Axogenic effect of estrogen in male rat hypothalamic neurons involves Ca(2+), protein kinase C, and extracellular signal-regulated kinase signaling

17-beta-Estradiol (E2) stimulates the growth of axons in male-derived hypothalamic neurons in vitro. This effect is not exerted through the classical intracellular estrogen receptor (ER) but depends on a membrane mechanism involving TrkB. In the present study, we investigate the intracellular signaling cascade that mediates the axogenic effect of E2. Treatment with an intracellular Ca(2+) chelator, a Ca(2+)-dependent protein kinase C (PKC) inhibitor, or two specific inhibitors of extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) completely inhibited the E2-induced axogenesis. E2 and the membrane-impermeant construct E2BSA rapidly induced phosphorylation of ERK, which was blocked by the specific inhibitor of the ERK pathway UO126 but not by the ER antagonist ICI 182,780. Decrease of intracellular free Ca(2+) or disruption of PKC activation by Ro 32-0432 attenuated ERK activation, indicating the confluence of signals in the MAPK pathway. Subcellular analysis of ERK demonstrated that the phospho-ERK signal is augmented in the nucleus after 15 min of E2 stimulation. We have also shown that E2 increased phosphorylation of CREB via ERK signaling. In summary, this study demonstrates that E2, probably via a membrane-associated receptor, induces axonal growth by activating CREB phosphorylation through ERK signaling by a mechanism involving Ca(2+) and PKC activation.     

Evidence that D2 receptor-mediated activation of hypothalamic tuberoinfundibular dopaminergic neurons in the male rat occurs via inhibition of tonically active afferent dynorphinergic neurons

The purpose of the present study was to determine if D₂ receptor-mediated activation of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons occurs via afferent neuronal inhibition of tonically active inhibitory dynorphinergic neurons in the male rat. To this end, the effects of either surgical deafferentation of the mediobasal hypothalamus or administration of a κ opioid receptor agonist (U-50,488) or antagonist (nor-binaltorphimine (NOR-BNI)) on D₂ receptor-mediated activation of TIDA neurons were assessed. For comparison, the activity of mesolimbic DA neurons was also determined in these studies. TIDA and mesolimbic DA neuronal activities were estimated by measuring dopamine synthesis (accumulation of 3,4-dihydroxyphenylalanine (DOPA) following decarboxylase inhibition) and metabolism (concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC)) in terminals of these neurons in the median eminence and nucleus accumbens, respectively. Intraperitoneal administration of the D₂ receptor agonist quinelorane caused a dose-dependent increase in DOPAC in the median eminence and a decrease in DOPAC in the nucleus accumbens; surgical deafferentation of the mediobasal hypothalamus prevented the effect of quinelorane in the median eminence, but not the nucleus accumbens. Activation of κ opioid receptors with U-50,488 had no effect per se, but blocked quinelorane-induced increases in median eminence DOPA. In contrast, U-50,488 had no effect on DOPA in the nucleus accumbens of either vehicle- or quinelorane-treated rats. Blockade of κ opioid receptors with NOR-BNI increased median eminence DOPA, and prevented the stimulatory effects of quinelorane on dopamine synthesis. Administration of prolactin also increased median eminence DOPA, but did not alter the ability of quinelorane to stimulate dopamine synthesis. Neither NOR-BNI nor prolactin had any effect on DOPA in the nucleus accumbens of vehicle- or quinelorane-treated rats. These results suggest that D₂ receptor-mediated activation of TIDA neurons occurs via an afferent neuronal mechanism involving, at least in part, inhibition of tonically active inhibitory dynorphinergic neurons in the male rat.     

Molecular pathways mediating activation by kainate of mitogen-activated protein kinase in oligodendrocyte progenitors

Oligodendroglial cells express ionotropic glutamate receptors of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid hydrobromide (AMPA) and kainate (KA) subtypes. Recently, we reported that AMPA receptor agonists increased 45Ca2+ uptake and phospholipase C (PLC) activity. To further elucidate the intracellular signaling mechanisms, we examined the effects of AMPA and KA on mitogen-activated protein kinase (MAPK). KA caused a time- and concentration-dependent increase in MAPK activity (predominantly the p42mapk or ERK2) and the effect was blocked by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a competitive AMPA/KA receptor antagonist. Furthermore, the noncompetitive antagonists of AMPA receptor GYKI 52466 and LY 303070 prevented the actions of the agonists, indicating that the effect of KA on MAPK activation is mediated through AMPA receptors in oligodendrocyte progenitors. Chelation of extracellular Ca2+ by EDTA or inhibition of PLC with U73122 abolished MAPK activation by KA. In addition, KA-stimulated MAPK activation was reduced by the protein kinase C (PKC) inhibitors, H7 and bisindolylmaleimide, as well as downregulation of PKC by prolonged exposure to phorbol esters. The involvement of PKC in the signal transduction pathways was further supported by the ability of KA to induce translocation of PKC measured by [3H]PDBu binding. Interestingly, a wortmannin-sensitive phosphatidylinositol 3-kinase and a pertussis toxin (PTX)-sensitive G protein form part of the molecular pathways mediating MAPK activation by AMPA receptor. A specific inhibitor of MAPK kinase, PD 098059, blocked MAPK activation and reduced KA-induced c-fos gene expression. All together, these results indicate that MAPK is implicated in the transmission of AMPA signaling to the nucleus and requires extracellular Ca2+, and PLC/PKC activation.     

Comparison of the effects of the dopamine D2 agonist quinelorane on tuberoinfundibular dopaminergic neuronal activity in male and female rats

The purpose of the present study was to examine the effects of quinelorane (LY163502), a potent and selective D₂ dopaminergic (DA) receptor agonist, on the activity of tuberoinfundibular DA neurons in male and female rats as estimated by determining the concentration of the primary metabolite of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), in terminals of these neurons in the median eminence (ME). In males, quinelorane produced dose- and time-related increases in the concentration of DOPAC in the ME which was blocked by the D₂ receptor antagonist raclopride. The activity of tuberoinfundibular neurons in female rats is higher than it is in males because circulating levels of prolactin tonically stimulate these neurons in the female. In female rats, quinelorane markedly lowered plasma concentrations of prolactin but failed to alter DOPAC concentrations in the ME. Pretreatment of female rats with prolactin antiserum induced hypoprolactinemia and reduced DOPAC concentrations in the ME; in these animals quinelorane increased ME DOPAC concentrations. These results indicate that by acting on D₂ receptors quinelorane is able to stimulate tuberoinfundibular DA neurons in both male and female rats, but in female rats the ability of quinelorane to reduce circulating levels of prolactin indirectly reduces the activity of tuberoinfundibular DA neurons and thereby masks the stimulatory action of this drug on these neurons.     

N-methyl-D-aspartate and TrkB receptors protect neurons against glutamate excitotoxicity through an extracellular signal-regulated kinase pathway

N-Methyl-D-aspartate (NMDA) at a subtoxic concentration (100 microM) promotes neuronal survival against glutamate-mediated excitotoxicity via a brain-derived neurotrophic factor (BDNF) autocrine loop in cultured cerebellar granule cells. The signal transduction mechanism(s) underlying NMDA neuroprotection, however, remains elusive. The mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3-K) pathways alter gene expression and are involved in synaptic plasticity and neuronal survival. This study tested whether neuroprotective activation of NMDA receptors, together with TrkB receptors, coactivated the MAPK or PI3-K pathways to protect rat cerebellar neurons. NMDA receptor activation caused a concentration- and time-dependent activation of MAPK lasting 24 hr. This activation was blocked by the NMDA receptor antagonist MK-801 but was attenuated only partially by the tyrosine kinase inhibitor k252a, suggesting that activation of both NMDA and TrkB receptors are required for maximal neuroprotection. The MAPK kinase (MEK) inhibitor U0126 (10 microM) partially blocked NMDA neuroprotection, whereas LY294002, a selective inhibitor of the PI3-K pathway, did not affect the neuroprotective activity of NMDA. Glutamate excitotoxicity decreased bcl-2, bcl-X(L), and bax mRNA levels,. NMDA increases Bcl-2 and Bcl-X(L) protein levels and decreases Bax protein levels. NMDA and TrkB receptor activation thus converge on the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway to protect neurons against glutamate-mediated excitotoxicity. By increasing antiapoptotic proteins of the Bcl-2 family, NMDA receptor activation may also promote neuronal survival by preventing apoptosis.     

Insulin-like growth factor-I receptor and estrogen receptor crosstalk mediates hormone-induced neurite outgrowth in PC12 cells

Estradiol (E(2)) and insulin-like growth factor-I (IGF-I) can act independently or in concert to promote neurite outgrowth in vivo and in cultured neurons. This study examined the role of crosstalk between estrogen receptor (ER)alpha and the IGF-I receptor as a critical mediator of hormone- and growth factor-dependent neurite outgrowth in a homogenous cell system. We used control PC12 cells and PC12 cells stably transfected with ER alpha, both of which express IGF-I receptor. Cells were treated for 1 week with vehicle, 1 nM E(2) or 100 ng/ml IGF-I alone or with E(2) or IGF-I in the presence of either the IGF-I receptor antagonist JB1 or the ER antagonist ICI 182,780. IGF-I significantly increased neurite outgrowth, as measured by the percentage of process-bearing cells, and absolute neurite length per cell in both control and ER alpha-transfected PC12 cells. In contrast, E(2) increased process formation and extension only in PC12 cells that were stably transfected with ER alpha. ICI 182,780 and JB1 blocked the IGF-I-induced increases in neurite length in both cell types. The efficacy of ICI 182,780 in control PC12 cells may have been due to the upregulation of ER alpha in these cells by the 7-day treatment with IGF-I. The ER and IGF-I receptor antagonists similarly blocked the E(2)-induced increase in neurite lengths in ER alpha-transfected cells. Immunofluorescent analysis of the cellular distribution of an axonal marker, phospho-neurofilament, verified that the processes extended by PC12 cells were neurites. These data suggest that receptor crosstalk between IGF-I receptors and ER alpha has an important role in neurite formation and extension even in a single-cell system.     

Adrenoceptor subtype involvement in suppression of prolactin secretion by noradrenaline

In sheep, injection of noradrenaline suppresses prolactin secretion by a direct effect at the pituitary gland. The aims of this study were to use primary cultures of ovine pituitary cells to examine the receptor subtypes that mediate the inhibitory effect of noradrenaline on prolactin secretion and, by using receptor antagonists in vivo, determine whether noradrenaline acts as a prolactin release-inhibiting factor (PIF). Noradrenaline and dopamine suppressed prolactin secretion from ovine pituitary cells with ED50s of 60.9+/-46.6 and 1.5+/-1.0x10-9 mol/l, respectively (P<0.05). The in-vitro prolactin release-inhibiting effect of noradrenaline (10-7 mol/l) was not blocked by the dopamine antagonists pimozide (D2) or SCH23390 (D1) but was blocked by each of the adrenoceptor antagonists (alpha1-adrenoceptor antagonists prazosin and WB4101, the alpha2-adrenoceptor antagonist yohimbine and the beta-adrenoceptor antagonist propranolol). The response to adrenoceptor agonists was also tested in vitro. The alpha1-adrenoceptor agonists phenylephrine and cirazoline significantly suppressed prolactin. Of the alpha2-agonists, clonidine had no effect whereas oxymetazoline and p-aminoclonidine both suppressed prolactin. The beta-adrenoceptor agonist isoproterenol also suppressed prolactin while the specific beta3-antagonist BRL37344 had no effect. When the adrenoceptor antagonists were tested in vivo in ewes manipulated to be in the luteal phase, only WB4101 significantly (P<0.05) increased plasma prolactin concentrations but this response was small and only observed in one of two experiments. In summary, these experiments suggest that adrenoceptors and not dopamine receptors are responsible for the inhibitory effect of noradrenaline on prolactin secretion in vitro but do not implicate a particular adrenoceptor subtype. The in-vivo experiments do not provide convincing evidence for a role for noradrenaline as a physiologically important PIF.     

17β-Estradiol Protects Dopaminergic Neurons in Organotypic Slice of Mesencephalon by MAPK-Mediated Activation of Anti-apoptosis Gene BCL2

Both clinical and experimental studies provide growing evidences that marked sex differences in certain neurological disorders or disease models are largely attributed to the neuroprotective effects of estrogen. The purposes of this study were to assess the neuroprotective effect of 17β-estradiol (E2) on dopaminergic neurons against 6-hydroxydopamine (6-OHDA) in organotypic mesencephalic slice culture and to elucidate the possible mechanism underlying neuroprotection. It was found that long-term exposure to E2 exerted marked effects on restoring the number of dopaminergic neurons, maintaining normal morphology of dopaminergic neurons, and preserving their ability to release dopamine at the presence of 6-OHDA. The neuroprotective effect of E2 could be dramatically blocked by an estrogen receptor antagonist ICI 182, 780 (ICI). The expression of GFAP, TLR4, and anti-apoptosis gene BCL2 were elevated at the presence of E2, whereas only BCL2 activation was blocked by ICI, dominantly responsible for E2-induced neuroprotection. Furthermore, activation of BCL2 was speculated to be mainly mediated through mitogen-activated protein kinase (MAPK) pathways, yet phosphatidylinositol-3-kinase signaling contributed largely to GFAP and TLR4 upregulation. Taken together, MAPK pathway-mediated BCL2 expression accounted for one of the key mechanisms involved in E2 neuroprotective effect on dopaminergic neurons against 6-OHDA insult. This finding provides new insight into controversial estrogen replacement therapy.     

Dual action of estrogen on glutamate-induced calcium signaling: mechanisms requiring interaction between estrogen receptors and src/mitogen activated protein kinase pathway

Conjugated equine estrogens (CEE) is the most widely prescribed pharmaceutical estrogen replacement therapy (ERT) for postmenopausal women in the United States and is the ERT of the Women’s Health Initiative. Previous studies from our laboratory have demonstrated that CEE exerts neurotrophic and neuroprotective effects in neurons involved in learning and memory, and which are affected in Alzheimer’s disease. The present work demonstrates that CEE potentiated the rise in intracellular calcium ([Ca²⁺]_i) following exposure to physiological concentrations of glutamate. In contrast, the reverse effect occurred in the presence of excitotoxic levels of glutamate exposure, where CEE attenuated the rise in [Ca²⁺]_i. Potentiation of the glutamate response was mediated by the NMDA receptor, as the NMDA receptor antagonist MK-801 blocked the CEE-induced potentiation, whereas the L-type calcium channel blocker nifedipine did not. Further, the CEE-potentiated glutamate response was mediated by a src tyrosine kinase, as the tyrosine kinase inhibitor PP2 blocked the potentiation induced by CEE and neurons treated with CEE displayed increased phosphorylated tyrosine. The inhibition by CEE of [Ca²⁺]_i rise in the presence of excitotoxic levels of glutamate was mediated by mitogen activated protein kinase (MAPK), as the protective effect of CEE was blocked by inhibiting MAPK activation with PD98059. These data provide potential mechanisms to explain the cognitive enhancing and neuroprotective effects exerted by ERT.     

Dopamine stimulates redox-tyrosine kinase signaling and p38 MAPK in activation of astrocytic C6-D2L cells

An increase in dopamine (DA) availability in rat brain has been suggested to participate in certain neurodegenerative processes. However, the regulatory effects of DA on glial cells have not been extensively studied. Using a rat C6 glioma cell line stably expressing recombinant D2L receptors, we have found that micromolar levels of DA stimulate mitogenesis and glial fibrillary acidic protein (GFAP) expression, both serving as parameters of reactive gliosis. This mitogenesis occurs about 29 h after exposure to DA and requires D2-receptor-mediated intracellular redox–tyrosine kinase activation. Either DA or quinpirole, a D2 receptor agonist, stimulates protein tyrosine phosphorylation. Application of either DPI, a potent inhibitor of NADPH-dependent oxidase, or NAC, an anti-oxidant, effectively prevented DA-induced tyrosine phosphorylation and DNA synthesis. Preincubation of (+)-butaclamol, a D2 receptor antagonist, inhibits both DA-stimulated tyrosine phosphorylation and mitogenesis. DA at micromolar levels also stimulates GFAP expression. This DA-regulated GFAP expression can be completely inhibited by SB203580, a selective p38 MAPK inhibitor, but not influenced by (+)-butaclamol and genistein, a protein tyrosine kinase inhibitor. Thus, our data suggest that regulation of DNA synthesis and GFAP expression induced by DA is mediated by independent signaling pathways. The mitogenesis requires a D2-receptor-mediated protein tyrosine kinase cascade, while GFAP expression needs a D2-receptor-independent p38 MAPK activation. This observation may help to understand the processes of reactive gliosis in some dopaminergic-related neurodegenerative diseases.     

Glutamate receptor agonist kainate enhances primary dendrite number and length from immature mouse cortical neurons in vitro

Glutamate is an important regulator of dendrite development that may inhibit, (during ischemic injury), or facilitate (during early development) dendrite growth. Previous studies have reported mainly on the N-methyl-D-aspartate (NMDA) receptor-mediated dendrite growth-promoting effect of glutamate. In this study, we examined how the non-NMDA receptor agonist kainate influenced dendrite growth. E18 mouse cortical neurons were grown for 3 days in vitro and immunolabeled with anti-microtubule-associated protein 2 (MAP2) and anti-neurofilament (NF-H), to identify dendrites and axons, respectively. Exposure of cortical neurons to kainate increased dendrite growth without affecting neuron survival. This effect was dose-dependent, reversible and blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/kainate receptor antagonist NBQX and the low-affinity kainate receptor antagonist NS-102, but not by the AMPA receptor antagonist CFM-2. In addition, the NMDA receptor antagonist MK-801 had no effect on kainate-induced dendrite growth. Immunolabeling and Western blot analysis of kainate receptors using antibodies against the GluR6 and KA2 subunits, demonstrated that the immature cortical neurons used in this study express kainate receptor proteins. These results suggest that kainate-induced non-NMDA receptor activation promotes dendrite growth, and in particular primary dendrite number and length, from immature cortical neurons in vitro, and that kainate receptors may be directly involved in this process. Furthermore, these data support the possibility that like NMDA receptors, kainate receptor activation may also contribute to early neurite growth from cortical neurons in vitro.     

IGF-I prevents glutamate-induced motor neuron programmed cell death

Insulin-like growth factor I (IGF-I) is currently in clinical trials for treatment of amyotrophic lateral sclerosis (ALS), but little is known about how it promotes the survival of motor neurons. In the current study, we examined IGF-I-mediated neuroprotection in an in vitro model of ALS utilizing enriched cultures of embryonic rat spinal cord motor neurons. IGF-I binds to the IGF-I receptor (IGF-IR) in motor neurons and activates MAPK and the downstream effector of phosphatidylinositol 3-kinase (PI-3K) signaling, Akt. IGF-I:IGF-IR signaling involves phosphorylation of IRS-1 and Shc, but not IRS-2. Glutamate, which is elevated in the cerebrospinal fluid of ALS patients, induced DNA fragmentation and caspase-3 cleavage in the spinal cord motor neurons. These effects of glutamate were blocked by co-treatment with IGF-I. However, a delay of IGF-I treatment for as little as 30 min eliminated its neuroprotective effect. Finally, alone, neither the MAPK pathway inhibitor PD98059 nor the PI-3K inhibitor LY294002 blocked the neuroprotective effect of IGF-I, but both inhibitors together were effective in this regard. These results suggest that the dose and timing of IGF-I administration are critical for producing a neuroprotective effect, and also suggest that both the MAPK and PI-3K/Akt pathways can promote the survival of motor neurons. We discuss our results in terms of novel strategies for ALS therapy.     

Role of protein kinases in the prolactin-induced intracellular calcium rise in Chinese hamster ovary cells expressing the prolactin receptor

There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx.     

Estrogen stimulates brain-derived neurotrophic factor expression in embryonic mouse midbrain neurons through a membrane-mediated and calcium-dependent mechanism

We have provided evidence that 17beta-estradiol (E) synthesized in the midbrain promotes the differentiation of midbrain dopamine neurons through nonclassical steroid action. Because these developmental effects resemble those reported for brain-derived neurotrophic factor (BDNF), we hypothesized that E influences dopaminergic cell differentiation through a BDNF-dependent mechanism. Competitive RT-PCR and ELISA techniques were employed to study first the developmental pattern of BDNF and trkB expression in the mouse midbrain. BDNF protein/mRNA levels peaked postnatally, whereas trkB did not fluctuate perinatally. To prove the hypothesis that E regulates BDNF expression in vivo, fetuses and newborns were treated with the aromatase antagonist CGS 16949A. CGS 16949A exposure reduced midbrain BDNF mRNA/protein levels. The coapplication of CGS 16949A and E abolished this effect. Midbrain cultures from mouse fetuses were used to investigate intracellular signaling mechanisms involved in transmitting E effects. Estrogen increased expression of BDNF but not of other neurotrophins. As concerns the related signaling mechanism, these effects were antagonized by interrupting intracellular Ca(2+) signaling with BAPTA and thapsigargin but not by the estrogen receptor antagonist ICI 182,780. Insofar as E effects on BDNF mRNA expression were inhibited by cycloheximide, it appears likely that other, not yet characterized intermediate proteins take part in the estrogenic regulation of BDNF expression. We conclude that E exerts its stimulatory effect on the differentiation of dopaminergic neurons by coordinating BDNF expression. This particular E effect appears to be transmitted through Ca(2+)-dependent signaling cascades upon activation of putative membrane estrogen receptors.     

Estrogen receptors and insulin-like growth factor-I receptors mediate estrogen-dependent synaptic plasticity

Previous studies have shown that estradiol induces a transient disconnection of axo-somatic inhibitory synapses in the hypothalamic arcuate nucleus of adult ovariectomized rats. The synaptic disconnection is accompanied by an increase in the levels of insulin-like growth factor-I (IGF-I) in the arcuate nucleus, suggesting that IGF-I signaling may be involved in the estrogen-induced synaptic plasticity. The role of estrogen receptors and IGF-I receptors in the synaptic changes has been studied by assessing the number of axo-somatic synapses in ovariectomized rats treated with intracerebroventricular administration of the estrogen receptor antagonist ICI 182,780 and the IGF-I receptor antagonist JBI to ovariectomized rats. Estradiol administration resulted in a significant decrease in the number of axo-somatic synapses on arcuate neurons in control ovariectomized rats. Both the estrogen receptor antagonist and the IGF-I receptor antagonist blocked the estrogen-induced synaptic decrease. This finding suggest that estrogen-induced synaptic plasticity in the arcuate nucleus is dependent on the activation of both estrogen receptors and IGF-I receptors.     

An endogenous dopaminergic tone acting on dopamine D3 receptors may be involved in diurnal changes of tuberoinfundibular dopaminergic neuron activity and prolactin secretion in estrogen-primed ovariectomized rats

The diurnal rhythm of tuberoinfundibular dopaminergic (TIDA) neuron activity, i.e., high in the morning and low in the afternoon, is prerequisite for the afternoon prolactin (PRL) surge in proestrous and estrogen-primed ovariectomized (OVX) female rats. Whether dopamine acts via D(3) receptors in regulating the rhythmic TIDA neuron activity and PRL secretion in estrogen-primed OVX (OVX+E(2)) rats is the focus of this study. Intracerebroventricular (icv) injection of a D(3) receptor agonist, PD128907 (0.1-10 μg/3 μl), in the morning significantly reduced the basal activity of TIDA neurons and increased plasma PRL level. The effects of PD128907 were reversed by co-administration of U99194A, a D(3) receptor antagonist, but not by raclopride, a D(2) receptor antagonist. To determine whether endogenous dopamine acts on D(3) receptors involved in the diurnal changes of the activities, we used both U99194A, a D(3) receptor antagonist, and an antisense oligodeoxynucleotide (ODN) against D(3) receptor mRNA in the study. U99194A (0.1 μg/3 μl, icv) given at 1200 h significantly reversed the lowered TIDA neuron activity and the afternoon PRL surge at 1500 h. Moreover, OVX+E(2) rats pretreated with the antisense ODN (10 μg/3 μl, icv) for 2 days had the same effects as the D(3) receptor antagonist on TIDA neuron activity and the PRL surge. The same treatment with sense ODN had no effect. In conclusion, an endogenous DA tone may act on D(3) receptors to inhibit TIDA neuron activity and in turn stimulate the PRL surge in the afternoon of OVX+E(2) rats.     

Regulation of a Ca2+-activated K+ channel by calcium-sensing receptor involves p38 MAP kinase

By using pharmacological and molecular approaches, we previously showed that the G-protein-coupled, extracellular calcium (Ca2+(o))-sensing receptor (CaR) regulates a large-conductance (approximately 140 pS), Ca(2+)-activated K+ channel [IK(Ca); CAKC] in U87 astrocytoma cells. Here we show that elevated Ca2+(o) stimulates extracellular-signal-regulated kinase (ERK1/2) and p38 MAP kinase (MAPK). The effect of high Ca2+(o) on p38 MAPK but not ERK1/2 is CaR mediated, insofar as transduction with a dominant-negative CaR (R185Q) using recombinant adeno-associated virus (rAAV) attenuated the activation of p38 MAPK but not of ERK1/2. p38 MAPK activation by the CaR is likely to be protein kinase C (PKC) independent, in that the pan-PKC inhibitor GF109203X failed to abolish the high-Ca2+(o)-induced phosphorylation of p38 MAPK. Consistently with our data on the activation of this kinase, we observed that inhibiting p38 MAPK blocked the activation of the CAKC induced by the specific pharmacological CaR activator NPS R-467. In contrast, inhibiting MEK1 only transiently inhibited the activation of this K+ channel by NPS R-467, despite the continued presence of the antagonist. Similarly to the lack of any effect of the PKC inhibitor on the activation of ERK1/2 and p38 MAPK, inhibiting PKC had no effect on NPS R-467-induced activation of this channel. Therefore, our data show that the CaR, acting via p38 MAPK, regulates a large-conductance CAKC in U87 cells, a process that is PKC independent. Large-conductance CAKCs play an important role in the regulation of cellular volume, so our results have important implications for glioma cell volume regulation.