Travis P. Barr

Keratinocytes are implicated in sensory transduction and can influence nociception, but whether these contribute to chronic pain is not known. In neurons, voltage-gated sodium channels (Na(v)) are involved in neuropathic pain and are activated by depolarization. Since keratinocytes can also show changes in membrane potential, we used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the expression of sodium channels in these cells. Na(v)1.1, Na(v)1.6, and Na(v)1.8 were localized within keratinocytes in rat epidermis. In addition, sodium channels contribute to the release of ATP from rat keratinocytes in response to increased [K(+)](o), implicating sodium channels in keratinocyte ligand release and nociception. To examine whether keratinocytes may contribute to human pain states, we analyzed sodium channel expression in human skin biopsies from subjects with complex regional pain syndrome Type 1 (CRPS) and post-herpetic neuralgia (PHN) using immunohistochemistry. Control skin exhibited immunolabeling for Na(v)1.5, Na(v)1.6 and Na(v)1.7. In contrast, painful skin from CRPS and PHN subjects displayed Na(v)1.1, Na(v)1.2, and Na(v)1.8 immunolabeling, in addition to substantially increased signal for Na(v)1.5, Na(v)1.6, Na(v)1.7. These observations lead us to propose that pathological increases in keratinocyte sodium channel expression may contribute to pain by increasing epidermal ATP release, resulting in excessive activation of P2X receptors on primary sensory axons. Consistent with this hypothesis, animal models of neuropathic pain exhibit increases in subcutaneous ATP release and activity of primary sensory neurons, and peripheral administration of P2X antagonists has been shown to reduce neuropathic pain in humans.

Calcitonin gene-related peptide (CGRP) is a vasodilatory peptide that has been detected at high levels in the skin, blood, and cerebrospinal fluid (CSF) under a variety of inflammatory and chronic pain conditions, presumably derived from peptidergic C and Aδ innervation. Herein, CGRP immunolabeling (IL) was detected in epidermal keratinocytes at levels that were especially high and widespread in the skin of humans from locations afflicted with postherpetic neuralgia (PHN) and complex region pain syndrome type 1 (CRPS), of monkeys infected with simian immunodeficiency virus, and of rats subjected to L5/L6 spinal nerve ligation, sciatic nerve chronic constriction, and subcutaneous injection of complete Freund's adjuvant. Increased CGRP-IL was also detected in epidermal keratinocytes of transgenic mice with keratin-14 promoter driven overexpression of noggin, an antagonist to BMP-4 signaling. Transcriptome microarray, quantitative Polymerase Chain Reaction (qPCR), and Western blot analyses using laser-captured mouse epidermis from transgenics, monolayer cultures of human and mouse keratinocytes, and multilayer human keratinocyte organotypic cultures, revealed that keratinocytes express predominantly the beta isoform of CGRP. Cutaneous peptidergic innervation has been shown to express predominantly the alpha isoform of CGRP. Keratinocytes also express the cognate CGRP receptor components, Calcitonin receptor-like receptor (CRLR), Receptor activity-modifying protein 1 (RAMP1), CGRP-receptor component protein (RCP) consistent with known observations that CGRP promotes several functional changes in keratinocytes, including proliferation and cytokine production. Our results indicate that keratinocyte-derived CGRPβ may modulate epidermal homeostasis through autocrine/paracrine signaling and may contribute to chronic pain under pathological conditions.

OBJECTIVE: Pepducins are membrane-tethered, cell-penetrating lipopeptides that target the cytoplasmic surface of their cognate receptor. Here, we report the first human use of a protease-activated receptor-1-based pepducin, which is intended as an antiplatelet agent to prevent ischemic complications of percutaneous coronary interventions. APPROACH AND RESULTS: PZ-128 was administered by 1 to 2 hours continuous intravenous infusion (0.01-2 mg/kg) to 31 subjects with coronary artery disease or multiple coronary artery disease risk factors. Safety, antiplatelet efficacy, and pharmacokinetics were assessed at baseline and 0.5, 1, 2, 6, 24 hours, and 7 to 10 days postdosing. The inhibitory effects of PZ-128 on platelet aggregation stimulated by the protease-activated receptor-1 agonist SFLLRN (8 μmol/L) at 30 minutes to 6 hours were dose dependent with 20% to 40% inhibition at 0.3 mg/kg, 40% to 60% at 0.5 mg/kg, and ≥ 80% to 100% at 1 to 2 mg/kg. The subgroup receiving aspirin in the 0.5 and 1-mg/kg dose cohorts had 65% to 100% inhibition of final aggregation to SFLLRN at 30 minutes to 2 hours and 95% to 100% inhibition by 6 hours. The inhibitory effects of 0.5 mg/kg PZ-128 were reversible with 50% recovery of aggregation to SFLLRN by 24 hours. There were no significant effects of PZ-128 on aggregation induced by AYPGKF, ADP, or collagen, indicating that the observed effects were specific to protease-activated receptor-1. The plasma half-life was 1.3 to 1.8 hours, and PZ-128 was nondetectable in urine. There were no effects on bleeding, coagulation, clinical chemistry, or ECG parameters. CONCLUSIONS: PZ-128 is a promising antiplatelet agent that provides rapid, specific, dose dependent, and reversible inhibition of platelet protease-activated receptor-1 through a novel intracellular mechanism. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01806077.

Reception and interpretation of environmental stimuli is critical for the survival of all organisms. Here, we show that the ablation of BBS1 and BBS4, two genes mutated in Bardet-Biedl syndrome and that encode proteins that localize near the centrioles of sensory neurons, leads to alterations of s.c. sensory innervation and trafficking of the thermosensory channel TRPV1 and the mechanosensory channel STOML3, with concomitant defects in peripheral thermosensation and mechanosensation. The thermosensory phenotype is recapitulated in Caenorhabditis elegans, because BBS mutants manifest deficient thermosensory responses at both physiological and nociceptive temperatures and defective trafficking of OSM-9, a polymodal sensory channel protein and a functional homolog of TRPV1 or TRPV4. Our findings suggest a hitherto unrecognized, but essential, role for mammalian basal body proteins in the acquisition of mechano- and thermosensory stimuli and highlight potentially clinical features of ciliopathies in humans.