Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

Abstract

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air 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 represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.

Phosphoinositide signaling in somatosensory neurons

“…Keratinocytes for example may release ATP via connexin hemichannels (Barr et al, 2013), and heat activation of TRPV3 in keratinocytes was proposed to transmit information via ATP release (Mandadi et al, 2009), a notion consistent with reduced non-noxious warmth sensitivity of P2X3 −/− mice (Souslova et al, 2000). ATP can also be released from damaged cells in a non-specific manner, and P2X3 −/− mice showed reduced responses to formalin-induced pain.…”

Mechanosensitive ATP release from hemichannels and Ca2+ influx through TRPC6 accelerate wound closure in keratinocytes

“…These results indicated that the stretch-induced mechanosensitive ATP release occurs through hemichannels. Recent studies have demonstrated pharmacologically that when keratinocytes are exposed to air, it results in ATP release through connexin hemichannels (Barr et al, 2013), and when keratinocytes, including HaCaTs, are exposed to hapten, it results in ATP release through pannexin 1 (Onami et al, 2014).…”

Sensitization of cutaneous neuronal purinergic receptors contributes to endothelin-1-induced mechanical hypersensitivity

“…ET A receptor-expressing nociceptive neurites terminate in the epidermis where they are surrounded by keratinocytes. Keratinocytes release a variety of pro-algesic molecules that activate excitatory receptors on nociceptive nerve endings, including glutamate, CGRP and ATP [2; 11; 15; 20; 33]. In particular, cutaneous ATP release has been implicated in numerous types of acute and chronic pain [5; 9].…”