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.

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.

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.

Short term arterial remodelling in the aortae of cholesterol fed New Zealand white rabbits shown in vivo by high-resolution magnetic resonance imaging - implications for human pathology.

High-resolution, non-invasive imaging methods are required to monitor progression and regression of atherosclerotic plaques. We investigated the use of MRI to measure changes in plaque volume and vessel remodelling during progression and regression of atherosclerosis in New Zealand White rabbits. Atherosclerotic lesions were induced in the abdominal aorta by balloon injury and cholesterol feeding. MR images (2D) of the abdominal aorta were acquired with cardiac and respiratory gating using a fast spin echo sequence with and without fat-suppression. In an initial study on rabbits treated for 30 weeks we imaged the aortae with a spatial resolution of 250x250 micrometers with a slice thickness of 2 mm and achieved a close correlation between MRI-derived measurements and those made on perfusion pressure-fixed histological sections (r(1) = 0.83, slope p(1) < 0.01). We subsequently imaged 18 rabbits before and periodically during 12 weeks of cholesterol feeding (progression) followed by 12 weeks on normal diet (regression). Aortic wall (atherosclerotic lesion) volume increased significantly during progression and decreased during regression. In contrast, lumen volume increased during progression and did not change during regression. In conclusion, this study confirms that non-invasive, high-resolution MRI can be used to monitor progression and regression of atherosclerosis, each within 3 months and shows, for the first time in a short-term model, that positive remodelling occurs early during progression and persists through regression of atherosclerotic lesions.

Vasopressin receptor V1a regulates circadian rhythms of locomotor activity and expression of clock-controlled genes in the suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) serve as the principal circadian pacemakers that coordinate daily cycles of behavior and physiology for mammals. A network of transcriptional and translational feedback loops underlies the operating molecular mechanism for circadian oscillation within the SCN neurons. It remains unclear how timing information is transmitted from SCN neurons to eventually evoke circadian rhythms. Intercellular communication between the SCN and its target neurons is critical for the generation of coherent circadian rhythms. At the molecular level, neuropeptides encoded by clock-controlled genes have been indicated as important output mediators. Arginine vasopressin (AVP) is the product of one such clock-controlled gene. Previous studies have demonstrated a circadian rhythm of AVP levels in the cerebrospinal fluid and the SCN. The physiological effects of AVP are mediated by three types of AVP receptors, designated as V1a, V1b, and V2. In this study, we report that V1a mRNA levels displayed a circadian rhythm in the SCN, peaking during night hours. The circadian rhythmicity of locomotor activities was significantly reduced in V1a-deficient (V1a(-/-)) mice (50-75% reduction in the power of fast Fourier transformation). However, the light masking and light-induced phase shift effects are intact in V1a(-/-) mice. Whereas the expression of clock core genes was unaltered, the circadian amplitude of prokineticin 2 (PK2) mRNA oscillation was attenuated in the SCN of V1a(-/-) mice ( approximately 50% reduction in the peak levels). In vitro experiments demonstrated that AVP, acting through V1a receptor, was able to enhance the transcriptional activity of the PK2 promoter. These studies thus indicate that AVP-V1a signaling plays an important role in the generation of overt circadian rhythms.

Differential activation of "social" and "solitary" variants of the Caenorhabditis elegans G protein-coupled receptor NPR-1 by its cognate ligand AF9.

Natural variations of wild Caenorhabditis elegans isolates having either Phe-215 or Val-215 in NPR-1, a putative orphan neuropeptide Y-like G protein-coupled receptor, result in either "social" or "solitary" feeding behaviors (de Bono, M., and Bargmann, C. I. (1998) Cell 94, 679-689). We identified a nematode peptide, GLGPRPLRF-NH2 (AF9), as a ligand activating the cloned NPR-1 receptor heterologously expressed in mammalian cells. Shifting cell culture temperatures from 37 to 28 degrees C, implemented 24 h after transfections, was essential for detectable functional expression of NPR-1. AF9 treatments linked both cloned receptor variants to activation of Gi/Go proteins and cAMP inhibition, thus allowing for classification of NPR-1 as an inhibitory G protein-coupled receptor. The Val-215 receptor isoform displayed higher binding and functional activity than its Phe-215 counterpart. This finding parallels the in vivo observation of a more potent repression of social feeding by the npr-1 gene encoding the Val-215 form of the receptor, resulting in dispersing (solitary) animals. Since neuropeptide Y shows no sequence homology to AF9 and was functionally inactive at the cloned NPR-1, we propose to rename NPR-1 and refer to it as an AF9 receptor, AF9-R1.

Cloning and functional expression of the first Drosophila melanogaster sulfakinin receptor DSK-R1.

Described in this report is a successful cloning and characterization of a functionally active Drosophila sulfakinin receptor designated DSK-R1. When expressed in mammalian cells, DSK-R1 was activated by a sulfated, Met(7-->Leu(7)-substituted analog of drosulfakinin-1, FDDY(SO(3)H)GHLRF-NH(2) ([Leu(7)]-DSK-1S). The interaction of [Leu(7)]-DSK-1S with DSK-R1 led to a dose-dependent intracellular calcium increase with an EC(50) in the low nanomolar range. The observed Ca(2+) signal predominantly resulted from activation of pertussis toxin (PTX)-insensitive signaling pathways pointing most likely to G(q/11) involvement in coupling to the activated receptor. The unsulfated [Leu(7)]-DSK-1 was ca. 3000-fold less potent than its sulfated counterpart which stresses the importance of the sulfate moiety for the biological activity of drosulfakinin. The DSK-R1 was specific for the insect sulfakinin since two related vertebrate sulfated peptides, human CCK-8 and gastrin-II, were found inactive when tested at concentrations up to 10(-5) M. To our knowledge, the cloned DSK-R1 receptor is the first functionally active Drosophila sulfakinin receptor reported to date.