Robert O. Messing

A Novel Nociceptor Signaling Pathway Revealed in Protein Kinase C ε Mutant Mice

Abstract 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 PKCe 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 PKCe-selective inhibitor peptide. Our findings indicate that PKCe regulates nociceptor function and suggest that PKCe inhibitors could prove useful in the treatment of pain.

Supersensitivity to allosteric GABA A receptor modulators and alcohol in mice lacking PKCε

Several of the actions of ethanol are mediated by γ-aminobutyrate type A (GABAA) receptors. Here we demonstrated that mutant mice lacking protein kinase C epsilon (PKCε) were more sensitive than wild-type littermates to the acute behavioral effects of ethanol and other drugs that allosterically activate GABAA receptors. GABAA receptors in membranes isolated from the frontal cortex of PKCε null mice were also supersensitive to allosteric activation by ethanol and flunitrazepam. In addition, these mutant mice showed markedly reduced ethanol self-administration. These findings indicate that inhibition of PKCε increases sensitivity of GABAA receptors to ethanol and allosteric modulators. Pharmacological agents that inhibit PKCε may be useful for treatment of alcoholism and may provide a non-sedating alternative for enhancing GABAA receptor function to treat other disorders such as anxiety and epilepsy.

The type 1 equilibrative nucleoside transporter regulates ethanol intoxication and preference

Adenosine is an important mediator of ethanol intoxication. In vitro, ethanol stimulates adenosine signaling by inhibiting the type 1 equilibrative nucleoside transporter (ENT1), whereas chronic ethanol exposure downregulates ENT1. It is not known, however, whether ENT1 is important for ethanol intoxication or consumption in vivo. Here we report that ENT1-null mice show reduced hypnotic and ataxic responses to ethanol and greater consumption of alcohol as compared with their wild-type littermates. These features are associated with a decrease in adenosine tone, as measured indirectly as a reduction in A1 receptor–mediated inhibition of glutamate excitatory postsynaptic currents (EPSCs) in the nucleus accumbens, leading to increased phosphorylation of CRE-binding protein (CREB) in the striatum. Treatment with an A1 receptor agonist decreases EPSC amplitude and reduces ethanol consumption in ENT1-null mice. Our results indicate that ENT1 has a physiological role in ethanol-mediated behaviors and suggest that decreased A1 adenosine receptor function promotes alcohol consumption.

A semisynthetic epitope for kinase substrates

The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPγS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester–specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38α MAPK, Erk1, Erk2, Akt1, PKCδ, PKCε, Cdk1/cyclinB, CK1, Cdc5, GSK3β, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.NOTE: In the version of this article initially published online, a sentence was missing a word and did not make sense. The corrected sentence now reads, “Erk2 was immunoprecipitated from each of these cell lines and assayed with A*TPγS analogs; N6-phenethyl ATPγS was a preferred nucleotide substrate for AS Erk2 and was not accepted by wild-type Erk2 (data not shown).” The error has been corrected for all versions of the article.

Decreased anxiety-like behavior, reduced stress hormones, and neurosteroid supersensitivity in mice lacking protein kinase Cε

Mice lacking protein kinase Cepsilon (PKCepsilon) are supersensitive to positive allosteric modulators of gamma aminobutyrate type A (GABA(A)) receptors. Since many of these compounds are anxiolytic, we examined whether anxiety-like behavior is altered in these mice. PKCepsilon-null mice showed reduced anxiety-like behavior and reduced levels of the stress hormones corticosterone and adrenocorticotrophic hormone (ACTH). This was associated with increased sensitivity to neurosteroid modulators of GABA(A) receptors. Treatment of PKCepsilon-null mice with the GABA(A) receptor antagonist bicuculline restored corticosterone levels and anxiety-like behavior to wild-type levels. These results suggest that increased GABA(A) receptor sensitivity to neurosteroids contributes to reduced anxiety-like behavior and stress hormone responses in PKCepsilon-null mice. The findings also suggest PKCepsilon as a possible therapeutic target for development of anxiolytics.

Role of the Protein Kinase C-ε–Raf-1–MEK-1/2–p44/42 MAPK Signaling Cascade in the Activation of Signal Transducers and Activators of Transcription 1 and 3 and Induction of Cyclooxygenase-2 After Ischemic Preconditioning

Background—Although Janus kinase (JAK)–mediated Tyr phosphorylation of signal transducers and activators of transcription (STAT) 1 and 3 is essential for the upregulation of cyclooxygenase-2 (COX-2) and the cardioprotection of late preconditioning (PC), the role of Ser phosphorylation of STAT1 and STAT3 in late PC and the upstream signaling mechanisms responsible for mediating Ser phosphorylation of STAT1 and STAT3 remain unknown. Methods and Results—In mice preconditioned with six 4-minute coronary occlusion/4-minute reperfusion cycles, we found that (1) ischemic PC activates the Raf1–mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase kinase (MEK) 1/2–p44/42 MAPK signaling pathway, induces phosphorylation of STAT1 and STAT3 on the Ser-727 residue, and upregulates COX-2 expression; (2) pSer-STAT1 and pSer-STAT3 form complexes with pTyr-p44/42 MAPKs in preconditioned myocardium, supporting the concept that Ser phosphorylation of these 2 factors is mediated by activated p44/42 MAPKs; and (3) activation of the Raf-1-MEK-1/2–p44/42 MAPK-pSer-STAT1/3 pathway and induction of COX-2 during ischemic PC are dependent on protein kinase C (PKC)-&egr; activity, as determined by both pharmacological and genetic inhibition of PKC&egr;. Conclusions—To our knowledge, this is the first study to demonstrate that ischemic PC causes Ser phosphorylation of STAT1 and STAT3 and that this event is governed by PKC&egr; via a PKC&egr;–Raf1-MEK1/2-p44/42 MAPK pathway. Furthermore, this is the first report that COX-2 expression in the heart is controlled by PKC&egr;. Together with our previous findings, the present study implies that STAT-dependent transcription of the genes responsible for ischemic PC is modulated by a dual signaling mechanism that involves both JAK1/2 (Tyr phosphorylation) and PKC&egr; (Ser phosphorylation).

Regulation of neuronal voltage-gated calcium channels by ethanol ☆

Voltage-gated calcium channels are key regulators of neuronal excitability. Several studies indicate that intoxicating concentrations of ethanol inhibit L-type, N-type and possibly T-type channels. The effects of ethanol on other channel subtypes are not yet clear. Chronic exposure to ethanol is associated with increases in functional L-type channels and this may contribute to signs of ethanol withdrawal. Preclinical studies in animals suggest that L-type calcium channel antagonists decrease ethanol consumption and signs of alcohol withdrawal. Although L-type channel antagonists do not appear to alter the performance impairing or psychological effects of acute ethanol administration, clinical trials will be needed to determine if L-type channel antagonists reduce ethanol consumption in humans.

Protein Kinase C Inhibits Adenylyl Cyclase Type VI Activity during Desensitization of the A2a-Adenosine Receptor-mediated cAMP Response

We have previously reported that phosphorylation of adenylyl cyclase type VI (AC6) may result in the suppression of adenylyl cyclase activity during desensitization of the A2a-adenosine receptor-mediated cAMP response (A2a desensitization) in rat pheochromocytoma PC12 cells. In the present study, we demonstrate that protein kinase C (PKC) is responsible for the phosphorylation and inhibition of AC6 during A2a desensitization. Inhibition of PKC by several independent methods markedly blocked the suppression of AC6 during A2a desensitization. Purified PKC from rat brain directly phosphorylated and inhibited recombinant AC6 expressed in Sf21 cells. Substantially lower AC6 activities were also observed in PC12 cells overexpressing PKCδ or PKCε. Stimulation of A2a-R in PC12 cells under the same conditions as those required for A2a desensitization resulted in an increase in Ca2+-independent PKC activity. Most importantly, exogenous PKC did not further suppress AC6 activity in A2a-desensitized membranes. In vitro PKC phosphorylation of AC6 isolated from A2a-desensitized cells was also profoundly lower than that from control cells, suggesting a specific role for PKC in regulating AC6 during A2a desensitization in PC12 cells. Taken together, our data demonstrate that a calcium-independent, novel PKC inhibits AC6 activity during A2a desensitization in PC12 cells. Independent regulation of AC6 by calcium-independent PKC and by Ca2+ provides an exquisite mechanism for integrating signaling pathways to fine-tune cAMP synthesis.

Neutrophil protein kinase Cδ as a mediator of stroke-reperfusion injury

Thrombolysis is widely used to intervene in acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Here we describe studies with mice lacking protein kinase Cδ (PKCδ) showing that absence of this enzyme markedly reduces reperfusion injury following transient ischemia. This was associated with reduced infiltration of peripheral blood neutrophils into infarcted tissue and with impaired neutrophil adhesion, migration, respiratory burst, and degranulation in vitro. Total body irradiation followed by transplantation with bone marrow from PKCδ-null mice donors reduced infarct size and improved neurological outcome in WT mice, whereas marrow transplantation from WT donors increased infarction and worsened neurological scores in PKCδ-null mice. These results indicate an important role for neutrophil PKCδ in reperfusion injury and strongly suggest that PKCδ inhibitors could prove useful in the treatment of stroke.

Sex hormones regulate the contribution of PKCε and PKA signalling in inflammatory pain in the rat

We have evaluated the contribution of differences in second messenger signalling to sex differences in inflammatory pain and its control by sex hormones. In normal male but not female rats, epinephrine‐induced mechanical hyperalgesia was antagonized by inhibitors of protein kinase Cε (PKCε), protein kinase A (PKA) and nitric oxide synthetase (NOS). Similarly, in PKCε knockout mice, a contribution of PKCε to epinephrine‐dependent mechanical hyperalgesia occurred in males only. In contrast, hyperalgesia induced by prostaglandin E2, in both females and males, was dependent on PKA and NO. In both sexes, inhibitors of mitogen‐activated protein kinase/extracellular‐signal related kinase kinase (MEK) inhibited epinephrine hyperalgesia. In gonadectomized females, the second messenger contributions to epinephrine hyperalgesia demonstrated the pattern seen in males. Administration of oestrogen to gonadectomized females fully reconstituted the phenotype of the normal female. These data demonstrate gender differences in PKCε, PKA and NO signalling in epinephrine‐induced hyperalgesia which are oestrogen dependent and appear to be exerted at the level of the β‐adrenergic receptor or the G‐protein to which it is coupled.