Troy A. Roepke

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.

Physiological consequences of membrane-initiated estrogen signaling in the brain

Many of the actions of 17beta-estradiol (E2) in the central nervous system (CNS) are mediated via the classical nuclear steroid receptors, ER(alpha) and ERbeta, which interact with the estrogen response element to modulate gene expression. In addition to the nuclear-initiated estrogen signaling, E2 signaling in the brain can occur rapidly within minutes prior to any sufficient effects on transcription of relevant genes. These rapid, membrane-initiated E2 signaling mechanisms have now been characterized in many brain regions, most importantly in neurons of the hypothalamus and hippocampus. Furthermore, our understanding of the physiological effects of membrane-initiated pathways is now a major field of interest in the hypothalamic control of reproduction, energy balance, thermoregulation and other homeostatic functions as well as the effects of E2 on physiological and pathophysiological functions of the hippocampus. Membrane signaling pathways impact neuronal excitability, signal transduction, cell death, neurotransmitter release and gene expression. This review will summarize recent findings on membrane-initiated E2 signaling in the hypothalamus and hippocampus and its contribution to the control of physiological and behavioral functions.

Genes Associated with Membrane-Initiated Signaling of Estrogen and Energy Homeostasis

During the reproductive cycle, fluctuations in circulating estrogens affect multiple homeostatic systems controlled by hypothalamic neurons. Two of these neuronal populations are arcuate proopiomelanocortin and neuropeptide Y neurons, which control energy homeostasis and feeding. Estradiol modulates these neurons either through the classical estrogen receptors (ERs) to control gene transcription or through a G protein-coupled receptor (mER) activating multiple signaling pathways. To differentiate between these two divergent ER-mediated mechanisms and their effects on homeostasis, female guinea pigs were ovariectomized and treated systemically with vehicle, estradiol benzoate (EB) or STX, a selective mER agonist, for 4 wk, starting 7 d after ovariectomy. Individual body weights were measured after each injection day for 28 d, at which time the animals were euthanized, and the arcuate nucleus was microdissected. As predicted, the body weight gain was significantly lower for EB-treated females after d 5 and for STX-treated females after d 12 compared with vehicle-treated females. Total arcuate RNA was extracted from all groups, but only the vehicle and STX-treated samples were prepared for gene microarray analysis using a custom guinea pig gene microarray. In the arcuate nucleus, 241 identified genes were significantly regulated by STX, several of which were confirmed by quantitative real-time PCR and compared with EB-treated groups. The lower weight gain of EB-treated and STX-treated females suggests that estradiol controls energy homeostasis through both ERalpha and mER-mediated mechanisms. Genes regulated by STX indicate that not only does it control neuronal excitability but also alters gene transcription via signal transduction cascades initiated from mER activation.

Contribution of a Membrane Estrogen Receptor to the Estrogenic Regulation of Body Temperature and Energy Homeostasis

The hypothalamus is a key region of the central nervous system involved in the control of homeostasis, including energy and core body temperature (Tc). 17β-Estradiol (E2) regulates Tc, in part, via actions in the basal hypothalamus and preoptic area. E2 primarily controls hypothalamic functions via the nuclear steroid receptors, estrogen receptor α/β. However, we have previously described an E2-responsive, Gq-coupled membrane receptor that reduces the postsynaptic inhibitory γ-aminobutyric acid-ergic tone and attenuates postovariectomy body weight gain in female guinea pigs through the administration of a selective Gq-mER ligand, STX. To determine the role of Gq-mER in regulating Tc, energy and bone homeostasis, ovariectomized female guinea pigs, implanted ip with temperature probes, were treated with STX or E2 for 7-8 wk. Tc was recorded for 4 wk, whereas food intake and body weight were monitored daily. Bone density and fat accumulation were determined postmortem. Both E2 and STX significantly reduced Tc in the females compared with controls. STX, similar to E2, reduced food intake and fat accumulation and increased tibial bone density. Therefore, a Gq-mER-coupled signaling pathway appears to be involved in maintaining homeostatic functions and may constitute a novel therapeutic target for treatment of hypoestrogenic symptoms.

Cross-talk between membrane-initiated and nuclear-initiated oestrogen signalling in the hypothalamus.

It is increasingly evident that 17β‐oestradiol (E2), via a distinct membrane oestrogen receptor (Gq‐mER), can rapidly activate kinase pathways to have multiple downstream actions in central nervous system (CNS) neurones. We have found that E2 can rapidly reduce the potency of the GABAB receptor agonist baclofen and mu‐opioid receptor agonist DAMGO to activate G‐protein‐coupled, inwardly rectifying K+ (GIRK) channels in hypothalamic neurones, thereby increasing the excitability (firing activity) of pro‐opiomelanocortin (POMC) and dopamine neurones. These effects are mimicked by the membrane impermeant E2‐BSA and a new ligand (STX) that is selective for the Gq‐mER that does not bind to ERα or ERβ. Both E2 and STX are fully efficacious in attenuating the GABAB response in ERα, ERβ and GPR 30 knockout mice in an ICI 182u2003780 reversible manner. These findings are further proof that E2 signals through a unique plasma membrane ER. We have characterised the coupling of this Gq‐mER to a Gq‐mediated activation of phospholipase C leading to the up‐regulation of protein kinase Cδ and protein kinase A activity in these neurones, which ultimately alters gene transcription. Finally, as proof of principle, we have found that STX, similar to E2, reduces food intake and body weight gain in ovariectomised females. STX, presumably via the Gq‐mER, also regulates gene expression of a number of relevant targets including cation channels and signalling molecules that are critical for regulating (as a prime example) POMC neuronal excitability. Therefore, E2 can activate multiple receptor‐mediated pathways to modulate excitability and gene transcription in CNS neurones that are critical for controlling homeostasis and motivated behaviors.

Oestrogen Modulates Hypothalamic Control of Energy Homeostasis Through Multiple Mechanisms

The control of energy homeostasis in women is correlated with the anorectic effects of oestrogen, which can attenuate body weight gain and reduce food intake in rodent models. This review investigates the multiple signalling pathways and cellular targets that oestrogen utilises to control energy homeostasis in the hypothalamus. Oestrogen affects all of the hypothalamic nuclei that control energy homeostasis. Oestrogen controls the activity of hypothalamic neurones through gene regulation and neuronal excitability. Oestrogen’s primary cellular pathway is the control of gene transcription through the classical oestrogen receptors (ERs) (ERα and ERβ) with ERα having the primary role in energy homeostasis. Oestrogen also controls energy homeostasis through membrane‐mediated events via membrane‐associated ERs or a novel, putative membrane ER that is coupled to G‐proteins. Therefore, oestrogen is coupled to at least two receptors with multiple signalling and transcriptional pathways to mediate immediate and long‐term anorectic effects. Ultimately, it is the interactions of all the receptor‐mediated processes in hypothalamus and other areas of the central nervous system that will determine the anorectic effects of oestrogen and its control of energy homeostasis.

Fasting and 17β-Estradiol Differentially Modulate the M-Current in Neuropeptide Y Neurons

Multiple K+ conductances are targets for many peripheral and central signals involved in the control of energy homeostasis. Potential K+ channel targets are the KCNQ subunits that form the channels underlying the M-current, a subthreshold, non-inactivating K+ current that is a common target for G-protein-coupled receptors. Whole-cell recordings were made from GFP (Renilla)-tagged neuropeptide Y (NPY) neurons from the arcuate nucleus of the hypothalamus using protocols to isolate and characterize the M-current in these orexigenic neurons. We recorded robust K+ currents in the voltage range of the M-current, which were inhibited by the selective KCNQ channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) (40 μm), in both intact males and ovariectomized, 17β-estradiol (E2)-treated females. Since NPY neurons are orexigenic and are active during fasting, the M-current was measured in fed and fasted male mice. Fasting attenuated the XE991-sensitive current by threefold, which correlated with decreased expression of the KCNQ2 and KCNQ3 subunits as measured with quantitative real-time PCR. Furthermore, E2 treatment augmented the XE991-sensitive M-current by threefold in ovariectomized (vs oil-treated) female mice. E2 treatment increased the expression of the KCNQ5 subunit in females but not KCNQ2 or KCNQ3 subunits. Fasting in females abrogated the effects of E2 on M-current activity, at least in part, by decreasing KCNQ2 and KCNQ3 expression. In summary, these data suggest that the M-current plays a pivotal role in the modulation of NPY neuronal excitability and may be an important cellular target for neurotransmitter and hormonal signals in the control of energy homeostasis in both males and females.

Gonadotropin-Releasing Hormone (GnRH) Activates the M-Current in GnRH Neurons: An Autoregulatory Negative Feedback Mechanism?

GnRH autoregulates GnRH neurons through an ultrashort feedback loop. One potential mechanism is the regulation of K(+) channel activity through the GnRH receptor. Whereas GnRH inhibits the activity of the M-current in peripheral neurons, there is no direct evidence that the M-current is involved in the autoregulatory pathway of GnRH or if the M-current is expressed in GnRH neurons. The M-current is a noninactivating, subthreshold K(+) current that inhibits cell excitability and is ubiquitously expressed in the central nervous system. We found that GnRH neurons expressed the neuronal M-current subunits, KCNQ2, -3, and -5 in addition to GnRH receptor (GnRH R1). Therefore, using whole-cell patch clamp recording and single-cell RT-PCR, we explored the effects of mammalian GnRH peptide on enhanced green fluorescent protein-tagged GnRH neurons acutely dispersed as well as in slice preparations. GnRH (100nm) inhibited GnRH neuronal excitability by hyperpolarizing the membrane. In the presence of CdCl(2), BaCl(2), and tetrodotoxin, GnRH activated an outward current in a dose-dependent manner (EC(50) 11 nm) in 30% of GnRH neurons. In voltage clamp, the selective M-channel blocker, XE-991, inhibited a K(+) current in GnRH neurons. XE-991 also antagonized the outward K(+) current induced by GnRH. Moreover, the GnRH effects on the M-current were blocked by the GnRH R1 antagonist antide. Therefore, these findings indicate that GnRH activates the M-current in a subpopulation of GnRH neurons via GnRH R1. This ultrashort circuit is one potential mechanism by which GnRH could modulate its own neuronal excitability through an autoreceptor.

Increase in multidrug transport activity is associated with oocyte maturation in sea stars

In this study, we report on the presence of efflux transporter activity before oocyte maturation in sea stars and its upregulation after maturation. This activity is similar to the multidrug resistance (MDR) activity mediated by ATP binding cassette (ABC) efflux transporters. In sea star oocytes the efflux activity, as measured by exclusion of calcein‐am, increased two‐fold 3 h post‐maturation. Experiments using specific and non‐specific dyes and inhibitors demonstrated that the increase in transporter activity involves an ABCB protein, P‐glycoprotein (P‐gp), and an ABCC protein similar to the MDR‐associated protein (MRP)‐like transporters. Western blots using an antibody directed against mammalian P‐gp recognized a 45 kDa protein in sea star oocytes that increased in abundance during maturation. An antibody directed against sea urchin ABCC proteins (MRP) recognized three proteins in immature oocytes and two in mature oocytes. Experiments using inhibitors suggest that translation and microtubule function are both required for post‐maturation increases in transporter activity. Immunolabeling revealed translocation of stored ABCB proteins to the plasma cell membrane during maturation, and this translocation coincided with increased transport activity. These MDR transporters serve protective roles in oocytes and eggs, as demonstrated by sensitization of the oocytes to the maturation inhibitor, vinblastine, by MRP and PGP‐specific transporter inhibitors.

Serotonin 5-HT2C receptor-mediated inhibition of the M-current in hypothalamic POMC neurons

Hypothalamic proopiomelanocortin (POMC) neurons are controlled by many central signals, including serotonin. Serotonin increases POMC activity and reduces feeding behavior via serotonion [5-hydroxytryptamine (5-HT)] receptors by modulating K(+) currents. A potential K(+) current is the M-current, a noninactivating, subthreshold outward K(+) current. Previously, we found that M-current activity was highly reduced in fasted vs. fed states in neuropeptide Y neurons. Because POMC neurons also respond to energy states, we hypothesized that fasting may alter the M-current and/or its modulation by serotonergic input to POMC neurons. Using visualized-patch recording in neurons from fed male enhanced green fluorescent protein-POMC transgenic mice, we established that POMC neurons expressed a robust M-current (102.1 ± 6.7 pA) that was antagonized by the selective KCNQ channel blocker XE-991 (40 μM). However, the XE-991-sensitive current in POMC neurons did not differ between fed and fasted states. To determine if serotonin suppresses the M-current via the 5-HT(2C) receptor, we examined the effects of the 5-HT(2A)/5-HT(2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on the M-current. Indeed, DOI attenuated the M-current by 34.5 ± 6.9% and 42.0 ± 5.3% in POMC neurons from fed and fasted male mice, respectively. In addition, the 5-HT(1B)/5-HT(2C) receptor agonist m-chlorophenylpiperazine attenuated the M-current by 42.4 ± 5.4% in POMC neurons from fed male mice. Moreover, the selective 5-HT(2C) receptor antagonist RS-102221 abrogated the actions of DOI in suppressing the M-current. Collectively, these data suggest that although M-current expression does not differ between fed and fasted states in POMC neurons, serotonin inhibits the M-current via activation of 5-HT(2C) receptors to increase POMC neuronal excitability and, subsequently, reduce food intake.