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 PKCε 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 INa) in cultured rat dorsal root ganglion (DRG) neurons, are inhibited by a PKCε-selective inhibitor peptide. Our findings indicate that PKCε regulates nociceptor function and suggest that PKCε inhibitors could prove useful in the treatment of pain.

The bchH gene of Rhodobacter capsulatus has been cloned into an expression strain of Escherichia coli. Following induction of expression of the BchH protein, it was found that the E. coli strain also accumulated porphyrins with the fluorescence properties of protoporphyrin and zinc protoporphyrin. It was also found that the soluble BchH protein increased the activity of S-adenosyl-l-methionine:magnesium protoporphyrin IX methyltransferase, when mixed with membranes of an expression strain of E. coli into which the bchM gene (which encodes the methyltransferase) had been cloned, as well as membranes of a bchH mutant of R. capsulatus.

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 PKCδ and PKCε, 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 PKCε (amino acids 2–144), which acts as an isozyme-specific inhibitor of PKCε in cardiac myocytes. Phorbol ester-stimulated translocation of PKCε 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 PKCδ. These results demonstrate that PKCε mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells.

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 beta, delta, and epsilon protein kinase C (PKC) isozymes. Since chronic ethanol exposure specifically upregulates delta and epsilon PKC, these findings suggest that ethanol promotes neurite growth by enhancing growth factor signal transduction through a delta or epsilon PKC-regulated pathway.

Protein kinase C (PKC) activation enhances neurite outgrowth in several cell lines and primary neurons. The PKC isozymes that mediate this response are unknown. One clue to their identity has come from studies using PC12 cells treated with ethanol. In these cells, ethanol increases levels of delta-PKC and epsilon-PKC and markedly enhances nerve growth factor (NGF)-induced neurite outgrowth and activation of mitogen-activated protein (MAP) kinases by a PKC-dependent mechanism. Since these findings suggest that delta-PKC or epsilon-PKC can promote neural differentiation, we studied neurite outgrowth in stably transfected PC12 cell lines that overexpress these isozymes. Overexpression of epsilon-PKC markedly increased NGF-induced neurite outgrowth. This effect was blocked by down-regulating PKC or by treating cells with the PKC inhibitor GF 109203X. In addition, overexpression of epsilon-PKC enhanced NGF-induced phosphorylations of MAP kinases. In contrast, overexpression of delta-PKC did not alter responses to NGF. These results demonstrate that epsilon-PKC promotes NGF-induced neurite outgrowth by enhancing NGF signal transduction. These findings suggest a role for epsilon-PKC in neural differentiation and plasticity.

Adaptation to chronic ethanol exposure results in a decrease in sensitivity to the intoxicating effects of ethanol. Recent evidence indicates that changes in the expression and function of certain proteins involved in signal transduction are important for adaptation to ethanol. Using the neural cell line PC12, we found that chronic exposure to ethanol increases the expression and function of L-type voltage-gated calcium channels and enhances neural differentiation induced by nerve growth factor. Both of these responses to ethanol require protein kinase C (PKC). Chronic ethanol exposure activates PKC-mediated phosphorylation, in part, by increasing the expression of two PKC isozymes, delta and epsilon. The PKC family of enzymes may be important targets for the development of drugs that could modify adaptive and toxic consequences of chronic ethanol exposure.