KATP Expression Research Articles

Models Of Paraventricular Nucleus (PVN) Sympathetic Neurone Modulation by Glucose and Hypoglycaemia

Hypoglycaemia activates much of the sympathetic nervous system (Fagius 2003. Acta Physiol. Scand. 177:337-343) and this is likely to be important to glucose counter-regulation. The PVN is ideally suited to mediate this response since it contains a high density of spinally-projecting sympathetic control neurones. Furthermore, inactivation of the PVN with lignocaine blunts counter-regulation (Evans et al. 2003 Am. J. Physiol. 284:R57-65).Our in vitro data show paradoxical responses of PVN sympathetic control neurones (SPNs) to hypoglycaemia: They express KATP channels and most are inhibited or unaffected by hypoglycaemia, whereas in vivo they appear to be activated by hypoglycaemia. There could be several possible explanations, however, in this study we explored the possibility that the paradox is accounted for by network properties in the PVN, and differential expression of KATP channels.We constructed very simple Neuron (Hines & Carnevale 1997. Neural Comput. 9:1179-1209) models of SPNs with inputs from both excitatory “Netstim” neurones and inhibitory interneurones. The interneurones are also driven by excitatory “Netstim” neurones. Both interneurones and SPNs incorporate identical KATP channels, but the latter with a lower density. We modelled the situation where this network is intact (in vivo) and where the inhibitory interneurones were lost (in vitro).In the in vitro model, SPN KATP conductance was sufficient for the expected (dose-related) decrease in action potential frequency with hypoglycaemia. Interestingly, however, with no changes to the set-up of the SPN neurones, but re-introduction of input from the inhibitory interneurones, the effect of hypoglycaemia was reversed. Hypoglycaemia now activated SPNs. This model also reproduced the common observation that whilst SPNs in brain slice experiments tend to be “spontaneously” active, they tend to be silent in vivo, but activated by GABAA inhibition.

Caveolae are an essential component of the pathway for endothelial cell signaling associated with abrupt reduction of shear stress

Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm2 followed by stop of flow (“ischemia”). Preconditioning resulted in a 2.7-fold increase in cellular expression of KATP (KIR 6.2) channels but no change in expression level of caveolin-1, gp91phox, or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of KIR 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G2/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow.

Effects of Aβ1–42 on the Subunits of KATP Expression in Cultured Primary Rat Basal Forebrain Neurons

ATP-sensitive potassium channels (KATP) play a crucial role in coupling metabolic energy to the membrane potential of cells, thereby functioning as cellular "metabolic sensors." Recent evidence has showed a connection between the amyloid neurotoxic cascade and metabolic impairment. With regard to their neuroprotection in other neuronal preparations, KATP channels may mediate a potential neuroprotective role in Alzheimer's disease (AD). To investigate the effects of Abeta1-42 on the subunits of KATP expression in cultured primary rat basal forebrain cholinergic neurons, primary rat basal forebrain neurons were cultured and evaluated. The subunits of KATP: Kir6.1, Kir6.2, SUR1 and SUR2 expressing changes were observed by double immunofluorescence and immunoblotting when the neurons were exposed to Abeta1-42(2 microM) for different time (0, 24, 72 h). We found a significant increase in the expression of Kir6.1 and SUR2 in the cultured neurons being exposed to Abeta1-42 for 24 h, while Kir6.2 and SUR1 showed no significant change. However, after being treated with Abeta1-42 for 72 h, the expression of the four subunits was all increased significantly compared with the control. These findings suggest that being exposed to Abeta1-42 for different time (24 and 72 h) induces differential regulations of KATP subunits expression in cultured primary rat basal forebrain cholinergic neurons. The change in composition of KATP may contribute to resist the toxicity of Abeta1-42.

Long-term inhibition of protein tyrosine kinase impairs electrophysiologic activity and a rapid component of exocytosis in pancreatic β-cells

Dysfunction of pancreatic beta-cells is a fundamental feature in the pathogenesis of type 2 diabetes. As insulin receptor signaling occurs via protein tyrosine kinase (PTK), we investigated the role of PTK activity in the etiology of beta-cell dysfunction by inhibiting PTK activity in primary cultured mouse pancreatic beta-cells and INS-1 cells with genistein treatment over 24 h. Electrophysiologic recordings showed genistein treatment significantly attenuated ATP-sensitive K(+) (K(ATP)) and voltage-dependent Ca(2+) currents, and depolarized the resting membrane potential in primary beta-cells. When stimulated by high glucose, genistein-treated beta-cells exhibited a time delay of both depolarization and Ca(2+) influx, and were unable to fire action potentials, as well as displaying a reduced level of Ca(2+) influx and a loss of Ca(2+) oscillations. Semiquantitative PCR analysis revealed decreased expression of K(ATP) and L-type Ca(2+) channel mRNA in genistein-treated islets. PTK inhibition also significantly reduced the rapid component of secretory vesicle exocytosis, as indicated by membrane capacitance measurements, and this is likely to be due to the reduced Ca(2+) current amplitude in these cells. These results illustrate that compromised PTK activity contributes to pancreatic beta-cell dysfunction and may be involved in the etiology of type 2 diabetes.

Titration of K ATP Channel Expression in Mammalian Cells Utilizing Recombinant Baculovirus Transduction

A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells. We have used the baculovirus transduction system, BacMam, to demonstrate transient expression of multi-subunit KATP channels in CHO-K1 and HEK-293 EBNA cells using sulfonylurea receptor 1 (SUR), SUR2A, SUR2B, and KIR 6.2 genes. [3H]-glyburide binding, patch clamp, and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression. BacMam delivery of each SUR isoform with KIR6.2 was demonstrated based on its pharmacological profiles. Expression levels of SUR1 and KIR6.2 were titrated by varying the viral concentration or time of virus addition, with functional activity measured in as little as 4-6 hours posttransduction. Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel. Independently altering treatment with virus containing either the SUR1 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane. This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function.