed a significant reduction in the number of mature Purkinje neurons and reduced expression of the protein marker calbindin D28k within individual Purkinje neurons. Western immunoblot analyses of cerebellar extracts also established significant deficits in the phosphorylation of cAMP response element-binding protein at serine-133, a proposed target of CaMKIV. Additionally, the absence of CaMKIV markedly altered neurotransmission at excitatory synapses in Purkinje cells. Multiple innervation by climbing fibers and enhanced parallel fiber synaptic currents suggested an immature development of Purkinje cells in the Camk4(-/-) mice. Together, these findings demonstrate that CaMKIV plays key roles in the function and development of the cerebellum.

Monoaminergic dysregulation in glutathione-deficient mice: possible relevance to schizophrenia?

Several lines of research have implicated glutathione (GSH) in schizophrenia. For instance, GSH deficiency has been reported in the prefrontal cortex of schizophrenics in vivo. Further, in rats postnatal GSH-deficiency combined with hyperdopaminergia led to cognitive impairments in the adult. In the present report we studied the effects of 2-day GSH-deficiency with L-buthionine-(S,R)-sulfoximine on monoaminergic function in mice. The effect of GSH-deficiency per se and when combined with the amphetamine and phencyclidine (PCP) models of schizophrenia was investigated. GSH-deficiency significantly altered tissue levels of dopamine (DA), 5-hydroxytryptamine (5-HT) and their respective metabolites homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in a region-specific fashion. The effects of GSH-deficiency on tissue monoamines were distinct from and, generally, did not interact with the effects of amphetamine (5 mg/kg; i.p.) on tissue monoamines. Microdialysis studies showed that extracellular DA-release after amphetamine (5 mg/kg, i.p.) was two-fold increased in the nucleus accumbens of GSH-deficient mice as compared with control mice. Basal DA was unaltered. Further, extracellular levels of HVA in the frontal cortex and hippocampus and 5-HIAA in the nucleus accumbens were elevated by GSH-deficiency per se. Spontaneous locomotor activity in the open field was unchanged in GSH-deficient mice. In contrast, GSH-deficiency modulated the locomotor responses to mid-range doses of amphetamine (1.5 and 5 mg/kg, i.p.). Further, GSH-deficient mice displayed an increased locomotor response to low (2 and 3 mg/kg, i.p.) doses of phencyclidine (PCP). In conclusion, the data presented here show that even short-term GSH-deficiency has consequences for DA and 5-HT function. This was confirmed on both neurochemical and behavioral levels. How GSH and the monoamines interact needs further scrutiny. Moreover, the open field findings suggest reduced or altered N-methyl-d-aspartate (NMDA) receptor function in GSH-deficient mice. Thus, GSH-deficiency can lead to disturbances in DA, 5-HT and NMDA function, a finding that may have relevance for schizophrenia.

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat.

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.

Endogenous protein kinase-C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels.

The osteoblast-like osteosarcoma cell line ROS 17/2.8, which expresses very low levels of estrogen receptor (ER), was stably transfected with the mouse ER in order to more easily evaluate the physiological role of estrogens in bone cell homeostasis. These transfected ROS.SMER 14 cells are highly responsive to estrogenic stimulation at subconfluence, but become refractory to estrogenic stimulation when postconfluency is reached. The purpose of these studies was to determine the mechanisms underlying this loss of responsiveness in these ER stably transfected cells at postconfluence. When proliferative capacity was evaluated by bromodeoxyuridine immunocytochemistry, approximately 70% of the subconfluent cells were actively dividing, whereas none of the postconfluent cells underwent division. Subconfluent cells were found to contain 2500-3000 ER-binding sites/cell, whereas the ER in postconfluent cells was low and often undetectable. Steady state ER mRNA levels were not significantly modified by postconfluency. ER protein levels were also unaffected by confluency status. Since protein kinase-C (PKC) has been reported to influence cell proliferation and steroid hormone receptor binding, PKC activity was measured in sub- and postconfluent cells. Calcium-dependent PKC activity was approximately about 2-fold higher in postconfluent compared to subconfluent cells, whereas no differences were discerned in calcium-independent PKC activity. In an effort to examine the role of PKC in greater detail, postconfluent cells were treated with PKC inhibitors (H-7 or staurosporine) or with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) to down-regulate PKC activity, and changes in ER were evaluated. Inhibition or down-regulation of the PKC activity in postconfluent cells enhanced ER-binding capacity in a dose-dependent manner and estrogen responsiveness of an exogenous reporter gene and of the endogenous alkaline phosphatase, representing an endogenous estrogen-stimulated gene. These data indicate that there is an interaction between the PKC and ER signaling systems in bone cells and that this interaction may be influenced by the proliferative and/or differentiative state of the cells, resulting in modulation of hormone responsiveness.

Testosterone selectively influences protein kinase-C-coupled secretion of proluteinizing hormone-releasing hormone-derived peptides.

LHRH and GnRH-associated peptide (GAP) are two major pro-LHRH-derived peptides which are secreted from median eminence (ME) nerve terminals in vitro. The purpose of the present experiment was to determine whether manipulation of gonadal steroid levels in vivo influenced selectively the in vitro secretion of LHRH and GAP under basal or K+ and phorbol ester (PDBu) stimulation. Secretion of both peptides under each of these three conditions was reduced at least 2-fold in 2-week orchidectomized (ORDX) rats relative to the level in intact controls. Tissue stores of LHRH and GAP were also depressed in the ME of ORDX relative to control rats. When the data were expressed in terms of the percentage of peptide secreted per ME, both groups secreted similar percentages of the peptides into the medium under basal and K+-stimulated conditions. Interestingly, PDBu-activated secretion of LHRH and GAP remained depressed in ORDX animals. The nerve terminals from ORDX animals were not susceptible to a more rapid depletion of releasable peptides, since both groups secreted similar percentages of the peptides during repeated K+ depolarization. By comparison, protein kinase C (PKC)-coupled secretion from ORDX rats was selectively affected, since secretion of pro-LHRH-derived peptides became even more depressed with successive activation with PDBu. Immediate replacement with testosterone after ORDX fully restored the peptide levels in tissue and the LHRH and GAP secretory response to PKC activation. Since testosterone influenced both tissue stores and PDBu-stimulated secretion of LHRH and GAP, this steroid may selectively regulate biosynthesis and secretion of pro-LHRH-derived peptides through activation of the metabolic cascade involving the PKC system.

In vivo biosynthesis of arginine vasopressin and oxytocin in hypothalami from intact and hypophysectomized rats.

The rates of incorporation of [35S]cysteine into arginine vasopressin (AVP) and oxytocin (OXT) were studied concurrently in hypothalami from intact and hypophysectomized male rats. After label injection into the third ventricle, rats were killed 0.5, 1, 2, 4, or 8 h later. The hypothalamic peptides were quantitated by specific RIA and separated by HPLC to allow quantitation of label incorporation into each peptide. In intact rats, labeling of both OXT and AVP rose rapidly to peak at 2 h; thereafter, radioactivity in both peptides declined slowly to 8 h. In hypophysectomized rats, labeling of AVP fell below that in intact animals at all time points. Labeling of OXT was somewhat below normal 2 h after label injection, but considerably above normal at 4 and 8 h. For comparison, label incorporation into somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28) was also studied in the same animals. In intact rats, labeling of both SRIF peptides rose slowly to maximal values at 8 h. In hypophysectomized animals, labeling of each peptide was reduced substantially at all times tested. Endogenous cysteine specific activity and protein specific activity did not differ between intact and hypophysectomized animals. Immunoreactive levels of AVP and OXT in the hypothalamus were unaffected by hypophysectomy, though total SRIF-like immunoreactivity was depressed. Together these results suggest that hypothalamic neurons synthesize OXT and AVP at rates much faster than those for the SRIF peptides, and that hypophysectomy has differential effects on the syntheses of cysteine-containing peptides in the hypothalamus. Specifically, the syntheses of SRIF-14 and SRIF-28 appear to be sizeably reduced in hypophysectomized rats, while that of AVP only modestly diminished. OXT synthesis may be increased.

Differential expression of gamma-aminobutyric acid receptors in immortalized luteinizing hormone-releasing hormone neurons.

gamma-Aminobutyric acid (GABA) has been shown both to stimulate and inhibit LH secretion in vivo. GABA apparently exerts these effects at the hypothalamic level by regulating the release of LHRH. In this study, we have investigated the effect of GABAergic agents on LHRH secretion from an immortalized hypothalamic neuronal cell line (GT1-7). LHRH secretion was stimulated in a dose-dependent manner with increasing concentrations of GABA. This effect was mimicked by the GABAA receptor agonist, muscimol, and was blocked by the selective antagonist, bicuculline. The stimulatory effect of muscimol on LHRH secretion was synergistic with low concentrations of [K+]. By comparison, neither activation of the GABAB receptors with baclofen nor blockade with phaclofen influenced basal LHRH secretion. Baclofen, however, did depress [K+]-induced LHRH release. Binding studies confirmed the presence of GABAA and GABAB receptors on GT1-7 cells. In addition, Northern blots with probes to the GABAA receptor alpha 1, beta 3, and gamma 2L subunits revealed that only the beta 3 messenger RNA (mRNA) was expressed in the GT1-7 cells. These data provide the first demonstration that immortalized LHRH neurons are directly responsive to GABAergic agents. To the extent that these immortalized neurons may resemble those in vivo, our results suggest that GABAergic agents may play a dual role in reproductive physiology by exerting both stimulatory and inhibitory control over LHRH release.

Basic fibroblast growth factor regulates the conversion of pro-luteinizing hormone-releasing hormone (Pro-LHRH) to LHRH in immortalized hypothalamic neurons.

Growth factors are commonly associated with the regulation of cellular proliferation and differentiation. In established cells, growth factors can also serve as trophic agents. Immortalized LHRH neurons contain basic fibroblast growth factor (bFGF) receptors. Although these receptors are coupled to activation of protein kinase C, and phorbol esters are strong activators of protein kinase C-stimulated LHRH release, bFGF did not influence LHRH secretion from these cells. To clarify this discrepancy, the effects of bFGF and phorbol ester on pro-LHRH biosynthesis, protein processing, and secretion were examined in GT1-7 cells. Phorbol ester stimulated LHRH secretion, whereas bFGF either had no effect or stimulated LHRH release depending upon the antiserum used. Pro-LHRH levels in lysate and medium were depressed by phorbol esters; concentrations in bFGF-treated cells were somewhat lower than those in unstimulated controls. HPLC analyses revealed that both agents enhanced the release of LHRH intermediate products into the medium. C-Terminally extended forms of LHRH, especially LHRH-[Gly11], were prominent in medium from bFGF-stimulated neurons. Levels of LHRH were depressed relative to those in the control or phorbol ester groups. These data indicate that phorbol esters control the biosynthesis, secretion, and, to some extent, processing of pro-LHRH. The effects of bFGF are novel because this factor regulates processing of the prohormone so that LHRH-intermediate products are predominantly secreted instead of LHRH. By enhancing the secretion of these intermediates over that of LHRH, bFGF can control the biological activity of the decapeptide and regulate LHRH neuronal function.

Metabolism of pro-luteinizing hormone-releasing hormone in immortalized hypothalamic neurons.

An immortalized hypothalamic neuronal cell line was recently developed by genetically targeting the expression of the simian virus-40 large T-antigen in LHRH neurons. These GT1 cells were subcloned to GT1-1, GT1-3, and GT1-7 cells, and they have been shown to express the mRNA for pro-LHRH and secrete LHRH-like immunoreactive (IR) materials into the media. The purpose of our study was to biochemically and immunologically characterize the IR materials within and secreted from these cells. Both LHRH- and GnRH-associated peptide (GAP)-like IR materials were present and were secreted from these four cell lines. Up to 3% of the total cellular protein was composed of LHRH and GAP materials. When materials from the cell lysate and media were separated according to mol wt (Mr), at least three different pro-LHRH species were detected. These precursors contained both LHRH- and GAP-like IR determinants, and they eluted in the void volume and at approximately 10,000-12,000 and 8,400-8,500 Mr. A material that contained GAP-like IR eluted at approximately 6,500-6,800 Mr. This species is probably mouse GAP-(1-56) because it eluted on a reverse phase column in the approximate position of rat GAP-(1-56). Cell lysates contained a single LHRH-like IR form which coeluted on a size-exclusion column with synthetic LHRH. This material stimulated secretion of LH from anterior pituitary cells in a dose-response manner. By comparison, two different molecular forms of LHRH were detected in media at approximately 1,500 and 540 Mr. HPLC analyses revealed these peaks to be heterogeneous and to contain at least (Gln1)LHRH-(Gly11,Lys12,Arg13), (Gln1)LHRH-(Gly1,Lys12), LHRH-(Gly11), and LHRH. These experiments demonstrate that the cells contain and secrete multiple molecular forms of the pro-LHRH and that processing of the prohormone must involve 1) cleavage by an endopeptidase to give GAP-(1-56) and a C-terminally extended LHRH, 2) removal of C-terminal basic amino acids by a carboxypeptidase, 3) amidation of LHRH-(Gly11) to LHRH, and 4) cyclization of glutamine to pyroglutamate at the N-terminal of LHRH. These results provide the first evidence for intermediates in the metabolic pathway of pro-LHRH to LHRH.

Hypothalamic astrocytes respond to transforming growth factor-alpha with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone.

Previous studies demonstrated the involvement of transforming growth factor-alpha (TGF alpha), a member of the epidermal growth factor (EGF) family, in the developmental regulation of hypothalamic LHRH release. Although both TGF alpha and EGF stimulate LHRH release, they do not appear to act directly on LHRH neurons, as no EGF/TGF alpha receptors are detected on these cells in vivo. Instead, the stimulatory effect of TGF alpha on LHRH release seems to require a glial intermediacy. The present study identifies one of the glial molecules involved in this process. In vitro exposure of purified hypothalamic astrocytes to TGF alpha or EGF in a defined medium led to activation of the cyclooxygenase-mediated pathway of arachidonic acid metabolism, as indicated by an increase in PGE2 release, but failed to affect lipooxygenase-mediated metabolism, as assessed by the lack of increase in leukotriene C4 production; addition of TGF alpha- (T-CM) or EGF-conditioned medium to cultures of LHRH-producing GT1-1 cells stimulated LHRH release. In contrast, direct exposure of GT1-1 cells to the growth factors was ineffective. Incubation of the cells in medium conditioned by untreated astrocytes (CM) was also ineffective. Blockade of either EGF receptor signal transduction or cyclooxygenase activity in the astrocytic cultures prevented both TGF alpha-induced PGE2 formation in astrocytes and the stimulatory effect of T-CM on LHRH release. Immunoneutralization of PGE2 actions or selective removal of the PG from T-CM also prevented T-CM-induced LHRH release. Addition of exogenous PGE2 restored the effect. Thus, PGE2 is one of the glial molecules involved in mediating the stimulatory effect of TGF alpha on LHRH release. The effectiveness of PGE2 in eliciting LHRH release was, however, greatly reduced when PG was delivered to GT1-1 cells in astrocyte-defined medium instead of CM. Thus, astrocytes appear to produce a yet to be identified substance(s) that facilitates the stimulatory effect of PGE2 on LHRH output. We postulate that the ability of TGF alpha to enhance LHRH release depends on the potentiating interaction of PGE2 with these additional glial-derived molecules.