RNA was converted to cDNA using an iScript kit (Biorad, Hercules, CA) with 2 G total RNA in 20 L reactions with an Ependorf Mastercycler (Hamburg, Germany) using the following protocol: 5 minutes at 25C, 30 minutes at 42C and 5 minutes at 85C

RNA was converted to cDNA using an iScript kit (Biorad, Hercules, CA) with 2 G total RNA in 20 L reactions with an Ependorf Mastercycler (Hamburg, Germany) using the following protocol: 5 minutes at 25C, 30 minutes at 42C and 5 minutes at 85C. results indicate that air flow exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins symbolize a novel, peripheral target for the treatment of chronic pain and dermatological disease. Introduction Unlike most cells in the body, keratinocytes Pipequaline lie at the interface with the external environment where they form the outermost layer of the skin, the epidermis. The epidermis is a dynamic, stratified structure formed by continually proliferating and differentiating keratinocytes that surround the sensory nerve endings of several subtypes of Pipequaline C- and A-fibers [1]. These fibers play an important role in tactile sensation and nociception and express numerous ligand-gated receptors that can regulate their signaling [2], [3]. Keratinocytes have been implicated in mechano- and thermosensation, as well as peripheral pain mechanisms due to their release of molecules that activate such receptors, including -endorphin, calcitonin gene-related peptide (CGRP) and Rabbit Polyclonal to MEF2C (phospho-Ser396) ATP [4], [5], [6]. Cutaneous ATP release is an important transmission for epidermal homeostasis as well as the generation of acute and chronic pain. Signaling among keratinocytes through the release of ATP influences their proliferation and differentiation, thereby playing a major role in the creation of the stratified structure of the epidermis and maintaining epidermal homeostasis [7]. During acute tissue injury, such as cuts and abrasions, excessive ATP release from damaged keratinocytes causes pain by activating excitatory purinergic receptors on nociceptive sensory nerve endings [8], [9], [10]. Lower levels of ATP released by keratinocytes during epidermal homeostasis and in response to moderate mechanical and thermal activation may participate in normal tactile sensation and also contribute to the spontaneous pain and tactile hypersensitivity that occurs under chronic pain conditions, when nociceptive nerve endings become sensitized [11], [12]. Release of ATP from keratinocytes may also be increased during chronic pain [5]. Consistent with a contribution of epidermal ATP release to chronic pain, cutaneous administration of purinergic receptor antagonists reduces nociceptive behavior in a variety of animal models of chronic pain [13], [14], [15], [16]. Despite the importance of ATP in epidermal homeostasis, tactile sensation and nociception, little is known about the mechanisms of keratinocyte ATP Pipequaline release. Mechanical and thermal activation, low pH and hypo-osmotic activation have all been shown to result in ATP release from keratinocytes, but the relevant mechanisms were not recognized [11], [12], [17], [18]. Recently, we showed that activation of keratinocyte voltage-gated sodium channels triggers ATP release and that this mechanism appears to be up-regulated under chronic pain conditions [5]. These results may indirectly suggest vesicular release, although such a mechanism has never been exhibited in keratinocytes. Several non-vesicular ATP release mechanisms have been proposed, but many remain controversial and are complicated by the non-specificity of available inhibitors [19], [20]. Air flow exposure has also been shown to cause ATP release from cultured keratinocytes, though this release mechanism was not previously investigated [21]. Keratinocyte interactions Pipequaline with air flow may be an important transmission to trigger epidermal stratification, as Pipequaline cultured keratinocytes will not form a fully stratified epidermis unless they are brought to the air interface [22], [23]. Given the importance of keratinocyte ATP release in epidermal stratification and nociception, combined with the lack of information about keratinocyte ATP release mechanisms, the goal of the present study was to characterize air-stimulated ATP release by analyzing its time course, the.