BackgroundStress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the β2-adrenergic receptor (β2AR), another study objective was to determine whether β2AR antagonists could block epinephrine effects on healing and improve wound repair.Methods and FindingsMigratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the β2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver β2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%–95% in humans, p < 0.001, and by 36%, 95% CI 24%–49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%–36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by β2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the β2AR, and is reproduced by incubation of keratinocytes with other β2AR-specific agonists. Activation of the β2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein ± standard error of the mean: unburned control, 0.6 ± 0.36; immediately postburn, 9.6 ± 1.58; 2 h postburn, 3.1 ± 1.08; 24 h post-burn, 6.7 ± 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with βAR antagonists results in a significant increase (44%, 95% CI 27%–61%, p < 0.00000001) in the rate of burn wound re-epithe...

We tested the hypothesis that eccentric contractions activate mechanosensitive or stretch-activated ion channels (SAC) in skeletal muscles, producing increased cation conductance. Resting membrane potentials and contractile function were measured in rat tibialis anterior muscles after single or multiple exposures to a series of eccentric contractions. Each exposure produced a significant and prolonged (>24 h) membrane depolarization in exercised muscle fibers. The magnitude and duration of the depolarization were related to the number of contractions. Membrane depolarization was due primarily to an increase in Na(+) influx, because the estimated Na(+)-to-K(+) permeability ratio was increased in exercised muscles and resting membrane potentials could be partially repolarized by substituting an impermeant cation for extracellular Na(+) concentration. Neither the Na(+)/H(+) antiport inhibitor amiloride nor the fast Na(+) channel blocker TTX had a significant effect on the depolarization. In contrast, addition of either of two nonselective SAC inhibitors, streptomycin or Gd(3+), produced significant membrane repolarization. The results suggest that muscle fibers experience prolonged depolarization after eccentric contractions due, principally, to the activation of Na(+)-selective SAC.

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The previous demonstration of an increase and redistribution of adenylate cyclase activity in injured peripheral nerve suggests that an increase in neuronal cyclic AMP concentration could play a role in peripheral nerve regeneration. We report our finding that accumulating adenylate cyclase activity was translated into a twofold increase in cyclic AMP concentration in the regenerating nerve stump, coincident with the initiation and elongation of regenerative nerve sprouts. We sought to magnify the role of cyclic AMP in regeneration by using forskolin, a robust activator of adenylate cyclase, to produce an additional increase in neuronal cyclic AMP in situ. Forskolin in vitro produced an approximately 40-fold greater elevation in neuronal cyclic AMP than an equimolar (10(-5] concentration of isoprenaline. Moreover, the elevated cyclic AMP concentration persisted for at least 60 min in the continued presence of forskolin. Daily injection of forskolin into the dorsal lymph sac of Rana pipiens, or delivery of forskolin through an implanted osmotic pump produced a sustained 40% increase in the rate of sensory nerve regeneration in freeze-lesioned sciatic nerves. We conclude that an increase in cyclic AMP concentration and, presumably, the activation of appropriate protein kinases stimulates regenerative nerve growth following trauma.

Gated31P-nuclear magnetic resonance followed the metabolic fluctuation in rat gastrocnemius muscle during a contraction cycle. Within 16 ms after stimulation, the phosphocreatine (PCr) level drops 11.3% from its reference state. The PCr minimum corresponds closely to the time of maximum force contraction. Pi increases stoichiometrically, while ATP remains constant. During a twitch, PCr hydrolysis produces 3.1 μmol ATP/g tissue, which is substantially higher than the reported 0.3 μmol ATP ⋅ twitch−1 ⋅ g tissue−1 derived from steady-state experiments. The results reveal that a substantial energy fluctuation accompanies a muscle twitch.