Inhibition of beta-cell sodium-calcium exchange enhances glucose-dependent elevations in cytoplasmic calcium and insulin secretion.

OBJECTIVE: The sodium-calcium exchanger isoform 1 (NCX1) regulates cytoplasmic calcium (Ca(2+)(c)) required for insulin secretion in beta-cells. NCX1 is alternatively spliced, resulting in the expression of splice variants in different tissues such as NCX1.3 and -1.7 in beta-cells. As pharmacological inhibitors of NCX1 splice variants are in development, the pharmacological profile of beta-cell NCX1.3 and -1.7 and the cellular effects of NCX1 inhibition were investigated. RESEARCH DESIGN AND METHODS: The patch-clamp technique was used to examine the pharmacological profile of the NCX1 inhibitor KB-R7943 on recombinant NCX1.3 and -1.7 activity. Ca(2+) imaging and membrane capacitance were used to assess the effects of KB-R7943 on Ca(2+)(c) and insulin secretion in mouse and human beta-cells and islets. RESULTS: NCX1.3 and -1.7 calcium extrusion (forward-mode) activity was approximately 16-fold more sensitive to KB-R7943 inhibition compared with cardiac NCX1.1 (IC(50s) = 2.9 and 2.4 vs. 43.0 micromol/l, respectively). In single mouse/human beta-cells, 1 micromol/l KB-R7943 increased insulin granule exocytosis but was without effect on alpha-cell glucagon granule exocytosis. KB-R7943 also augmented sulfonylurea and glucose-stimulated Ca(2+)(c) levels and insulin secretion in mouse and human islets, although KB-R7943 was without effect under nonstimulated conditions. CONCLUSIONS: Islet NCX1 splice variants display a markedly greater sensitivity to pharmacological inhibition than the cardiac NCX1.1 splice variant. NCX1 inhibition resulted in glucose-dependent increases in Ca(2+)(c) and insulin secretion in mouse and human islets. Thus, we identify beta-cell NCX1 splice variants as targets for the development of novel glucose-sensitive insulinotropic drugs for type 2 diabetes.

Splice variant-dependent regulation of beta-cell sodium-calcium exchange by acyl-coenzyme As.

The sodium-calcium exchanger isoform 1 (NCX1) is intimately involved in the regulation of calcium (Ca(2+)) homeostasis in many tissues including excitation-secretion coupling in pancreatic beta-cells. Our group has previously found that intracellular long-chain acyl-coenzyme As (acyl CoAs) are potent regulators of the cardiac NCX1.1 splice variant. Despite this, little is known about the biophysical properties of beta-cell NCX1 splice variants and the effects of intracellular modulators on their important physiological function in health and disease. Here, we show that the forward-mode activity of beta-cell NCX1 splice variants is differentially modulated by acyl-CoAs and is dependent both upon the intrinsic biophysical properties of the particular NCX1 splice variant as well as the side chain length and degree of saturation of the acyl-CoA moiety. Notably, saturated long-chain acyl-CoAs increased both peak and total NCX1 activity, whereas polyunsaturated long-chain acyl-CoAs did not show this effect. Furthermore, we have identified the exon within the alternative splicing region that bestows sensitivity to acyl-CoAs. We conclude that the physiologically relevant forward-mode activity of NCX1 splice variants expressed in the pancreatic beta-cell are sensitive to acyl-CoAs of different saturation and alterations in intracellular acyl-CoA levels may ultimately lead to defects in Ca(2+)-mediated exocytosis and insulin secretion.

The reliability of 2D and colour Doppler ultrasound in localising longline position.

BACKGROUND: The position of percutaneously inserted central venous catheters (longlines) in neonates is critical, as malpositioned longlines are associated with potentially fatal complications. AIM: To determine if cardiac ultrasound (two-dimensional (2D) and colour Doppler) is useful in evaluating longline position, when compared with the position identified by contrast radiography. SETTING: Single level 3 neonatal unit. PARTICIPANTS: Forty-four neonates undergoing insertion of 24-gauge silastic longlines between July 2004 and September 2005. METHODS: Infants who had a longline inserted underwent echocardiography by a novice and an experienced operator. Operators identified longline position using a 2D then colour Doppler echocardiography during a rapid bolus infusion of saline. The position was identified from contrast radiography by two independent observers. RESULTS: Using 2D echocardiography, the novice and experienced operators could identify 41 and 59% of longlines, respectively. However, only 34% of longlines were identified by both operators. In 15 infants whose longline positions were identified by both operators, there was agreement in only eight infants (53%). Colour Doppler improved the experienced operator's success but did not assist the novice operator. For radiographs, there was 68% agreement on longline position between observers. The experienced echocardiographer located three (7%) longlines within the heart that from radiographs were thought to be in a proximal central vessel. CONCLUSIONS: This technique is experience-dependent and complements rather than replaces the use of contrast radiography. However, some infants with an apparently acceptable longline position on contrast radiography have longlines located within the heart on echocardiography.

Elevation in intracellular long-chain acyl-coenzyme A esters lead to reduced beta-cell excitability via activation of adenosine 5'-triphosphate-sensitive potassium channels.

Closure of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long-chain acyl-coenyzme A esters (acyl-CoAs) can potently activate K(ATP) channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced beta-cell excitability and insulin secretion observed in obesity and type 2 diabetes. However, reduced beta-cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic beta-cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse beta-cells using virally delivered overexpression of long-chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on beta-cell K(ATP) channel activity and cell excitability was assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in K(ATP) channel activity in AdACSL-1-infected beta-cells cultured in medium supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected beta-cells (with palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mm glucose. These results suggest a direct link between intracellular fatty ester accumulation and K(ATP) channel activation, which may contribute to beta-cell dysfunction in type 2 diabetes.

Amyloid beta peptide as a physiological modulator of neuronal 'A'-type K+ current.

Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K(+) channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid beta protein (Abeta) can increase the 'A'-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Abeta is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones. A-type K(+) currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1-40 and 1-42 forms of Abeta (100nM, 2-24h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in 'A'-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Abeta, rather than protein degredation. Endogenous Abeta production in cerebellar granule neurones was blocked using inhibitors of either gamma- or beta-secretase and resulted in decreased K(+) current. Crucially this could be prevented by co-application of exogenous Abeta (1nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred. These data show that Abeta is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Abeta is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

A central role for ROS in the functional remodelling of L-type Ca2+ channels by hypoxia.

Periods of prolonged hypoxia are associated clinically with an increased incidence of dementia, the most common form of which is Alzheimer's disease. Here, we review recent studies aimed at providing a cellular basis for this association. Hypoxia promoted an enhanced secretory response of excitable cells via formation of a novel Ca2+ influx pathway associated with the formation of amyloid peptides of Alzheimer's disease. More strikingly, hypoxia potentiated Ca2+ influx specifically through L-type Ca2+ channels in three distinct cellular systems. This effect was post-transcriptional, and evidence suggests it occurred via increased formation of amyloid peptides which alter Ca2+ channel trafficking via a mechanism involving increased production of reactive oxygen species by mitochondria. This action of hypoxia is likely to contribute to dysregulation of Ca2+ homeostasis, which has been proposed as a mechanism of cell death in Alzheimer's disease. We suggest, therefore, that our data provide a cellular basis to account for the known increased incidence of Alzheimer's disease in patients who have suffered prolonged hypoxic episodes.

Digital imaging does not improve localization of percutaneously inserted central lines in neonates.

OBJECTIVE: To determine whether the use of digital imaging improves the localization of percutaneously inserted central (PIC) line tips compared to standard radiographs, with contrast being used in both cases. METHODS: Digital radiographs taken for localization of PIC lines between October 2002 and September 2003 were identified from the neonatal database. Two reporters independently reviewed the radiographs and stated (i) whether the tip could confidently be seen and (ii) the position. The observers were encouraged to use all the image modification modalities available. The formal radiology report was taken as the third 'reporter'. RESULTS: Overall 117 digital radiographs from 98 infants were reviewed. Reporter 1 identified the tip in 83 (71%); reporter 2 in 84 (72%); and the radiologist in 95 (81%), although all agreed that the tip could be identified in only 55 (47%) radiographs. In radiographs where the reporters agreed that the tip could be identified, they agreed on the position in 37 (67%) cases. In 61 (52%) radiographs the reporters did not agree that the tip could be seen, but they agreed on its position in 36 (59%) cases. The kappa coefficient for agreement on line position was 0.63. The level of agreement between reporters in this study using digital imaging was not significantly different from our previous findings using standard radiography with contrast (74/117, 63% vs 57/96, 59%). CONCLUSION: The use of digital imaging, with its ability to manipulate images in order to identify PIC line tips, is not significantly better than using standard radiography with contrast.

Altered processing of the amyloid precursor protein and decreased expression of ADAM 10 by chronic hypoxia in SH-SY5Y: no role for the stress-activated JNK and p38 signalling pathways.

Clinical studies suggest that the incidence of Alzheimer's disease (AD) is increased following an ischaemic or hypoxic episode, such as stroke. Furthermore, levels of the AD-associated amyloid beta-peptides (Abeta) and the amyloid precursor protein (APP) are enhanced in experimental ischaemia. In our previous study [Webster, N.J., Green, K.N., Peers, C., Vaughan, P.F., Altered processing of amyloid precursor protein in the human neuroblastoma SH-SY5Y by chronic hypoxia, J. Neurochem., 83 (2002) 1262-1271] we reported that exposing cells of neuronal origin to a period of chronic hypoxia (CH; 2.5% O(2), 24 h) led to a decrease in processing of the amyloid precursor protein (APP) by the alternative and neuroprotective alpha-secretase pathway. In SH-SY5Y cells, the most likely mechanism was that CH inhibits the protein level of ADAM 10, a disintegrin metalloprotease widely believed to be the alpha-secretase. One effect of CH is to alter the activity of the stress-activated protein kinases (SAPKs) c-Jun amino terminal kinase (JNK) and p38. Thus, the main aims of this study were to investigate the effect of CH on (1) the activity of these SAPKs in SH-SY5Y and (2) whether changes in the activity of these kinases may account for the CH-induced decreases in ADAM 10 expression and sAPPalpha secretion. We demonstrated that the phosphorylation (activity) of JNK was decreased approximately 50% following a period of CH. An inhibitor of JNK did not mimic the effects of CH on either ADAM 10 expression or sAPPalpha secretion under conditions in which the phosphorylation of c-Jun was inhibited by approximately 80%. Thus the loss of JNK activity does not appear to be linked to the decrease in expression of ADAM 10 and secretion of sAPPalpha. In contrast, phosphorylation (activity) of p38 was enhanced approximately 300% following a period of CH. However, inhibitors of p38 were unable to reverse the loss of sAPPalpha in CH cells, indicating that this increase in activity was not linked to the altered processing of APP.

Altered processing of amyloid precursor protein in the human neuroblastoma SH-SY5Y by chronic hypoxia.

Alzheimer's disease (AD) is more prevalent following an ischemic or hypoxic episode, such as stroke. Indeed, brain levels of amyloid precursor protein (APP) and the cytotoxic amyloid beta peptide (Abeta) fragment are enhanced in these patients and in animal models following experimental ischaemia. We have investigated the effect of chronic hypoxia (CH; 2.5% O2, 24 h) on processing of APP in the human neuroblastoma, SH-SY5Y. We demonstrate that constitutive and muscarinic-receptor-enhanced secretion of the alpha-secretase cleaved fragment of APP, sAPPalpha, was reduced by approximately 60% in CH cells. The caspase inhibitor BOC-D(Ome)FMK did not reverse this effect of CH, and CH cells were as viable as controls, based on MTT assays. Thus, loss of sAPPalpha is not related to cell death or caspase processing of APP. Pre-incubation with antioxidants did not reverse the effect of CH, and the effect could not be mimicked by H2O2, discounting the involvement of reactive oxygen species in hypoxic loss of sAPPalpha. CH did not affect muscarinic activation of extracellular-signal regulated kinase. However, expression of ADAM 10 (widely believed to be alpha-secretase) was decreased approximately 50% following CH. Thus, CH selectively decreases processing of APP by the alpha-secretase pathway, most likely by decreasing levels of ADAM 10.