Neurobehavioral and neurophysiological actions of the peptide endothelin-1 (ET-1) were investigated after subcutaneous plantar hindpaw injections in adult male Sprague Dawley rats. Hindpaw flinching developed within minutes after ET-1 (8-16 nmol) injection, peaked at 30 min, lasted for 60 min, and was strongly inhibited by the endothelin-A (ET A ) receptor antagonist, BQ-123 (3.2 M). In separate experiments, impulse activity of single, physiologically characterized sensory C-, A␦-, and A␤-fibers was recorded from the sciatic nerve in anesthetized rats after subcutaneous injections of endothelin-1 (1-20 nmol), alone or together with BQ-123 (3.2 M), into the plantar hindpaw receptive fields of these units. All nociceptive C-fibers (31 of 33 C-fibers studied) were excited by ET-1 (1-20 nmol) in a dosedependent manner. For doses of 16-20 nmol, the mean latency for afferent activation after injection of ET-1 was 3.16 Ϯ 0.31 min, and the mean and maximum response frequency were 2.02 Ϯ 0.48 impulses (imp)/sec and 14.0 Ϯ 3.2 imp/sec, respectively. All 10 nociceptive A␦-fibers (of 12 A␦-fibers studied) also responded to 1-20 nmol of ET-1 in a dose-dependent manner with a mean latency of 3.5 Ϯ 0.12 min and mean response frequency of 3.3 Ϯ 2.3 imp/sec. In contrast, most A␤-fibers (9 of 12) did not respond to ET-1. BQ-123, when coinjected with ET-1, blocked ET-1-induced activation in all Cand A␦-fibers tested. These data demonstrate that subcutaneous administration of ET-1 to the rat plantar hindpaw produces pain-like behavior and selective excitation of nociceptive fibers through activation of ET A receptors. Key words: excitability; peripheral nerve; algogenic; C-fiber; nociceptor; cancerPain is a frequent and disabling consequence of metastatic prostate and breast cancer in humans. The cause of this pain is unknown but may involve mediator-dependent signaling by tumor cells to spinal nerve roots. One candidate mediator, the potent vasoconstrictive peptide and mitogen endothelin-1 (ET-1), is secreted in high concentrations by metastatic prostate and breast cancer cells and is known to induce pain-like behavior in animals and pain in humans (Ferreira et al

The influence of glucocorticoids on the expression and activity of the transcription factors CCAAT/enhancer binding protein (C/EBP)beta and delta in skeletal muscle was examined by treating rats or cultured L6 myotubes with dexamethasone. Treatment of rats with 10 mg/kg of dexamethasone resulted in increased C/EBPbeta and delta DNA binding activity in the extensor digitorum longus muscle as determined by electrophoretic mobility shift assay (EMSA) and supershift analysis. A similar response was noticed in dexamethasone-treated myotubes. In other experiments, myocytes were transfected with a plasmid containing a promoter construct consisting of multiple C/EBP binding elements upstream of a luciferase reporter gene. Treatment of these cells with dexamethasone resulted in a fourfold increase in luciferase activity, suggesting that glucocorticoids increase C/EBP-dependent gene activation in muscle cells. In addition, dexamethasone upregulated the protein and gene expression of C/EBPbeta and delta in the myotubes in a time- and dose-dependent fashion as determined by Western blotting and real-time PCR, respectively. The results suggest that glucocorticoids increase C/EBPbeta and delta activity and expression through a direct effect in skeletal muscle.

We examined whether endothelin-1 (ET-1), a potent vasoconstrictive peptide secreted in high concentration by metastatic prostate cancer cells, produces endothelin receptor-dependent pain behavior when applied to rat sciatic nerve. ET-1 (200-800 microM) applied to the epineurial surface of rat sciatic nerve produced reliable, robust, unilateral hindpaw flinching lasting 60 min. Pre-emptive systemic morphine completely blocked this effect in a naloxone-reversible manner, suggesting that this behavior was pain-related. Equipotent doses of epineurially applied epinephrine had no effect, suggesting that ET-1 effects are on tissue sites other than sciatic nerve microvessels. Prior and co-administration of BQ-123, an endothelin-A (ET(A)) receptor antagonist, also blocked ET-1-induced hindpaw flinching establishing that pain behavior induced by ET-1 application to rat sciatic nerve is ET(A) receptor mediated.

-Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting. ubiquitin ligases; calpains MUSCLE WASTING DURING SEPSIS is mainly caused by an increase in protein breakdown, in particular, breakdown of the myofibrillar proteins actin and myosin (17,36). Previous studies from our and other laboratories suggest that muscle proteolysis in sepsis and a number of other catabolic conditions, such as burn injury, cancer, and uremia, reflects ubiquitin-proteasomedependent proteolysis (11,13,17,36,44). Proteins degraded by this mechanism are first conjugated to multiple molecules of ubiquitin followed by degradation by the 26S proteasome (22). Ubiquitination of the protein substrates is regulated by multiple enzymes, including the ubiquitin-activating enzyme E1, ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). Among these enzymes, the E3s are particularly important because they account for substrate specificity in the system. In recent studies, the expression of two newly discovered musclespecific ubiquitin ligases, atrogin-1/MAFbx and MuRF1, was substantially increased in skeletal muscle during various muscle-wasting conditions, including sepsis (5, 15, 45), and increased mRNA levels for atrogin-1/MAFbx and MuRF1 have been suggested to be reliable molecular markers for muscle atrophy (28).Although ubiquitin-proteasome-dependent protein degradation plays an important ...

, and Per-Olof Hasselgren. Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3. Am J Physiol Regul Integr Comp Physiol 288: R580 -R590, 2005. First published November 24, 2004 doi:10.1152/ajpregu.00341.2004.-We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of -and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.

We showed previously that subcutaneous injection of the injury-associated peptide mediator endothelin-1 (ET-1) into the rat plantar hindpaw produces pain behavior and selective excitation of nociceptors, both through activation of ET(A) receptors likely on nociceptive terminals. The potential role of ET(B) receptor activation in these actions of ET-1-has not been examined. Therefore, in these experiments, we studied the effect of blocking or activating ET(B) receptors on ET-1-induced hindpaw flinching and excitation of nociceptors in rats. An ET(B) receptor-selective antagonist, BQ-788 (3 mm), coinjected with ET-1 (200 microm) reduced the time-to-peak of flinching and significantly enhanced the average maximal flinch frequency (MFF). In contrast, coinjection of an ET(B) receptor selective agonist, IRL-1620 (100 or 200 microm), with ET-1 reduced the average MFF and the average total number of flinches. Interestingly, this unexpected inhibitory effect of IRL-1620 was prevented by the nonselective opioid receptor antagonist naloxone (2.75 mm). To confirm these inhibitory actions, we studied the effects of IRL-1620 on ET-1-induced spike responses in single, physiologically characterized nociceptive C-fibers. IRL-1620 suppressed spike responses to ET-1 in all (n = 12) C-units, with mean and maximum response frequencies of 0.08 +/- 0.02 and 1.5 +/- 0.4 impulses/sec versus 0.32 +/- 0.07 and 4.17 +/- 0.17 impulses/sec for ET-1 alone. In additional support of the behavioral results, coinjection of naloxone (2.75 mm) completely prevented this inhibitory action of IRL-1620. These results establish that ET(B) receptor activation inhibits ET-1-induced pain behavior and nociception in a naloxone-sensitive manner and point to a previously unrecognized dual modulation of acute nociceptive signaling by ET(A) and ET(B) receptors in cutaneous tissues.

Muscle wasting in various catabolic conditions is, at least in part, regulated by glucocorticoids. The role of glucocorticoids in muscle wasting is complex and reflects regulation at the molecular level of multiple mechanisms influencing both synthesis and degradation of muscle proteins.

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