Evidence-Based View of Safety and Effectiveness of Prokineticin Receptors Antagonists during Pregnancy.

Endocrine gland derived vascular endothelial growth factor (EG-VEGF) is a canonical member of the prokineticin (PROKs) family. It acts via the two G-protein coupled receptors, namely PROKR1 and PROKR2. We have recently demonstrated that EG-VEGF is highly expressed in the human placenta; contributes to placental vascularization and growth and that its aberrant expression is associated with pregnancy pathologies including preeclampsia and fetal growth restriction. These findings strongly suggested that antagonization of its receptors may constitute a potential therapy for the pregnancy pathologies. Two specific antagonists of PROKR1 (PC7) and for PROKR2 (PKRA) were reported to reverse PROKs adverse effects in other systems. In the view of using these antagonists to treat pregnancy pathologies, a proof of concept study was designed to determine the biological significances of PC7 and PKRA in normal pregnancy outcome. PC7 and PKRA were tested independently or in combination in trophoblast cells and during early gestation in the gravid mouse. Both independent and combined treatments uncovered endogenous functions of EG-VEGF. The independent use of antagonists distinctively identified PROKR1 and PROKR2-mediated EG-VEGF signaling on trophoblast differentiation and invasion; thereby enhancing feto-placental growth and pregnancy outcome. Thus, our study provides evidence for the potential safe use of PC7 or PKRA to improve pregnancy outcome.

Overexpression of Prokineticin 2 in Transgenic Mice Leads to Reduced Circadian Behavioral Rhythmicity and Altered Molecular Rhythms in the Suprachiasmatic Clock.

In mammals, the master pacemaker driving circadian rhythms is thought to reside in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. A clear view of molecular clock mechanisms within the SCN neurons has been elucidated. In contrast, much less is known about the output mechanism by which the SCN circadian pacemaker sends timing information for eventual control of physiological and behavioral rhythms. Two secreted molecules, prokineticin 2 (PK2) and vasopressin, that are encoded by respective clock-controlled genes, have been indicated as candidate SCN output molecules. Several lines of evidence have emerged that support the role of PK2 as an output signal for the SCN circadian clock, including the reduced circadian rhythms in mice that are deficient in PK2 or its receptor, PKR2. In the current study, transgenic mice with the overexpression of PK2 have been generated. These transgenic mice displayed reduced oscillation of the PK2 expression in the SCN and decreased amplitude of circadian locomotor rhythm, supporting the important signaling role of PK2 in the regulation of circadian rhythms. Altered molecular rhythms were also observed in the SCN in the transgenic mice, indicating that PK2 signaling also regulates the operation of core clockwork. This conclusion is consistent with recent reports showing the likely signaling role of PK2 from the intrinsically photosensitive retinal ganglion cells to SCN neurons. Thus, PK2 signaling plays roles in both the input and the output pathways of the SCN circadian clock.

Prokineticin-2 upregulation during neuronal injury mediates a compensatory protective response against dopaminergic neuronal degeneration.

Prokineticin-2 (PK2), a recently discovered secreted protein, regulates important physiological functions including olfactory biogenesis and circadian rhythms in the CNS. Interestingly, although PK2 expression is low in the nigral system, its receptors are constitutively expressed on nigrostriatal neurons. Herein, we demonstrate that PK2 expression is highly induced in nigral dopaminergic neurons during early stages of degeneration in multiple models of Parkinson's disease (PD), including PK2 reporter mice and MitoPark mice. Functional studies demonstrate that PK2 promotes mitochondrial biogenesis and activates ERK and Akt survival signalling pathways, thereby driving neuroprotection. Importantly, PK2 overexpression is protective whereas PK2 receptor antagonism exacerbates dopaminergic degeneration in experimental PD. Furthermore, PK2 expression increased in surviving nigral dopaminergic neurons from PD brains, indicating that PK2 upregulation is clinically relevant to human PD. Collectively, our results identify a paradigm for compensatory neuroprotective PK2 signalling in nigral dopaminergic neurons that could have important therapeutic implications for PD.

Differential arousal regulation by prokineticin 2 signaling in the nocturnal mouse and the diurnal monkey.

The temporal organization of activity/rest or sleep/wake rhythms for mammals is regulated by the interaction of light/dark cycle and circadian clocks. The neural and molecular mechanisms that confine the active phase to either day or night period for the diurnal and the nocturnal mammals are unclear. Here we report that prokineticin 2, previously shown as a circadian clock output molecule, is expressed in the intrinsically photosensitive retinal ganglion cells, and the expression of prokineticin 2 in the intrinsically photosensitive retinal ganglion cells is oscillatory in a clock-dependent manner. We further show that the prokineticin 2 signaling is required for the activity and arousal suppression by light in the mouse. Between the nocturnal mouse and the diurnal monkey, a signaling receptor for prokineticin 2 is differentially expressed in the retinorecipient suprachiasmatic nucleus and the superior colliculus, brain projection targets of the intrinsically photosensitive retinal ganglion cells. Blockade with a selective antagonist reveals the respectively inhibitory and stimulatory effect of prokineticin 2 signaling on the arousal levels for the nocturnal mouse and the diurnal monkey. Thus, the mammalian diurnality or nocturnality is likely determined by the differential signaling of prokineticin 2 from the intrinsically photosensitive retinal ganglion cells onto their retinorecipient brain targets.

Expression of prokineticin 2 and its receptor in the macaque monkey brain.

Prokineticin 2 (PK2) has been indicated as an output signaling molecule for the suprachiasmatic nucleus (SCN) circadian clock. Most of these studies were performed with nocturnal animals, particularly mice and rats. In the current study, the PK2 and its receptor, PKR2, was cloned from a species of diurnal macaque monkey. The macaque monkey PK2 and PKR2 were found to be highly homologous to that of other mammalian species. The mRNA expression of PK2 and PKR2 in the macaque brain was examined by in situ hybridization. The expression patterns of PK2 and PKR2 in the macaque brain were found to be quite similar to that of the mouse brain. Particularly, PK2 mRNA was shown to oscillate in the SCN of the macaque brain in the same phase and with similar amplitude with that of nocturnal mouse brain. PKR2 expression was also detected in known primary SCN targets, including the midline thalamic and hypothalamic nuclei. In addition, we detected the expression of PKR2 mRNA in the dorsal raphe nucleus (DR) of both macaque and mouse brains. As a likely SCN to dorsal raphe projection has previously been indicated, the expression of PKR2 in the raphe nuclei of both macaque and mouse brain signifies a possible role of DR as a previously unrecognized primary SCN projection target.

PPARγ controls pregnancy outcome through activation of EG-VEGF: new insights into the mechanism of placental development.

PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EG-VEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants (n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ(+/-) and PPARγ(-/-) mice. Major processes of placental development that are known to be controlled by PPARγ, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ(-/-) mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion.

Rhythmic Trafficking of TRPV2 in the Suprachiasmatic Nucleus is Regulated by Prokineticin 2 Signaling.

The mammalian circadian clock is composed of single-cell oscillators. Neurochemical and electrical signaling among these oscillators is important for the normal expression of circadian rhythms. Prokineticin 2 (PK2), encoding a cysteine-rich secreted protein, has been shown to be a critical signaling molecule for the regulation of circadian rhythms. PK2 expression in the suprachiasmatic nucleus (SCN) is highly rhythmic, peaking during the day and being essentially absent during the night. Mice with disrupted PK2 gene or its receptor PKR2 display greatly reduced rhythmicity of broad circadian parameters such as locomotor activity, body temperature and sleep/wake patterns. PK2 has been shown to increase the firing rate of SCN neurons, with unknown molecular mechanisms. Here we report that TRPV2, an ion channel belonging to the family of TRP, is co-expressed with PKR2 in the SCN neurons. Further, TRPV2 protein, but not TRPV2 mRNA, was shown to oscillate in the SCN in a PK2-dependent manner. Functional studies revealed that TRPV2 enhanced signaling of PKR2 in calcium mobilization or ion current conductance, likely via the increased trafficking of TRPV2 to the cell surface. Taken together, these results indicate that TRPV2 is likely part of the downstream signaling of PK2 in the regulation of the circadian rhythms.

Functional rescue of Kallmann syndrome-associated prokineticin receptor 2 (PKR2) mutants deficient in trafficking.

Mutations in the G protein-coupled prokineticin receptor 2 (PKR2) are known to cause Kallmann syndrome and idiopathic hypogonadotropic hypogonadism manifesting with delayed puberty and infertility. Some of the mutant receptors are not routed to the cell surface; instead, they are trapped in the cellular secretory pathway. The cell-permeant agonists/antagonists have been used to rescue some membrane receptors that are not targeted onto the cell membrane. Here, we chose three disease-associated mutations (W178S, G234D, and P290S), which all resulted in retention of PKR2 intracellularly. We show that a small molecule PKR2 antagonist (A457) dramatically increased cell surface expression and rescued the function of P290S PKR2, but had no effect on W178S and G234D PKR2. Furthermore, we also tested chemical chaperone glycerol on the cell surface expression and function of PKR2 mutants. Treatment with 10% glycerol significantly increased the cell surface expression and signaling of P290S and W178S PKR2. These data demonstrate that some Kallmann syndrome-associated, intracellularly retained mutant PKR2 receptors can be functionally rescued, suggesting a potential treatment strategy for patients bearing such mutations.

CARL lncRNA inhibits anoxia-induced mitochondrial fission and apoptosis in cardiomyocytes by impairing miR-539-dependent PHB2 downregulation.

Abnormal mitochondrial fission participates in the pathogenesis of many diseases. Long non-coding RNAs (lncRNAs) are emerging as new players in gene regulation, but how lncRNAs operate in the regulation of mitochondrial network is unclear. Here we report that a lncRNA, named cardiac apoptosis-related lncRNA (CARL), can suppress mitochondrial fission and apoptosis by targeting miR-539 and PHB2. The results show that PHB2 is able to inhibit mitochondrial fission and apoptosis. miR-539 is responsible for the dysfunction of PHB2 and regulates mitochondrial fission and apoptosis by targeting PHB2. Further, we show that CARL can act as an endogenous miR-539 sponge that regulates PHB2 expression, mitochondrial fission and apoptosis. Our present study reveals a model of mitochondrial fission regulation that is composed of CARL, miR-539 and PHB2. Modulation of their levels may provide a new approach for tackling apoptosis and myocardial infarction.

Oxytocin inhibits the activity of acid-sensing ion channels through the vasopressin, V1A receptor in primary sensory neurons.

BACKGROUND AND PURPOSE: A growing number of studies have demonstrated that oxytocin (OT) plays an analgesic role in modulation of nociception and pain. Most work to date has focused on the central mechanisms of OT analgesia, but little is known about whether peripheral mechanisms are also involved. Acid-sensing ion channels (ASICs) are distributed in peripheral sensory neurons and participate in nociception. Here, we investigated the effects of OT on the activity of ASICs in dorsal root ganglion (DRG) neurons. EXPERIMENTAL APPROACH: Electrophysiological experiments were performed on neurons from rat DRG. Nociceptive behaviour was induced by acetic acid in rats and mice lacking vasopressin, V1A receptors. KEY RESULTS: OT inhibited the functional activity of native ASICs. Firstly, OT dose-dependently decreased the amplitude of ASIC currents in DRG neurons. Secondly, OT inhibition of ASIC currents was mimicked by arginine vasopressin (AVP) and completely blocked by the V1A receptor antagonist SR49059, but not by the OT receptor antagonist L-368899. Thirdly, OT altered acidosis-evoked membrane excitability of DRG neurons and significantly decreased the amplitude of the depolarization and number of action potentials induced by acid stimuli. Finally, peripherally administered OT or AVP inhibited nociceptive responses to intraplantar injection of acetic acid in rats. Both OT and AVP also induced an analgesic effect on acidosis-evoked pain in wild-type mice, but not in V1A receptor knockout mice. CONCLUSIONS AND IMPLICATIONS: These results reveal a novel peripheral mechanism for the analgesic effect of OT involving the modulation of native ASICs in primary sensory neurons mediated by V1A receptors.

Signaling role of prokineticin 2 on the estrous cycle of female mice.

The possible signaling role of prokineticin 2 (PK2) and its receptor, prokineticin receptor 2 (PKR2), on female reproduction was investigated. First, the expression of PKR2 and its co-localization with estrogen receptor (ERα) in the hypothalamus was examined. Sexually dimorphic expression of PKR2 in the preoptic area of the hypothalamus was observed. Compared to the male mice, there was more widespread PKR2 expression in the preoptic area of the hypothalamus in the female mice. The likely co-expression of PKR2 and ERα in the preoptic area of the hypothalamus was observed. The estrous cycles in female PK2-null, and PKR2-null heterozygous mice, as well as in PK2-null and PKR2-null compound heterozygous mice were examined. Loss of one copy of PK2 or PKR2 gene caused elongated and irregular estrous cycle in the female mice. The alterations in the estrous cycle were more pronounced in PK2-null and PKR2-null compound heterozygous mice. Consistent with these observations, administration of a small molecule PK2 receptor antagonist led to temporary blocking of estrous cycle at the proestrous phase in female mice. The administration of PKR2 antagonist was found to blunt the circulating LH levels. Taken together, these studies indicate PK2 signaling is required for the maintenance of normal female estrous cycles.

A pre-microRNA-149 (miR-149) genetic variation affects miR-149 maturation and its ability to regulate the Puma protein in apoptosis.

MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs that function as negative regulators of gene expression. They are transcribed from endogenous DNA and form hairpin structures (termed as pre-miRNAs) that are processed to form mature miRNAs. It remains largely unknown as to the molecular consequences of the natural genetic variation in pre-miRNAs. Here, we report that an A→G polymorphism (rs71428439) is located in Homo sapiens miR-149 stem-loop region. This polymorphism results in a change in the structure of the miR-149 precursor. Our results showed that the genotype distribution of this polymorphism in myocardial infarction cases was significantly different from that in the control subjects. We examined the biological significance of this polymorphism on the production of mature miR-149, and we observed that the G-allelic miR-149 precursor displayed a lower production of mature miR-149 compared with the A-allelic one. Further investigations disclosed that miR-149 could withstand mitochondrial fission and apoptosis through targeting the pro-apoptotic factor p53-up-regulated modulator of apoptosis (Puma). Enforced expression of miR-149 promoted cell survival, whereas knockdown of miR-149 rendered cells to be sensitive to apoptotic stimulation. Intriguingly, the A to G variation led pre-miR-149 to elicit an attenuated effect on the inhibition of mitochondrial fission and apoptosis. Finally, this polymorphism exerts its influence on cardiac function in the mouse model of myocardial infarction. These data suggest that this polymorphism in the miR-149 precursor may result in important phenotypic traits of myocardial infarction. Our findings warrant further investigations on the relationship between miR-149 polymorphism and myocardial infarction.

A PK2/Bv8/PROK2 antagonist suppresses tumorigenic processes by inhibiting angiogenesis in glioma and blocking myeloid cell infiltration in pancreatic cancer.

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy.

Prokineticin 2 is involved in the thermoregulation and energy expenditure.

Animals have developed adaptive strategies to survive tough situations such as food shortage. However, the underlying molecular mechanism is not fully understood. Here, we provided evidence that the regulatory peptide prokineticin 2 (PK2) played an important role in such an adaptation. The PK2 expression was rapidly induced in the hypothalamic paraventricular nucleus (PVN) after fasting, which can be mimicked by 2-deoxy-D-glucose (2-DG) injection. The fasting-induced arousal was absent in the PK2-deficient (PK2(-/-)) mice. Furthermore, PK2(-/-) mice showed less energy expenditure and body weight loss than wild-type (WT) controls upon fasting. As a result, PK2(-/-) mice entered torpor after fasting. Supply of limited food (equal to 5% of body weight) daily during fasting rescued the body weight loss and hypothermal phenotype in WT mice, but not in PK2(-/-) mice. Our study thus demonstrated PK2 as a regulator in the thermoregulation and energy expenditure.

The circadian output signals from the suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus comprise a self-sustained biological clock generating an endogenous ∼24-h circadian rhythm, driving many overt daily rhythms in the body. An important remaining question is how the SCN neurons communicate with their efferent targets to control the daily oscillations in behavior and physiology. In this chapter, we summarize several signaling factors that may serve as such SCN output factors. Whereas vasopressin may be involved in the regulation of circadian hormone rhythms, SCN-derived prokineticin 2 (PK2), TGF-α, and cardiotrophin-like cytokine (CLC) may serve as output factors for other circadian rhythms, including locomotor activity, body temperature, and energy metabolism. The circadian rhythm in firing activity of SCN neurons is also likely to be a critical output signaling mechanism. The likely involvement of these output factors in the generation of the circadian rhythm in SCN neuronal firing activity is also discussed.

Prokineticin 2 potentiates acid-sensing ion channel activity in rat dorsal root ganglion neurons.

BACKGROUND: Prokineticin 2 (PK2) is a secreted protein and causes potent hyperalgesia in vivo, and is therefore considered to be a new pronociceptive mediator. However, the molecular targets responsible for the pronociceptive effects of PK2 are still poorly understood. Here, we have found that PK2 potentiates the activity of acid-sensing ion channels in the primary sensory neurons. METHODS: In the present study, experiments were performed on neurons freshly isolated from rat dorsal root ganglion by using whole-cell patch clamp and voltage-clamp recording techniques. RESULTS: PK2 dose-dependently enhanced proton-gated currents with an EC50 of 0.22 ± 0.06 nM. PK2 shifted the proton concentration-response curve upwards, with a 1.81 ± 0.11 fold increase of the maximal current response. PK2 enhancing effect on proton-gated currents was completely blocked by PK2 receptor antagonist. The potentiation was also abolished by intracellular dialysis of GF109203X, a protein kinase C inhibitor, or FSC-231, a protein interacting with C-kinase 1 inhibitor. Moreover, PK2 enhanced the acid-evoked membrane excitability of rat dorsal root ganglion neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, PK2 exacerbated nociceptive responses to the injection of acetic acid in rats. CONCLUSION: These results suggest that PK2 increases the activity of acid-sensing ion channels via the PK2 receptor and protein kinase C-dependent signal pathways in rat primary sensory neurons. Our findings support that PK2 is a proalgesic factor and its signaling likely contributes to acidosis-evoked pain by sensitizing acid-sensing ion channels.

Prokineticin 2 is an endangering mediator of cerebral ischemic injury.

Stroke causes brain dysfunction and neuron death, and the lack of effective therapies heightens the need for new therapeutic targets. Here we identify prokineticin 2 (PK2) as a mediator for cerebral ischemic injury. PK2 is a bioactive peptide initially discovered as a regulator of gastrointestinal motility. Multiple biological roles for PK2 have been discovered, including circadian rhythms, angiogenesis, and neurogenesis. However, the role of PK2 in neuropathology is unknown. Using primary cortical cultures, we found that PK2 mRNA is up-regulated by several pathological stressors, including hypoxia, reactive oxygen species, and excitotoxic glutamate. Glutamate-induced PK2 expression is dependent on NMDA receptor activation and extracellular calcium. Enriched neuronal culture studies revealed that neurons are the principal source of glutamate-induced PK2. Using in vivo models of stroke, we found that PK2 mRNA is induced in the ischemic cortex and striatum. Central delivery of PK2 worsens infarct volume, whereas PK2 receptor antagonist decreases infarct volume and central inflammation while improving functional outcome. Direct central inhibition of PK2 using RNAi also reduces infarct volume. These findings indicate that PK2 can be activated by pathological stimuli such as hypoxia-ischemia and excitotoxic glutamate and identify PK2 as a deleterious mediator for cerebral ischemia.

Genetic overlap in Kallmann syndrome, combined pituitary hormone deficiency, and septo-optic dysplasia.

CONTEXT: Kallmann syndrome (KS), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) all result from development defects of the anterior midline in the human forebrain. OBJECTIVE: The objective of the study was to investigate whether KS, CPHD, and SOD have shared genetic origins. DESIGN AND PARTICIPANTS: A total of 103 patients with either CPHD (n = 35) or SOD (n = 68) were investigated for mutations in genes implicated in the etiology of KS (FGFR1, FGF8, PROKR2, PROK2, and KAL1). Consequences of identified FGFR1, FGF8, and PROKR2 mutations were investigated in vitro. RESULTS: Three patients with SOD had heterozygous mutations in FGFR1; these were either shown to alter receptor signaling (p.S450F, p.P483S) or predicted to affect splicing (c.336C>T, p.T112T). One patient had a synonymous change in FGF8 (c.216G>A, p.T72T) that was shown to affect splicing and ligand signaling activity. Four patients with CPHD/SOD were found to harbor heterozygous rare loss-of-function variants in PROKR2 (p.R85G, p.R85H, p.R268C). CONCLUSIONS: Mutations in FGFR1/FGF8/PROKR2 contributed to 7.8% of our patients with CPHD/SOD. These data suggest a significant genetic overlap between conditions affecting the development of anterior midline in the human forebrain.

Prokineticin 2 regulates the electrical activity of rat suprachiasmatic nuclei neurons.

Neuropeptide signaling plays roles in coordinating cellular activities and maintaining robust oscillations within the mammalian suprachiasmatic nucleus (SCN). Prokineticin2 (PK2) is a signaling molecule from the SCN and involves in the generation of circadian locomotor activity. Prokineticin receptor 2 (PKR2), a receptor for PK2, has been shown to be expressed in the SCN. However, very little is known about the cellular action of PK2 within the SCN. In the present study, we investigated the effect of PK2 on spontaneous firing and miniature inhibitory postsynaptic currents (mIPSCs) using whole cell patch-clamp recording in the SCN slices. PK2 dose-dependently increased spontaneous firing rates in most neurons from the dorsal SCN. PK2 acted postsynaptically to reduce γ-aminobutyric acid (GABA)-ergic function within the SCN, and PK2 reduced the amplitude but not frequency of mIPSCs. Furthermore, PK2 also suppressed exogenous GABA-induced currents. And the inhibitory effect of PK2 required PKC activation in the postsynaptic cells. Our data suggest that PK2 could alter cellular activities within the SCN and may influence behavioral and physiological rhythms.

The puzzles of the prokineticin 2 pathway in human reproduction.

Prokineticin, 1 (PROK1) and prokineticin 2 (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. MIT-1 was initially identified as a non-toxic constituent in the venom of the black mamba snake (Dendroaspis polylepis) (Joubert and Strydom, 1980) while Bv8 was identified in the skin secretion of the toad, Bombina variegate (Mollay et al., 1999). All three homologs stimulate gastrointestinal motility thus accounting for their family name "prokineticins" (Schweitz et al., 1990, 1999). However, since its initial description, both PROK1 and PROK2 have been found to regulate a dazzling array of biological functions throughout the body. In particular, PROK1 acts as a potent angiogenic mitogen on endocrine vascular epithelium, thus earning its other name, Endocrine gland-vascular endothelial factor (EG-VEGF) (LeCouter et al., 2002). In contrast, the PROK2 signaling pathway is a critical regulator of olfactory bulb morphogenesis and sexual maturation in mammals and this function is the focus of this review.