Pre-treatment with a PKC or PKA inhibitor prevents the development of morphine tolerance but not physical dependence in mice.

We previously demonstrated that intracerebroventricular (i.c.v.) administration of protein kinase C (PKC) or protein kinase A (PKA) inhibitors reversed morphine antinociceptive tolerance in 3-day morphine-pelleted mice. The present study aimed at evaluating whether pre-treating mice with a PKC or PKA inhibitor prior to pellet implantation would prevent the development of morphine tolerance and physical dependence. Antinociception was assessed using the warm-water tail immersion test and physical dependence was evaluated by quantifying/scoring naloxone-precipitated withdrawal signs. While drug-naïve mice pelleted with a 75 mg morphine pellet for 3 days developed a 5.8-fold tolerance to morphine antinociception, mice pre-treated i.c.v. with the PKC inhibitors bisindolylmaleimide I, Go-7874 or Go-6976, or with the myristoylated PKA inhibitor, PKI-(14-22)-amide failed to develop any tolerance to morphine antinociception. Experiments were also conducted to determine whether morphine-pelleted mice were physically dependent when pre-treated with PKC or PKA inhibitors. The same inhibitor doses that prevented morphine tolerance were evaluated in other mice injected s.c. with naloxone and tested for precipitated withdrawal. The pre-treatment with PKC or PKA inhibitors failed to attenuate or block the signs of morphine withdrawal including jumping, wet-dog shakes, rearing, forepaw tremor, increased locomotion, grooming, diarrhea, tachypnea and ptosis. These data suggest that elevations in the activity of PKC and PKA in the brain are critical to the development of morphine tolerance. However, it appears that tolerance can be dissociated from physical dependence, indicating a role for PKC and PKA to affect antinociception but not those signs mediated through the complex physiological processes of withdrawal.

mGluR5 antagonists that block calcium mobilization in vitro also reverse (S)-3,5-DHPG-induced hyperalgesia and morphine antinociceptive tolerance in vivo.

The present study comparatively evaluated the potency of a series of new phenylethyl[1,2,4]methyltriazines which are analogues of the classical metabotropic glutamate (mGlu) receptor subtype 5 (mGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) in blocking hyperalgesia induced by the group I mGlu receptor agonist (S)-3,5-DHPG as well as in reversing morphine antinociceptive tolerance in mice. Hyperalgesia was assessed in mice using the tail immersion test. Intrathecal (i.t.) pre-treatment with the test compounds 5-methyl-3-phenylethynyl-[1,2,4]triazine (RTI-4229-707), 5-methyl-3-(4-phenoxy-phenylethynyl-[1,2,4]triazine (RTI-4229-766), and 3-(3-methylphenylethynyl)-5-methyl-[1,2,4]triazine (RTI-4229-787) resulted in a dose-dependent blockade of (S)-3,5-DHPG-induced hyperalgesia. The inhibitory dose-50 (ID(50)) values were 0.49, 0.72 and 0.44 nmol/mouse, for RTI-4229-707, RTI-4229-766 and RTI-4229-787, respectively, compared to 18.63 nmol/mouse for MPEP. The other two compounds tested 3-(2,5-dimethylphenylethynyl)-5-methyl[1,2,4]triazine (RTI-4229-785) and 3-(2-methylphenylethynyl)-5-methyl[1,2,4]triazine (RTI-4229-828) were totally inactive. Morphine tolerance was induced in mice by implanting a 75 mg morphine pellet and assessing morphine-induced antinociception 72-h later. The morphine-pelleted mice showed a 5.5-fold tolerance to the antinociceptive effect of acute morphine compared to placebo-pelleted mice in the tail immersion test. Intracerebroventricular (i.c.v.) administration of the three active mGluR5 antagonists dose-dependently reversed morphine antinociceptive tolerance. The ID(50) values were 57.7, 25.8 and 64.3 nmol/mouse, for RTI-4229-707, RTI-4229-766 and RTI-4229-787, respectively, compared to 1050 nmol/mouse for MPEP. Similar to the hyperalgesia study, test compounds RTI-4229-785 and RTI-4229-828 were totally inactive in reversing morphine tolerance. These results are in agreement with our previous study in which we demonstrated that the same active mGluR5 antagonists blocked glutamate-mediated mobilization of internal calcium in a selective mGluR5 in vitro efficacy assay.

How important is protein kinase C in mu-opioid receptor desensitization and morphine tolerance?

The repeated administration of opiate drugs such as morphine results in the development of tolerance to their analgesic, rewarding (euphoric) and respiratory-depressant effects; thus, to obtain the same level of response with subsequent administrations, a greater dose must be used. Tolerance can limit the clinical efficacy of opiate drugs and enhance the social problems that are inherent in recreational opioid abuse. Surprisingly, the mechanism (or mechanisms) underlying the development of morphine tolerance remains controversial. Here, we propose that protein kinase C could have a crucial role in the desensitization of mu-opioid receptors by morphine and that this cellular process could contribute to the development and maintenance of morphine tolerance in vivo.

Synthesis and pharmacological evaluation of phenylethynyl[1,2,4]methyltriazines as analogues of 3-methyl-6-(phenylethynyl)pyridine.

Procedures were developed for the synthesis of 3-methyl-5-phenylethynyl[1,2,4]triazine (4), 6-methyl-3-phenylethynyl[1,2,4]triazine (5), and 5-methyl-3-phenylethynyl[1,2,4]triazine (6a) as analogues of 2-methyl-6-(phenylethynyl)pyridine (2). The compounds were evaluated for antagonism of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro efficacy assay. The most potent of the three analogues was 6a. Twenty additional analogues of 6a were synthesized and evaluated for mGluR5 antagonist efficacy. The most potent compounds were 3-(3-methylphenylethynyl)-5-methyl[1,2,4]triazine (6b), 5-(3-chlorophenylethynyl)-5-methyl[1,2,4]triazine (6c), and 3-(3-bromophenylethynyl)-5-methyl[1,2,4]triazine (6d).

Role of kappa and delta opioid receptors in mediating morphine-induced antinociception in morphine-tolerant infant rats.

We have previously noted that the antinociceptive efficacy of morphine was significantly decreased in rat pups chronically infused with morphine from implanted osmotic minipumps. In this study, morphine was fully efficacious (i.e., 100% maximum possible effect, %MPE) in the 52 degrees C tail-immersion test after a 72-h infusion from implanted saline-filled osmotic minipumps. However, administration of up to 1000 mg/kg, s.c. morphine failed to elicit greater than a 27% MPE in rats infused with morphine at 2 mg/kg/h. Morphine was more efficacious when the water bath temperature was decreased to 49 degrees C. Experiments were conducted to determine the mechanisms whereby chronic morphine administration leads to a decrease in antinociceptive efficacy. The kappa-opioid antagonist nor-binalorphimine completely blocked the antinociceptive effects of morphine in morphine-infused rat pups. The kappa agonist U50,488 elicited antinociception; however, the requirement to use higher doses in morphine- than saline-infused rats indicates that kappa cross-tolerance was present. Thus, in tolerant rats the antinociceptive effects of high doses of morphine appear to be mediated through kappa-opioid receptors. The delta-opioid antagonist naltrindole was inactive in both treatment groups. DAMGO-stimulated [(35)S]GTPgammaS and [(3)H]naloxone binding reveals that the anatomical distribution of the mu-opioid receptor was consistent with that of the adult rat brain. In adult rats, the mu-opioid receptor is desensitized during morphine tolerance. However, desensitization was not evident in P17 rats based on the lack of significant decreases in [(35)S]GTPgammaS binding. Furthermore, [(3)H]naloxone binding indicated a lack of mu receptor downregulation in morphine-tolerant rat pups.

Evidence for an important role of protein phosphatases in the mechanism of morphine tolerance.

Acute morphine antinociception has been shown to be blocked by very low picogram doses of okadaic acid indicating that inhibition of protein phosphatase PP2A allows for increases in phosphorylation to inhibit antinociception. Comparative studies in morphine tolerant animals have not been reported. In the present study, we showed a significant increase in the total phosphatase activity in the periaqueductal gray matter (PAG) from morphine-pelleted versus placebo-pelleted mice, 72-h after pellet implantation. This supports our hypothesis that phosphatase activity is increased in tolerance as a compensatory mechanism for the increase in kinase activity during the development of tolerance. We also demonstrated that i.c.v. administration of the phosphatase inhibitor okadaic acid (3 pmol/mouse; a dose tested to be inert in placebo-pelleted mice) enhanced the level of morphine antinociceptive tolerance assessed by the tail immersion test, 72-h following pellet implantation. This was supported by the fact that the same treatment with okadaic acid blocked the increase in phosphatase activity in PAG of morphine tolerant mice indicating that selective inhibition of PP2A contributes to enhanced levels of morphine tolerance. We have previously reported that PKC or PKA inhibitors reversed morphine antinociceptive tolerance in mice. The current study shows that i.c.v. administration of the PKC inhibitors bisindolylmaleimide I or Go6976 reversed the enhanced level of morphine tolerance induced by okadaic acid treatment to the same level of tolerance observed in non-okadaic acid-treated tolerant mice. However, the PKA inhibitor PKI-(14-22)-amide only partially reversed the enhancement of morphine tolerance induced by okadaic acid. Our data suggest an important role for the balance between kinases and phosphatases in modulating tolerance levels. Further studies will be directed towards a better understanding of the role of different phosphatase isoforms in morphine tolerance.

Enhancement of bupivacaine local anesthesia with the potassium channel blocker ibutilide.

In some clinical settings it is necessary to inject large volumes of local anesthetic--and consequently very high doses--in order to provide an adequate level of block. Subsequent absorption of these high doses, or inadvertent intravenous administration of even small doses, has led to systemic toxicity. Thus, it is desirable to develop adjuvants that are inert alone, but would enhance the potency and/or efficacy of local anesthetics to improve their safety. Adelta/C fibers possess K(+) channels identified as sustained delayed rectifier type K(DR) currents and transient A-type K(A) currents. In the heart, the class III antiarrhythmic drug ibutilide blocks the cardiac component of the rapid delayed rectifying K(+) current (IKr). Experiments were conducted to determine whether co-administration of the K(+) channel blocker ibutilide would enhance the local anesthetic bupivacaine in mice. After injecting bupivacaine mixed with vehicle or ibutilide in the popliteal region of mice, paw withdrawal latencies were determined by applying the plantar aspect of a single hind-paw to the surface a 55 degrees C hot-plate device. 0.5% Bupivacaine+ibutilide (7.8x10(-5) M) elicited significantly longer hot-plate latencies than 0.5% bupivacaine+vehicle. In addition, bupivacaine was 2.6-fold more potent when co-administered with ibutilide rather than vehicle. Epinephrine extends the tissue concentrations of local anesthetics by inducing localized vasoconstriction. Epinephrine augmented the enhancement by ibutilide of bupivacaine's potency by 6.8-fold. In summary, ibutilide may enhance the effects of bupivacaine by blocking K(+) channels on sensory nociceptive nerves.

Alterations in brain Protein Kinase A activity and reversal of morphine tolerance by two fragments of native Protein Kinase A inhibitor peptide (PKI).

Two peptide fragments of native Protein Kinase A inhibitor (PKI), PKI-(6-22)-amide and PKI-(Myr-14-22)-amide, significantly reversed low-level morphine antinociceptive tolerance in mice. The inhibition of Protein Kinase A (PKA) activity by both peptide fragments was then measured in specific brain regions (thalamus, periaqueductal gray (PAG), and medulla) and in lumbar spinal cord (LSC), which in previous studies have been shown to play a role in morphine-induced analgesia. In drug naive animals, cytosolic PKA activity was greater than particulate PKA activity in each region, while cytosolic and particulate PKA activities were greater in thalamus and PAG compared to medulla and LSC. The addition of both peptides to homogenates from each region completely abolished cytosolic and particulate PKA activities in vitro. Following injection into the lateral ventricle of the brain of drug naive mice and morphine-tolerant mice, both peptides inhibited PKA activity in the cytosolic, but not the particulate fraction of LSC. In addition, cytosolic and particulate PKA activities were inhibited by both peptides in thalamus. These results demonstrate that the inhibition of PKA reverses morphine tolerance. Moreover, the inhibition of PKA activity in specific brain regions and LSC from morphine-tolerant mice by PKI analogs administered i.c.v. is evidence that PKA plays a role in morphine tolerance.

Chronic Delta9-tetrahydrocannabinol treatment produces antinociceptive tolerance in mice without altering protein kinase A activity in mouse brain and spinal cord.

The present study investigated the effect of different levels of Delta-9-tetrahydrocannabinol (Delta(9)-THC) antinociceptive tolerance on Protein Kinase A (PKA) activity in mouse brain and spinal cord. To strengthen this investigation, a positive control was developed to demonstrate the assay utilized in this study was sensitive enough to detect an increase in PKA activity in the anatomical regions utilized in this study. The membrane-permeant and phosphodiesterase-resistant cAMP analog 8-Bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-8-Br-cAMPS) was utilized for the development of this positive control and this compound produced an increase in PKA activity in several mouse brain regions (i.c.v.) and lumbar spinal cord (i.t.) following its administration. Models were then developed in which mice expressed either a 13-fold or 49-fold level of Delta(9)-THC antinociceptive tolerance following chronic treatment with 10mg/kg Delta(9)-THC or 80mg/kg Delta(9)-THC for 6.5 days. Basal and total cytosolic and particulate PKA activities were measured directly in homogenates from the striatum, hippocampus, cerebellum, cortex and lumbar spinal cord. Results from this study indicate that chronic exposure to Delta(9)-THC does not produce an increase in PKA activity in these mouse brain regions or spinal cord. Future work is needed to determine the role of PKA in cannabinoid tolerance in mice.

Enhancement of transdermal fentanyl and buprenorphine antinociception by transdermal delta9-tetrahydrocannabinol.

Previous studies have demonstrated that delta9-tetrahydrocannabinol (THC) enhances the antinociceptive potency of many opioids administered by a variety of different routes of administration. We hypothesized that THC would enhance fentanyl or buprenorphine analgesia via the transdermal route of administration. THC was first demonstrated to enhance opioid antinociception when both drugs were administered parenterally in a hairless guinea pig model using the pin prick test. A low dose of THC (50 mg/kg, i.p.) produced no antinociception. However, THC enhanced the potency of s.c. fentanyl by 6.7-fold, and s.c. buprenorphine in a non-parallel fashion. For the transdermal studies, THC, fentanyl or buprenorphine was applied by pipette to the skin of the dorsum between the fore- and hind-flanks and covered with individual Tegederm patches. THC (400 mg/kg) produced no antinociception. However, THC enhanced fentanyl's potency by 3.7-fold at 2-h, and 5.8-fold at 4-h. Buprenophine's potency was increased 8.2-fold at 2-h and 7.2-fold at 4-h when co-administered with THC. These results indicate that the enhancement of transdermal opioids by THC could lead to the design of an effective combination analgesic patch.

Task specificity of cross-tolerance between Delta9-tetrahydrocannabinol and anandamide analogs in mice.

Relatively few studies have compared the effects of tetrahydrocannabinols and anandamide-like cannabinoids following repeated dosing. Whereas pronounced tolerance develops to many of the in vivo pharmacological effects of Delta9-tetrahydrocannabinol with repeated dosing, tolerance to anandamide-induced effects is typically less noted. In the present study, we examined cross-tolerance between Delta9-tetrahydrocannabinol and anandamide-like compounds (anandamide, 2-methylanandamide, and O-1812) in a tetrad of in vivo tests sensitive to cannabinoid action, including spontaneous activity, tail flick, rectal temperature, and a ring immobility test of catalepsy. Six intraperitoneal injections of Delta9-tetrahydrocannabinol 10 mg/kg over a period of 4 days resulted in the development of pronounced tolerance to all of its in vivo effects. In contrast, task specificity was observed in cross-tolerance to anandamide and its analogs: antinociception (all three compounds), suppression of spontaneous activity (2-methylanandamide and O-1812), catalepsy (O-1812), and hypothermia (none of the compounds). Furthermore, when it occurred, the magnitude of cross-tolerance was notably smaller. These results suggest that anandamide-like cannabinoids may have a unique pharmacology that only partially overlaps with that of Delta9-tetrahydrocannabinol and other traditional cannabinoids. Although the basis for this unique pharmacology has not as yet been determined, it is possible that regional specificity of cannabinoid CB1 receptor downregulation and endocannabinoid release induced by repeated dosing with Delta9-tetrahydrocannabinol may play a role.

Time-kill Kinetics of a Novel Antimicrobial Silver Wound Gel Against Select Wound Pathogens.

UNLABELLED: New treatments are needed as infection risk associated with diabetic, venous, and pressure ulcers are becoming more prevalent as comorbidities of obesity, aging, and major disease. Postsurgical, burn, and immunocompromised patients are also at an increased risk of wounds and infection. Silver has been utilized in treating various wounds associated with infections and, although highly effective, caution is required for use beyond 2 weeks due to potential silver cytotoxicity. To overcome this obstacle, an antimicrobial wound gel (CelaCare Technologies, Inc, Dallas, TX) was designed to allow low concentrations of a proprietary silver salt combined with acemannan, which has been demonstrated to aid wound healing. MATERIALS AND METHODS: This study's objective was to determine the time-kill kinetics of the antimicrobial wound gel vs 4 commercial topical silver products against 6 common wound pathogens and Bacillus subtilis as a spore-forming bacteria. RESULTS: The antimicrobial wound gel achieved a 2.9 log reduction in growth of Pseudomonas aeruginosa within 30 minutes, a 2.3 log reduction in Streptococcus pyogenes within 8 hours, a 2.1 log reduction in methicillin-resistant Staphylococcus aureus within 48 hours, a 2.3 log reduction in S. aureus within 24 hours, a 4.1 log reduction in Escherichia coli within 30 minutes, a 2.9 log reduction in B. subtilis within 60 minutes, and a 3.4 log reduction in Candida albicans within 90 minutes. Overall, the antimicrobial wound gel demonstrated broad antimicrobial coverage against all wound pathogens evaluated, and it was comparable to, or better than, other tested topical silver products containing substantially higher silver concentrations. CONCLUSION: The broad-spectrum antimicrobial activity of the wound gel indicates it could become a product alternative to current commercial products.

Evidence that the antinociceptive tail-flick response is produced independently from changes in either tail-skin temperature or core temperature.

It has recently been hypothesized that tail-skin temperature may exert a profound influence on the latency of the tail-flick response to radiant heat. Several recent reports in the literature urge investigators to assess tail temperatures concurrently when using the tail-flick test and to adjust the tail-flick latency by a coefficient when a change in tail temperature is detected. Because much of the supporting evidence of this hypothesis was strictly correlational, the purpose of the present study was to determine whether tail-skin temperature is an important factor contributing to the latency of the tail-flick response to radiant heat. The effects of a series of pharmacological and non-pharmacological manipulations on tail-skin temperature and response latencies were assessed using either a low-intensity or high-intensity tail-flick stimulus. In addition, colonic temperature was evaluated. None of the drug treatments yielded a significant correlation between tail temperature and tail-flick latency. Of the seven drugs tested, only mecamylamine produced a consistent change in tail-skin temperature. Although mecamylamine significantly elevated tail temperature by more than 2 degrees C, it failed to alter response latencies. Similarly tail submergence into 5 degrees C water for 10 sec led to profound decreases in tail temperature ranging from -6.5 to -7.6 degrees C while producing only minimal increases in tail-flick latency. Conversely, submerging the tail in 38 degrees C water or placing the animals over a heating pad maintained at 38 degrees C increased tail temperatures at least 2 degrees C without affecting response latencies. Inverse correlations were found between tail-flick latency and colonic temperature after morphine, delta 9-tetrahydrocannabinal (delta 9-THC), and nicotine administration; however, these relationships do not appear to be causal. Sodium barbital produced far more hypothermia than any other agent, but did not produce any antinociception. Moreover, placing subjects in heated cages increased tail-skin temperature between 2 and 4 degrees C and blocked the hypothermic effects of morphine and delta 9-THC without reducing the antinociceptive potencies of these agents. These findings indicate that tail-skin and core temperatures have a negligible influence on the tail-flick response. We conclude that monitoring tail-skin or core temperatures when employing the tail-flick test is unnecessary and altering tail-flick latencies to account for changes in tail temperature is unwarranted.

The expression of a high level of morphine antinociceptive tolerance in mice involves both PKC and PKA.

We have previously reported that intracerebroventricular (i.c.v.) injection of either a PKC or PKA inhibitor completely reversed the expression of 5- to 8-fold morphine antinociceptive tolerance. We developed a model of 45-fold morphine tolerance that included a 75-mg morphine pellet and twice daily morphine injections. PKC inhibitor doses of bisindolylmaleimide I and Gö-7874 that completely reversed 8-fold tolerance only partly reversed the 45-fold level of antinociceptive tolerance. A component of tolerance was resistant to PKC inhibition, since even higher inhibitor doses failed to further reverse the high level of morphine tolerance. In addition, the 45-fold tolerance was only partly reversed by the PKA inhibitor KT-5720 at a dose previously cited by others to reverse 5-fold tolerance. Another PKA inhibitor 4-cyano-3-methylisoquinoline only partly reversed the morphine tolerance as well. In other experiments PKC and PKA inhibitors were co-administered together to determine their effectiveness for completely reversing the 45-fold level of morphine tolerance. Co-administering either bisindolylmaleimide I with KT-5720, or Gö-7874 with KT-5720, completely reversed the high level of tolerance. The high level of morphine tolerance was also completely reversed by co-administering Gö-7874 with 4-cyano-3-methylisoquinoline. Thus, high levels of morphine tolerance may reflect increases in protein phosphorylation by the terminal kinases of both the adenylyl cyclase and phosphatidylinositol cascades in brain and spinal cord areas critical to the expression of antinociception.

Prolonged reversal of morphine tolerance with no reversal of dependence by protein kinase C inhibitors.

The phosphatidylinositol (PI) cascade plays a pivotal role in mediating behavioral tolerance to the antinociceptive effects of morphine. Earlier we reported that antinociceptive tolerance was completely reversed 30 min after the administration of inhibitors of each step in the PI cascade. The aim of this study was to determine whether injection of a single dose of protein kinase C (PKC) inhibitor would elicit a prolonged reversal of morphine tolerance for up to 24 h. Three days after implantation of placebo- or 75-mg morphine pellets, mice received intracerebroventricular (i.c.v.) injections of vehicle or PKC inhibitor drug. Morphine challenge doses were then administered 4, 8 and 24 h later to test for tolerance reversal. In non-tolerant mice, Gö-7874 and sangivamycin had no effect on the potency of morphine. However, Gö-7874 and sangivamycin significantly reversed morphine tolerance at 4, 8 and 24 h. In addition, the role of PKC in morphine physical dependence was determined. Gö-7874 and sangivamycin by themselves did not precipitate spontaneous morphine withdrawal. Therefore, experiments were conducted to determine whether the PKC inhibitors would block naloxone-precipitated withdrawal. However, neither a 30-min nor a 24-h pretreatment with Gö-7874 or sangivamycin blocked naloxone withdrawal. Our results along with other publications indicate that PKC is a pivotal kinase essential for maintaining animals in an opioid tolerant state. Finally, the use of persistent PKC inhibitors that lasted for 24 h demonstrated that the neuronal systems in these animals did not adapt by increasing the activity of other protein kinase cascades to re-establish morphine tolerance.

Effects of mGlu1 and mGlu5 metabotropic glutamate antagonists to reverse morphine tolerance in mice.

Intracerebroventricular (i.c.v.) injection of phospholipase C inhibitors and structurally dissimilar PKC inhibitors were shown to completely reverse morphine antinociceptive tolerance in mice. Since Group I metabotropic glutamate receptors (mGlu(1) and mGlu(5)) activate phospholipase C through Galpha(q) Galpha(11) proteins, we hypothesized that morphine tolerance could occur through an increase in mGlu(1) and mGlu(5) receptor stimulation. Seventy-two hours after implantation of placebo or 75 mg morphine pellets, mice were tested in the 56 degrees C warm-water tail-withdrawal test following i.c.v. injection of vehicle or test drug. The mGlu(1) receptor antagonist CPCCOEt (7-(Hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) partly but significantly reversed morphine tolerance. The mGlu(5) receptor antagonist MPEP (2-Methyl-6-(phenylethynyl)pyridine hydrochloride) also partly reversed the antinociceptive tolerance. Co-administering CPCCOEt with MPEP completely reversed the tolerance. Furthermore, the mixed mGlu(1)/mGlu(5) antagonist AIDA ((RS)-1-Aminoindan-1,5-dicarboxylic acid) also completely reversed the tolerance. Thus, greater mGlu(1) and mGlu(5) receptor stimulation during morphine tolerance may lead to persistent activation of the phosphatidylinositol cascade.

PKC and PKA inhibitors reverse tolerance to morphine-induced hypothermia and supraspinal analgesia in mice.

Morphine antinociceptive tolerance in the tail-flick test is completely reversed by inhibitors of protein kinase C (PKC) or cAMP-dependent protein kinase (PKA). The effects of these inhibitors on tolerance to supraspinally mediated antinociception, such as the hot-plate test was unknown, as well as their effects in tests of mechanical nociception. The PKC inhibitors bisinolylmaleimide I ((2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) and Gö-7874 [2[1[(3-Dimethylaminopropyl)-5-methozyindol-3-yl]-3-(1H-indol-3-yl) hydrochloride] completely reversed the tolerance to morphine in both the hot-plate and tail-pinch tests. Similarly, the PKA inhibitor KT-5720 (8R, 9S, 11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one also reversed tolerance in both tests. The role of PKC and PKA in mediating tolerance to morphine-induced hypothermia was also investigated. Bisinolylmaleimide I, Gö-7874 and KT-5720 only partly reversed the 32-fold level of tolerance induced by the morphine pellets. However, co-administration of bisinolylmaleimide I with KT-5720 or Gö-7874 with KT-5720 completely reversed the tolerance. This demonstrates that tolerance in a non-behavioral system involves the actions of PKC and PKA.

Influence of intracellular Ca2+ release modulating drugs on bupivacaine infiltration anesthesia in mice.

The endoplasmic reticulum inside neurons can provide enormous amounts of releasable Ca2+ to increase cytosolic Ca2+ levels through the activation of endoplasmic membrane ion channels. Ryanodine (RyR) channels release Ca2+ into the cytosol when activated by Ca2+ influx through voltage-gated channels, or by cyclicADP ribose. Inositol tris-phosphate (IP3) channels are stimulated by phospolipid metabolism and the release of IP3. The hypothesis was tested that drugs that bind RyR or IP3 channels would affect the anesthetic potency of bupivacaine. The radiant heat tail-flick test was used to assess for anesthesia following subcutaneous infiltration of bupivacaine and Ca2+ modulating drugs in the tails of mice. No musculature is contained in the tail that could result in motor block. The RyR channel agonists 4-chloro-m-cresol and poly-L-lysine significantly reduced the anesthetic potency of bupivacaine. The plant alkaloid ryanodine elicited a bi-phasic effect, with low concentrations blocking bupivacaine anesthesia, and a high concentration enhancing anesthesia. Alternatively, the RyR channel antagonist dantrolene sodium dose-dependently increased bupivacaine's potency. However, the IP3 channel drugs were inactive. The IP3 agonist adenophostin A failed to affect bupivacaine anesthesia. Furthermore, bupivacaine was unaffected by the IP3 channel antagonists xestospongin C or low molecular weight heparin. Our results indicate that only the RyR channel drugs modulated the anesthetic effects of bupivacaine. Electrophysiological and molecular studies of sensory dorsal root ganglia neurons, the source of Adelta and C-fiber nociceptors, have demonstrated the presence of RyR3 Ca2+ release channels. This provides the first evidence that RyR channels might affect bupivacaine anesthesia in some fashion.

A pilot study to assess cognition and pillbox fill accuracy by community-dwelling older adults.

OBJECTIVE: To assess pillbox fill accuracy and cognition among community-dwelling older adults. DESIGN: A descriptive, cross-sectional study. SETTING: Retail pharmacy. PARTICIPANTS: Convenience sample of English-speaking adults older than 60 years of age without dementia, taking more than four medications, and naive to Mediset use. INTERVENTIONS: In face-to-face interviews, subjects provided demographic, medical, and medication information, completed the Mini-Cog and Medi-Cog (combination of Mini-Cog and medication-transfer screen [MTS]), and filled their own medications in a pillbox. Data were analyzed using descriptive statistics and stepwise regression analysis with correctly filled pill count (PC) as the dependent variable and the cognitive screens as independent variables. MAIN OUTCOME MEASURES: Accuracy of the Mini-Cog, MTS, and Medi-Cog in predicting PC. RESULTS: Among 50 subjects (58% female, mean age 76.4 years), only one subject failed to pass the Mini-Cog and two failed to reach the criterion level of correctly filled PC. The mean (standard deviation) Mini-Cog score for the sample was 4.38 (0.81), MTS score was 4.1 (1.31), Medi-Cog score was 8.48 (1.82), and the mean PC was 97% (8%). The Mini-Cog and MTS individually accounted for about 30% of the variance (P < 0.001); the Medi-Cog accounted for 44% of the variance (P < 0.001), indicating strongest PC prediction. CONCLUSION: Nearly all study participants filled pillboxes accurately. The Medi-Cog was the strongest predictor of pillbox fill accuracy. Future studies of medication self-management abilities among community-dwelling older adults should include representative samples of this population, comprehensive assessment of health status, cognitive screening, pillbox fill accuracy, and the utilization of medications in filled pillboxes.

Determination of the role of conventional, novel and atypical PKC isoforms in the expression of morphine tolerance in mice.

This study comprehensively determines the role of all the major PKC isoforms in the expression morphine tolerance. Pseudosubstrate and receptors for activated C-kinase (RACK) peptides inhibit only a single PKC isoform, while previously tested chemical PKC inhibitors simultaneously inhibit multiple isoforms making it impossible to determine which PKC isoform mediates morphine tolerance. Tolerance can result in a diminished effect during continued exposure to the same amount of substance. In rodents, morphine pellets provide sustained exposures to morphine leading to the development of tolerance by 72 h. We hypothesized that administration of the PKC isoform inhibitors i.c.v. would reverse tolerance and reinstate antinociception in the tail immersion and hot plate tests from the morphine released solely from the pellet. Inhibitors to PKC alpha, gamma and epsilon (100-625 pmol) dose-dependently reinstated antinociception in both tests. The PKC beta(I), beta(II), delta, theta, epsilon, eta and xi inhibitors were inactive (up to 2500 pmol). In other mice, the degree of morphine tolerance was determined by calculating ED50 and potency-ratio values following s.c. morphine administration. Morphine s.c. was 5.6-fold less potent in morphine-pelleted vs. placebo-pelleted mice. Co-administration of s.c. morphine with the inhibitors i.c.v. to either PKC alpha (625 pmol), gamma (100 pmol) or epsilon (400 pmol) completely reversed the tolerance so that s.c. morphine was equally potent in both placebo- and morphine-pelleted mice. The PKC beta(I), beta(II), delta, theta, epsilon, eta and xi inhibitors were inactive. Thus, PKC alpha, gamma and epsilon appear to contribute to the expression of morphine tolerance in mice.