KATP Channel Activity Research Articles

Effect of fenofibrate and selective PPARα modulator (SPPARMα), pemafibrate on KATP channel activity and insulin secretion

ObjectiveInsulin secretion is regulated by ATP-sensitive potassium (KATP) channels in pancreatic beta-cells. Peroxisome proliferator-activated receptors (PPAR) α ligands are clinically used to treat dyslipidemia. A PPARα ligand, fenofibrate, and PPARγ ligands troglitazone and 15-deoxy-∆12,14-prostaglandin J2 are known to close KATP channels and induce insulin secretion. The recently developed PPARα ligand, pemafibrate, became a new entry for treating dyslipidemia. Because pemafibrate is reported to improve glucose intolerance in mice treated with a high fat diet and a novel selective PPARα modulator, it may affect KATP channels or insulin secretion.ResultsThe effect of fenofibrate (100 µM) and pemafibrate (100 µM) on insulin secretion from MIN6 cells was measured by using batch incubation for 10 and 60 min in low (2 mM) and high (10 mM) glucose conditions. The application of fenofibrate for 10 min significantly increased insulin secretion in low glucose conditions. Pemafibrate failed to increase insulin secretion in all of the conditions experimented in this study. The KATP channel activity was measured by using whole-cell patch clamp technique. Although fenofibrate (100 µM) reduced the KATP channel current, the same concentration of pemafibrate had no effect. Both fenofibrate and pemafibrate had no effect on insulin mRNA expression.

The effect of protocatechuic acid on neuropathic pain and possible mechanism.

The goal of the research is to investigate the protocatechuic acid (PCA) potential action, a phenolic acid derivative, on pain induced by neuropathy and to determine its efficacy on activation of KATP type channels and A1 receptors. Neuropathic pain by cause of sciatic nerve damage was induced in Sprague-Dawley rats. Anti-allodynic and anti-hyperalgesic effects were evaluated with von Frey apparatus and Hargreave's plantar test apparatus, respectively. The effects of PCA at the doses of 75, 150 and 300 mg/kg, carbamazepine at the doses of 50 and 100 mg/kg, combination of low effective doses of PCA and carbamazepine were tested. Pretreatments 3 μg/kg DPCPX as adenosine A1 receptor antagonist and 60.7 nmol glibenclamide as KATP channel blocker were applied for mechanistic studies. PCA showed anti-allodynic and anti-hyperalgesic effects without impairing locomotor activity. In addition, the combination treatment was found to be more effective than the separate individual treatments of drugs. KATP channel activation related with A1 receptor stimulation makes a significant contribution to the anti-allodynia and anti-hyperalgesia induced by PCA. It can be said that PCA has similar effects with carbamazepine, which is used in clinical practice, and that PCA can take place as an adjuvant drug in neuropathic pain with the combination group. In addition, it is seen that the undesirable effects that drugs can cause alone can be avoided and a more effective treatment potential can be created with multiple mechanisms.

ATP-sensitive inward rectifier potassium channels regulate secretion of pro-feeding salivary proteins in the lone star tick (Amblyomma americanum)

Understanding the physiological and molecular regulation of tick feeding is necessary for developing intervention strategies to curb disease transmission by ticks. Pharmacological activation of ATP-gated inward rectifier potassium (KATP) channels reduced fluid secretion from isolated salivary gland and blood feeding in the lone star tick, Amblyomma americanum, yet the temporal expression pattern of KATP channel proteins remained unknown. KATP channels were highly expressed in type II and III acini in off-host stage and early feeding phase ticks, yet expression was reduced in later stages of feeding. We next assessed KATP channel regulation of the secreted proteome of tick saliva. LC-MS/MS analysis identified 40 differentially secreted tick saliva proteins after exposure to KATP activators or inhibitors. Secretion of previously validated tick saliva proteins that promote tick feeding, AV422, AAS27, and AAS41 were significantly reduced by upwards of 8 log units in ticks exposed to KATP channel activators when compared to untreated ticks. Importantly, activation of KATP channels inhibited tick feeding and vice versa for KATP channel inhibitors. Data indicate KATP channels regulate tick feeding biology by controlling secretion of pro-feeding proteins that are essential during early feeding phases, which provides insights into physiological and molecular regulation of tick feeding behavior.

Rapid Characterization of the Functional and Pharmacological Consequences of Cantú Syndrome KATP Channel Mutations in Intact Cells.

Gain-of-function of KATP channels, resulting from mutations in either KCNJ8 (encoding inward rectifier sub-family 6 [Kir6.1]) or ABCC9 (encoding sulphonylurea receptor [SUR2]), cause Cantú syndrome (CS), a channelopathy characterized by excess hair growth, coarse facial appearance, cardiomegaly, and lymphedema. Here, we established a pipeline for rapid analysis of CS mutation consequences in Landing pad HEK 293 cell lines stably expressing wild type (WT) and mutant human Kir6.1 and SUR2B. Thallium-influx and cell membrane potential, reported by fluorescent Tl-sensitive Fluozin-2 and voltage-sensitive bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) dyes, respectively, were used to assess channel activity. In the Tl-influx assay, CS-associated Kir6.1 mutations increased sensitivity to the ATP-sensitive potassium (KATP) channel activator, pinacidil, but there was strikingly little effect of pinacidil for any SUR2B mutations, reflecting unexpected differences in the molecular mechanisms of Kir6.1 versus SUR2B mutations. Compared with the Tl-influx assay, the DiBAC4(3) assay presents more significant signal changes in response to subtle KATP channel activity changes, and all CS mutants (both Kir6.1 and SUR2B), but not WT channels, caused marked hyperpolarization, demonstrating that all mutants were activated under ambient conditions in intact cells. Most SUR2 CS mutations were markedly inhibited by <100 nM glibenclamide, but sensitivity to inhibition by glibenclamide, repaglinide, and PNU37883A was markedly reduced for Kir6.1 CS mutations. Understanding functional consequences of mutations can help with disease diagnosis and treatment. The analysis pipeline we have developed has the potential to rapidly identify mutational consequences, aiding future CS diagnosis, drug discovery, and individualization of treatment. SIGNIFICANCE STATEMENT: We have developed new fluorescence-based assays of channel activities and drug sensitivities of Cantú syndrome (CS) mutations in human Kir6.1/SUR2B-dependent KATP channels, showing that Kir6.1 mutations increase sensitivity to potassium channel openers, while SUR2B mutations markedly reduce K channel opener (KCO) sensitivity. However, both Kir6.1 and SUR2B CS mutations are both more hyperpolarized than WT cells under basal conditions, confirming pathophysiologically relevant gain-of-function, validating DiBAC4(3) fluorescence to characterize hyperpolarization induced by KATP channel activity under basal, non KCO-activated conditions.

Dosage of the pseudoautosomal gene SLC25A6 is implicated in QTc interval duration

The genetic architecture of the QT interval, defined as the period from onset of depolarisation to completion of repolarisation of the ventricular myocardium, is incompletely understood. Only a minor part of the QT interval variation in the general population has been linked to autosomal variant loci. Altered X chromosome dosage in humans, as seen in sex chromosome aneuploidies such as Turner syndrome (TS) and Klinefelter syndrome (KS), is associated with altered QTc interval (heart rate corrected QT), indicating that genes, located in the pseudoautosomal region 1 of the X and Y chromosomes may contribute to QT interval variation. We investigate the dosage effect of the pseudoautosomal gene SLC25A6, encoding the membrane ADP/ATP translocase 3 in the inner mitochondrial membrane, on QTc interval duration. To this end we used human participants and in vivo zebrafish models. Analyses in humans, based on 44 patients with KS, 44 patients with TS, 59 male and 22 females, revealed a significant negative correlation between SLC25A6 expression level and QTc interval duration. Similarly, downregulation of slc25a6 in zebrafish increased QTc interval duration with pharmacological inhibition of KATP channels restoring the systolic duration, whereas overexpression of SLC25A6 shortened QTc, which was normalized by pharmacological activation of KATP channels. Our study demonstrate an inverse relationship between SLC25A6 dosage and QTc interval indicating that SLC25A6 contributes to QT interval variation.

A novel hydrogen sulfide donor reduces neuroinflammation and seizures by activating ATP-sensitive potassium channels

Epilepsy is a common neurological disorder worldwide. Hydrogen sulfide (H2S) has been found to have anti-seizure effects. However, its mechanism remains to be explored. In the present study, we showed that a novel H2S donor attenuated neuroinflammation by up-regulating ATP-sensitive potassium channel (KATP) expression to reduce seizures. The novel H2S donor significantly reduced the expression of TNF-α and increased the expression of IL-10 in LPS-treated BV2 cells and the hippocampus of pilocarpine-induced epileptic mice. The modulatory effects of the H2S donor on inflammatory cytokines were prevented by glibenclamide, a common KATP channels blocker. The H2S donor promoted the expression of KATP channel subunits SUR2 and Kir6.1 in LPS-treated BV2 cells and the hippocampus of pilocarpine-induced epileptic mice. In addition, the H2S donor reduced the electroencephalography amplitude of hippocampal epileptic waves and reduced seizures in pilocarpine-induced epileptic mice, which were also attenuated by glibenclamide. These results indicated that the novel H2S donor reduced seizures and regulated microglial inflammatory cytokines by activating KATP channels, which may provide a prospective therapeutic strategy for the anti-seizure effects of H2S donor.

ATP-gated potassium channels contribute to ketogenic diet-mediated analgesia in mice

Chronic pain is a substantial health burden and options for treating chronic pain remain minimally effective. Ketogenic diets are emerging as well-tolerated, effective therapeutic strategies in preclinical models of chronic pain, especially diabetic neuropathy. We tested whether a ketogenic diet is antinociceptive through ketone oxidation and related activation of ATP-gated potassium (KATP) channels in mice. We demonstrate that consumption of a ketogenic diet for one week reduced evoked nocifensive behaviors (licking, biting, lifting) following intraplantar injection of different noxious stimuli (methylglyoxal, cinnamaldehyde, capsaicin, or Yoda1) in mice. A ketogenic diet also decreased the expression of p-ERK, an indicator of neuronal activation in the spinal cord, following peripheral administration of these stimuli. Using a genetic mouse model with deficient ketone oxidation in peripheral sensory neurons, we demonstrate that protection against methylglyoxal-induced nociception by a ketogenic diet partially depends on ketone oxidation by peripheral neurons. Injection of tolbutamide, a KATP channel antagonist, prevented ketogenic diet-mediated antinociception following intraplantar capsaicin injection. Tolbutamide also restored the expression of spinal activation markers in ketogenic diet-fed, capsaicin-injected mice. Moreover, activation of KATP channels with the KATP channel agonist diazoxide reduced pain-like behaviors in capsaicin-injected, chow-fed mice, similar to the effects observed with a ketogenic diet. Diazoxide also reduced the number of p-ERK+ cells in capsaicin-injected mice. These data support a mechanism that includes neuronal ketone oxidation and activation of KATP channels to provide ketogenic diet-related analgesia. This study also identifies KATP channels as a new target to mimic the antinociceptive effects of a ketogenic diet.

Pulmonary Microcirculation in Experimental Model of Pulmonary Thromboembolism after Pretreatment with Chloroquine.

Changes of pulmonary microcirculation in response to pulmonary artery embolization after pretreatment with chloroquine were studied on the model of isolated perfused rabbit lungs. The increase in the pulmonary vascular resistance and pre- and postcapillary resistance was less pronounced than after pulmonary thromboembolism after pretreatment with mibefradil (T-type Ca2+ channels blocker) or nifedipine (L-type Ca2+ channels blocker). The shifts of capillary filtration coefficient correlated with changes in the precapillary resistance. When modeling pulmonary thromboembolism after pretreatment with chloroquine combined with glibenclamide (KATP channels blocker), the studied hemodynamics parameters increased to the same extent as after pretreatment with nifedipine. The results indicate that chloroquine exhibits the properties of an L- and T-type Ca2+ channels blocker and an activator of KATP channels.

Dose Dependent Effect of Sulfamethoxazole on Inhibiting KATP Channel of Mouse Pancreatic β Cell.

Sulfamethoxazole (SMX) is widely used as an antibiotic in the clinical application with side effects of hypoglycemia. This is because SMX contains the sulfonamide structure, which closes ATP-sensitive potassium (KATP) channels and induces insulin secretion. However, there are no detail reports that measure the effective dose that can close KATP channels and induce insulin secretion. In this study, whole-cell patch clamp recording was utilized to measure the effect of SMX on KATP channel activity on pancreatic β cells. Also, the static incubation assay with mice islets was assessed to measure the insulin secretion capacity of SMX. SMX was shown to inhibit the KATP channel in pancreatic β cell membrane and induce insulin secretion in relatively high concentration. The half maximal inhibitory concentration (IC50) for KATP channel activity of SMX was .46 ± .08mM. It was also shown that a near IC50 concentration of SMX (.5mM) was able to nearly fully block the KATP channel when simultaneously applied with low concentration sulfonylurea, tolbutamide (.01mM). Our present data provide important information for the clinical use of SMX to treat infection in diabetic patients using sulfonylureas.

Potassium ion channels as a molecular target to reduce virus infection and mortality of honey bee colonies

Declines in managed honey bee populations are multifactorial but closely associated with reduced virus immunocompetence and thus, mechanisms to enhance immune function are likely to reduce viral infection rates and increase colony viability. However, gaps in knowledge regarding physiological mechanisms or ‘druggable’ target sites to enhance bee immunocompetence has prevented therapeutics development to reduce virus infection. Our data bridge this knowledge gap by identifying ATP-sensitive inward rectifier potassium (KATP) channels as a pharmacologically tractable target for reducing virus-mediated mortality and viral replication in bees, as well as increasing an aspect of colony-level immunity. Bees infected with Israeli acute paralysis virus and provided KATP channel activators had similar mortality rates as uninfected bees. Furthermore, we show that generation of reactive oxygen species (ROS) and regulation of ROS concentrations through pharmacological activation of KATP channels can stimulate antiviral responses, highlighting a functional framework for physiological regulation of the bee immune system. Next, we tested the influence of pharmacological activation of KATP channels on infection of 6 viruses at the colony level in the field. Data strongly support that KATP channels are a field-relevant target site as colonies treated with pinacidil, a KATP channel activator, had reduced titers of seven bee-relevant viruses by up to 75-fold and reduced them to levels comparable to non-inoculated colonies. Together, these data indicate a functional linkage between KATP channels, ROS, and antiviral defense mechanisms in bees and define a toxicologically relevant pathway that can be used for novel therapeutics development to enhance bee health and colony sustainability in the field.

1539-P: Effects of Free Fatty Acids (FFA) on Central Regulation of Glucose Metabolism

Activation of hypothalamic KATP channels reduces endogenous glucose production (EGP) in nondiabetic rodents and humans, but this response is lost in T2D. Lowering FFA with nicotinic acid (NA) restored central regulation of EGP in T2D subjects (Diabetes 2019; 1799P). High FFA levels, typical of T2D, may increase endoplasmic reticulum (ER) stress and thereby impair hypothalamic control of EGP. To explore how increased FFA might impact central regulation of EGP, we harvested hypothalamic wedges from n=10 Zucker Diabetic Fatty (ZDF) rats (glucose ~ 370 mg/dL) following infusion of NA or saline (Alzet minipump) for 18 h. Nondiabetic rats (n=5) were studied as controls. Gene expression of KATP channel subunit Kir6.2 was decreased in ZDF rats. Upon lowering FFA with NA, hypothalamic expression of this major subunit was restored (Fig 1a). Conversely, increased expression of genes regulating ER stress was reversed by NA (Fig 1b). Directly treating ZDF rat brain slices with NA did not alter the expression of these genes, suggesting that NA's effects were mediated by lowering FFA (Fig 1c). In conclusion, hypothalamic effects of elevated FFA include reduced KATPchannel expression and increased expression of ER stress genes, both of which may contribute reversibly to the loss of central regulation of EGP in T2D. These findings are consistent with our observations in humans noted above, suggesting that FFA could be a therapeutic target in T2D. Disclosure M. Nogueira Cordeiro: None. V.H. Routh: None. A. Manavalan: None. P.M. Mathias: None. C.E. Boyle: None. A.C. Rios Chen: None. N. Jiang: None. N. Friedman: None. Y. Demirhan: None. M. Hawkins: None. K. Zhang: None. Funding American Diabetes Association (7-08-CR-28 to M.H.); National Institutes of Health (R01DK069861); Diabetes Action Research and Education Foundation

1772-P: Alpha-Cell Heterogeneity Contributes to Phasic Glucagon Release

Glucagon, produced by α-cells of pancreatic islets, is one of the body’s primary tools for elevating blood glucose during hypoglycemia. The exact mechanism linking hypoglycemia to glucagon release by islet α-cells is, however, unclear; we therefore aimed to investigate it using optogenetics. We generated mice harboring the light-activated ion channel channelrhodopsin-2 (ChR2) specifically in α-cells and imaged the effect of optically induced depolarization of plasma membrane (10-ms 470-nm light pulses at 20Hz) at the level of membrane potential, cytosolic Ca2+ ([Ca2+]i) and ATP/ADP ratio. At 1 mmol/L glucose, optostimulation depolarized the electrically active α-cells from -76mV to -8mV, induced a transient surge in [Ca2+]i and elevated glucagon secretion by 200%. In contrast, the glucagonostatic effect of high glucose (&amp;gt;6 mmol/L) was mitigated by optostimulation of α-cells. At 1 mmol/L glucose, the stimulatory effects were abolished by the KATP channel activator diazoxide (100μmol/L) and L-type Ca2+ channel blocker isradipine (10μmol/L). Notably, KATP channel inhibitor tolbutamide (100μmol/L) induced electrical activity in the fraction of α-cells insensitive to low glucose (35%). As stimulation of electrical activity persisted significantly beyond optoactivation, we probed the interplay between electrical activity and energy metabolism. The depolarizing stimulus induced by a cocktail of amino acids ((AA) 2 mmol/L of alanine + arginine +glutamine) or a moderate increase of extracellular K+ (from 4 to 15 mmol/L) reduced ATP/ADP in α-cells exhibiting spontaneous Ca2+ oscillations at low glucose. Surprisingly, in the population of inactive α-cells, the stimulation increased ATP/ADP ratio. These data indicate that α-cells are functionally heterogeneous due to metabolic differences. The observed secretory responses reflect the combination of stimulatory and inhibitory effects in electrically active and silent cells, which may contribute to the phasic nature of AA-induced glucagon secretion. Disclosure C.A.Miranda: None. H.Dou: None. J.Tolö: None. A.I.Tarasov: None. P.Rorsman: None. Funding Svenska Sällskapet för Medicinsk Forskning (PD20-0056); Swedish Vetenskap Rådet

1540-P: Diazoxide (DZ) May Lower Endogenous Glucose Production (EGP) via Mitochondrial ATP-Sensitive K+ (mt KATP) Channels

Rodent and human studies suggest that ventromedial hypothalamus (VMH) KATP channels suppress EGP. The VMH possesses glucose-sensing neurons that are either excited (GE) or inhibited (GI) by glucose. Since administration of the KATP channel activator DZ reduces EGP in rodents and humans, we examined the impact of DZ on VMH glucose sensing neurons. DZ opens both plasma membrane and mt KATP channels; the former inhibits neurons while the latter maintains mitochondrial metabolic function. We hypothesize that the concentration of brain DZ that lowers EGP affects the mt and not the plasma membrane KATP channel of VMH glucose sensing neurons. VMH glucose-excited (GE) neurons were exposed to 0.1 µM DZ in 2.5 mM glucose for 20 minutes prior to lowering glucose to 0.1 mM in the presence of DZ. DZ did not alter membrane potential (MP; 2.5 glucose: −63 ± 4 mV; DZ: −64 ± 5 mV, P = 0.3807, N = 7) or cell resistance (R; 2.5 glucose: 658 ± 136 MΩ; DZ 656 ± 146 MΩ, P = 0.9526, N = 7) in 2.5 mM glucose. There was also no difference in the MP or R response to lowering glucose from 2.5 to 0.1 mM in the presence or absence of DZ (% change MP control: −20.8 ± 3, diazoxide: −19.9 ± 3, P = 0.9044, N = 5; R control: −59.0 ± 5, diazoxide: −55.3 ± 6, P = 0.7134, N = 5). Similar results were seen with VMH glucose-inhibited neurons (N=4; Fig. 1). Our results are consistent with an action of DZ on mt but not plasma membrane KATP channels of VMH glucose sensing neurons. Disclosure G.Vail: None. V.Patel: None. P.Sarkar: None. K.Zhang: None. M.Hawkins: None. V.H.Routh: None. Funding National Institutes of Health (2R01DK069681-11)

Attenuation of ST-segment elevation by ischemic preconditioning: Reflection of cardioprotection in Göttingen but not in Ossabaw minipigs

BackgroundIschemic preconditioning (IPC; brief cycles of coronary occlusion/ reperfusion) reduces myocardial infarct size. The ST-segment elevation during coronary occlusion is progressively attenuated with increasing number of IPC cycles. Progressive attenuation of ST-segment elevation is considered a result of sarcolemmal KATP channel activation and has been considered to reflect and predict IPC's cardioprotection. We have recently demonstrated that IPC failed to reduce infarct size in minipigs of a particular strain (Ossabaw), which had a genetic predisposition to develop, but not yet established a metabolic syndrome. To determine whether or not Ossabaw minipigs nevertheless had attenuated ST-segment elevation over repetitive IPC cycles, we compared Göttingen vs. Ossabaw minipigs in which IPC reduces infarct size. Methods and resultsWe analyzed surface chest electrocardiographic (ECG) recordings of anesthetized open-chest contemporary Göttingen (n = 43) and Ossabaw minipigs (n = 53). Both minipig strains were subjected to 60 min coronary occlusion and 180 min reperfusion without or with IPC (3 × 5 min/ 10 min coronary occlusion/ reperfusion). ST-segment elevations during the repetitive coronary occlusions were analyzed. In both minipig strains, IPC attenuated ST-segment elevation with increasing number of coronary occlusions. IPC reduced infarct size in Göttingen minipigs (45 ± 10% without vs. 25 ± 13% of area at risk with IPC), whereas such cardioprotection was absent in Ossabaw minipigs (54 ± 11% vs. 50 ± 11%). ConclusionApparently, the block of signal transduction of IPC in Ossabaw minipigs occurs distal to the sarcolemma, where KATP channel activation still attenuates ST-segment elevation as it does in Göttingen minipigs.

KATP channels are necessary for glucose-dependent increases in amyloid-β and Alzheimer's disease-related pathology.

Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-β (Aβ) release, offering a mechanistic link between type 2 diabetes and Alzheimer's disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits Kir6.2/KCNJ11 and SUR1/ABCC8 were expressed on excitatory and inhibitory neurons in the human brain, and cortical expression of KCNJ11 and ABCC8 changed with AD pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aβ pathology in a potentially novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (i.e., amyloid precursor protein [APP]/PS1 Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemia-dependent increases in interstitial fluid levels of Aβ, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a potentially new role for Kir6.2-KATP channels in AD and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aβ pathology in patients with diabetes or prediabetes.

The Impact of T- and L-Type Calcium Channels Blockers on Pulmonary Microhemodynamics in Experimental Model of Pulmonary Thromboembolism

In acute experiments on isolated perfused rabbit’s Сhinchilla lungs changes of pulmonary microhemodynamics were studied in case of pulmonary embolization in the comparison group and after pretreatment with ethosuximide, mibefradil, chloroquine and nifedipine. In response to administration of T-type Ca2+-channel blockers ethosuximide and mibefradil, pulmonary artery pressure, precapillary and pulmonary vascular resistance decreased approximately to the same extent, postcapillary resistance did not change. After pretreatment with chloroquine pulmonary artery pressure, precapillary and pulmonary vascular resistance decreased more than in response to the administration of ethosuximide and mibefradil and the antagonist of L-type Ca2+-channels nifedipine; postcapillary resistance decreased. In the case of chloroquine administration combined with infusion of the KATP- channel blocker glibenclamide, most parameters of pulmonary microcirculation decreased almost to the same extent as in response to mibefradil administration, and postcapillary resistance did not change. After pretreatment with chloroquine in response to pulmonary embolization, pulmonary vascular resistance, pre- and postcapillary resistance increased less pronounced than with thromboembolism after pretreatment with ethosuximide, mibefradil and nifedipine. When modeling thromboembolism after pretreatment with chloroquine combined with KATP-channels blocker glibenclamide, the studied hemodynamics parameters increased to the same extent as after nifedipine pretreatment. Thus, chloroquine exhibits the properties of L- and T-type Ca2+-channels blocker, as well as an activator of KATP-channels, whereas ethosuximide has a blocking effect mainly on T-type Ca2+-channels of smooth muscle cells of pulmonary arterial vessels. Shifts of capillary filtration coefficient under these conditions depend more on changes of precapillary resistance than from the changes of permeability of endothelium of pulmonary vessels.

Recovery of the Functional Activity of K&lt;sub&gt;ATP&lt;/sub&gt;-Channels of Pial Arteries after Ischemia/Reperfusion Using Cell Therapy

This study aims to examine the efficiency of intravenous transplantation of human mesenchymal stem cells (hMSCs) performed 7 days after cerebral ischemia/reperfusion (I/R) for recovery of the functional activity of KATP-channels of cerebral arteries. Using a device for intravital visualization of pial vessels, the reaction of arteries to the KATP-channel blocker glibenclamide (GB), the activator of the same channels of pinacidil (PI), acetylcholine (ACh), and ACh against a background of GB action (ACh/GB) 14 and 21 days after I/R and intravenous hMSC transplantation performed 7 days after ischemic exposure. On exposure to GB 14 days after I/R, 1.5–1.8 times fewer arteries narrowed than in the sham–operated (SO) rats. By day 21 after I/R, the constriction reaction was completely restored, except for arteries with a diameter more 40 μm. In the cell–therapy group, the constrictor response to GB was completely recovered to the level of SO animals in arteries with a diameter less than 40 μm by 14 day after I/R exposure; in arteries with a diameter of more than 40 μm, the constriction reaction did not recover until 21 days. The number of dilations per ACh/GB compared to a clear ACh in SO rats was reduced in 1.6–1.8 times on 14 day after I/R and in 1.6–6.6 after 21 days. In I/R animals on 14 day, the number of dilatations per ACh/GB compared to clear ACh was significantly increased in arteries with a diameter of more than 20 μm by 1.5–1.7 times, and after 21 days in arteries with a diameter of more than 40 μm by 1.2 times. After the introduction of hMSC, GB blocked ACh–mediated dilation in arteries less than 40 μm in diameter both on days 14 and 21 after I/R. In arteries with a diameter of more than 40 μm the functional activity of KATP-channels did not recover until 21 days. Conclusion. I/R of the rat cerebral cortex reduces the contribution of KATP-channels to maintaining the basal tone of the pial arteries and almost completely excludes these channels from the formation of ACh–mediated dilation during 21 days of the postischemic period. Practically did not participate in the dilatory response. Intravenous transplantation of hMSC, performed 7 days after I/R, results in restoration of participation of SMC KATP-channels in maintaining the basal tone and ACh–mediated dilatation of pial arteries with a diameter less than 40 μm already 14 days after I/R.

Two distinct calcium signaling behaviors are present in interstitial cells of Cajal of the gastroesophageal junction

Background/Objectives: The lower esophageal sphincter (LES) is located within the gastroesophageal junction (GEJ) and generates tone to prevent gastric reflux into the esophagus. Previously, we demonstrated that the generation and maintenance of tone in the LES clasp muscle involves intramuscular interstitial cells of Cajal (ICC-IM) (Drumm et al., 2022; PMID: 35229888). The aim of the current study was to further characterize Ca2+ signaling in ICC-IM within the GEJ. We hypothesize that Ca2+ release from the endoplasmic reticulum (ER) activates Ca2+-activated Cl- channels (ANO1) in ICC leading to depolarization that is conducted to smooth muscle cells (SMCs) via gap junctions; this in turn leads to Ca2+ influx via L-type Ca2+ channels (CavL) and the development of tone. Methods: Confocal spinning disc microscopy was used to image Ca2+ transients in the LES and distal esophagus of mice expressing GCaMP6f in ICC (Kit-GCaMP6f). Muscles of the GEJ imaged with the submucosal surface upwards at 20x. Ani9 (3mM), nifedipine (1mM) and CPA (10mM) were used to inhibit ANO1, CavL and SERCA, respectively. Pinacidil (10mM) was used to activate KATP channels. Results: ICC-IM within the GEJ displayed two types of Ca2+ signaling behaviors. Ca2+ transients in ICC-IM of the LES clasp muscle were stochastic, arising from multiple firing sites within the same cell. These Ca2+ transients were not synchronized within the same cell or between adjacent cells. In contrast, two Ca2+ signaling patterns were observed in ICC-IM of the distal esophagus where smooth muscle transitions to skeletal muscle. One ICC-IM population (Type I) shared the Ca2+ signaling behaviors of ICC-IM in the LES clasp. Another ICC population (Type II) exhibited rhythmic whole-cell Ca2+ transients that were synchronized between adjacent Type II ICC. Whole cell Ca2+ transients in Type II ICC were abolished by Ani9, nifedipine or pinacidil, revealing underlying Ca2+ transients similar to those observed in Type I ICC. In contrast, Ca2+ transients in Type I ICC were resistant to Ani9, nifedipine and pinacidil. Ca2+ transients were abolished in both types of ICC by removal of extracellular Ca2+, however the activity in Type I ICC persisted longer than that of Type II ICC. Discussion/Conclusions: This study demonstrates two distinct Ca2+ signaling behaviors in ICC-IM of the GEJ with differing pharmacology. This is similar to what has been previously described within the internal anal sphincter (IAS) (Hannigan et al., 2020; PMID: 32587396). In the IAS, it has been suggested that Type II ICC-IM generate slow waves however their role in the GEJ remains unclear as slow waves have not been described within this region of the mouse. Thus, the functional role of ICC-IM in the GEJ, particularly Type II ICC, warrants further investigation. NIH DK129528 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A plasma membrane-associated glycolytic metabolon is functionally coupled to KATP channels in pancreatic α and β cells from humans and mice

The ATP-sensitive K+ (KATP) channel is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATP channel activity in pancreatic β cells and the lesser-studied α cells, from both humans and mice, we provide evidence that a glycolytic metabolon locally controls KATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further show the presence of a plasma membrane-associated NAD+/NADH cycle whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.

Rapamycin treatment unmasks a sex-specific pattern of scar expansion of the infarcted rat heart: The relationship between mTOR and KATP channel.

Inhibition of the mammalian target of rapamycin (mTOR) with the macrolide rapamycin or pharmacological suppression of KATP channel opening translated to scar expansion of the myocardial infarcted (MI) adult female rodent heart. The present study tested the hypotheses that rapamycin-mediated scar expansion was sex-specific and that mTOR signaling directly influenced KATP channel subunit expression/activity. Scar size was significantly larger in post-MI male rats as compared to the previous data reported in post-MI female rats. The reported scar expansion of rapamycin-treated post-MI female rats was not observed following the administration of the macrolide to post-MI male rats. Protein levels of the KATP channel subunits Kir6.2 and SUR2A and phosphorylation of the serine2448 residue of mTOR were similar in the normal heart of adult male and female rats. By contrast, greater tuberin inactivation characterized by the increased phosphorylation of the threonine1462 residue and reduced raptor protein levels were identified in the normal heart of adult female rats. Rapamycin pretreatment of phorbol 12,13-dibutyrate (PDBu)-treated neonatal rat ventricular cardiomyocytes (NNVMs) suppressed hypertrophy, inhibited p70S6K phosphorylation, and attenuated SUR2A protein upregulation. In the presence of low ATP levels, KATP channel activity detected in untreated NNVMs was significantly attenuated in PDBu-induced hypertrophied NNVMs via a rapamycin-independent pathway. Thus, rapamycin administration to post-MI rats unmasked a sex-specific pattern of scar expansion and mTOR signaling in PDBu-induced hypertrophied NNVMs significantly increased SUR2A protein levels. However, the biological advantage associated with SUR2A protein upregulation was partially offset by an mTOR-independent pathway that attenuated KATP channel activity in PDBu-induced hypertrophied NNVMs.