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.

Chronic alcohol consumption produces a painful peripheral neuropathy for which there is no reliably successful therapy, attributable to, in great part, a lack of understanding of the underlying mechanisms. We tested the hypothesis that neuropathic pain associated with chronic alcohol consumption is a result of abnormal peripheral nociceptor function. In rats maintained on a diet to simulate chronic alcohol consumption in humans, mechanical hyperalgesia was present by the fourth week and maximal at 10 weeks. Thermal hyperalgesia and mechanical allodynia were also present. Mechanical threshold of C-fibers in ethanol fed rats was lowered, and the number of action potentials during sustained stimulation increased. The hyperalgesia was acutely attenuated by intradermal injection of nonselective protein kinase C (PKC) or selective PKC⑀ inhibitors injected at the site of nociceptive testing. Western immunoblot analysis indicated a higher level of PKC⑀ in dorsal root ganglia from alcohol-fed rats, supporting a role for enhanced PKC⑀ secondmessenger signaling in nociceptors contributing to alcohol-induced hyperalgesia.Key words: protein kinase C ⑀; alcoholic peripheral neuropathy; pain; hyperalgesia; allodynia; primary afferent nociceptor Ethanol consumption is the most common cause of peripheral nervous system, as well as CNS, neurotoxicity. Ethanol is thought to exert a direct neurotoxic action on the peripheral nervous system, resulting in a neuropathy that mostly involves smalldiameter fibers (Diamond and Messing, 1994;Monforte et al., 1995;Kielhorn, 1996;Ortiz-Plata et al., 1998;Tredici et al., 1999). The peripheral neuropathy is a potentially incapacitating complication of chronic consumption of ethanol, characterized by pain and dysesthesias, primarily in the lower extremities, and is poorly relieved by available therapies (Ratcliff, 1979;Monforte et al., 1995;Ortiz-Plata et al., 1998).Whereas enhanced nociception and primary afferent nociceptor hypersensitivity have been demonstrated in animal models of other neuropathic pain states, such as those induced by diabetes (Ahlgren and Levine, 1994), chemotherapy (Tanner et al., 1998; Authier et al., 1999), or trauma (Bennett andXie, 1988;Campbell et al., 1988;Seltzer et al., 1990;Xie and Xiao, 1990;Kim and Chung, 1992;Kim et al., 1993;Sheen and Chung, 1993;Yoon et al., 1996;Pedersen and Kehlet, 1998;Zahn and Brennan, 1999), an animal model for alcohol-induced neuropathy does not exist, nor has it even been demonstrated that primary afferent nociceptor function is altered by chronic exposure to alcohol.In animal models of other painful peripheral neuropathies, enhanced nociception involves alterations in intracellular signaling. Specifically, protein kinase C (PKC) (Ahlgren and Levine, 1994) [particularly the epsilon (⑀) isoform (Gerstin et al., 1998; Khasar et al., 1999] and protein kinase A (PKA) (Ahlgren and Levine, 1994) signaling pathways have been implicated in enhancing nociceptor function. Because alcohol has been shown to activate PKC and PKA (Coe et al., 1...

Inflammatory pain, characterized by a decrease in mechanical nociceptive threshold (hyperalgesia), arises through actions of inflammatory mediators, many of which sensitize primary afferent nociceptors via G-protein-coupled receptors. Two signaling pathways, one involving protein kinase A (PKA) and one involving the epsilon isozyme of protein kinase C (PKCepsilon), have been implicated in primary afferent nociceptor sensitization. Here we describe a third, independent pathway that involves activation of extracellular signal-regulated kinases (ERKs) 1 and 2. Epinephrine, which induces hyperalgesia by direct action at beta(2)-adrenergic receptors on primary afferent nociceptors, stimulated phosphorylation of ERK1/2 in cultured rat dorsal root ganglion cells. This was inhibited by a beta(2)-adrenergic receptor blocker and by an inhibitor of mitogen and extracellular signal-regulated kinase kinase (MEK), which phosphorylates and activates ERK1/2. Inhibitors of G(i/o)-proteins, Ras farnesyltransferases, and MEK decreased epinephrine-induced hyper-algesia. In a similar manner, phosphorylation of ERK1/2 was also decreased by these inhibitors. Local injection of dominant active MEK produced hyperalgesia that was unaffected by PKA or PKCepsilon inhibitors. Conversely, hyperalgesia produced by agents that activate PKA or PKCepsilon was unaffected by MEK inhibitors. We conclude that a Ras-MEK-ERK1/2 cascade acts independent of PKA or PKCepsilon as a novel signaling pathway for the production of inflammatory pain. This pathway may present a target for a new class of analgesic agents.

Site-directed mutagenesis is an essential tool for the study of structure-function relationships. A variety of methods have been developed for modifying specific amino acids at predetermined sites in proteins. We present here an improvement of the method based on homologous recombination described by Jones and Howard (1) which not only largely diminishes the background of non-mutated clones but also greatly simplifies the whole mutagenesis procedure. The method combines both polymerase chain reaction (PCR) technology and homologous recombination. Briefly, an entire circular plasmid is amplified by PCR using mutagenic primers with overlapping sequences (25-base overlap). The linear fragment thus obtained bears homologous ends. After separation from the circular template by electrophoresis on an agarose gel, the linear fragment is transformed into competent DH5a E.coli cells. Homologous recombination occurs in the bacteria and yields clones harbouring a mutated circular plasmid. We have applied this technique with success to a pUC19 plasmid containing the sequence coding for mature BDNF (brain-derived neurotrophic factor). The size of the plasmid is 3.1 kilobases (kb), and 100% of mutated clones were obtained. However when the same strategy was applied to a pUC19 plasmid containing the prepro-BDNF sequence (ppBDpUC3), which is 3.6 kb long, the efficiency of mutagenesis dropped to 20-25%. This decrease in efficiency was explained by the difficulty of separating linear from supercoiled DNA above a certain size. Since the transformation efficiency is much higher with supercoiled than with linear plasmid, the low amount of contaminating supercoiled template plasmid yields a high background of non-mutated plasmid after transformation. To circumvent this problem we decreased the amount of template plasmid in the PCR reaction. An appropriate ratio between linear mutated PCR product and circular wild-type template is thus obtained and allows direct transformation into bacteria. The primers BDXHOA 5'-CTC GAG GAC TGT GAC CGT CCC GCC AGA CAT GTC CAC TGC-3' and BDXHO1 5'-TGG CGG GAC GGT CAC AGT CCT CGA GAA AGT CCC GGT ATC C-3' were designed to create a silent XhoI restriction site in the mouse BDNF sequence at position 582-587 in the sequence available in GenBank under the accession number X55573. The NI -; Figure 1. Effect of decreasing amounts of template on PCR yield. Electrophoresis was performed on a 1% agarose gel stained with ethidium bromide at 0.5 jg/ml. M: k HindIIllEcoRI marker (Appligene). Lanes 1-3: 10 .l of PCR amplification performed respectively with 1 ng, 0.1 ng, 0.01 ng of the 3.6 kb-long ppBDpUC3 plasmid.mutagenic nucleotides are in italic bold characters and the overlapping regions are underlined. Amplification of full-length plasmid was performed in a 100 ,l incubation volume containing 1 x buffer (10 mM Tris-HCI pH 9; 50 mM KCI; 1.5 mM MgCl2; 0.1% Triton X-100; 0.2 mg/ml BSA or gelatin), 200 ,uM of each deoxynucleoside triphosphate, 1 FM of each primer and 2.5 U of Taq polymerase (Appligene) using a Per...

The tumour-associated D294G mutant of protein kinase Calpha (PKCalpha) was recently shown not to be translocated to the plasma membrane on stimulation with PMA, in contrast with the wild-type enzyme. Using recombinant wild-type and mutant PKCalpha, we establish here that, although the PKCalpha intrinsic lipid-dependent catalytic activity remains unaltered by the D294G mutation, the mutant enzyme exhibits a selective loss of substrate recognition. Indeed, whereas the mutant enzyme is still able to phosphorylate histone IIIS with comparable efficiency to that of the wild-type enzyme, it exhibits a lack of kinase activity towards the previously cloned 35F and 35H substrates for PKC. Overlay experiments demonstrate that this selective loss of kinase activity is correlated with a decrease in binding of D294G PKCalpha to the 35F and 35H proteins compared with that of the wild-type enzyme. Because the 35H and 35F proteins are predicted to be PKCalpha-anchoring proteins, these findings suggest a selective loss of PKCalpha-protein interactions that might fail to stabilize the location of the PKCalpha mutant at the plasma membrane.

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A point mutation in the protein kinase Cα (PKCα) gene has been discovered in a subpopulation of human pituitary tumors characterized by their invasive phenotype. Here we show that: (1) thyroid tumors can express the PKCα mutation detected in a subpopulation of follicular adenomas and carcinomas, and (2) mutated PKCα has modified enzymatic properties as compared to wild-type PKCα. It has lost its capacity to phosphorylate the S17R substrate and exhibits a higher sensitivity to degradation as compared to wild-type PKCα. In conclusion, the presence of the PKCα mutant in tumors other than pituitary tumors and the observation that the presence of the point mutation induces changes in PKCα properties suggest the involvement of this mutant in tumorigenesis.

The in vitro collagenolytic and proteoglycanasic activity from human fibrillated osteoarthritic cartilage was determined using labelled proteoglycans and type II collagen as substrates. In vitro, a glycosaminoglycan-peptide complex (GP-C Rumalon) induced a dose-dependent inhibition of both collagenolytic and proteoglycanasic activities while sodium salicylate and indomethacin had only a weak suppressive effect on proteoglycanase. Phospholipase A2 activity was unmodified by GP-C suggesting that the effect of the drug on degradative enzymes was unrelated to prostaglandin formation.