Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a neonatal disease characterized by dysregulation of insulin secretion accompanied by profound hypoglycemia. We have discovered that islet cells, isolated from the pancreas of a PHHI patient, proliferate in culture while maintaining a beta celllike phenotype. The PHHI-derived cell line (NES2Y) exhibits insulin secretory characteristics typical of islet cells derived from these patients, i.e. they have no K ATP channel activity and as a consequence secrete insulin at constitutively high levels in the absence of glucose. In addition, they exhibit impaired expression of the homeodomain transcription factor PDX1, which is a key component of the signaling pathway linking nutrient metabolism to the regulation of insulin gene expression. To repair these defects NES2Y cells were triple-transfected with cDNAs encoding the two components of the K ATP channel (SUR1 and Kir6.2) and PDX1. One selected clonal cell line (NISK9) had normal K ATP channel activity, and as a result of changes in intracellular Ca 2؉ homeostasis ([Ca 2؉ ] i ) secreted insulin within the physiological range of glucose concentrations. This approach to engineering PHHI-derived islet cells may be of use in gene therapy for PHHI and in cell engineering techniques for administering insulin for the treatment of diabetes mellitus. Persistent hyperinsulinemic hypoglycemia of infancy (PHHI)1 is a potentially lethal disease of the newborn. It is characterized by inappropriate insulin release in relation to the corresponding levels of glycemia (1, 2). Affected children run the risk of severe neurological damage unless immediate and adequate steps are taken to avoid profound hypoglycemia. Treatment involves administration of glucose along with drugs such as diazoxide and somatostatin that inhibit insulin secretion. However, in many cases this is not effective, and within the first few weeks of birth a near total (ϳ95%) pancreatectomy is required to control blood glucose levels.Recently, it has been shown that PHHI arises from defects in the regulation of insulin secretion. This is due principally to the loss of function of ATP-regulated potassium (K ATP ) channels. Genetic linkage has identified a susceptibility locus for PHHI within a region of chromosome 11 that encodes subunits of these channels (3, 4), while direct recordings of beta cells isolated from PHHI patients (following pancreatectomy) have documented the absence of K ATP channels (5). In beta cells these channels are composed of at least two subunits as follows: a K ϩ channel pore, Kir6.2, and an ATP-binding cassette protein, SUR1 (6, 7). Open K ATP channels set the resting membrane potential for the beta cell and a change in the intracellular ATP/ADP ratio following glucose metabolism results in their closure and the initiation of a depolarization of the cell membrane. This in turn activates voltage-dependent calcium channels and the ensuing influx of calcium stimulates membrane docking and fusion of preformed insulin granules resulting ...
Regulation of the kinetics of intracellular Ca(2+) signals with a novel, membrane-penetrable, inositol 1,4,5-trisphosphate (InsP(3)) receptor/Ca(2+) channel modulator, 2-amino-ethoxydiphenyl borate (2APB), has been investigated using patch-clamp, whole-cell recording to monitor Ca(2+)-activated Cl(-) currents in single isolated pancreatic acinar cells. 2APB itself fails to evoke a detectable current response but it dramatically changes the kinetics of agonist-induced Ca(2+) release from pulsatile spikes to long-lasting, huge Ca(2+) waves, suggesting that 2APB coordinates local Ca(2+) release to generate global Ca(2+) signals. The regulation by 2APB can be elicited by internal perfusion of InsP(3) in a concentration-dependent manner, indicating that this regulation is not mediated through membrane receptors or G protein signal transduction. The InsP(3) receptor blocker heparin, but not the ryanodine-sensitive receptor blockers ruthenium red or ryanodine, abolishes 2APB-mediated regulation of Ca(2+) release. This results also suggest that 2APB effects are mediated through InsP(3) receptors. 2APB substantially modifies single inward Cl(-) current pulse evoked by the photolytic release of caged InsP(3) but not by caged Ca(2+). These data indicate that 2APB-induced regulation is mediated neither by Ca(2+)-induced Ca(2+) release nor by affecting Cl(-) channel activity directly. We conclude that 2APB regulates the kinetics of intracellular Ca(2+) signals, represented as the change in the Ca(2+) oscillation patterns from brief pulsatile spikes to huge, long-lasting Ca(2+) waves. Moreover, this regulation seems to be mediated through InsP(3)-sensitive Ca(2+) pools. 2APB may act as a novel, useful pharmacological tool to study the genesis of intracellular Ca(2+) signals.
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