Removing cold water from wet fur or feathers can help endothermic animals to thermoregulate. The “wet dog shakes” (WDS) behavior has been largely characterized in mammals but to a much lesser extent in birds. Although it is known that TRPM8 is the main molecular transducer of low temperature in mammals, it is not clear if wetness-induced shaking in furred and feathered animals is dependent on TRPM8. Here, we show that a novel TRPM8 agonist induces WDS in rodents, and also in birds, similar to the shaking behavior evoked by water-spraying. Furthermore, the WDS onset depends on TRPM8, as we show in water-sprayed mice. Overall, our results provide multiple evidence for a TRPM8 dependence of WDS behaviors in all tested species. These suggest that a convergent evolution selected similar shaking behaviors to expel water from fur and feathers, with TRPM8 being involved in cold wetness sensing in both mammals and birds. Shaking evoked by wet fur or plumage is reproduced by a synthetic agonist of the cold and menthol receptor TRPM8, in rats, mice, and chickens. In water sprayed mice, the onset of the shaking behavior depends on water temperature and TRPM8 expression.

Cool Esthesia airway hygiene: Stop stuffiness, cough and clear phlegm Cryosim is a molecule that produces sensations of coolness but does not affect tissue temperatures. It can be delivered as a liquid to the surfaces of the nasal cavity and throat with an immediate cooling effect. We want our airways to feel cool and clean. In the nose, the result is cool breathing and the absence of stuffiness. For the throat, it is the absence of irritation and urge to cough. For inflamed airways, the goal is to get rid of phlegm or gunk. All of this can be done with Cryosims, molecules designed to produce cool esthesia. The cooling is immediate, so there is Instant Gratification (and adherence to use). Stopping stuffiness in rhinitis has value because 10-15% of the world's population has rhinitis. Control of cough can treat upper respiratory tract infections. Symptomatic relief facilitates sleep, which has economic value in improved work productivity.

Heat impairs human learning and work output when environmental temperatures increase from 18 °C → 25+°C. The hypothesis here is that if the localized, ambient milieu around the upper eyelid margin is perceived as being ~15 °C in coolness, then the adverse effects of heat can be attenuated. This is achieved by topical wipe of a solution to the closed eyelid skin above the eyelashes. The receptor target is TRPM8, an integral membrane protein that transduces the sense of coolness/cold to the central nervous system. The wipe solution contains a TRPM8 agonist called cryosim-3 (1-diisopropylphosphorylnonane) at 1-3 mg/mL in water and designed for delivery to the eyelid margin. Other sensory systems negatively affected by heat, such as the surfaces of the nasal cavity, can also be treated with the TRPM8 agonist as an adjunct to relieve discomfort from the heat.

Transient receptor potential (TRP) channels transduce signals of chemical irritation and temperature change from the ocular surface to the brain. Dry eye disease (DED) is a multifactorial disorder wherein the eyes react to trivial stimuli with abnormal sensations, such as dryness, blurring, presence of foreign body, discomfort, irritation, and pain. There is increasing evidence of TRP channel dysfunction (i.e., TRPV1 and TRPM8) in DED pathophysiology. Here, we review some of this literature and discuss one strategy on how to manage DED using a TRPM8 agonist.

The carotid body (CB) detects the arterial partial pressure of O2 and CO2. Previous studies suggested that nicotinic ACh receptors (nAChRs) play a role in these processes. We investigated if deletion of the nAChR α3 subunit influences gene expression, morphology and function of the CB in 2 week old mice. Real‐time PCR analysis was performed on CB from wild type (WT), heterozygous (HET), and knockout (KO) mice. Cholinergic traits (enzymes for ACh metabolism and receptors) were well preserved in all mice, except for the α3 subunit in KO mice. Dopaminergic traits and BK channels were down regulated in KO mice. Morphometry studies were performed in plastic embedded and serial‐sectioned CBs. The volumes and cell morphology between WT and HET CBs were similar. CB function was assessed by the ventilatory response to short exposures of 100% O2 and 15% O2 using whole body plethysmography. Both WT and HET have similar ventilatory responses to hyperoxia and hypoxia, hypoventilating in response to hyperoxia and hyperventilating in response to hypoxia. The results suggest that the α3 subunit is linked to the expression of BK channels and dopaminergic traits in the CB. Similarities between the phenotypes of WT and HET mice may be due to compensation occurring in the HET.Supported by HL72293. Transgenic mice were originally provided by Dr. De Biasi, Baylor College of Medicine.

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin-1 receptor (MC-1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melanocytes in culture. Much lower levels of alpha-MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and alpha-MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone convertases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and alpha-MSH, and this was enhanced in response to corticotrophin-releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably transfected with the MC-1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, alpha-MSH, and beta-MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC-1R highlight the importance of POMC processing as a key regulatory event in the skin.

Human skin is a local source of corticotropin-releasing hormone (CRH) and expresses CRH and CRH receptors (CRH-R) at mRNA and protein levels. Epidermal melanocytes respond to CRH by induction of cAMP with up-regulation of pro-opiomelanocortin gene expression and subsequent production of adrenocorticotropin hormone. However, the role of CRH/CRH-R in melanocyte biology is complicated by the significant heterogeneity of cutaneous melanocyte subpopulations, from continuously active and UV-responsive melanocytes in epidermis to UV nonresponsive, hair growth cycle-coupled melanogenesis in hair follicles. In the present study we report that normal human scalp hair follicle melanocytes express CRH at the mRNA level. Furthermore, CRH, urocortin and CRH-R 1 and 2 were differentially expressed in follicular melanocytes, fibroblasts, and keratinocytes depending on anatomic location and differentiation status in situ and in vitro. Stimulation of follicular melanocytes with CRH and CRH peptides, modified for selectivity for CRH-R1 and/or CRH-R2, variably induced cell melanogenesis, dendricity, and proliferation. CRH-peptides also stimulated the expression and activity of Tyrosinase, and expression of Tyrosinase-related protein-1 and-2. However, a modified urocortin peptide highly selective for CRH-R2 down-regulated melanocyte differentiation phenotype. This study indicates that CRH peptides can differentially influence hair follicle melanocyte behavior not only via CRH-R1 signaling but also by complex cross-talk between CRH-R1 and CRH-R2.

Background Corticotropin-releasing hormone (CRH) is proposed to be involved in the regulation of the proliferative capacity of keratinocytes, based on its significant actions in the skin. These are mediated by CRH-R1 alpha and represented by adenylate cyclase activation, Ca2+ influx, inhibition of cell proliferation and modifications in intracellular signal transduction by NF-kappa B.Objectives To define CRH action in the cell cycle we investigated its effects on the differentiation programme using the HaCaT keratinocytes model.Methods HaCaT keratinocytes were incubated with CRH in Dulbecco's modified Eagles's medium (containing 1.8 mmol L-1 calcium) or EpiLife (containing 0.06 mmol L-1 calcium) medium. Cell proliferation was assessed with the MTT assay. Flow cytometry was used for the measurement of DNA content, cell size and granularity and the expression of cytokeratin 14, cytokeratin 1 and involucrin. The electrophoretic mobility shift assay was used to determine DNA binding activity by AP-1 transcription factor. Expression of cytokeratin 1 was also assessed with immunofluorescence microscopy.Results CRH did produce inhibition of proliferation, which was dose-dependent; the shape of the inhibition curve was determined by the media calcium concentration. CRH action was pinpointed at inhibition of the G0/1 to the S phase transition of the cell cycle. CRH also increased AP-1 binding activity, cell granularity, cytokeratin 1 and involucrin expression, and inhibited cytokeratin 14 expression.Conclusions These results are consistent with CRH induction of the keratinocyte differentiation programme. Thus, the overall CRH cutaneous actions connote protective functions for the epidermis, that appear to include the triggering or acceleration of the differentiation programme.

Injury to soft tissue results in the lowering of interstitial fluid pressure (P(if)), plasma protein extravasation, and increased total tissue volume. In this study, the effects of N-acetyl neurotensin(8-13) [AcNT(8-13)] on P(if) in rat trachea were examined after electrical stimulation (ES) of the vagus nerve. P(if) was measured with glass capillaries connected to a servocontrolled counterpressure system. In pentobarbital-anesthetized female Wistar rats, the P(if) after intravenous saline was -1.8 +/- 0.3 mmHg (means +/- SD) and decreased to -5.0 +/- 0.6 mmHg (P < 0.01, n = 9) after ES. AcNT(8-13) (10 microg/kg) blocked the fall in P(if) after ES (-2.5 +/- 2.3 mmHg, P < 0.01, n = 8). In tracheal tissue from animals pretreated with AcNT(8-13) at the same dose and immersed in phosphate-buffered saline (0.15 M, pH 7.4), the rate of fluid accumulation in excised tissues was significantly reduced after 2 h. The ability of AcNT(8-13) to modulate the fluid mechanics of tracheal interstitium after inflammation suggests that it may be a useful tool for studying cell adhesion and related factors that maintain structural integrity of connective tissue after injury.

Hyaluronan and its associated water of hydration are the basis of the swelling and edema of acute inflammation. Mystixins are small, synthetic peptides that suppress the acute inflammatory response. Mystixin-7, a prototype of these peptides, has the structure p-anisoyl-Arg-Lys-Leu-Leu-d-Thi-Ile-d-Leu-NH2. As shown previously by this laboratory, the mystixin-7 peptide inhibits edema formation in vivo following intravenous administration at doses of less than 1.0 mg/kg. Mechanisms by which this peptide might suppress edema were examined here in vitro using cultured cells. Normal human dermal fibroblasts normally secrete large quantities of hyaluronan in response to inflammatory stimuli. Mystixin-7 reduced hyaluronan deposition by up to 80% in such cultures. Stimulation of hyaluronidase activity was observed. Mystixins represent a novel class of anti-inflammatory peptides that suppress the edema associated with inflammation. We propose that stimulation of hyaluronidase activity, with a decrease in net hyaluronan deposition and its associated water of hydration, is among the mechanisms of the anti-inflammatory effect of mystixin peptides.

Soft tissue injury is accompanied by lowering of interstitial fluid pressure (P(if)), plasma protein extravasation, and edema. Inflammation was produced by electrical stimulation (ES) of the vagus and the effects of the synthetic peptide mystixin-7 (p-anisoyl-Arg-Lys-Leu-Leu-D-Thi-Ile-D-Leu-NH(2)) on P(if) were examined. Micropuncture measurement of P(if) in submucosa, without opening the trachea, was conducted on rats anesthetized with pentobarbital sodium (50 mg/kg) and euthanized with intravenous KCl. P(if) in control (intravenous saline) was -1.2 +/- 0.7 mmHg before ES and decreased to -4.7 +/- 1.0 mmHg (P < 0.01, n = 8) after ES. Mystixin-7 (10 and 20 microg/kg iv) blocked the fall in P(if) after ES (-1.1 +/- 0.3 and -0.8 +/- 0.2 mmHg, P < 0.01, n = 8 and n = 4). The 1 microg/kg dose was without effect. When trachea from animals pretreated with mystixin-7 (20 microg/kg iv) were soaked in phosphate-buffered saline (0.15 M, pH 7.4), the rate of fluid accumulation was significantly reduced. This study suggests that mystixin peptides, which have structural similarity to a fragment from laminin-alpha1 chain, may be useful tools for studying cell adhesion and factors that maintain the structural integrity of connective tissue after injury.

Following previous findings in human skin of the functional expression of genes for the corticotropin releasing hormone (CHR) receptor type 1 (CRH-R1) and CRH itself, we searched for local phenotypic effects for peptides related to CRH. We now report that CRH, sauvagine, and urocortin inhibit proliferation of human HaCaT keratinocytes in a dose-dependent manner. The peptides produced variable cyclic adenosine 3′∶5′-monophosphate stimulation with CRH having the highest potency. Binding of iodine 125 CRH to intact keratinocytes was inhibited by increasing doses of CRH, sauvagine, or urocortin, all showing equal inhibitory potency. Immunocytochemistry identified CRH-R1 immunoreactivity in HaCaT keratinocytes. In conclusion, CRH (exogenous or produced locally) and the related urocortin and sauvagine peptides can modify human keratinocyte phenotype through a receptor-mediated pathway.

ABSTRACT: The classical neuroendocrine pathway for response to systemic stress is by hypothalamic release of corticotropin releasing hormone (CRH), subsequent activation of pituitary CRH receptors (CRH‐R), and production and release of proopiomelanocortin (POMC) derived peptides. It has been proposed that an equivalent to the hypothalamic‐pituitary‐adrenal axis functions in mammalian skin, in response to local stress (see Reference 1 ). To further define such system we used immunocytochemistry, RP‐HPLC separation, and RIA techniques, in rodent and human skin, and in cultured normal and malignant melanocytes and keratinocytes. Production of mRNA for CRH‐R1 was documented in mouse and human skin using RT‐PCR and Northern blot techniques; CRH binding sites and CRH‐R1 protein were also identified. Addition of CRH to immortalized human keratinocytes, and to rodent and human melanoma cells induced rapid, specific, and dose‐dependent increases in intracellular Ca 2+ . The latter were inhibited by the CRH antagonist α‐helical‐CRH(9–41) and by the depletion of extracellular calcium with EGTA. CRH production was enhanced by ultraviolet light radiation and forskolin (a stimulator for intracellular cAMP production), and inhibited by dexamethasone. Thus, evidence that skin cells, both produce CRH and express functional CRH‐R1, supports the existence of a local CRH/CRH‐R neuroendocrine pathway that may be activated within the context of a skin stress response system .