Spinal Protein Kinase C Research Articles

Sex differences in the contributions of spinal atypical PKCs and downstream targets to the maintenance of nociceptive sensitization.

BackgroundChronic pain has been shown to depend on nociceptive sensitization in the spinal cord, and while multiple mechanisms involved in the initiation of plastic changes have been established, the molecular targets which maintain spinal nociceptive sensitization are still largely unknown. Building upon the established neurobiology underlying the maintenance of long-term potentiation in the hippocampus, this present study investigated the contributions of spinal atypical protein kinase C (PKC) isoforms PKCι/λ and PKMζ and their downstream targets (p62/GluA1 and NSF/GluA2 interactions, respectively) to the maintenance of spinal nociceptive sensitization in male and female rats.ResultsPharmacological inhibition of atypical PKCs by ZIP reversed established allodynia produced by repeated intramuscular acidic saline injections in male animals only, replicating previously demonstrated sex differences. Inhibition of both PKCι/λ and downstream substrates p62/GluA1 resulted in male-specific reversals of intramuscular acidic saline-induced allodynia, while female animals continued to display allodynia. Inhibition of NSF/GluA2, the downstream target to PKMζ, reversed allodynia induced by intramuscular acidic saline in both sexes. Neither PKCι/λ, p62/GluA1 or NSF/GluA2 inhibition had any effect on formalin response for either sex.ConclusionThis study provides novel behavioural evidence for the male-specific role of PKCι/λ and downstream target p62/GluA1, highlighting the potential influence of ongoing afferent input. The sexually divergent pathways underlying persistent pain are shown here to converge at the interaction between NSF and the GluA2 subunit of the AMPA receptor. Although this interaction is thought to be downstream of PKMζ in males, these findings and previous work suggest that females may rely on a factor independent of atypical PKCs for the maintenance of spinal nociceptive sensitization.

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Antisense oligonucleotide knockdown of mGlu5 receptor attenuates the antinociceptive tolerance and up-regulated expression of spinal protein kinase C associated with chronic morphine treatment

Spinal metabotropic glutamate receptor 5 (mGlu5 receptor) is known to influence the development of intrathecal morphine antinociceptive tolerance. However, the signaling mechanisms remain unknown. We carried out intrathecal administration of an antisense oligodeoxynucleotide (ODN), which results in reduced expression of spinal mGlu5 receptor, to determine its effects on morphine tolerance and spinal protein kinase C (PKC) expression. Rats were treated intrathecally with saline, morphine, mGlu5 receptor antisense ODN or mGlu5 receptor mismatched ODN. Behavioral tests were used to test the thermal and mechanical pain thresholds. Eight days later, rats were sacrificed and spinal cords were harvested to assess the expression of spinal PKC (α, γ and ε) by Western blotting and real-time polymerase chain reaction (PCR). Compared to control, intrathecal mGlu5 receptor antisense ODN resulted in a ~53.9% reduction of spinal mGlu5 receptor after 8days treatment. The mGlu5 receptor antisense ODN prevented the development of morphine tolerance. Expression of spinal PKC (α, γ and ε) was up-regulated at the mRNA and protein levels during the development of tolerance. Meanwhile, antisense ODN but not mismatched ODN reduced the spinal dorsal horn levels of PKC (α, γ and ε) which had been up-regulated after morphine exposure. We conclude that mGlu5 receptor participates in the development of morphine tolerance. Expression of spinal PKC (α, γ and ε) at the mRNA and protein levels increased during morphine tolerance. Antisense ODN of mGlu5 receptor prevented the tolerance and inhibited the altered expression of spinal PKC (α, γ and ε) during the development of tolerance.

Implication of endogenous β-endorphin in the inhibition of the morphine-induced rewarding effect by the direct activation of spinal protein kinase C in mice

It has often been proposed that opioid addiction does not arise as a consequence of opioid treatment for pain. Recently, we demonstrated that activated protein kinase C (PKC) in the spinal cord associated with chronic pain-like hyperalgesia suppressed the morphine-induced rewarding effect in mice. In the present study, we investigated whether a gene deletion for an endogenous mu-opioid peptide beta-endorphin could affect pain-like behavior and the suppression of the morphine-induced rewarding effect by the direct activation of PKC in the spinal cord. We found that activation of spinal PKC by intrathecal (i.t.) treatment with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused thermal hyperalgesia, pain-like behaviors and suppression of the morphine-induced rewarding effect. This suppression of morphine reward was eliminated in mice that lacked beta-endorphin. In contrast, thermal hyperalgesia and pain-like behaviors were not affected in beta-endorphin knockout mice. These results suggest that the activation of PKC in the spinal cord may play an essential role in the suppression of the morphine-induced rewarding effect in mice with neuropathic pain through the constant release of beta-endorphin.

Implication of spinal protein kinase Cγ isoform in activation of the mouse brain by intrathecal injection of the protein kinase C activator phorbol 12,13-dibutyrate using functional magnetic resonance imaging analysis

The aim of the present study was to investigate whether direct activation of protein kinase C (PKC) in the spinal cord could change brain activation using a functional magnetic resonance imaging (fMRI) analysis in mice that lack the PKCgamma gene. The activation of spinal PKC by intrathecal (i.t.) injection with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused a time-dependent decrease in paw-withdrawal latency to the heat thermal stimulus. In contrast, i.t. injection of PDBu failed to cause thermal hyperalgesia in mice which lacked the PKCgamma gene. We found that the i.t. injection with PDBu caused a remarkable increase in the activity of several brain regions in wild-type mice compared with vehicle injection. In the somatosensory cortex and lateral and medial thalamus, i.t. injection of PDBu produced a dramatic and time-dependent increase in signal intensity at 1-6h after i.t. PDBu injection. In contrast, i.t. injection of PDBu produced a delayed but significant increase in signal intensity at 3-6h in the cingulate cortex, at 4-6h in the nucleus accumbens and at 3-6h in the ventral tegmental area. In addition, all effects of PDBu were abolished in mice that lacked the PKCgamma gene. These results suggest that the activation of spinal PKCgamma associated with the activation of ascending pain transmission may be an important factor in chronic pain-like hyperalgesia with changes in emotionality.

Activation of Protein Kinase C in the Spinal Cord Produces Mechanical Hyperalgesia by Activating Glutamate Receptors, but does not Mediate Chronic Muscle-Induced Hyperalgesia

BackgroundProtein kinase C (PKC) in the spinal cord appears to mediate chronic injury-induced pain, but not acute nociceptive pain. Muscle insult results in increased release of glutamate spinally, and hyperalgesia that is reversed by spinal blockade of NMDA and non-NMDA glutamate receptors. Therefore, we hypothesized that spinal activation of PKC 1) mediates the late phase of hyperalgesia 1 week after muscle insult, and 2) produces mechanical hyperalgesia through activation of NMDA and non-NMDA glutamate receptors.ResultsRats were implanted with intrathecal catheters for delivery of drugs directly to the spinal cord. Mechanical withdrawal thresholds of the paw were determined using von Frey filaments. Intrathecal phorbol 12,13 dibutyrate (PDBu) produced a dose-dependent decrease in the mechanical withdrawal threshold of the paw that was prevented by pretreatment with the PKC inhibitor, GF109203X. Pretreatment with an NMDA receptor antagonist (AP5) or a AMPA/kainate receptor antagonist (NBQX) prevented the decrease in mechanical withdrawal threshold by PDBu. Two injections of acidic saline in the gastrocnemius muscle decreased the mechanical withdrawal thresholds of the paw bilaterally 24 h and 1 week after the second injection. However, blockade PKC in the spinal cord had no effect on the decreased withdrawal thresholds of the paw when compared to vehicle controls.ConclusionSpinal activation of PKC produces mechanical hyperalgesia of the paw that depends on activation of NMDA and non-NMDA receptors. Chronic muscle-induced mechanical hyperalgesia, on the other hand, does not utilize spinal PKC.

Induction of c- fos expression in the mouse brain associated with hyperalgesia induced by intrathecal injection of protein kinase C activator

Here, we found that a single intrathecal (i.t.) administration of a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), induced pain-like behaviors in mice. Furthermore, i.t.-administered PDBu caused the increased c- fos-like immunoreactivity in the parafascicular nuclei (PF), amygdala and cingulate cortex (CG), but not hippocampus. These findings suggest that the stimulation of spinal PKC results in an enhancement of neuronal activity in the PF, amygdala and CG associated with hyperalgesia.

Implication of spinal protein kinase C in the suppression of morphine-induced rewarding effect under a neuropathic pain-like state in mice

We previously demonstrated that spinal protein kinase C (PKC) is involved in the development of a neuropathic pain-like state induced by sciatic nerve ligation, and the morphine-induced rewarding effect is attenuated by sciatic nerve ligation in rodents. Here we first investigated whether sciatic nerve injury could change the activity of a conventional PKC (cPKC) and an atypical PKC isoform PKCζ in the mouse spinal cord. The second experiment was to investigate whether direct inhibition of spinal PKC by intrathecal (i.t.) administration of a specific PKC inhibitor, 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methyl-1 H-indole-3-yl)maleimide (RO-32-0432), could affect the rewarding effect induced by morphine following sciatic nerve ligation in mice. We found here that the activities of both cPKC and PKCζ in the spinal cord were clearly increased following sciatic nerve ligation. Furthermore, i.t. administration of RO-32-0432 reversed a long-lasting pain-like syndrome as indicated by thermal hyperalgesia following sciatic nerve ligation in mice. These data provide direct evidence that activated cPKC and PKCζ in the spinal cord may contribute to the development and maintenance of neuropathic pain. In the present study, we confirmed that the morphine-induced place preference was significantly suppressed by sciatic nerve ligation. It should be mentioned that i.t. pretreatment with RO-32-0432 significantly reversed the attenuation of morphine-induced rewarding effect following sciatic nerve ligation. These results suggest that activation of PKCs, including cPKC and PKCζ, within the spinal cord is directly responsible for the attenuation of the morphine-induced rewarding effect under a neuropathic pain-like state following sciatic nerve ligation in mice.

Differential involvement of spinal protein kinase C and protein kinase A in neuropathic and inflammatory pain in mice

In the present study, we demonstrated the differential role of spinal protein kinases in neuropathic and inflammatory pain. Mice with sciatic nerve ligation exhibited a spinal protein kinase C (PKC)-dependent neuropathic pain-like state. In contrast, an intraplanter injection of inflammatory agent caused a protein kinase A (PKA)-related thermal hyperalgesia. These findings suggest that the substantial activation of spinal PKC and PKA may differentially contribute to the development of respective chronic pain-like state in mice.

Differential Roles of Spinal Protein Kinases C and A in Development of Primary Heat and Mechanical Hypersensitivity Induced by Subcutaneous Bee Venom Chemical Injury in the Rat

It has been demonstrated that subcutaneous injection of bee venom (BV) can produce different types of pain and hypersensitivity including persistent spontaneous nociception (PSN), primary heat and mechanical hypersensitivity (hyperalgesia) and mirror-image heat (MIH) hypersensitivity in an individual animal, and the changes of spinal neurons are likely to be responsible for the production of these pain-related behaviors. In this study, we examined the roles of spinal protein kinase C (PKC) and protein kinase A (PKA) in the BV-induced different types of pain and hypersensitivity in conscious rats. We found that: (1) BV-induced primary heat hypersensitivity could be blocked by intrathecal pre- or posttreatment with a PKC inhibitor, chelerythrine chloride (CH), while a PKA inhibitor, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89), had no effect. (2) BV-induced primary mechanical hypersensitivity could be blocked by pre- or posttreatment with H89, whereas CH had no effect. (3) Both pre- and posttreatment with H89 produced suppressive effects on both induction and maintenance of the BV-induced PSN and MIH hypersensitivity. Based on the present findings, we proposed that spinal PKC might be activated during the central processes of primary heat hypersensitivity, while spinal PKA is likely to be involved in primary mechanical hypersensitivity induced by subcutaneous BV chemical injury. Taken together with our previous report however, spinal PKC and PKA are likely to be simultaneously involved in the central processes of both PSN and MIH hypersensitivity.

Knockdown of spinal metabotropic glutamate receptor 1 (mGluR(1)) alleviates pain and restores opioid efficacy after nerve injury in rats.

1. Nerve injury often produces long-lasting spontaneous pain, hyperalgesia and allodynia that are refractory to treatment, being only partially relieved by clinical analgesics, and often insensitive to morphine. With the aim of assessing its therapeutic potential, we examined the effect of antisense oligonucleotide knockdown of spinal metabotropic glutamate receptor 1 (mGluR(1)) in neuropathic rats. 2. We chronically infused rats intrathecally with either vehicle, or 50 microg day(-1) antisense or missense oligonucleotides beginning either 3 days prior to or 5 days after nerve injury. Cold, heat and mechanical sensitivity was assessed prior to any treatment and again every few days after nerve injury. 3. Here we show that knockdown of mGluR(1) significantly reduces cold hyperalgesia, heat hyperalgesia and mechanical allodynia in the ipsilateral (injured) hindpaw of neuropathic rats. 4. Moreover, we show that morphine analgesia is reduced in neuropathic rats, but not in sham-operated rats, and that knockdown of mGluR(1) restores the analgesic efficacy of morphine. 5. We also show that neuropathic rats are more sensitive to the excitatory effects of intrathecally injected N-methyl-D-aspartate (NMDA), and have elevated protein kinase C (PKC) activity in the spinal cord dorsal horn, two effects that are reversed by knockdown of mGluR(1). 6. These results suggest that activity at mGluR(1) contributes to neuropathic pain through interactions with spinal NMDA receptors and PKC, and that knockdown of mGluR(1) may be a useful therapy for neuropathic pain in humans, both to alleviate pain directly, and as an adjunct to opioid analgesic treatment.

Inhibition of spinal protein kinase C blocks substance P-mediated hyperalgesia

Substance P (SP) is an important neuromediator in the spinal processing of nociceptive afferent information. Our previous study has shown that spinal (intrathecal, IT) application of SP produces thermal hyperalgesia that is mediated by activation of the G-protein coupled NK1 receptor. The activation of some classes of the G-protein coupled receptors is known to produce diacylglycerol with consequent activation of protein kinase C (PKC). In the present study, we have demonstrated that intrathecal administration of a selective PKC inhibitor GF109203X (GF, 0.73 nmol) in rats chronically implanted with intrathecal catheters 15 min prior to IT-SP (48 nmol) completely blocked the SP-induced thermal hyperalgesia. The effect of GF was dose-dependent (0.073–0.73 nmol). Bisindolymaleimide V, the inactive homolog of GF, had no effect. Pretreatment with GF 3 h, but not 24 h, prior to SP still produced antinociception. Moreover, intrathecal treatment with GF (0.73 nmol) attenuated the formalin paw injection-induced flinching, preferentially at the 2nd phase, that is known to be associated with the release of endogenous SP at the spinal cord. These data suggest that activation of spinal PKC is involved in the SP-mediated hyperalgesia. Thus, SP, which is released in the spinal cord subsequent to persistent stimulation of small sensory afferents after tissue injury, may contribute to spinal hyperexcitability and persistent pain by enhancement of PKC-mediated phosphorylation of target molecules such as NMDA receptors.

Spinal PKC activity and expression: role in tolerance produced by continuous spinal morphine infusion

It has been hypothesized that spinal morphine tolerance results from protein kinase C (PKC) mediated phosphorylation. Chronic lumbar intrathecal (i.t.) infusion of morphine (20 nmol/μl/h) was shown to produce antinociception on day 1 (d1) that disappeared by d5 (tolerance). On d6, a bolus i.t. probe dose of morphine (60 nmol) produced a more profound antinociception in saline-infused rats than in morphine-infused rats. Coinfusion of morphine with a PKC inhibitor, chelerythrine, prevented tolerance to the probe morphine dose. Bolus i.t. chelerythrine or GF109203X (GF), another PKC inhibitor, on d5, but not the inactive homologue of GF Bisindolymaleimide V, also blocked development of tolerance after 24 h. I.t. morphine infusion, but not saline, produced a 2-fold increase in dorsal horn PKC phosphorylating activity and in the expression of PKC α/ γ. Bolus chelerythrine on d5 after spinal morphine infusion blocked upon an increase in PKC activity, confirming that at the behaviorally active dose the drug had the intended biochemical effect upon spinal PKC activity. PKC activity and protein expression did not change when assessed 1 h after bolus i.t. morphine in naive rats. Thus, tolerance produced by morphine infusion is dependent upon an increase in local phosphorylating activity by PKC. Blocking the PKC activity prevents expression of the morphine tolerance.

Alteration of spinal protein kinase C expression and kinetics in morphine, but not clonidine, tolerance

Antinociceptive synergism between spinally administered morphine and clonidine decreases to an additive interaction in morphine- and clonidine-tolerant mice. Spinally administered protein kinase C (PKC) inhibitors also decrease the synergism to addition. To determine whether chronic morphine or clonidine treatment alters spinal PKC activity, the present studies measured PKC activity and expression of PKC isoform proteins in spinal cord cytosol and membrane fractions. Mice were treated for 4 days with either placebo pellets, morphine pellets, s.c. saline, or s.c. clonidine. Morphine pellet-implanted mice were tolerant to morphine-induced tail flick antinociception, but not cross-tolerant to clonidine. Clonidine-pretreated mice were tolerant to clonidine, but not cross-tolerant to morphine. Induction of morphine tolerance produced a 2-fold lower K m value for PKC (8.24 ± 1.67 μM in placebo pellet vs 4.43 ± 1.24 μM in morphine pellet) in cytosol, but not membrane fractions from spinal cord. V max values were not different. No difference in K m or V max values was found between proteins from saline- and clonidine-pretreated animals. Immunoreactive cPKCα, βI, and γ isoforms decreased 14, 26, and 17%, respectively, in cytosol from morphine-tolerant animals. No difference in PKC isoforms was found in the membranes or in fractions from clonidine-tolerant mice. Morphine tolerance, but not clonidine tolerance, enhanced PKC activity while decreasing protein expression.

Effect of protein kinase C modulation on outcome of experimental CNS ischemia

Protein kinase C (PKC) is an important intracellular regulator, and its activity may play a central role in the modulation of neuronal ischemic damage. Staurosporine and the compound H-7 are potent in vitro inhibitors of PKC, and 1,2-oleoylacetylglycerol (OAG) is an effective activator. We administered these compounds through a spinal subarachnoid catheter and demonstrated in vivo alteration of spinal cord PKC activity. We then tested the effects of altering PKC activity in a well-established rabbit model of reversible spinal cord ischemia. Animals within each experimental group were subjected to a range of spinal cord ischemic durations by temporary occlusion of the infrarenal abdominal aorta. Compared to control, both staurosporine and H-7 significantly shortened the duration of ischemia that the animals could tolerate, without developing permanent paraplegia. OAG resulted in an insignificant lengthening of the ischemic duration that the animals could withstand. The worsening of ischemic outcome by PKC inhibitors suggests that the enzyme is important for maintaining neurologic function under ischemic conditions, possibly secondary to modulation of intracellular calcium levels.

Blockade of the spinal excitatory effect of cAMP on the startle reflex by intrathecal administration of the isoquinoline sulfonamide H-8: comparison to the protein kinase C inhibitor H-7

Drugs thought to inhibit the actions of protein kinase C (PKC) and cAMP dependent protein kinase (A-kinase) were infused intrathecally into the subarachnoid space of the lumbar region of the spinal cord, and the effects on acoustic startle were measured. Previous work has shown that intrathecal infusion of drugs thought to increase cAMP increase the startle response. The present experiment evaluated whether inhibition of A-kinase would prevent this effect. Rats were infused with the isoquinoline sulfonamide, H-8 (360 nmol) or vehicle (50% dimethyl sulfoxide), 30 min prior to infusion of 100 nmol of dibutyryl cAMP. By itself, H-8 had little effect on startle, but completely blocked the normal excitatory effect of dibutyryl cAMP on startle. In contrast, the isoquinoline sulfonamide, H-7, which is less active in blocking A-kinase, but more active in blocking PKC, did not block dibutyryl cAMP. Moreover, H-8 did not block the excitatory effect of intrathecal infusion of the 5-HT 1A receptor agonist, 8-OH-dipropylaminotetraline (8-OH-DPAT). Thus, the blockade of dibutyryl cAMP by H-8 appears somewhat specific and suggests an involvement of A-kinase in the excitatory effects of dibutyryl cAMP on the acoustic startle response. In a second experiment, it was found that administration of the isoquinoline sulfonomide H-7 caused a marked, dose-dependent (150–800 nmol) facilitation of the startle reflex in comparison with its vehicle, Tris buffer (0.1 M). Like H-7, another PKC inhibitor, GT 1b (20 nmol) produced a marked increase in the startle reflex versus its vehicle, 0.01 M phosphate buffer. Other isoquinoline sulfonamides, HA1004 (400 nmol) and H-8 (360 nmol), which have less PKC inhibitory activity than H-7, caused only modest increases in startle. Thus, the increases in startle caused by intrathecal infusions of the isoquinoline sulfonamides and the ganglioside GT 1b indicate that spinal PKC inhibition elevates startle, perhaps because PKC tonically inhibits startle at the spinal level.