A Tale of Switched Functions: From Cyclooxygenase Inhibition to M-Channel Modulation in New Diphenylamine Derivatives

Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K+ channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K+ channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers

Statin or fibrate chronic treatment modifies the proteomic profile of rat skeletal muscle

Statins and fibrates can cause myopathy. To further understand the causes of the damage we performed a proteome analysis in fast-twitch skeletal muscle of rats chronically treated with different hypolipidemic drugs. The proteomic maps were obtained from extensor digitorum longus (EDL) muscles of rats treated for 2-months with 10 mg/kg atorvastatin, 20 mg/kg fluvastatin, 60 mg/kg fenofibrate and control rats. The proteins differentially expressed were identified by mass spectrometry and further analyzed by immunoblot analysis. We found a significant modification in 40 out of 417 total spots analyzed in atorvastatin treated rats, 15 out of 436 total spots in fluvastatin treated rats and 21 out of 439 total spots in fenofibrate treated rats in comparison to controls. All treatments induced a general tendency to a down-regulation of protein expression; in particular, atorvastatin affected the protein pattern more extensively with respect to the other treatments. Energy production systems, both oxidative and glycolytic enzymes and creatine kinase, were down-regulated following atorvastatin administration, whereas fenofibrate determined mostly alterations in glycolytic enzymes and creatine kinase, oxidative enzymes being relatively spared. Additionally, all treatments resulted in some modifications of proteins involved in cellular defenses against oxidative stress, such as heat shock proteins, and of myofibrillar proteins. These results were confirmed by immunoblot analysis. In conclusions, the proteomic analysis showed that either statin or fibrate administration can modify the expression of proteins essential for skeletal muscle function suggesting potential mechanisms for statin myopathy

Emerging role of calcium-activated potassium channel in the regulation of cell viability following potassium ions challenge in HEK293 cell and pharmacological modulation

Emerging evidences suggest that Ca(2+)activated-K(+)-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10(-11)-10(-3)M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2 x 10(-7)M-5 x 10(-3)M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the cell proliferation induced by hyperkalemia. These findings may have relevance in disorders associated with abnormal K(+) ion homeostasis including periodic paralysis and myotonia

Dual response of the KATP channels to staurosporine: a novel role of SUR2B, SUR1 and Kir6.2 subunits in the regulation of the atrophy in different skeletal muscle phenotypes

We investigated on the role of the genes encoding for the ATP-sensitive K+-channel(KATP) subunits(SUR1-2A/B, Kir6.2) in the atrophy induced "in vitro" by staurosporine (STS) in different skeletal muscle phenotypes of mouse. Patch-clamp and gene expression experiments showed that the expression/activity of the sarcolemma KATP channel subunits was higher in the fast-twitch than in the slow-twitch fibers. After 1 to 3h of incubation time, the STS(2.14×10-6M) treatment enhanced the expression/activity of the SUR2B, SUR1 and Kir6.2 subunit genes, but not SUR2A, in the slow-twitch muscle fibers, induced the caspase-3-9, Atrogin-1 and Murf-1 gene expression without affecting protein content. After 3 to 6h, the STS-related atrophy markedly down-regulated the SUR2B, SUR1 and Kir6.2 genes reducing the KATP currents and reduced the protein content/muscle weight ratio of the slow-twitch muscle by -36.4±6%(p<0.05). After 6 to 24h, no additional changes of the SUR1-2B and Kir6.2 gene expression and muscle protein were observed. In the fast-twitch muscles, STS mildly affected the atrophic genes and protein content, but potentiated the KATP currents down-regulating the Bnip-3 gene. Diazoxide(250-500×10-6M), a SUR1-2B/Kir6.2 channel opener, prevented the protein loss induced by STS in the slow-twitch muscle after 6h showing an EC50 of 1.35×10-7M and Emax of 75%, down-regulated the caspase-9 gene and enhanced the KATP currents. The enhanced expression/activity of the SUR2B, SUR1 and Kir6.2 genes are cytoprotective against STS-induced atrophy in the slow-twitch muscle; their reduced expression/activity is associated with proteolysis and atrophy in skeletal muscl

Statins and fenofibrate affect skeletal muscle chloride conductance in rats by differently impairing ClC-1 channel regulation and expression

Background and purpose: Statins and fibrates can produce mild to life-threatening skeletal muscle damage. Resting chloride channel conductance (gCl), carried by the ClC-1 channel, is reduced in muscles of rats chronically treated with fluvastatin, atorvastatin or fenofibrate, along with increased resting cytosolic calcium in statin-treated rats. A high gCl, controlled by the Ca2+-dependent protein kinase C (PKC), maintains sarcolemma electrical stability and its reduction alters muscle function. Here, we investigated how statins and fenofibrate impaired gCl. Experimental approach: In rats treated with fluvastatin, atorvastatin or fenofibrate, we examined the involvement of PKC in gCl reduction by the two intracellular microelectrodes technique and ClC-1 mRNA level by quantitative real time-polymerase chain reaction. Direct drug effects were tested by patch clamp analysis on human ClC-1 channels expressed in human embryonic kidney (HEK) 293 cells. Key results: Chelerythrine, a PKC inhibitor, applied in vitro on muscle dissected from atorvastatin-treated rats fully restored gCl, suggesting the involvement of this enzyme in statin action. Chelerythrine partially restored gCl in muscles from fluvastatin-treated rats but not in those from fenofibrate-treated rats, implying additional mechanisms for gCl impairment. Accordingly, a decrease of ClC-1 channel mRNA was found in both fluvastatin- and fenofibrate-treated rat muscles. Fenofibric acid, the in vivo metabolite of fenofibrate, but not fluvastatin, rapidly reduced chloride currents in HEK 293 cells. Conclusions and implications: Our data suggest multiple mechanisms underlie the effect of statins and fenofibrate on ClC-1 channel conductance. While statins promote Ca2+-mediated PKC activation, fenofibrate directly inhibits ClC-1 channels and both fluvastatin and fenofibrate impair expression of mRNA for ClC-1

Pharmacological evaluation of NADPH oxidase involvement in pathophysiology of mdx mouse, an animal model of muscular dystrophy

Oxidative stress, caused by reactive oxygen species (ROS), has been implicated on disease progression and chronic inflammation in Duchenne Muscular Dystrophy (DMD). NADPH oxidase 2 (NOX2) is currently considered to be a major source of ROS and it is over-expressed in skeletal muscle and heart of mdx mice, the most widely used model for DMD. Its activation in mdx myofibers via stretch-sensitive pathways has also been shown (Whitehead et al., 2010; Khairallah et al., 2012). Consequently, drugs able to reduce ROS production by inhibition of NOX-2 are potential treatment for muscular dystrophy. In line with this view, we have recently shown that enalapril, by inhibiting the production of angiotensin II (Ang II), one of the main endogenous activator of NOX, reduces the signs of oxidative stress and the percentage of p65-NF-kB positive nuclei in the mdx muscles. In this frame we also observed, in the myofibers of mdx mice treated with enalapril, a dose-dependent restoration of macroscopic chloride conductance (gCl), a sensitive biomarker of inflammation in skeletal muscle (Cozzoli et al., 2011). The aim of the present study was to investigate the involvement of NOX-2 dependent-ROS production in relation to the aberrant mechano-transduction occurring in dystrophic muscle. RT-PCR experiments confirmed a higher expression of β-tubulin and NOX2 (gp91phox) mRNA in gastrocnemius (GC) muscle of mdx mice. Interestingly, this increased expression was maintained in GC muscles of mdx mice that underwent a standard chronic (1-2 months) exercise protocol on treadmill. Then, we tested the effect of a chronic treatment with apocynin (38 mg/kg in drinking water/day for 5-9 weeks), a natural compound able to directly inhibit NOX-2, on exercised mdx mice (De Luca et al., 2003). Treatment started at 4-5 weeks of age and the outcome was evaluated by a multidisciplinary approach on pathology-related in vivo and ex vivo endpoints. In vivo, apocynin significantly increased mouse strength, with normalized forelimb force values of 6.4 ± 0.16 (n=9) vs. 5.6 ± 0.19 (n=10; p<0.05) of untreated mice, but did not improve exercise performance. Furthermore, no effect was observed on plasma creatine kinase and lactate dehydrogenase. However, the treatment with apocynin counteracted the exercise-induced impairment of total membrane conductance (gm), which is mainly sustained by the reduction of gCl, in extensor digitorum longus (EDL) muscle fibers, gm being 2536 ±105 µS/cm2 (n =37) vs. 1886 ± 92 µS/cm2 (n = 42, p <0.0001) of untreated mdx myofibers. Then the recovery score for this parameter, considering the value of 2607 ± 23 µS/cm2 (n =19) of wild-type C57BL10 myofibers, was 90% in apocynin-treated myofibers. This latter effect prompted us to investigate the possibility that the channels underlying macroscopic gCl could be target of the redox-dependent NOX actions in skeletal muscle. Parallel experiments on EDL muscle fibers of C57BL10 mouse showed that Ang II decreases gCl in a concentration-dependent manner (IC50 = 60nM) and this effect was fully contrasted by the prior incubation of apocynin (10 µM) or with a known anti-oxidant N-acetyl cysteine (5 mM). Markers of oxidative stress and inflammation, RT-PCR, histo-morphology are currently under evaluation. However, this preliminary data support the hypothesis that pharmacological targeting of NOX-2, providing protection from cross-talk between ROS production and inflammation, may represent a valuable approach in DMD. (Supported by DPP/NL and MIUR-PRIN n° 20108YB5W3). Whitehead et al. (2010). PLoS One, 5(12):e15354. Khairallah et al. (2012). Sci Signal.5(236):ra56. Cozzoli et al. (2011). Pharmacol Res. 64(5):482-92. De Luca et al. (2003) J Pharmacol Exp Ther.304(1):453-63