Increased activity of calcium leak channels caused by proteolysis near sarcolemmal ruptures.

Dystrophin, a 427 kD membrane-associated structural protein in muscle cells, is thought to confer strength to the myofiber sarcolemma and protect the membrane from rupture during the stresses of contraction. Dystrophin is absent in muscle cells from Duchenne muscular dystrophy (DMD) patients and mdx mice, a DMD model. Dystrophic muscle membranes undergo more frequent transient, nonlethal tears than normal cell membranes, especially during exercise. In addition, the mean open probability of a background ("leak") calcium channel is higher in dystrophic muscle cells, which leads to higher intracellular free calcium levels. Because elevated calcium levels may contribute to the eventual necrosis of muscle cells in DMD, we examined the possibility that the history of sarcolemmal rupture at a specific location on the membrane affects the open probability of nearby calcium leak channels. Membrane ruptures left by the excision of cell-attached patch-clamp electrodes were used to mimic natural tears. Patches made within 5 microns of excision sites contained channels with a fourfold greater mean open probability than channels in patches 50 microm away from ruptures. The increased leak channel activity near ruptures was seen continuously through the duration of the recordings and was not seen if the rupture was made in the presence of the protease inhibitor leupeptin. Calcium background channels proteolytically activated near ruptures, perhaps in a calcium-dependent manner, may thus be the lasting consequence of the weaker dystrophic sarcolemma, leading to chronically raised intracellular free calcium, increased calcium-dependent proteolysis and, eventually, necrosis.

Mechanical transduction by rat dorsal root ganglion neurons in vitro.

Although it is generally presumed that mechanical sensitivity of somatosensory nerve fibers results from the activation of mechanosensitive ion channels, a mechanically-gated whole-cell current has never been demonstrated in dorsal root ganglion (DRG) neurons. We performed patch clamp experiments on rat DRG neurons in culture, and report the first mechanically-activated current in somatosensory neurons (I(mech)). This whole-cell current is observed in most dorsal root ganglion neurons but not in non-sensory sympathetic ganglion neurons. The current-voltage relation of I(mech) indicates that it is a non-selective cation current. Sensitivity of I(mech) to block by gadolinium suggests that it may be mediated by a member of a family of mechanosensitive non-selective cation channels observed in many cell types. Sensitivity to benzamil supports this idea, and further suggests that the current might be mediated by a member of the degenerin/ epithelial sodium channel (DEG/ENaC) family.

Epinephrine produces a beta-adrenergic receptor-mediated mechanical hyperalgesia and in vitro sensitization of rat nociceptors.

Hyperalgesic and nociceptor sensitizing effects mediated by the beta-adrenergic receptor were evaluated in the rat. Intradermal injection of epinephrine, the major endogenous ligand for the beta-adrenergic receptor, into the dorsum of the hindpaw of the rat produced a dose-dependent mechanical hyperalgesia, quantified by the Randall-Selitto paw-withdrawal test. Epinephrine-induced hyperalgesia was attenuated significantly by intradermal pretreatment with propranolol, a beta-adrenergic receptor antagonist, but not by phentolamine, an alpha-adrenergic receptor antagonist. Epinephrine-induced hyperalgesia developed rapidly; it was statistically significant by 2 min after injection, reached a maximum effect within 5 min, and lasted 2 h. Injection of a more beta-adrenergic receptor-selective agonist, isoproterenol, also produced dose-dependent hyperalgesia, which was attenuated by propranolol but not phentolamine. Epinephrine-induced hyperalgesia was not affected by indomethacin, an inhibitor of cyclo-oxygenase, or by surgical sympathectomy. It was attenuated significantly by inhibitors of the adenosine 3',5'-cyclic monophosphate signaling pathway (the adenylyl cyclase inhibitor, SQ 22536, and the protein kinase A inhibitors, Rp-adenosine 3',5'-cyclic monophosphate and WIPTIDE), inhibitors of the protein kinase C signaling pathway (chelerythrine and bisindolylmaleimide) and a mu-opioid receptor agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Consistent with the hypothesis that epinephrine produces hyperalgesia by a direct action on primary afferent nociceptors, it was found to sensitize small-diameter dorsal root ganglion neurons in culture, i. e., to produce an increase in number of spikes and a decrease in latency to firing during a ramped depolarizing stimulus. These effects were blocked by propranolol. Furthermore epinephrine, like several other direct-acting hyperalgesic agents, caused a potentiation of tetrodotoxin-resistant sodium current, an effect that was abolished by Rp-adenosine 3',5'-cyclic monophosphate and significantly attenuated by bisindolylmaleimide. Isoproterenol also potentiated tetrodotoxin-resistant sodium current. In conclusion, epinephrine produces cutaneous mechanical hyperalgesia and sensitizes cultured dorsal root ganglion neurons in the absence of nerve injury via an action at a beta-adrenergic receptor. These effects of epinephrine are mediated by both the protein kinase A and protein kinase C second-messenger pathways.

A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice.

There is great interest in discovering new targets for pain therapy since current methods of analgesia are often only partially successful. Although protein kinase C (PKC) enhances nociceptor function, it is not known which PKC isozymes contribute. Here, we show that epinephrine-induced mechanical and thermal hyperalgesia and acetic acid-associated hyperalgesia are markedly attenuated in PKCepsilon mutant mice, but baseline nociceptive thresholds are normal. Moreover, epinephrine-, carrageenan-, and nerve growth factor- (NGF-) induced hyperalgesia in normal rats, and epinephrine-induced enhancement of tetrodotoxin-resistant Na+ current (TTX-R I(Na)) in cultured rat dorsal root ganglion (DRG) neurons, are inhibited by a PKCepsilon-selective inhibitor peptide. Our findings indicate that PKCepsilon regulates nociceptor function and suggest that PKCepsilon inhibitors could prove useful in the treatment of pain.

Nitric oxide signaling in pain and nociceptor sensitization in the rat.

We investigated the role of nitric oxide (NO) in inflammatory hyperalgesia. Coinjection of prostaglandin E2 (PGE2) with the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (L-NMA) inhibited PGE2-induced hyperalgesia. L-NMA was also able to reverse that hyperalgesia. This suggests that NO contributes to the maintenance of, as well as to the induction of, PGE2-induced hyperalgesia. Consistent with the hypothesis that the NO that contributes to PGE2-induced sensitization of primary afferents is generated in the dorsal root ganglion (DRG) neurons themselves, L-NMA also inhibited the PGE2-induced increase in tetrodotoxin-resistant sodium current in patch-clamp electrophysiological studies of small diameter DRG neurons in vitro. Although NO, the product of NOS, often activates guanylyl cyclase, we found that PGE2-induced hyperalgesia was not inhibited by coinjection of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. We then tested whether the effect of NO depended on interaction with the adenylyl cyclase-protein kinase A (PKA) pathway, which is known to mediate PGE2-induced hyperalgesia. L-NMA inhibited hyperalgesia produced by 8-bromo-cAMP (a stable membrane permeable analog of cAMP) or by forskolin (an adenylyl cyclase activator). However, L-NMA did not inhibit hyperalgesia produced by injection of the catalytic subunit of PKA. Therefore, the contribution of NO to PGE2-induced hyperalgesia may occur in the cAMP second messenger pathway at a point before the action of PKA. We next performed experiments to test whether administration of exogenous NO precursor or donor could mimic the hyperalgesic effect of endogenous NO. Intradermal injection of either the NOS substrate L-arginine or the NO donor 3-(4-morphinolinyl)-sydnonimine hydrochloride (SIN-1) produced hyperalgesia. However, this hyperalgesia differed from PGE2-induced hyperalgesia, because it was independent of the cAMP second messenger system and blocked by the guanylyl cyclase inhibitor ODQ. Therefore, although exogenous NO induces hyperalgesia, it acts by a mechanism different from that by which endogenous NO facilitates PGE2-induced hyperalgesia. Consistent with the hypothesis that these mechanisms are distinct, we found that inhibition of PGE2-induced hyperalgesia caused by L-NMA could be reversed by a low dose of the NO donor SIN-1. The following facts suggest that this dose of SIN-1 mimics a permissive effect of basal levels of NO with regard to PGE2-induced hyperalgesia: (1) this dose of SIN-1 does not produce hyperalgesia when administered alone, and (2) the effect was not blocked by ODQ. In conclusion, we have shown that low levels of NO facilitate cAMP-dependent PGE2-induced hyperalgesia, whereas higher levels of NO produce a cGMP-dependent hyperalgesia.

Lipofection of a cDNA plasmid containing the dystrophin gene lowers intracellular free calcium and calcium leak channel activity in mdx myotubes.

Muscle cells from Duchenne muscular dystrophy (DMD) patients and the dystrophic mdx mouse lack the protein dystrophin. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myofibers and cultured myotubes and is correlated with abnormally active calcium-specific leak channels. Higher [Ca2+]i results in greater calcium-dependent proteolysis, which may eventually lead to necrosis. We performed liposome-mediated transfection of a cDNA plasmid containing the full-length dystrophin gene into mdx myoblasts and examined the resting [Ca2+]i and leak channel activity of the resulting differentiated myotubes. Many myotubes from transfected cultures expressed dystrophin at levels similar to normal myotubes as determined by immunostaining. The intracellular free calcium, measured by emission ratio microfluorimetry using the calcium indicator fura-PE3, was significantly lower in the dystrophin-positive mdx myotubes than in untransfected control mdx myotubes. The mean open probability of the calcium leak channel was also reduced to a level similar to normal myotubes and significantly less than that for untransfected mdx myotubes. These results show that introduction of extrachromosomal copies of the full dystrophin gene to originally dystrophic muscle cells can correct the defect in calcium homeostasis that is hypothesized to lead to the muscle cell necrosis seen in DMD.

Validity of the laser refraction technique for meridional measurement.

This study assessed the validity of the laser refraction technique for determining the refractive status of a given meridian of the eye. A subjective-to-BVA finding was taken on a total of 75 eyes, and the laser technique was then used to refract each of the 2 principal meridians that had been located by the subjective procedure. Correlations between laser and nonlaser findings indicated that the laser technique is a valid method for meridional refraction. Differential findings for against-the-rule astigmatism are also discussed.