A novel brain receptor is expressed in a distinct population of olfactory sensory neurons

Three novel G-protein-coupled receptor genes related to the previously described RA1c gene have been isolated from the mouse genome. Expression of these genes has been detected in distinct areas of the brain and also in the olfactory epithelium of the nose. Developmental studies revealed a differential onset of expression: in the brain at embryonic stage 17, in the olfactory system at stage E12. In order to determine which cell type in the olfactory epithelium expresses this unique receptor type, a transgenic approach was employed which allowed a coexpression of histological markers together with the receptor and thus visualization of the appropriate cell population. It was found that the receptor-expressing cells were located very close to the basal membrane of the epithelium; however, the cells extended a dendritic process to the epithelial surface and their axons projected into the main olfactory bulb where they converged onto two or three glomeruli in the dorsal and posterior region of the bulb. Thus, these data provide evidence that this unique type of receptor is expressed in mature olfactory neurons and suggests that it may be involved in the detection of special odour molecules

β-lapachone regulates mammalian inositol pyrophosphate levels in an NQO1- and oxygen-dependent manner

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involve PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H 2 O 2 ) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly β-lapachone (β-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that β-lap treatment caused PP-InsP changes that are oxygen-dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that β-lap requires NQO1 bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with β-lap. The data presented here unveil unique aspects of β-lap pharmacology and its impact on PP-InsP levels. The identification of different quinone drugs as modulators of PP-InsP synthesis will allow the overall impact on cellular function of such drugs to be better appreciated

BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo

Bdnf exon-IV and exon-VI transcripts are driven by neuronal activity and are involved in pathologies related to sleep, fear or memory disorders. However, how their differential transcription translates activity changes into long-lasting network changes is elusive. Aiming to trace specifically the network controlled by exon-IV and -VI derived BDNF during activity-dependent plasticity changes, we generated a transgenic reporter mouse for BDNF-live-exon-visualization (BLEV), in which expression of Bdnf exon-IV and -VI can be visualized by co-expression of CFP and YFP. CFP and YFP expression was differentially activated and targeted in cell lines, primary cultures and BLEV reporter mice without interfering with BDNF protein synthesis. CFP and YFP expression, moreover, overlapped with BDNF protein expression in defined hippocampal neuronal, glial and vascular locations in vivo. So far, activity-dependent BDNF cannot be explicitly monitored independent of basal BDNF levels. The BLEV reporter mouse therefore provides a new model, which can be used to test whether stimulus-induced activity-dependent changes in BDNF expression are instrumental for long-lasting plasticity modifications

Interdomain Interactions Control Ca2+-Dependent Potentiation in the Cation Channel TRPV4

Several Ca2+-permeable channels, including the non-selective cation channel TRPV4, are subject to Ca2+-dependent facilitation. Although it has been clearly demonstrated in functional experiments that calmodulin (CaM) binding to intracellular domains of TRP channels is involved in this process, the molecular mechanism remains elusive. In this study, we provide experimental evidence for a comprehensive molecular model that explains Ca2+-dependent facilitation of TRPV4. In the resting state, an intracellular domain from the channel N terminus forms an autoinhibitory complex with a C-terminal domain that includes a high-affinity CaM binding site. CaM binding, secondary to rises in intracellular Ca2+, displaces the N-terminal domain which may then form a homologous interaction with an identical domain from a second subunit. This represents a novel potentiation mechanism that may also be relevant in other Ca2+-permeable channels

Osmosensitivity of Transient Receptor Potential Vanilloid 1 Is Synergistically Enhanced by Distinct Activating Stimuli Such as Temperature and Protons

In animals, body-fluid osmolality is continuously monitored to keep it within a narrow range around a set point (∼300 mOsm/kg). Transient receptor potential vanilloid 1 (TRPV1), a cation channel, has been implicated in body-fluid homeostasis in vivo based on studies with the TRPV1-knockout mouse. However, the response of TRPV1 to hypertonic stimuli has not been demonstrated with heterologous expression systems so far, despite intense efforts by several groups. Thus, the molecular entity of the hypertonic sensor in vivo still remains controversial. Here we found that the full-length form of TRPV1 is sensitive to an osmotic increase exclusively at around body temperature using HEK293 cells stably expressing rat TRPV1. At an ambient temperature of 24°C, a slight increase in the intracellular calcium concentration ([Ca2+]i) was rarely observed in response to hypertonic stimuli. However, the magnitude of the osmosensitive response markedly increased with temperature, peaking at around 36°C. Importantly, the response at 36°C showed a robust increase over a hypertonic range, but a small decrease over a hypotonic range. A TRPV1 antagonist, capsazepine, and a nonspecific TRP channel inhibitor, ruthenium red, completely blocked the increase in [Ca2+]i. These results endorse the view that the full-length form of TRPV1 is able to function as a sensor of hypertonic stimuli in vivo. Furthermore, we found that protons and capsaicin likewise synergistically potentiated the response of TRPV1 to hypertonic stimuli. Of note, HgCl2, which blocks aquaporins and inhibits cell-volume changes, significantly reduced the osmosensitive response. Our findings thus indicate that TRPV1 integrates multiple different types of activating stimuli, and that TRPV1 is sensitive to hypertonic stimuli under physiologically relevant conditions

The Increased Activity of TRPV4 Channel in the Astrocytes of the Adult Rat Hippocampus after Cerebral Hypoxia/Ischemia

The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult