Light detected in the retina modulates several physiological processes including circadian photo-entrainment and pupillary light reflex. Intrinsically photosensitive retinal ganglion cells (ipRGCs) convey rod-cone and melanopsin-driven light input to the brain. Using EEGs and electromyograms, we show that acute light induces sleep in mice during their nocturnal active phase whereas acute dark awakens mice during their diurnal sleep phase. We used retinal mutant mouse lines that lack (i) the ipRGCs, (ii) the photo-transduction pathways of rods and cones, or (iii) the melanopsin protein and showed that the influence of light and dark on sleep requires both rod-cone and melanopsin signaling through ipRGCs and is independent of image formation. We further show that, although acute light pulses overcome circadian and homeostatic drives for sleep, upon repeated light exposures using a 3.5 h/3.5 h light/dark cycle, the circadian and homeostatic drives override the light input. Thus, in addition to their known role in aligning circadian physiology with day and night, ipRGCs also relay light and dark information from both rod-cone and melanopsin-based pathways to modulate sleep and wakefulness.

Transient receptor potential vanilloid 1 (TRPV1) is an ion channel that is gated by noxious heat, capsaicin and other diverse stimuli. It is a nonselective cation channel that prefers Ca2+ over Na+. These permeability characteristics, as in most channels, are widely presumed to be static. On the contrary, we found that activation of native or recombinant rat TRPV1 leads to time- and agonist concentration-dependent increases in relative permeability to large cations and changes in Ca2+ permeability. Using the substituted cysteine accessibility method, we saw that these changes were attributable to alterations in the TRPV1 selectivity filter. TRPV1 agonists showed different capabilities for evoking ionic selectivity changes. Furthermore, protein kinase C-dependent phosphorylation of Ser800 in the TRPV1 C terminus potentiated agonist-evoked ionic selectivity changes. Thus, the qualitative signaling properties of TRPV1 are dynamically modulated during channel activation, a process that probably shapes TRPV1 participation in pain, cytotoxicity and neurotransmitter release.

In the absence of functional rod and cone photoreceptors, mammals retain the ability to detect light for a variety of physiological functions such as circadian photoentrainment and pupillary light reflex. This is attributed to a third class of photoreceptors, the intrinsically photosensitive retinal ganglion cells that express the photopigment melanopsin. Even though in the absence of rods and cones, mammals retain the ability to detect light for various nonimage-forming visual functions, rods and cones can compensate for the absence of the melanopsin protein in nonvisual light-dependent physiological behaviors. Several studies have addressed the relative contribution of each photoreceptor type to nonimage-forming visual functions; however, a comprehensive model for these interactions is far from complete. Under conditions where melanopsin-containing retinal ganglion cells were genetically ablated, image formation is maintained, whereas circadian photoentrainment and pupillary light reflex are severely impaired. The findings indicate that multiple photoreceptors contribute to nonimage-forming visual functions through signaling via melanopsin-containing retinal ganglion cells. Future studies will aim to determine more quantitatively the relative contributions of each retinal photoreceptor in signaling light for nonimage-forming visual functions.

Aquaporin-5 (AQP5) is expressed in epithelia of lung, cornea, and various secretory glands, sites where extracellular osmolality is known to fluctuate. Hypertonic aquaporin (AQP) induction has been described, but little is known about the effects of a hypotonic environment on AQP abundance. We report that, when mouse lung epithelial cells were exposed to hypotonic medium, a dose-responsive decrease in AQP5 abundance was observed. Hypotonic reduction of AQP5 was blocked by ruthenium red, methanandamide, and miconazole, agents that inhibit the cation channel transient receptor potential vanilloid (TRPV) 4 present in lung epithelial cells. Several observations indicate that TRPV4 participates in hypotonic reduction of AQP5, including a requirement for extracellular calcium to achieve AQP5 reduction; an increase in intracellular calcium in mouse lung epithelial (MLE) cells after hypotonic stimulation; and reduction of AQP5 abundance after addition of the TRPV4 agonist 4alpha-Phorbol-12,13-didecanoate (4alpha-PDD). Similarly, addition of hypotonic PBS to mouse trachea in vivo decreased AQP5 within 1 h, an effect blocked by ruthenium red. To confirm a functional interaction, AQP5 was expressed in control or TRPV4-expressing human embryonic kidney (HEK) cells. Hypotonic reduction of AQP5 was observed only in the presence of TRPV4 and was blocked by ruthenium red. Combined with earlier studies, these observations indicate that AQP5 abundance is tightly regulated along a range of osmolalities and that AQP5 reduction by extracellular hypotonicity can be mediated by TRPV4. These findings have direct relevance to regulation of membrane water permeability and water homeostasis in epithelia of the lung and other organs.

2-Aminoethyl diphenylborinate was recently identified as a chemical activator of TRPV1, TRPV2, and TRPV3, three heat-gated members of the transient receptor potential vanilloid (TRPV) ion channel subfamily. Here we demonstrated that two structurally related compounds, diphenylboronic anhydride (DPBA) and diphenyltetrahydrofuran (DPTHF), can also modulate the activity of these channels. DPBA acted as a TRPV3 agonist, whereas DPTHF exhibited prominent antagonistic activity. However, all three diphenyl-containing compounds promoted some degree of channel activation or potentiation, followed by channel block. Strong TRPV3 activation by DPBA often leads to the appearance of a secondary, enhanced, current phase. A similar biphasic response was observed during TRPV3 heat stimulation; an initial, gradually sensitizing phase (I(1)) was followed by an abrupt transition to a secondary phase (I(2)). I(2) was characterized by larger current amplitude, loss of outward rectification, and alterations in the following properties: permeability among cations; ruthenium red and DPTHF sensitivity; temperature dependence; and voltage-dependent gating. The I(1) to I(2) transition depended strongly on TRPV3 current density. Removal of extracellular divalent cations resulted in heat-evoked currents resembling I(2), whereas mutation of a putative Ca(2+)-binding residue in the pore loop domain, aspartate 641, facilitated detection of the I(1) to I(2) transition, suggesting that the conversion to I(2) resulted from the agonist- and time-dependent loss of divalent cationic inhibition. Primary keratinocytes overexpressing exogenous TRPV3 also exhibited biphasic agonist-evoked currents. Thus, strong activation by either chemical or thermal stimuli led to biphasic TRPV3 signaling behavior that may be associated with changes in the channel pore.

The mammalian nervous system constantly evaluates internal and environmental temperatures to maintain homeostasis and to avoid thermal extremes. Several members of the transient receptor potential (TRP) family of ion channels have been implicated as transducers of thermal stimuli, including TRPV1 and TRPV2, which are activated by heat, and TRPM8, which is activated by cold. Here we demonstrate that another member of the TRP family, TRPV4, previously described as a hypo-osmolarity-activated ion channel, also can be activated by heat. In response to warm temperatures, TRPV4 mediates large inward currents in Xenopus oocytes and both inward currents and calcium influx into human embryonic kidney 293 cells. In both cases these responses are observed at temperatures lower than those required to activate TRPV1 and can be inhibited reversibly by ruthenium red. Heat-evoked TRPV4-mediated responses are greater in hypo-osmotic solutions and reduced in hyperosmotic solutions. Consistent with these functional properties, we observed TRPV4 immunoreactivity in anterior hypothalamic structures involved in temperature sensation and the integration of thermal and osmotic information. Together, these data implicate TRPV4 as a possible transducer of warm stimuli within the hypothalamus.

Aberrant TNF alpha expression in adipocytes is a molecular mechanism by which insulin action is modulated in adipose tissue. While this might be a compensatory response to limit adipose expansion, neither the mechanisms underlying this local effect nor its systemic biological consequences have been studied. It is also not clear whether TNF alpha-induced insulin resistance in adipocyte alone is responsible for systemic insulin resistance in the absence of obesity. In a transgenic mouse model deficient in endogenous TNF alpha, we demonstrate that specific expression of the transmembrane TNF alpha (mTNF alpha) in adipocytes leads to decreased whole body adipose mass, and local, but not systemic insulin resistance. These data demonstrate that exclusive action of TNF alpha in adipose tissue strongly inhibits insulin action at this site and leads to reduced adiposity in mice. However, this isolated adipocyte insulin resistance in the context of reduced fat mass and/or the absence of obesity is insufficient to alter systemic glucose homeostasis.