M Prentki

Neomycin: a specific drug to study the inositol-phospholipid signalling system?

Neomycin, an antibiotic previously thought to interact specifically with inositol‐containing phospholipids, was found to inhibit IP3‐mediated Ca2+ release from the intracellular stores of permeabilized insulinoma and liver cells. This inhibition could be relieved by increasing the IP3 concentration. Radiolabelled IP3 was found to bind tightly to columns prepared from neomycin covalently attached to glass beads. ATP was also bound by these colums. It is concluded that neomycin acts in biological systems as a weak anion exchanger and is therefore unsuitable for use as a specific tool to study the role of inositol phospholipids in intracellular signalling.

Cyclic AMP raises cytosolic Ca2+ and promotes Ca2+ influx in a clonal pancreatic β-cell line (HIT T-15)

The effect on cytosolic Ca2+ concentration ([Ca2+]i) of cAMP analogues and the adenylate cyclase‐stimulating agents forskolin, isoproterenol and glucagon has been examined in an insulin‐secreting β‐cell line (HIT T‐15) using fura 2. All these manipulations of the cAMP messenger system promoted a rise in [Ca2+]i which was blocked by the Ca2+ channel antagonists verapamil and nifedipine or by removal of extracellular Ca2+. The action of the adenylate cyclase activator forskolin was glucose‐dependent. The results suggest that cAMP elevates [Ca2+]i in HIT cells by promoting Ca2+ entry through voltage‐sensitive Ca2+ channels, not through mobilization of stored Ca2+. Activation of Ca2+ influx may be an important component of the mechanisms by which cAMP potentiates fuel‐induced insulin release.

Epidermal growth factor‐induced increases in inositol trisphosphates, inositol tetrakisphosphates, and cytosolic Ca2+ in a human hepatocellular carcinoma‐derived cell line

A human hepatocellular carcinoma‐derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5‐bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml) a rapid (10–15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 andIns(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 2O s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.

Biochemical Design Features of the Pancreatic Islet Cell Glucose-Sensory System

Our understanding of the biochemical basis of fuelstat function of the pancreatic islet cells is still fragmentary in contrast to the comprehensiveness of our knowledge about the morphology and physiology of pancreatic islets. The present account addresses several fundamental issues of β-cell biochemistry, namely the manner in which these cells keep track of the blood glucose level and how those blood glucose measurements might be coupled to insulin secretion. Expressed in the jargon of the field the presentation will deal with the pancreatic β-cell glucose-sensor-device and with the problem of stimulus-secretion-coupling.

EGF receptor down-regulation attenuates ligand-induced second messenger formation

Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, and in an EGF receptor-overexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within 10-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at 1-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/5 cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/5 cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors.

Hormone-Induced Inositol Lipid Breakdown and Calcium-Mediated Cellular Responses in Liver

Although it has been recognized for many years that changes of the intracellular free Ca2+ concentration by a variety of agonists form an important signaling device for regulation of cell function, the source of the Ca2+ and the molecular events regulating receptor-mediated changes of cellular calcium homeostasis have remained recalcitrant problems despite much effort directed towards their elucidation. However, advances made along a number of different lines have contributed towards the rapid increase of knowledge in this area. These include on the one hand the development of fluorescent Ca2+ indicators such as Quin 2 (Tsien, 1983) and more recently Fura 2 (Grynkiewicz et al., 1985), which allow kinetic measurements of changes in the cytosolic free Ca2+ concentration of isolated cells, and on the other hand the elucidation of the signaling roles of two new intracellular second messengers, namely, inositol trisphosphate and diacylglycerol (for reviews see Nishizuka et al., 1984; Nishizuka, 1984a; Berridge and Irvine, 1984; Williamson et al., 1985; Williamson, 1986).

Integrated Mitochondrial and Microsomal Regulation of Free Ca2+ in Permeabilized Insulinoma Cells

Permeabilized RINm5F insulinoma cells (Joseph et al., 1984, Prentki et al., 1984, 1985) were used to evaluate the effect of stable levels of ATP, ADP and orthophosphate (Pi) on mitochondrial and non-mitochondrial, presumably endoplasmic reticulum (ER) Ca2+ handling. We sought to determine the nature of changes that affect Ca2+ handling, the range of values where such effects are apparent and the intracellular organelle exhibiting responsiveness.

Metabolic Regulation of Ca 2+ Handling in Permeabilized Insulinoma Cells

Signal generation in the pancreaticscell requires metabolism of the stimulatory fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+ (Hedeskov, 1980; Matschinskyet al., 1983; Meglasson and Matschinsky, 1986; Prentki and Matschinsky, 1987). We hypothesized that fuel phosphorylation decreased the cytosolic MgATP/MgADP ratio sufficiently to stimulate O2consumption, and simultaneously reduced the activity of Ca2+ -ATPase with resulting increases in free-Ca2+ levels. To explore this hypothesis, we varied the MgATP/MgADP ratio in permeabilized RINm5F insulinoma cells that maintain a low-medium Ca2+ concentration in the presence of MgATP. Either of the following was added: (1) creatine phosphokinase plus various fixed ratios of creatine/creatine phosphate, or (2) a cell-free extract of rat skeletal muscle that exhibits spontaneous oscillatory behavior of glycolysis, and linked oscillations in the MgATP/MgADP ratio when provided with glucose and a hexokinase (Tornheim, 1979). We found that the free-Ca2+ level maintained by the permeabilized cells varied inversely with the MgATP/MgADP ratio, regardless of the mechanism used to vary MgATP/MgADP. In addition, free Ca2+ was decreased by increasing levels of orthophosphate (Pi). Ca2+ levels oscillated in phase with glycolytic oscillations and correlated closely with the MgATP/MgADP ratio. Ca2+ oscillations were evoked by increasing glucose levels from 2.5 to 10 or 20 mM in the presence of glucokinase, whereas oscillations occurred at 2.5 mM glucose in the presence of hexokinase and were unaffected by increasing the glucose concentration. These results provide the first demonstration of a link between metabolite changes and free-Ca2+ levels, and suggest a mechanism by which fuel metabolism might be coupled to activation of the Ca2+ messenger system in pancreaticβcells.