Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca2+. Thapsigargin (Fig. 1), a naturally occurring sesquiterpene lactone, promotes tumorigenesis in mouse skin (2) but does MATERIALS AND METHODSMeasurements of [Ca2+J1. Hepatocytes were prepared by perfusion of collagenase (type I; Sigma) (16) through livers from fed 250-g male rats and were kept in a Krebs-Henseleit buffer (119 mM NaCl/4.7 mM KCI/1.1 mM KH2PO4/1.2 mM MgSO4/25 mM NaHCO3, buffered with CO2 to pH 7.4) containing 1.0 mM Ca2+. The cells were stored at 3.0 x 106 per ml and continuously gassed with 02/CO2, 19:1 (vol/vol).After 5 min of incubation at 37°C, 10 uM indo-1 acetoxymethyl ester (Molecular Probes) was added and the incubation continued for a further 30 min. After centrifugation at 50 x g for 2 min, the indo-1-loaded hepatocytes were resuspended at 3.0 x 106 per ml in Krebs-Henseleit buffer including 1.0 mM CaC12 or 1.0 mM EGTA. The tTo whom reprint requests should be addressed. 2466The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be Ϸ30 M. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be Ϸ7 M. Likewise, the NAD(P)H increase of the mitochondrial domain is Ϸ60 M and is delayed with respect to the change in cytoplasmic NAD(P)H by Ϸ20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.G lucose-induced insulin secretion is coupled to the metabolic state of the ␤ cell. After transport into the cell, glucose is phosphorylated and shunted into glycolysis, which increases metabolic flux. This altered metabolic state, which can be monitored by NAD(P)H autofluorescence increase, leads to an increase in the ATP͞ADP ratio that closes the plasma membrane-associated ATP-sensitive potassium (K ATP ) channel. Closure of this channel depolarizes the membrane, leading to the activation of voltage-sensitive calcium (Ca 2ϩ ) channels, Ca 2ϩ influx, and insulin secretion (1).Glucose usage in stimulated pancreatic ␤ cells is principally glycolytic, with the polyol pathway, glycogen synthesis, and pentose phosphate pathway accounting for Ͻ10% of total usage (2). Thus, the glucose metabolic signal is derived from glycolysis in the cytoplasm and pyruvate metabolism in the mitochondria. The absence of stimulated insulin secretion with nonmetabolizable glucose derivatives (1) and the abolition of glucosestimulated insulin secretion in a pancreatic ␤ cell line lacking mitochondrial DNA (3) suggest roles for both cytoplasmic and mitochondrial metabolism in normal secretion. Additionally, both glycolytic intermediates, such as glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (2), and mitochondrial substrates, such as leucine (4) or methyl pyruvate (5), stimulate insulin secretion, which further supports a role for metabolism in both compartments in glucose signaling.Attempts to resolve the glycolytic and mitochondrial contributions to glucose-stimulated insulin secretion have relied on nutrient secretagogues that couple at various points into glycolysis or Kreb's cycle or on pharmacological inhibition at various points along each pathway (1, 6). Although various nutrient supplements indicate that couplings of the pathway can lead to insulin secretion, they seldom mimic the effects of glucose. For example, pyruvate potentiates glucose-stimulated secretion but does not cause s...

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Thapsigargin, a sesquiterpene lactone with potent irritant and tumour-promoting activities, stimulates a rapid (within 15 s) transient increase in intracellular [Ca2+] in the NG115-401L neural cell line, as measured by the fluorescent indicator dye fura-2. This increase in cytoplasmic free [Ca2+] is concentration-dependent (ED50 around 20 nM) and occurs in the absence of extracellular Ca2+. Activation of NG115-401L cells by the inflammatory peptide bradykinin generates inositol phosphates, which parallel increases in intracellular [Ca2+]. However, the rise in cytoplasmic [Ca2+] stimulated by thapsigargin occurs in the absence of detectable production of inositol phosphates. Thapsigargin is unlike phorboid tumour promoters in that it has no action on two non-invasive indicators of phorbol stimulation of these cells, i.e. [3H]choline metabolite production and rise in intracellular pH. These data suggest that thapsigargin releases Ca2+ from an intracellular store by a novel mechanism, independent of the hydrolysis of phosphoinositides and concomitant activation of protein kinase C. Thus thapsigargin may provide a valuable tool for the analysis of intracellular signalling mechanisms.

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