Thomas McMahon

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.

Prkcz null mice show normal learning and memory

Protein kinase M-ζ (PKM-ζ) is a constitutively active form of atypical protein kinase C that is exclusively expressed in the brain and implicated in the maintenance of long-term memory. Most studies that support a role for PKM-ζ in memory maintenance have used pharmacological PKM-ζ inhibitors such as the myristoylated zeta inhibitory peptide (ZIP) or chelerythrine. Here we use a genetic approach and target exon 9 of the Prkcz gene to generate mice that lack both protein kinase C-ζ (PKC-ζ) and PKM-ζ (Prkcz−/− mice). Prkcz−/− mice showed normal behaviour in a cage environment and in baseline tests of motor function and sensory perception, but displayed reduced anxiety-like behaviour. Notably, Prkcz−/− mice did not show deficits in learning or memory in tests of cued fear conditioning, novel object recognition, object location recognition, conditioned place preference for cocaine, or motor learning, when compared with wild-type littermates. ZIP injection into the nucleus accumbens reduced expression of cocaine-conditioned place preference in Prkcz−/− mice. In vitro, ZIP and scrambled ZIP inhibited PKM-ζ, PKC-ι and PKC-ζ with similar inhibition constant (Ki) values. Chelerythrine was a weak inhibitor of PKM-ζ (Ki = 76 μM). Our findings show that absence of PKM-ζ does not impair learning and memory in mice, and that ZIP can erase reward memory even when PKM-ζ is not present.

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.

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.

Protein Kinase Cϵ Regulates γ-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of γ2 Subunits

Ethanol enhances γ-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABAA receptors, especially at low concentrations achieved during social drinking. We postulated that the ϵ isoform of protein kinase C (PKCϵ) regulates the ethanol sensitivity of GABAA receptors, as mice lacking PKCϵ show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCϵ mutant to selectively inhibit the catalytic activity of PKCϵ. We used this mutant and PKCϵ-/- mice to determine that PKCϵ phosphorylates γ2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at α1β2γ2 receptors, which is the most abundant GABAA receptor subtype in the brain. Our findings indicate that PKCϵ phosphorylation of γ2 regulates the response of GABAA receptors to specific allosteric modulators, and, in particular, PKCϵ inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

Protein Kinase Cδ Mediates Ethanol-induced Up-regulation of L-type Calcium Channels

Brief ethanol exposure inhibits L-type, voltage-gated calcium channels in neural cells, whereas chronic exposure increases the number of functional channels. In PC12 cells, this adaptive response is mediated by protein kinase C (PKC), but the PKC isozyme responsible is unknown. Since chronic ethanol exposure increases expression of PKCδ and PKCε, we investigated the role these isozymes play in up-regulation of L-type channels by ethanol. Incubation with the PKC inhibitor GF 109203X or expression of a PKCδ fragment that inhibits phorbol ester-induced PKCδ translocation largely prevented ethanol-induced increases in dihydropyridine binding and K+-stimulated 45Ca2+uptake. A corresponding PKCε fragment had no effect on this response. These findings indicate that PKCδ mediates up-regulation of L-type channels by ethanol. Remaining responses to ethanol in cells expressing the PKCδ fragment were not inhibited by GF 109203X, indicating that PKCδ-independent mechanisms also contribute. PKCδ overexpression increased binding sites for dihydropyridine and L-channel antagonists, but did not increase K+-stimulated45Ca2+ uptake, possibly because of homeostatic responses that maintain base-line levels of channel function. Since L-type channels modulate drinking behavior and contribute to neuronal hyperexcitability during alcohol withdrawal, these findings suggest an important role for PKCδ in alcohol consumption and dependence.

Protein Kinase Cδ Regulates Ethanol Intoxication and Enhancement of GABA-Stimulated Tonic Current

Ethanol alters the distribution and abundance of PKCδ in neural cell lines. Here we investigated whether PKCδ also regulates behavioral responses to ethanol. PKCδ−/− mice showed reduced intoxication when administered ethanol and reduced ataxia when administered the nonselective GABAA receptor agonists pentobarbital and pregnanolone. However, their response to flunitrazepam was not altered, suggesting that PKCδ regulates benzodiazepine-insensitive GABAA receptors, most of which contain δ subunits and mediate tonic inhibitory currents in neurons. Indeed, the distribution of PKCδ overlapped with GABAA δ subunits in thalamus and hippocampus, and ethanol failed to enhance tonic GABA currents in PKCδ−/− thalamic and hippocampal neurons. Moreover, using an ATP analog-sensitive PKCδ mutant in mouse L(tk−) fibroblasts that express α4β3δ GABAA receptors, we found that ethanol enhancement of GABA currents was PKCδ-dependent. Thus, PKCδ enhances ethanol intoxication partly through regulation of GABAA receptors that contain δ subunits and mediate tonic inhibitory currents. These findings indicate that PKCδ contributes to a high level of behavioral response to ethanol, which is negatively associated with risk of developing an alcohol use disorder in humans.

Protein kinase C isozymes that mediate enhancement of neurite outgrowth by ethanol and phorbol esters in PC12 cells.

Using PC12 cells to study ethanols effects on growth of neural processes, we found that ethanol enhances NGF- and basic FGF-induced neurite outgrowth. Chronic ethanol exposure selectively up-regulates delta and epsilon protein kinase C (PKC) and increases PKC-mediated phosphorylation in PC12 cells. Since PKC regulates differentiation, we investigated the role of PKC in enhancement of neurite outgrowth by ethanol. Like ethanol, 0.3-10 nM phorbol 12-myristate, 13-acetate (PMA) increased NGF-induced neurite outgrowth. However, higher concentrations did not, and immunoblot analysis demonstrated that 100 nM PMA markedly depleted cells of beta, delta and epsilon PKC. PMA (100 nM) also down-regulated beta, delta and epsilon PKC in ethanol-treated cells and completely prevented enhancement of neurite outgrowth by ethanol. In contrast, the cAMP analogue 8-bromoadenosine cAMP did not completely mimic the effects of ethanol on neurite outgrowth, and ethanol was able to enhance neurite formation in mutant PC12 cells deficient in protein kinase A (PKA). These findings implicate beta, delta or epsilon PKC, but not PKA, in the neurite-promoting effects of ethanol and PMA. Since chronic ethanol exposure up-regulates delta and epsilon, but not beta PKC, these findings suggest that delta or epsilon PKC regulate neurite outgrowth.