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.

We have studied nerve growth factor (NGF)-induced differentiation of PC12 cells to identify PKC isozymes important for neuronal differentiation. Previous work showed that tumor-promoting phorbol esters and ethanol enhance NGF-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth by a PKC-dependent mechanism. Ethanol also increases expression of PKC␦ and PKC⑀, suggesting that one these isozymes regulates responses to NGF. To examine this possibility, we established PC12 cell lines that express a fragment encoding the first variable domain of PKC⑀ (amino acids 2-144), which acts as an isozyme-specific inhibitor of PKC⑀ in cardiac myocytes. Phorbol esterstimulated translocation of PKC⑀ was markedly reduced in these PC12 cell lines. In addition, phorbol ester and ethanol did not enhance NGF-induced MAP kinase activation or neurite outgrowth in these cells. In contrast, phorbol ester and ethanol increased neurite outgrowth and MAP kinase phosphorylation in cells expressing a fragment derived from the first variable domain of PKC␦. These results demonstrate that PKC⑀ mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells. Protein kinase C (PKC)1 is a multigene family of phospholipiddependent, serine-threonine kinases that plays a central role in cell growth and differentiation. Molecular cloning studies have identified 10 isozymes encoded by 9 different mRNAs (1, 2). Based on sequence homology and biochemical properties, the PKC gene family has been divided into three groups: "conventional" PKCs (␣, ␤I, ␤II, and ␥) regulated by calcium and diacylglycerols or phorbol esters; "novel" PKCs (⑀, ␦, , and ), which are calcium-independent but diacylglycerol-and phorbol ester-sensitive; and "atypical" PKCs (, and /), which are insensitive to calcium, diacylglycerol, and PMA. In addition, two related phospholipid-dependent kinases, PKC and protein kinase D, share sequence homology in their regulatory domains to novel PKCs and may constitute a new subgroup (3, 4).Several studies with tumor-promoting phorbol esters suggest that PKC modulates neural differentiation. Phorbol esters induce neural tissue from ectoderm in Xenopus embryos (5) and elicit neurite outgrowth from chick sensory ganglia (6, 7), chick ciliary ganglion neurons (8), several human neuroblastoma cell lines (9, 10), and rat PC12 cells (11, 12). Studies using purified isozymes, kinase-defective mutants, and transgenic or mutant cell lines have implicated PKC␣, -␤, -␦, -⑀, and -in the differentiation of nonneural cells (13-17). Overexpression of PKC␣ or -␤ in Xenopus embryos enhances neural induction (18), but little else is known about the identity of specific PKC isozymes that regulate neural differentiation.Recent evidence suggests that PKC⑀ plays a role in neural differentiation and plasticity. PKC⑀ is expressed predominantly in the nervous system and is particularly abundant in the hippocampus, olfactory tubercle, and layers I and II of cerebral cortex (19). Within immunoreactiv...

Excessive alcohol consumption alters neuronal growth and causes striking elongation of axons and dendrites in several brain regions. This could result from increased sensitivity to neurotrophic factors, since ethanol markedly enhances nerve growth factor (NGF)-and basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth in the neural cell line PC12. The mechanism by which ethanol enhances growth factor responses was investigated by examining activation of mitogen-activated protein kinases (MAP kinases), a key event in growth factor signaling. Ethanol (100 mM) increased NGF-and bFGF-induced activation of MAP kinases. This increase, like ethanol-induced increases in neurite outgrowth, was prevented by down regulation of .3, 6, and E protein kinase C (PKC) isozymes. Since chronic ethanol exposure specifically upregulates 6 and E PKC, these findings suggest that ethanol promotes neurite growth by enhancing growth factor signal transduction through a a or E PKC-regulated pathway.Medical complications of alcohol abuse contribute to more than 20% of hospital admissions in the United States (1). Several neurological disorders are associated with alcoholism and many appear to result from a direct neurotoxic effect of alcohol (2). One mechanism by which alcohol injures the nervous system is by altering the growth of axons and dendrites (neurites). Although early studies showed that ethanol inhibited neurite growth in some neurons (3, 4), several reports (5-10) indicate that ethanol markedly enhances neurite growth in several brain regions. For example, in adult rats, chronic ethanol exposure increases the length of dendrites in cerebellar Purkinje cells and hippocampal dentate granule cells and the number of dendritic spines on dentate granule cells and somatosensory cortical neurons (5-8). Prenatal ethanol exposure increases dendritic arborization in layer Vb somatosensory corticospinal neurons and causes hyperdevelopment of hippocampal dentate granule cell axons (9, 10). Ethanol also enhances neurite outgrowth in cultured cerebellar neurons (11) and in PC12 cells treated with nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) (12, 13). Increases in neurite length could disturb neuronal function by delaying nerve conduction and by interfering with remodeling of neurites and synapses during development and learning.We have used PC12 cells as a model system to study mechanisms by which ethanol alters neurite outgrowth. We previously reported that enhancement of NGF-induced neurite outgrowth by ethanol was inhibited by downregulation of ,B, 6, and s isoforms of protein kinase C (PKC) (14). We also found that exposure to 25-100 mM ethanol for 2-6 days increased levels of mRNA (15) and protein (16) for 8 and s PKC and activated PKC in PC12 cells (16). Taken together, these findings suggest that ethanol enhances neurite formation by activating specific isozymes of PKC.One mechanism by which ethanol and PKC could modulate NGF-induced neurite outgrowth is by altering NGF signal transduction. A k...