Increases in extracellular potassium concentration during metabolic stress are thought to be mediated primarily by the ATP-sensitivepotassiumchannel. Anion-coupled potassium efflux, i.e. with lactate or phosphate, also occurs but is thought to be less important (Weiss et al. 1992). The experiments in which the ATP-sensitivepotassiumchannel agonists were infused are somewhat difficult to interpret. ATP-sensitivepotassium channels were activated first using pinacidil. Infusion of pinacidil into hearts showed there were no species differences in the effects it had on heart rate, ventricular pressure and aortic pressure, and that even at the highest concentrations of agonist (100 m mol l 21 ) less than a 20 % inhibition of heart rate was seen. A different agonist, Lemakalim, administered to another group of animals, gave rather different results. Heart rate decreased in both species in response to Lemakalim infusion but the decrease was significantly greater in the muskrat than the guinea pig. Lemakalim was more effective than pinacidil in the inhibition of heart rate for both muskrats and guinea pigs, with almost 40 % inhibition being seen at the highest concentrations (near the 10 24 mol l 21 level) of agonist tested. Less than 20 % inhibition of heart rate was achieved by this same concentration of pinacidil. One muskrat heart responded with a greater decline in heart rate at the five highest concentrations of infused Lemakalim, but the reason for this augmented response in this animal is not known. Guinea pigs responded similarly and consistently to both ATP-sensitivepotassiumchannel agonists with respect to heart rate but, as can be seen by the data points on Figs 4 and 5, there was considerable scatter in the muskrat heart rate data. This scatter was seen within animals. The reason for inconsistent effects on heart rate between the two drugs is not known. Considerable scatter was also observed in the measurement of ventricular pressure in both species, with some values being observed above control values at lower concentrations of Lemakalim. Again, the reason for this scatter and for the rise in ventricular pressure in response to Lemakalim infusion is unknown.
Fig. 2 Extracellular HSP70 triggers inflammatory response dependent on TLR4 in microglia. a, b Recombinant mouse HSP70 (100 ng/mL, 12 h) upregulated the phosphorylation of p38 MAPK and NF- κ B p65 in BV-2 cells. Cell extracts were collected and analyzed by western blot ( n = 3). c, d The levels of Il1b and Tnfa mRNAs in response to HSP70 under treatment of TLR4 antagonist or p38 inhibitor were assessed in BV-2 cells. Cells were pretreated with TLR4 antagonist (TAK242, 10 μ M) or p38 inhibitor (SB202190, 10 μ M) for 15 min, followed by recombinant mouse HSP70 (100 ng/mL) treatment. Then, cell extracts were collected 12 h after HSP70 treatment and analyzed by qPCR ( n = 3). e Recombinant mouse HSP70 (100 ng/mL, 12 h) increased the levels of pro-IL-1 β and NLRP3 in BV-2 cells. Cell extracts were collected and analyzed by western blot ( n = 3). f BV-2 cells were stimulated by recombinant mouse HSP70 (100 ng/mL) for 12 h, and then, the inflammasome was activated with 5 mM of ATP for 0.5 h, inducing the maturation of caspase-1 and IL-1 β . Supernatants of BV-2 cells were collected and analyzed by western blot ( n = 3). g, h Condi- tional medium collected from morphine-treated (200 μ M, 12 h) SH-SY5Y cells incubated BV-2 cells for 12 h in presence of anti-HSP70 antibody (100 ng/mL) or normal IgM (100 ng/mL), and then, the cell extracts were collected and analyzed by qPCR ( n = 3). a, b, e, and f Data were ana- lyzed by Student ’ s t test. c, d, g, and h Data were analyzed by one-way ANOVA.* P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle, ##
Six novel, heterozygous missense mutations were identified in 10 of the 29 patients. In two patients the diabetes was familial, and in eight it arose from a spontaneous muta- tion. Their neonatal diabetes was characterized by ketoacidosis or marked hyperglyce- mia and was treated with insulin. Patients did not secrete insulin in response to glucose or glucagon but did secrete insulin in response to tolbutamide. Four of the patients also had severe developmental delay and muscle weakness; three of them also had epilepsy and mild dysmorphic features. When the most common mutation in Kir6.2 was coex- pressed with sulfonylurea receptor 1 in Xenopus laevis oocytes, the ability of ATP to block mutant K ATP channels was greatly reduced.
will have comparable ATP sensitivity to the channel contain- ing all wild-type subunits, so the resulting ATP sensitivity of heterozygous population is very close, but not identical, to that of a pure wild-type channel population. However, this small shift of ATP sensitivity in the heterozygous channel population leads to NDM for the following reasons. Under physiological conditions, intracellular concentration of ATP is in the range of 1–5 mM, such that K ATP channels exhibit very low activity. Additionally, the β -cell membrane possesses a high electrical resistance such that only a small reduction in ATP sensitivity to the channel results in a small increase in K ATPchannel activity that holds the β -cell membrane potential in a more polarized state and suppresses insulin secretion. 23
Oxidative stress was induced by rotenone. ROS generation was measured by 2′,7′-dichloro- fluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochon- drial membrane was also detected with the use of the potential-sensitive dye DiOC 6 (3,3′-dihexy- loxacarbocyanine iodide). Diazoxide, a selective activator of mitoK ATP , depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoK ATP blocker glyben- clamide effectively restored mitochondrial poten- tial in rat myocytes. Estimated <K 1/2 > value for diazoxide to mitoK ATP under normoxia was four times higher than under oxidative stress condi- tions: 5.01 ± 1.47∙10 -6 м and 1.24 ± 0.21 ∙10 -6 м
expected, the Kir6.0 subunit forms the ion conducting pore, although due to the fact that Kir6.0 subunits do not form fimctional channels when expressed alone (Inagaki et. al., 1995b) confirmation was only obtained by mutagenesis studies where site- directed mutations in residues important for inward rectification or potassium selectivity were shown to produce channels with altered rectification properties or non-fimctional channels (Shyng et. al., 1997a). Definitive proof was obtained when a truncated mutant of Kir6.2 where 36 C-terminal amino acids were removed was found to express current with the same conductance as the heteromeric channel (Tucker et. al., 1997). Furthermore, it was found that ATP"*" inhibited the potassium current carried by the C-terminal truncation mutant, without any requirement for Mg^^, suggesting that the inhibitory effect of ATP is independent o f ATP hydrolysis. Therefore, the site for ATP inhibition is present on the Kirô.O subunit. Further evidence was provided by studies showing that azido derivatives o f ATP directly bind to Kir6.2 (Tanabe et. al., 1999; Tanabe et. al., 2000.) and that mutations in Kir6.2 decrease ATP inhibition of the Kir6.2 C-terminal truncation mutant (Reimann et. al., 1999). The putative site for ATP binding on Kir6.2 is located on the C- terminus (Drain et. al., 1998), although there is evidence that the N-terminus is also involved in ATP-mediated inhibitory gating (Babenko et. al., 1999a; Roster et. al., 1999). The consensus is that ATP binding stabilises a closed state of the channel (Enkvetchakul et. al., 2000). The effect o f ATP on the channel also depends upon the Kirô.O subtype, for example co-expression o f Kir6.2 with SUR2B results in a
Modern medical studies found that this bioactive agent, extracted from the rhizome of a herb named Curcuma Longa Linn, exerted potent anti-cancer activity by inhi- biting cell proliferation in vitro and in vivo . The mechanisms involved in curcumin’s proliferation inhib- ition were thought to be complicated: multiple pathways and molecular mediators were proved relevant. It was believed that three canonical apoptotic pathways, namely the death receptor , mitochondrial , and endo- plasmic reticulum stress  pathways, were activated to induce apoptosis of cancer cells. Some transcriptional factor-related mechanisms, such as downregulation of TNF-induced nuclear factor κB mediated gene expres- sions  by curcumin were also indicated . A re- cent report of the involvement of mitoK ATP in regulating
ATPsensitivepotassiumchannel and the depolarization induced by hypoxia . PAH is a severe disease and ac- counts for most deaths of chronic obstructive pulmonary diseases, and remodeling of the pulmonary vessel wall contribute to the increased pulmonary vascular resistance. In the present study, we successfully established rat model of PAH. Values of mPAP significantly increased compared with controls. Results showed that pretreatment of rats with 5-HD could block hypoxia-induced increase of mPAP, the expression of smooth muscle-specific a-actin in pulmonary arteries, and alterations of PASMC ultrastruc- ture. The present study in vitro showed that hypoxia in- duced depolarization of Δψm and promote PASMCs proliferation, which was significantly inhibited by 5-HD treatment. In the current study, Changes in Δψm, as indi- cated by the intensity of R-123 fluorescence. It indicated that mitoK ATP channels was involved in and/or respon-
recording of the membrane patch revealed the existence of ATP-sensitivepotassiumchannel in activin-treated cells. These results indicate that activin A converts amylase- secreting AR42J cells to neuronlike cells. Given that pancreatic endocrine cells possess neuronlike properties and express ATP-sensitivepotassiumchannel as well as
Glucose modulates beta cell insulin secretion via effects on ATP-sensitivepotassium (KATP) channels. To test the hypothesis that glucose exerts a similar effect on neuronal function, local glucose availability was varied in awake rats using microdialysis in the substantia nigra, the brain region with the highest density of KATP channels. 10 mM glucose perfusion increased GABA release by 111 +/- 42%, whereas the sulfonylurea, glipizide, increased GABA release by 84 +/- 20%. In contrast, perfusion of the KATP channel activator, lemakalim, or depletion of ATP by perfusion of 2-deoxyglucose with oligomycin inhibited GABA release by 44 +/- 8 and 45 +/- 11%, respectively. Moreover, the inhibition of GABA release by 2-deoxyglucose and oligomycin was blocked by glipizide. During systemic insulin-induced hypoglycemia (1.8 +/- 0.3 mM), nigral dialysate GABA concentrations decreased by 49 +/- 4% whereas levels of dopamine in striatal dialysates increased by 119 +/- 18%. We conclude that both local and systemic glucose availability influences nigral GABA release via an effect on KATP channels and that inhibition of GABA release may in part mediate the hyperexcitability associated with hypoglycemia. These data support the hypothesis that glucose acts as a signaling molecule, and not simply as an energy-yielding fuel, for neurons.
observed during the short periods used to study acute renal preconditioning, thus post-operative stress may be a contributing factor. Further investigation is required to assess whether a delayed phase of '^remote organ" preconditioning exists. The use of the K^^-p channel blocker 5HD, n ow thought to be specific for mito-K^jp, channels, suggests a role for these particular channels in the adaptation of the myocardium to stress. H ow opening of these channels might lead to cardioprotection, however, rem ains unclear. Under normal conditions mito-K^^p channels function, together with the K^/H^ exchanger, to regulate flux and hence m itochondrial matrix volum e. Opening of mito-K^jp channels allows influx thus increasing matrix volum e. A delay occurs before the increased rate of influx is compensated for by the K^'/H^ exchanger resulting in a higher resting volum e . Fatty acid oxidation and respiration are known to be stimulated by increases in matrix volum e [216, 217]. The accumulation of fatty acids during ischaemia would inhibit opening of the mito-K^jp channels. Perhaps preconditioning over-rides this inhibition allow ing
1991; Thiemermann, 1997). In addition, Hall et al., (1996) found that the LPS- induced hyporeactivity to the a-adrenergic agonist, phenylephrine couid be fuliy reversed with tetraethylammonium (TEA), a non-selective channel inhibitor. Part of this effect is likely to be mediated by BKca channels, since iberiotoxin and charybdotoxin, both highly potent and selective inhibitors of these channels partially reversed the vascular hyporeactivity in the same tissue (Taguchi et al., 1996; Chen et al., 1999). This conclusion is further supported by electrophysiological studies in cultured vascular smooth muscle cells where application of L-arginine to LPS-treated cells resulted in persistent activation of BKca channels (Miyoshi and Nakaya, 1994). Interestingiy, only transient increases in BKca current were seen after application of an NO donor drug, suggesting that the inducible NO pathway may produce different biologfcal effects. Taken together, these observations imply that at least two, and possibly more, types of K* channel are activated during endotoxaemia.
currents have been reported for the reduced analgesic effectiveness of morphine (Cunha et al., 2010). Since diazoxide caused more decrease in analgesic effect of morphine, we propose that K+ATP channels in sen- sitized mice have been influenced by pretreatment of morphine. It has been reported that intracerebroven- tricular administrations of the K+ATPchannel block- ers antagonize the analgesic effects of opioids (Ocana, Del Pozo, Barrios & Baeyens, 1995; Ocana, Del Pozo, Barrios, Robles & Baeyens, 1990; Wild, Vanderah, Mosberg & Porreca, 1991). Our results also showed that diazoxide antagonized the analgesic effect of morphine in sensitized mice. Blockade of KATP chan- nels has been proposed to suppress the activation of descending noradrenergic system induced by i.c.v. injections of morphine (Narita et al., 1992). In this study, we examined the effects of K+ATP channels and L-type Ca 2+ channels in morphine-induced hyper-
Potassium channels are a diverse group of ion channels, which have a crucial role in the control of cell excitability. Cells in a resting state have an intracellular potassium ion (K+) concentration that is twenty five times higher than the external K+ concentration. When a potassiumchannel opens, an efflux of positive charge and an outward current results. The efflux of K+ is a mechanism to recover, maintain or enhance the resting membrane potential. The opening of K+ channels enables the membrane potential of the cell to move towards the equilibrium potential for potassium. The equilibrium potential for potassium can be defined as the membrane potential produced when the concentration of potassium ions across the membrane are in equilibrium.
the most abundant potassium channels and likely contri- butes to the resting membrane potential in smooth mus- cle tissues . The channel comprises heteromultimers of an inwardly rectifying K + channel (Kir) and a modula- tory sulphonylurea receptor subunit (SUR) which is responsible for the ATP sensitivity and pharmacological properties [7-10]. Functional studies indicated that K ATP * Correspondence: firstname.lastname@example.org
Involvement of the ATP-dependent potassiumchannel Kir6.1 in systemic circulation has been demonstrated in numerous studies. Kir6.1 expression is up-regulated in rats in LPS as well as in cecal ligation peritonitis (CLP) models [11, 19, 20]. This is not only restricted to rats but has been observed in mouse aortic smooth muscle cells and guinea pigs [6, 21]. Thus, increased Kir6.1 ex- pression is a specific pathophysiologic response to a broad inflammatory stimulus at least in rodents. Endo- toxin induced increase of Kir6.1 gene expression in rat mesenteric arteries contributes to systemic hypotension and can be restored by specific inhibition [6, 20].
Due to the difference between human and animal and/ or the stage of diabetes, the response of blood pressure is varied; changes of blood pressure in diabetic compli- cations seem not so simple. Numerous evidences sug- gest that diabetic heart is characterized by compromised ventricular contraction and prolonged relaxation attribu- table to multiple causative factors including calcium accumulation, oxidative stress and apoptosis. Previous study have demonstrated that blocking the calcium channel and oxidative stress could have advantage in diabetic heart . Another study also showed that hyperglycemia can cause systolic dysfunction and a
total volume of 30 m l. The reaction mixture was overlaid with 50 m l of mineral oil and incubated at 50 8 C for 16 h. The total amount of RNA in each sample was kept constant (100 m g) by adding the appropriate amount of yeast tRNA. The hybridization products were digested by adding 300 m l of an RNAase digestion mixture (300 mM NaCl, 10 mM Tris, 5 mM EDTA, 2 m g/ml RNAase T1, 40 m g/ml RNAase A, pH 7.4) and incubated for 1 h at 32 8 C. The mixture was then incu- bated for 30 min at 37 8 C with proteinase K (0.03 mg/ml) in the pres- ence of 0.6% SDS. The reaction was extracted with phenol/chloro- form and the RNAase-resistant products were precipitated by adding 1 m g of glycogen, 750 m l of ice-cold ethanol and incubating at 2 70 8 C for 1 h. The pellet was washed with 70% ethanol, dried at room tem- perature, and dissolved in formamide loading buffer (80% forma- mide, 10 mM EDTA, pH 8, 1 mg/ml xylene cyanol FF, 1 mg/ml bro- mophenol blue). The reaction products were size-fractionated by denaturing polyacrylamide (5%) gel electrophoresis (13). HaeIII di- gest fragments of l-bacteriophage were end-labeled with T4 polynu- cleotide kinase (Boehringer Mannheim, Indianapolis, IN) and [ 32 P] g ATP and used as size markers. The gel was dried and exposed
channel blocker glibenclamide (20 mg/kg i.v.) are presented in Table II. In the absence of glibenclamide, hindlimb contrac- tion significantly increased femoral blood flow velocity and vascular conductance. Glibenclamide increased baseline mean arterial pressure by 1464 mmHg (P , 0.05), but had no signif- icant effect on baseline femoral blood flow velocity or conduc- tance. In contrast, in these same rats, glibenclamide signifi- cantly attenuated the contraction-induced increases in femoral blood flow velocity (1151614% before vs. 193618% after, P , 0.05) and conductance (1148612% before vs. 182618% after, P , 0.05). The force produced by the contracting mus- cles was similar before and after glibenclamide (peak force Table I. Hemodynamic Responses to Infusion of Diazoxide