The effects of levosimendan and glibenclamide on circulatory and metabolic variables in a canine model of acute hypoxia

Purpose  

To study the effects of pretreatment with levosimendan (LEVO, a Ca²⁺-sensitizer and K_ATP⁺ channel opener) and/or the K_ATP⁺ channel antagonist glibenclamide (GLIB) on systemic hemodynamics, metabolism, and regional gastromucosal oxygenation during hypoxic hypoxemia.     

Role of K ATP channels in cephalic vasodilatation induced by calcitonin gene-related peptide, nitric oxide, and transcranial electrical stimulation in the rat

Objective.— The objective of this study was to explore the role of K_ATP channels in vasodilatation induced by calcitonin gene‐related peptide (CGRP), nitric oxide (NO), and transcranial electrical stimulation (TES) in intracranial arteries of rat. Background.— Dilatation of cerebral and dural arteries causes a throbbing, migraine‐like pain. Both CGRP and NO are potent vasodilators that can induce migraine. Their antagonists are effective in the treatment of migraine attacks. K_ATP channel openers cause headache in the majority of healthy subjects suggesting a role for K_ATP channels in migraine pathogenesis. We hypothesized that vasodilatation induced by CGRP and the NO donor glyceryltrinitrate (GTN) is mediated via K_ATP channels. Methods.— We examined the effects of the K_ATP channel inhibitor glibenclamide on dural and pial vasodilatation induced by CGRP, NO, and endogenously released CGRP by TES. A rat genuine closed cranial window model was used for in vivo studies and myograph baths for studying the effect in vitro. In the closed cranial window model the diameter of dural vessels was measured directly in anesthetized animals to investigate the vascular effects of infused CGRP, NO, and endogenous CGRP after electrical stimulation. Also diameter changes of pial arteries, mean arterial blood pressure and local cerebral blood flow by Laser Doppler flowmetry (LCBF_Flux) were measured. Results.— CGRP, NO, and TES caused dilatation of the 2 arteries in vivo and in vitro. In anesthetized rats glibenclamide significantly attenuated CGRP induced dural and TES induced dural/pial artery dilatation (P = .001; P = .001; P = .005), but had no effect on dural/pial vasodilatation induced by GTN. In vitro glibenclamide failed to significantly inhibit CGRP‐ and GTN‐induced vasodilatation. Conclusions.— These results show that a K_ATP channel blocker in vivo but not in vitro inhibits CGRP, but not GTN‐induced dilatation of dural and pial arteries, a mechanism thought to be important in migraine.     

Modulation of spinal GABAergic analgesia by inhibition of chloride extrusion capacity in mice

Spinal gamma-aminobutyric acid receptor type A (GABA_A) receptor modulation with agonists and allosteric modulators evokes analgesia and antinociception. Changes in K⁺-Cl^? cotransporter isoform 2 (KCC2) expression or function that occur after peripheral nerve injury can result in an impairment in the Cl^? extrusion capacity of spinal dorsal horn neurons. This, in turn, alters Cl^?-mediated hyperpolarization via GABA_A receptor activation, contributing to allodynia or hypersensitivity associated with nerve injury or inflammation. A gap in knowledge exists concerning how this loss of spinal KCC2 activity differentially impacts the analgesic efficacy or potency of GABA_A agonists and allosteric modulators. We utilized intrathecal drug administration in the tail flick assay to measure the analgesic effects of general GABA_A agonists muscimol and Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid (ZAPA), the ?-subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP), and allosteric modulators of the benzodiazepine (midazolam) and neurosteroid (ganaxolone) class, alone or in the presence of K⁺-Cl^? cotransporter isoform (KCC) blockade. Intrathecal muscimol, ZAPA, THIP midazolam, and ganaxolone all evoked significant analgesia in the tail flick test. Coadministration of either agonists or allosteric modulators with [(dihydroindenyl)oxy] alkanoic acid (DIOA) (a drug that blocks KCC2) had no effect on agonist or allosteric modulator potency. On the other hand, the analgesic efficacy of muscimol and ZAPA and the allosteric modulator ganaxolone were markedly reduced whereas THIP and midazolam were unaffected. Finally, in the spared nerve injury model, midazolam significantly reversed tactile hypersensitivity while ganaxolone had no effect. These results indicate that the KCC2-dependent Cl^? extrusion capacity differentially regulates the analgesic efficacy of agonists and allosteric modulators at the GABA_A receptor complex.PerspectiveOur work suggests that drug discovery efforts for the treatment of chronic pain disorders should target benzodiazepine or ?-subunit-containing sites at the GABA_A complex.     

The value of in vitro studies in a case of neonatal diabetes with a novel Kir6.2‐W68G mutation

In infants, especially with novel previously undescribed mutations of the K_ATP channel causing neonatal diabetes, in vitro studies can be used to both predict the response to sulphonylurea treatment and support a second trial of glibenclamide at higher than standard doses if the expected response is not observed.     

Interaction of morphine and potassium channel openers on experimental models of pain in mice

Combination of opioid and potassium channel openers holds immense potential for the treatment for most acute and chronic pain. Therefore, the study was performed to assess the interaction between morphine and K(+) -channel openers. Swiss albino mice of either sex weighing between 25 and 30 g were used for the study. The study assesses the interaction between morphine and K(+) -channel openers (cromakalim, diazoxide and minoxidil), when administered intraperitoneally, using formalin and tail-flick tests in mice. Both morphine and K(+) -channel openers produced significant antinociception at higher doses in both the behavioral tests. Lower doses of morphine and K(+) -channel openers had no significant effect on tail-flick latency, while the same drugs had significant antinociceptive effect on formalin test. The combination of lower doses of morphine and K(+) -openers was observed to have significant antinociceptive effect in both the behavioral tests. Administration of naloxone prior to morphine or K(+) -channel openers antagonized the analgesic effect of morphine but not of K(+) -channel openers, whereas prior administration of glibenclamide antagonized the effect of both morphine and K(+) -channel openers. The study, therefore, suggests that the common site of action of morphine and K(+) -channel openers is at the levels of K(+) -channels rather than at the level of receptors. However, such interaction depends on the differential sensitivity to different pain stimulus.     

The effect of glibenclamide on acetylcholine and sodium nitroprusside induced vasodilatation in human cutaneous microcirculation

Objective: K_ATP channels have an important regulatory role in resting vascular tone and during hypoxia. Their role in endothelium dependent and independent vasodilatation in human skin microcirculation is less known. Methods: We monitored the laser‐Doppler (LD) response in 14 healthy male volunteers on the skin of the forearm. In the case of endothelium dependent vasodilatation [acetylcholine (ACh) induced], a saline solution (used as control) or a solution of glibenclamide (K_ATP channel blocker) were randomly injected each into a distinct measurement site on different forearms followed by the iontophoresis of ACh. In the case of endothelium dependent vasodilatation with the inhibition of prostaglandin production, diclofenac (cyclooxygenase inhibitor) or the combination of diclofenac and glibenclamide were randomly injected each into a distinct measurement site on different forearms followed by the iontophoresis of ACh. In the case of endothelium independent vasodilatation [sodium nitroprusside (SNP) induced], a saline solution or glibenclamide were randomly injected each into a distinct measurement site on different forearms, iontophoresis of SNP followed. Results: Glibenclamide alone, diclofenac alone or the combination of glibenclamide and diclofenac reduced the LD flux values during rest and after ACh application (P<0·05). The reduction of LD flux in ACh mediated vasodilatation was greatest when using the combination of glibenclamide and diclofenac. In the case of SNP application, there was also a significantly lower LD flux rise for glibenclamide in comparison with the saline solution (P<0·05). Conclusions: K_ATP channels play an important role in human cutaneous vasodilatation induced by ACh and SNP.     

Anticonvulsant effect of melatonin through ATP-sensitive channels in mice

Melatonin is a neurohormone secreted principally by the pineal gland. This molecule has various pharmacological properties including improving immune system, prevent cancer, anti-aging, and anti-oxidant effects. The anticonvulsant effects of melatonin have been proved by previous studies. Adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channels are considered as an important target in the seizure modulation. The aim of the present study was to investigate the anticonvulsant effect of melatonin in pentylenetetrazole (PTZ)-induced seizures in mice, focusing on its ability to regulate K_ATP channels. Acute intraperitoneal administration of melatonin (40 and 80 mg/kg) increased clonic seizure threshold induced by intravenous administration of PTZ. Melatonin (40 and 80 mg/kg) increased the latency of clonic seizure and reduced its frequency in mice receiving an intraperitoneal injection of PTZ. Administration of glibenclamide, a K_ATP channels blocker, before intravenous injection of PTZ reduced melatonin anticonvulsant effect. Diazoxide and cromakalim, as K_ATP channels openers, increased antiseizure effect of melatonin in PTZ model of seizures. These findings suggest that the antiseizure effect of melatonin probably is gained through increasing the opening of K_ATP channels.     

Effects of Cannabinoid Type 2 Receptor Agonist AM1241 on Morphine-Induced Antinociception,Acute and Chronic Tolerance,and Dependence in Mice

Morphine is a potent opioid analgesic used to alleviate moderate or severe pain, but the development of drug tolerance and dependence limits its use in pain management. Previous studies showed that cannabinoid type 2 (CB₂) receptor ligands may modulate opioid effects. However, there is no report of the effect of CB₂ receptor agonist on acute morphine tolerance and physical dependence. We therefore investigated the effect of a CB₂ receptor agonist (AM1241) on morphine-induced morphine tolerance and physical dependence in mice. Repeated coadministration of AM1241 (1 or 3mg/kg intraperitoneally) and morphine (10mg/kg subcutaneously) for 7days increased the mechanical paw withdrawal threshold in mice as measured by the von Frey filament test, and 3mg/kg AM1241 in combination with morphine increased the thermal paw withdrawal latency as measured by the hot-plate test. Combination with 3mg/kg AM1241 and morphine increased acute morphine antinociception. Coadministration of 1 or 3mg/kg AM1241 and morphine reduced acute morphine tolerance, and 3mg/kg AM1241 reduced chronic morphine tolerance. Coadministration of 1 or 3mg/kg AM1241 and morphine reduced naloxone-precipitated withdrawal jumping, but not diarrhea. Coadministration of AM1241 and morphine did not inhibit spontaneous locomotor activity. Pretreatment with 3mg/kg AM1241 decreased the chronic morphine-induced Iba1 expression in spinal cord. Coadministration of AM1241 (3 mg/kg) reduced the production of interleukin-1β, tumor necrosis factor-α, and interleukin-6 induced by long-term and acute morphine treatment. Our findings suggest that the coadministration of the CB₂ receptor agonist and morphine could increase morphine antinociception and reduce morphine tolerance and physical dependence in mice.Perspective:The combination of a CB₂ agonist and morphine may provide a new strategy for better treatment of acute and chronic pain and prevention of opioid tolerance and dependence. This finding may also provide a clue for the treatment of opioid tolerance and dependence in clinics.     

Pharmacological mechanisms involved in the antinociceptive effects of dexmedetomidine in mice

Dexmedetomidine (DEX) is a α₂‐adrenoceptor (α₂‐AR) agonist used as an anesthetic adjuvant and as sedative in critical care settings. Typically, α₂‐AR agonists release nitric oxide (NO) and subsequently activate NO‐GMPc pathway and have been implicated with antinociception. In this study, we investigate the pharmacological mechanisms involved in the antinociceptive effects of DEX, using an acetic acid‐induced writhing assay in mice. Saline or DEX (1, 2, 5, or 10 μg/kg) was intravenously injected 5 min before ip administration of acetic acid and the resulting abdominal constrictions were then counted for 10 min. To investigate the possible mechanisms related to antinociceptive effect of DEX (10 μg/kg), the animals were also pretreated with one of the following drugs: 7‐nitroindazole (7‐NI; 30 mg/kg ip); 1H‐[1,2,4] oxadiazole [4,3‐a] quinoxaline‐1‐one (ODQ; 2.5 mg/kg, ip); yohimbine (YOH; 1 mg/kg, ip); atropine (ATRO; 2 mg/kg, ip); glibenclamide (GLIB; 1 mg/kg, i.p.) and naloxone (NAL; 0.2 mg/kg, ip). A rotarod and open‐field performance test were performed with DEX at 10 μg/kg dose. DEX demonstrated its potent antinociceptive effect in a dose‐dependent manner. The pretreatment with 7‐NI, ODQ, GLIB, ATRO, and YOH significantly reduced the antinociceptive affects of DEX. However, NAL showed no effecting DEX‐induced antinociception. The rotarod and open‐field tests confirmed there is no detectable sedation or even significant motor impairment with DEX at 10 μg/kg dose. Our results suggest that the α₂‐AR and NO‐GMPc pathways play important roles in the systemic antinociceptive effect of DEX in a murine model of inflammatory pain. Furthermore, the antinociceptive effect exerted by DEX appears to be dependent on K_ATP channels, independent of opioid receptor activity.     

High-fat diet may impair KATP channels in vascular smooth muscle cells

K_ATP channel in vascular smooth muscle cell (VSMC) is closely linked to the etiology of hypertension. The aim of this study was to investigate effect of the high-fat diet-induced obesity on K_ATP channel and blood pressure. Obesity was induced by a 24-week high-fat diet feeding in rats. Function and expression of K_ATP channel in mesenteric arteries were examined using myography system, patch clamp and Western blotting. We show that high-fat diet increased blood pressure, decreased K_ATP channel-mediated relaxation responses and currents, and down-regulated K_ATP expression in VSMC. In conclusion, diet-induced obesity impairs K_ATP channels in VSMC, which may underscore obesity-triggered increase in blood pressure.     

Intrathecal antinociceptive interaction between the NMDA antagonist ketamine and the opioids,morphine and biphalin

Biphalin is an opioid peptide analogue that currently is under clinical development as a new type of site‐directed analgesic. In rats, the intrathecal (i.t.) analgesic potency of biphalin is 1000‐fold greater than morphine. Such a high activity may reflect this compound's activation of three types of opioid receptors (μ, δ and κ). NMDA receptors also play an important role in nociceptive processing. Therefore, we investigated in rats whether an NMDA antagonist may influence biphalin‐induced antinociception. In the present study, ketamine was chosen because of the widespread safe use of this drug in clinical practice. I.t. application of ketamine alone had relatively little analgesic effect and its excitatory effects limited possible doses of the drug. Co‐administration of ketamine with biphalin or morphine produced markedly greater antinociception than biphalin or morphine alone in acute, thermal tail flick testing. These results suggest that NMDA antagonists may be useful potentiators of biphalin analgesia. Thus, to obtain the same spinal antinociceptive effect, lower doses of biphalin or morphine are required when ketamine is co‐administered.     

Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress

The enhanced oxidative stress associated with type 2 diabetes mellitus contributes to disease pathogenesis. We previously identified plasma membrane–associated ATP-sensitive K⁺ (K_ATP) channels of pancreatic β cells as targets for oxidants. Here, we examined the effects of genetic and pharmacologic ablation of K_ATP channels on loss of mouse β cell function and viability following oxidative stress. Using mice lacking the sulfonylurea receptor type 1 (Sur1) subunit of K_ATP channels, we found that, compared with insulin secretion by WT islets, insulin secretion by Sur1^–/– islets was less susceptible to oxidative stress induced by the oxidant H₂O₂. This was likely, at least in part, a result of the reduced ability of H₂O₂ to hyperpolarize plasma membrane potential and reduce cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) in the Sur1^–/– β cells. Remarkably, Sur1^–/– β cells were less prone to apoptosis induced by H₂O₂ or an NO donor than WT β cells, despite an enhanced basal rate of apoptosis. This protective effect was attributed to upregulation of the antioxidant enzymes SOD, glutathione peroxidase, and catalase. Upregulation of antioxidant enzymes and reduced sensitivity of Sur1^–/– cells to H₂O₂-induced apoptosis were mimicked by treatment with the sulfonylureas tolbutamide and gliclazide. Enzyme upregulation and protection against oxidant-induced apoptosis were abrogated by agents lowering [Ca²⁺]_c. Sur1^–/– mice were less susceptible than WT mice to streptozotocin-induced β cell destruction and subsequent hyperglycemia and death, which suggests that loss of K_ATP channel activity may protect against streptozotocin-induced diabetes in vivo.     

Cardioprotective effect of hemin in isoprenaline-induced myocardial infarction: role of ATP-sensitive potassium channel and endothelial nitric oxide synthase

Ischemic heart disease is a common cardiac health problem. Despite the significant advances in prevention and treatment of this disorder, its incidences and complications are very serious. So, the search for more antioxidants and anti-inflammatory agents with cardioprotective effects is an urgent task. We aimed to evaluate the effects of a heme oxygenase 1 (HO1) inducer, hemin (HEM), on isoprenaline (ISO)-induced myocardial damage. Forty-five Wistar albino rats were used. Animals were treated with HEM (25 mg/kg/day) i.p. for 5 days and injected with ISO (150 mg/kg/day) i.p. on 4th and 5th day of the experiment. Detection of the role of ATP-sensitive potassium channel (K_ATP) was performed by administration of glibenclamide (GP) (5 mg/kg/day) orally 2 h before HEM. Moreover, the role of endothelial nitric oxide synthase (eNOS) was detected by coadministration of Nitro- ω-L-arginine (L-NNA) (25 mg/kg/day) for 5 days. The ISO group showed increase in heart weight, cardiac enzymes, tumor necrosis factor alpha (TNFα), and malondialdehyde (MDA) with decrease in reduced glutathione (GSH), HO1, and total antioxidant capacity (TAC). In addition, there were increases in Bcl-2 associated X protein (Bax) and cleaved caspase-3, but decreases in B-cell lymphoma-2 (Bcl-2) and eNOS. Moreover, the histopathological examination of the ISO group showed degeneration of the cardiac muscle fibers and marked infiltration of the inflammatory cells. The biochemical and histopathological changes induced by ISO were markedly ameliorated in the HEM plus ISO group. This protective effect was diminished with coadministration of GP or L-NNA; thus, K_ATP and eNOS might mediate HEM cardioprotection.     

Spinal antinociception by adenosine analogs and morphine after intrathecal administration of the neurotoxins capsaicin, 6-hydroxydopamine and 5,7-dihydroxytryptamine

The effects of intrathecal pretreatment with the neurotoxins capsaicin, 6-hydroxydopamine and 5,7-dihydroxytryptamine on spinal antinociception by adenosine analogs (NECA, 5'-N-ethylcarboxamido adenosine and CHA, N6-cyclohexyl adenosine) and morphine were examined using the rat tail flick and hot plate tests. Pretreatment with 50 micrograms capsaicin for 7 to 11 days (which reduced substance P immunoreactivity in the superficial layers of the dorsal spinal cord) produced a slight increase in the action of NECA and CHA, and reduced the action on morphine in the hot plate test but not in the tail flick test. Pretreatment with 50 to 100 micrograms 6-hydroxydopamine for 7 to 14 days (which reduced spinal cord noradrenaline levels by 54-65%) reduced spinal antinociception by NECA and CHA but not that by morphine. Pretreatment with 50 micrograms 5,7-dihydroxytryptamine (which reduced spinal cord serotonin levels by 74-89%) had no effect on any agent. Acute pretreatment with 7.5-30 micrograms phentolamine reduced the spinal antinociceptive action of noradrenaline, NECA and CHA, primarily in the hot plate test. Phentolamine (30 micrograms) also reduced the action of morphine (hot plate greater than tail flick), but did not affect the action of L-baclofen. These results suggest that spinal antinociception by adenosine analogs: 1) occurs primarily at a postsynaptic site of action (capsaicin results), and 2) is dependent on release of endogenous noradrenaline and activation of spinal adrenergic receptors (6-hydroxydopamine and phentolamine results). The reduction in the effect of morphine by capsaicin (removes a source of adenosine release) and phentolamine (antagonizes the action of endogenously released adenosine) can be explained in terms of the adenosine release hypothesis of morphine action within the spinal cord.     

Tolerance to repeated microinjection of morphine into the periaqueductal gray is associated with changes in the behavior of off- and on-cells in the rostral ventromedial medulla of rats

Although the administration of opioids is the most effective treatment for pain, their efficacy is limited by the development of tolerance. The midbrain periaqueductal gray matter (PAG) participates in opioid analgesia and tolerance. Microinjection of morphine into PAG produces antinociception, probably through neurons in the rostral ventromedial medulla (RVM), namely through the activation of off-cells, which inhibit nociception, and the inhibition of on-cells, which facilitate nociception. After its repeated microinjection into the PAG morphine loses effectiveness. The present study sought to determine whether tolerance to PAG morphine administration is associated with changes in the behavior of RVM neurons. Morphine (0.5 microg/0.4 microl) or saline (0.4 microl) was microinjected into the ventrolateral PAG twice daily. Initially morphine caused a latency increase in the hot plate test (antinociception) but this effect disappeared by day 3 (tolerance). On day 4, each rat was anesthetized with halothane and recordings were made from off- and on-cells in the RVM, i.e. from neurons that decrease or increase their firing, respectively, just before a heat-elicited tail flick. In contrast to saline-pretreated rats, PAG microinjection of morphine in tolerant animals did not change the baseline activity of off- or on-cells, did not prevent the off-cell pause or the on-cell activation upon tail heating, and did not lengthen the tail flick latency. However, microinjection of kainic acid into the PAG (1) caused off-cells to become continuously active and on-cells to become silent, and (2) prevented the tail flick, i.e. exactly what morphine did before tolerance developed. These results demonstrate a correspondence between neuronal and behavioral measures of tolerance to PAG opioid administration, and suggest that tolerance is mediated by a change in opioid-sensitive neurons within the PAG.     

New insights on relaxant effects of (—)‐borneol monoterpene in rat aortic rings

The monoterpene alcohol (?)‐borneol has many biological effects such as sedative, anti‐inflammatory, analgesic, anti‐nociceptive, antithrombotic and vasorelaxant effects. Our objective in this study was to investigate the mechanism of action of (?)‐borneol and determine its vasorelaxant effect. (?)‐Borneol was tested on isolated aortic rings contracted with PE (10^?6 m ). This study was performed in the absence or in the presence of endothelium, L‐NAME (100 μm ), indomethacin (10 μm ), TEA (1 and 10 mm ), 4‐AP (1 mm ) or glibenclamide (1 mm ) to assess the participation of EDRF, nitric oxide, prostanoids and potassium channels on the relaxing effect of (?)‐borneol. In this work, (?)‐borneol induced a relaxant effect in aortic rings, with and without endothelium, in a concentration‐dependent manner. The pharmacological characterization obtained using L‐NAME, indomethacin, TEA, 4‐AP and glibenclamide demonstrates that the effect of (?)‐borneol was modified in the presence of L‐NAME, indomethacin and glibenclamide showing that these signal transduction pathways are involved in the relaxing effect of the monoterpene. (?)‐Borneol has a vasorelaxant effect that depends on the presence of vascular endothelium, with the participation of nitric oxide and prostanoids. Also, (?)‐borneol displayed a direct action on the vascular smooth muscle, greatly dependent on K_ATP channels.     

Mutations in the Kir6.2 subunit of the KATP channel and permanent neonatal diabetes: New insights and new treatment

Permanent neonatal diabetes (PNDM) is diagnosed in the first three months of life and is a major management problem as patients require lifelong insulin injections. Recently, activating mutations in the KCNJ11 gene which encodes the Kir6.2 subunit of the K_ATP channels in the pancreatic beta‐cells were found to be an important cause of PNDM. The mutated K_ATP channels do not close in the presence of adenosine triphosphate (ATP) so the beta‐cell membrane is hyperpolarized and insulin secretion does not occur. Some patients have DEND syndrome (developmental delay, epilepsy and neonatal diabetes) with the neurological features arising from mutated K_ATP channels in muscle, nerve and brain. Defining a genetic aetiology has not only given insights into clinical classification and disease mechanism, but has also influenced treatment. Sulphonylureas, by binding the sulphonylurea receptor, can close the K_ATP channel. This has led to patients who were insulin‐dependent being able to discontinue insulin injections and achieve excellent control with sulphonylurea tablets. In this article we discuss the work that established Kir6.2 mutations as a common cause of neonatal diabetes, the clinical features, the underlying mechanism and the impact on patient treatment.     

Reopening of ATP-sensitive potassium channels reduces neuropathic pain and regulates astroglial gap junctions in the rat spinal cord

Adenosine triphosphate–sensitive potassium (K_ATP) channels are suggested to be involved in pathogenesis of neuropathic pain, but remain underinvestigated in primary afferents and in the spinal cord. We examined alterations of K_ATP channels in rat spinal cord and tested whether and how they could contribute to neuropathic pain. The results showed that protein expression for K_ATP channel subunits SUR1, SUR2, and Kir6.1, but not Kir6.2, were significantly downregulated and associated with thermal hyperalgesia and mechanical allodynia after sciatic nerve injury. Spinal administration of a K_ATP channel opener cromakalim (CRO, 5, 10, and 20 μg, respectively) prevented or suppressed, in a dose-dependent manner, the hyperalgesia and allodynia. Nerve injury also significantly increased expression and phosphorylation of connexin 43, an astroglial gap junction protein. Such an increase of phosphorylation of connexin 43 was inhibited by CRO treatment. Furthermore, preadministration of an astroglial gap junction decoupler carbenoxolone (10 μg) completely reversed the inhibitory effects of CRO treatment on the hyperalgesia and allodynia and phosphorylation of NR1 and NR2B receptors and the subsequent activation of Ca²⁺-dependent signals Ca²⁺/calmodulin-dependent kinase II and cyclic adenosine monophosphate (cAMP) response element binding protein. These findings suggest that nerve injury–induced downregulation of the K_ATP channels in the spinal cord may interrupt the astroglial gap junctional function and contribute to neuropathic pain, thus the K_ATP channels opener can reduce neuropathic pain probably partly via regulating the astroglial gap junctions. This study may provide a new strategy for treating neuropathic pain using K_ATP channel openers in the clinic.     

Disruption of KATP Channel Expression in Skeletal Muscle by Targeted Oligonucleotide Delivery Promotes Activity-linked Thermogenesis

Despite the medical, social, and economic impact of obesity, only a few therapeutic options, focused largely on reducing caloric intake, are currently available and these have limited success rates. A major impediment is that any challenge by caloric restriction is counterbalanced by activation of systems that conserve energy to prevent body weight loss. Therefore, targeting energy-conserving mechanisms to promote energy expenditure is an attractive strategy for obesity treatment. Here, in order to suppress muscle energy efficiency, we target sarcolemmal ATP-sensitive potassium (K_ATP) channels which have previously been shown to be important in maintaining muscle energy economy. Specifically, we employ intramuscular injections of cell-penetrating vivo-morpholinos to prevent translation of the channel pore-forming subunit. This intervention results in significant reduction of K_ATP channel expression and function in treated areas, without affecting the channel expression in nontargeted tissues. Furthermore, suppression of K_ATP channel function in a group of hind limb muscles causes a substantial increase in activity-related energy consumption, with little effect on exercise tolerance. These findings establish a proof-of-principle that selective skeletal muscle targeting of sarcolemmal K_ATP channel function is possible and that this intervention can alter overall bodily energetics without a disabling impact on muscle mechanical function.     

Assessment of mechanisms involved in antinociception caused by sesquiterpene polygodial

Polygodial, a sesquiterpene isolated from the bark of Drymis winteri given systemically, intraplantarly, or by spinal or supraspinal sites, produced antinociception when assessed in both phases of the formalin test and against capsaicin-induced pain. Polygodial, even at high doses, had no antinociceptive or antihyperalgesic effect when assessed in hot-plate assay or in glutamate-induced hyperalgesia, nor did it significantly interfere with the motor coordination of animals when tested in the rota-rod test. The polygodial antinociception assessed in the formalin test was not affected by i.p. treatment of animals with cyprodime, yohimbine, phaclofen, bicuculine, or nitric oxide precursor or by intrathecal administration of potassium channel blockers such as apamin, charybdotoxin, glibenclamide, or tetraethylammonium. In contrast, polygodial antinociception was significantly attenuated by i.p. treatment of animals with naloxone, naltrindole, 2-(3, 4-dichlorophenyl)-n-methyl-n-[(1S)-1-(3-isothiocynatophenyl)-2-(1- pry rolidinyl)ethyl]acetamide, p-chlorophenylalanine, prazosin, or by i. c.v. treatment with pertussis toxin. In addition, polygodial antinociception was not cross-tolerant to morphine, nor was its effect affected by the adrenalectomy of animals. Together, these results show that polygodial produces pronounced systemic, spinal, and supraspinal antinociception in mice, mainly preventing the neurogenic pain produced by formalin and capsaicin. The mechanism by which polygodial produces antinociception seems likely to involve an interaction with the opioid system, mainly kappa and delta subtypes, depend on the activation of G(i/o) protein sensitive to pertussis toxin, alpha(1)-adrenoceptors, and the serotoninergic system. Collectively, these results suggest that polygodial itself or its derivatives may have potential therapeutic value for the development of new analgesic drugs.