Oline K. Rønnekleiv

Rapid effects of estrogen to modulate G protein-coupled receptors via activation of protein kinase A and protein kinase C pathways.

17Beta-estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic neurons. We have used intracellular recordings from female guinea pig hypothalamic slices to characterize the receptor and intracellular pathway(s) mediating E2s rapid effects. E2 acts stereospecifically with physiologically relevant concentration-dependence (EC50 = 8 nM) to cause a fourfold reduction in the potency of the mu-opioid agonist (D-Ala2-N-Me-Phe4-Gly5-ol)-enkephalin and the GABA(B) agonist baclofen to activate an inwardly rectifying K+ conductance in hypothalamic neurons. Both the nonsteroidal estrogen diethylstilbestrol and the anti-estrogen ICI 164,384 blocked E2 actions to uncouple mu-opioid receptors. Using a pharmacological Schild analysis, we found that ICI 164,384 competed for this E2 receptor with a Ke of approximately 0.3 nM. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a rapid, nongenomic mechanism of E2 action. The effects of E2 were mimicked by the bath application of the protein kinase A (PKA) activators, forskolin and Sp-cAMP, and the protein kinase C (PKC) activator phorbol-12,13-dibutyrate. Furthermore, the selective PKA antagonists Rp-cAMP and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. In addition, the actions of E2 were blocked by the selective PKC inhibitor Calphostin C. Therefore, it appears that E2 can activate both PKA and PKC to cause a heterologous desensitization of both mu-opioid and GABA(B) receptors, which has the potential to alter synaptic transmission in many regions of the CNS.

Kisspeptin Depolarizes Gonadotropin-Releasing Hormone Neurons through Activation of TRPC-Like Cationic Channels

Kisspeptin and its cognate receptor, GPR54, are critical for reproductive development and for the regulation of gonadotropin-releasing hormone (GnRH) secretion. Although kisspeptin has been found to depolarize GnRH neurons, the underlying ionic mechanism has not been elucidated. Presently, we found that kisspeptin depolarized GnRH neurons in a concentration-dependent manner with a maximum depolarization of 22.6 ± 0.6 mV and EC50 of 2.8 ± 0.2 nm. Under voltage-clamp conditions, kisspeptin induced an inward current of 18.2 ± 1.6 pA (Vhold = −60 mV) that reversed near −115 mV in GnRH neurons. The more negative reversal potential than EK+ (−90 mV) was caused by the concurrent inhibition of barium-sensitive, inwardly rectifying (Kir) potassium channels and activation of sodium-dependent, nonselective cationic channels (NSCCs). Indeed, reducing extracellular Na+ (to 5 mm) essentially eliminated the kisspeptin-induced inward current. The current–voltage relationships of the kisspeptin-activated NSCC currents exhibited double rectification with negative slope conductance below −40 mV in the majority of the cells. Pharmacological examination showed that the kisspeptin-induced inward currents were blocked by TRPC (canonical transient receptor potential) channel blockers 2-APB (2-aminoethyl diphenylborinate), flufenamic acid, SKF96365 (1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), and Cd2+, but not by lanthanum (100 μm). Furthermore, single-cell reverse transcription-PCR analysis revealed that TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 subunits were expressed in GnRH neurons. Therefore, it appears that kisspeptin depolarizes GnRH neurons through activating TRPC-like channels and, to a lesser extent, inhibition of Kir channels. These actions of kisspeptin contribute to the pronounced excitation of GnRH neurons that is critical for mammalian reproduction.

A G-Protein-Coupled Estrogen Receptor Is Involved in Hypothalamic Control of Energy Homeostasis

Estrogens are involved in the hypothalamic control of multiple homeostatic functions including reproduction, stress responses, energy metabolism, sleep cycles, temperature regulation, and motivated behaviors. The critical role of 17β-estradiol (E2) is evident in hypoestrogenic states (e.g., postmenopause) in which many of these functions go awry. The actions of E2 in the brain have been attributed to the activation of estrogen receptors α and β through nuclear, cytoplasmic, or membrane actions. However, we have identified a putative membrane-associated estrogen receptor that is coupled to desensitization of GABAB and μ-opioid receptors in guinea pig and mouse hypothalamic proopiomelanocortin neurons. We have synthesized a new nonsteroidal compound, STX, which selectively targets the Gαq-coupled phospholipase C–protein kinase C–protein kinase A pathway, and have established that STX is more potent than E2 in mediating this desensitization in an ICI 182, 780-sensitive manner in both guinea pig and mouse neurons. Both E2 and STX were fully efficacious in estrogen receptor α,β knock-out mice. Moreover, in vivo treatment with STX, similar to E2, attenuated the weight gain in hypoestrogenic female guinea pigs. Therefore, this membrane-delimited signaling pathway plays a critical role in the control of energy homeostasis and may provide a novel therapeutic target for treatment of postmenopausal symptoms and eating disorders in females.

Regulation of NKB pathways and their roles in the control of Kiss1 neurons in the arcuate nucleus of the male mouse.

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

Epigenetic control of female puberty

The timing of puberty is controlled by many genes. The elements coordinating this process have not, however, been identified. Here we show that an epigenetic mechanism of transcriptional repression times the initiation of female puberty in rats. We identify silencers of the Polycomb group (PcG) as principal contributors to this mechanism and show that PcG proteins repress Kiss1, a puberty-activating gene. Hypothalamic expression of two key PcG genes, Eed and Cbx7, decreased and methylation of their promoters increased before puberty. Inhibiting DNA methylation blocked both events and resulted in pubertal failure. The pubertal increase in Kiss1 expression was accompanied by EED loss from the Kiss1 promoter and enrichment of histone H3 modifications associated with gene activation. Preventing the eviction of EED from the Kiss1 promoter disrupted pulsatile gonadotropin-releasing hormone release, delayed puberty and compromised fecundity. Our results identify epigenetic silencing as a mechanism underlying the neuroendocrine control of female puberty.

Leptin Excites Proopiomelanocortin Neurons via Activation of TRPC Channels

Leptin can exert its potent appetite-suppressing effects via activation of hypothalamic proopiomelanocortin (POMC) neurons. It depolarizes POMC neurons via activation of a yet unidentified nonselective cation current. Therefore, we sought to identify the conductance activated by leptin using whole-cell recording in EGFP-POMC neurons from transgenic mice. The TRPC channel blockers SKF96365 (1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), flufenamic acid, and 2-APB (2-aminoethyl diphenylborinate) potently inhibited the leptin-induced current. Also, lanthanum (La3+) and intracellular Ca2+ potentiated the effects of leptin. Moreover, the diacylglycerol-permeable analog OAG (2-acetyl-1-oleoyl-sn-glycerol) failed to activate any TRPC current. Using a Cs+-gluconate-based internal solution, the leptin-activated current reversed near −20 mV. After replacement of external Na+ and K+ with Cs+, the reversal shifted to near 0 mV, and the I/V curve exhibited a negative slope conductance at voltages more negative than −40 mV. Based on scRT-PCR, TRPC1 and TRPC4–7 mRNA were expressed in POMC neurons, with TRPC5 being the most prevalent. The leptin-induced current was blocked by the Jak2 inhibitor AG490, the PI3 kinase inhibitor wortmannin, and the phospholipase C inhibitors, U73122 and ET-18-OCH3. Notably, we identified PLCγ1 transcripts in the majority of POMC neurons. Therefore, leptin through a Jak2–PI3 kinase–PLCγ pathway activates TRPC channels, and TRPC1, 4, and 5 appear to be the key channels mediating the depolarizing effects of leptin in POMC neurons.

Estrogen modulation of hypothalamic neurons: activation of multiple signaling pathways and gene expression changes.

Hypothalamic target neurons of estrogen include neurosecretory neurons such as gonadotropin-releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons such as proopiomelanocortin (POMC) and gamma-aminobutyric acid (GABA) neurons. These and other hypothalamic neurons are involved in regulating numerous homeostatic functions including reproduction, thermoregulation, stress responses, feeding and motivated behaviors. Using a combination of techniques to examine the molecular mechanisms leading to physiological changes induced by estrogen, we find that both rapid effects and transcriptional changes alter excitability of hypothalamic neurons. We have identified membrane-initiated, rapid signaling pathways through which 17beta-estradiol (E2) alters synaptic responses in these neurons using whole-cell patch recording in hypothalamic slices from ovariectomized female guinea pigs. E2 rapidly uncouples mu-opioid and GABA(B) receptors from G protein-gated inwardly rectifying K+ (GIRK) channels in POMC and dopamine neurons as manifested by a reduction in the potency of mu-opioid and GABA(B) receptor agonists to activate these channels. Inhibitors of phospholipase C, protein kinase C and protein kinase A block the actions of E2, indicative that the E2 receptor is G protein-coupled to activation of this cascade. Taking advantage of an animal model we developed to investigate estrogens feedback actions on secretion of gonadotropin-releasing hormone (GnRH), we studied the transcriptional changes induced by estrogen using suppression subtractive hybridization (SSH) and microarray analysis. Many of the observed mRNA expression changes include transcripts encoding proteins critical for neurotransmitter release and receptor dynamics. Some of these include gec-1, PI3-kinase p55gamma, rab11a GTPase, synaptobrevin2, synaptogyrin, taxilin, Ca2+-dependent activator protein for secretion (CAPS) and a number of proteins containing pleckstrin homology domains-domains that are involved in plasma membrane targeting of their host protein. In situ hybridization and quantitative film autoradiography analysis on selected transcripts show differential distribution and expression in hypothalamic nuclei. Furthermore, single-cell PCR analysis reveals these genes to be expressed in neurons such as POMC (and GnRH). Whether these expression changes are mediated by the classical or membrane estrogen receptors has yet to be delineated. More detailed investigations of transcript spatial localization within neurons and their temporal expression, i.e., within minutes or hours, will provide more insight regarding how estrogen alters neuronal excitability and synaptic efficacy that ultimately lead to changes in complex behavior.

Estrogen modulation of G-protein-coupled receptor activation of potassium channels in the central nervous system.

Abstract: Estrogen rapidly alters the excitability of hypothalamic neurons that are involved in regulating numerous homeostatic functions including reproduction, stress responses, feeding, and motivated behaviors. Neurosecretory neurons, such as gonadotropin‐releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons, such as pro‐opiomelanocortin (POMC) and γ‐aminobutyric acid (GABA) neurons, are among those involved. We have identified membrane‐initiated, rapid‐signaling pathways through which 17β‐estradiol (E2) alters synaptic responses in these neurons using whole‐cell patch recording in hypothalamic slices from ovariectomized female guinea pigs. E2 rapidly uncouples μ‐opioid and GABAB receptors from G‐protein‐gated inwardly rectifying K+ (GIRK) channels in POMC and dopamine neurons as manifested by a reduction in the potency of μ‐opioid and GABAB receptor agonists to activate these channels. These effects are mimicked by the selective E2 receptor modulators raloxifene and 4OH‐tamoxifen, the membrane impermeable E2‐bovine serum albumin (BSA), but not by 17α‐estradiol. Furthermore, the anti‐estrogen ICI 182,780 antagonizes these rapid effects of E2. Inhibitors of phospholipase C, protein kinase C, and protein kinase A block the actions of E2, indicating that the E2 receptor is G‐protein‐coupled to activation of this cascade. Conversely, estrogen enhances the efficacy of α1‐adrenergic receptor agonists to inhibit apamin‐sensitive small‐conductance, Ca2+‐activated K+ (SK) currents in preoptic GABAergic neurons; it does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid‐induced hyperpolarization of GnRH neurons. These findings indicate that E2 can modulate K+ channels in hypothalamic (POMC, dopamine, GABA, GnRH) neurons that are involved in regulating numerous homeostatic functions through multiple intracellular signaling pathways.

Rapid effects of estrogen on G protein-coupled receptor activation of potassium channels in the central nervous system (CNS) ☆

Estrogen rapidly alters the excitability of hypothalamic neurons that are involved in regulating numerous homeostatic functions including reproduction, stress responses, feeding and motivated behaviors. Some of the neurons include neurosecretory neurons such as gonadotropin-releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons such as proopiomelanocortin (POMC) and gamma-aminobutyric acid (GABA) neurons. We have elucidated several non-genomic pathways through which the steroid alters synaptic responses in these hypothalamic neurons. We have examined the modulation by estrogen of the coupling of various receptor systems to inwardly-rectifying and small-conductance, Ca(2+)-activated K(+) (SK) channels using intracellular sharp-electrode and whole-cell recording techniques in hypothalamic slices from ovariectomized female guinea pigs. Estrogen rapidly uncouples mu-opioid receptors from G protein-gated inwardly-rectifying K(+) (GIRK) channels in POMC neurons and GABA(B) receptors from GIRK channels in dopamine neurons as manifested by a reduction in the potency of mu-opioid and GABA(B) receptor agonists to hyperpolarize their respective cells. This effect is blocked by inhibitors of protein kinase A (PKA) and protein kinase C (PKC). In addition, after 24h following steroid administration in vivo, the GABA(B)/GIRK channel uncoupling observed in GABAergic neurons of the preoptic area is associated with reduced agonist efficacy. Conversely, estrogen enhances the efficacy of alpha(1)-adrenergic receptor agonists to inhibit apamin-sensitive SK currents in these preoptic GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid-induced hyperpolarization of GnRH neurons. These findings indicate a richly complex yet coordinated steroid modulation of K(+) channel activity in hypothalamic (POMC, dopamine, GABA, GnRH) neurons that are involved in regulating numerous homeostatic functions.

Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension-induced renal injury in mice

Renal cytochrome P450 (CYP)‐derived epoxyeicosatrienoic acids (EETs) regulate sodium transport and blood pressure. Although endothelial CYP‐derived EETs are potent vasodilators, their contribution to the regulation of blood pressure remains unclear. Consequently, we developed transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases to increase endothelial EET biosynthesis. Compared to wild‐type littermate controls, an attenuated afferent arteriole constrictor response to endothelin‐1 and enhanced dilator response to acetylcholine was observed in CYP2J2 and CYP2C8 transgenic mice. CYP2J2 and CYP2C8 transgenic mice demonstrated modestly, but not significantly, lower mean arterial pressure under basal conditions compared to wild‐type controls. However, mean arterial pressure was significantly lower in both CYP2J2 and CYP2C8 transgenic mice during coadministration of N‐nitro‐l‐arginine methyl ester and indomethacin. In a separate experiment, a high‐salt diet and subcutaneous angiotensin II was administered over 4 wk. The angiotensin/high‐salt‐induced increase in systolic blood pressure, proteinuria, and glomerular injury was significantly attenuated in CYP2J2 and CYP2C8 transgenic mice compared to wild‐type controls. Collectively, these data demonstrate that increased endothelial CYP epoxygenase expression attenuates afferent arteriolar constrictor reactivity and hypertension‐induced increases in blood pressure and renal injury in mice. We conclude that endothelial CYP epoxygenase function contributes to the regulation of blood pressure.—Lee, C. R., Imig, J. D., Edin, M. E., Foley, J., DeGraff, L. M., Bradbury, J. A., Graves, J. P., Lih, F. B., Clark, J., Myers, P., Perrow, A. L., Lepp, A. N., Kannon, M. A., Ronnekleiv, O. K., Alkayed, N.J., Falck, J. R., Tomer, K B., Zeldin, D. C. Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension‐induced renal injury in mice. FASEB J. 24, 3770–3781 (2010). www.fasebj.org