F. Di Virgilio

Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers.

Fura-2 is widely used to measure the concentration of cytosolic free calcium, but in many cells the dye does not remain localized within the cytoplasmic matrix. In these cells, Fura-2 is sequestered within intracellular organelles, secreted into the extracellular medium, or both. We have found that, in mouse peritoneal macrophages, J774 cells, PC12 cells, and N2A cells, Fura-2 sequestration and secretion are mediated by organic anion transport systems and are blocked by the inhibitors probenecid and sulfinpyrazone. Under appropriate conditions these agents have little affect on calcium transients, and may facilitate the use of Fura-2 in a variety of cell types.

Tumor promoter phorbol 12-myristate, 13-acetate inhibits phosphoinositide hydrolysis and cytosolic Ca2+ rise induced by the activation of muscarinic receptors in PC12 cells

Preincubation of PC12 cells (used both before and after differentiation by NGF) with phorbol myristate acetate (PMA) was without effect on the basal concentration of inositol phosphates (metabolites of phosphoinositide hydrolysis) and of free cytosolic Ca2+, but inhibited considerably the increases induced by the cholinergic agonist carbachol via the activation of the muscarinic receptor. Inasmuch as binding was unaffected, this inhibition might occur at the level of receptor coupling to its transduction mechanism(s). Inhibition appeared within 1 min and was maximal after 3 min. The concentrations of PMA needed (10(-9)-10(-8)M) were in the range believed to cause specifically the activation of protein kinase C. The muscarinic receptor, via the hydrolysis of phosphoinositides and the generation of diacylglycerol, participates in the regulation of the latter enzyme. Our data suggest therefore that the receptor operates under stringent feedback control by the metabolites generated as a consequence of its activation.

Second Messenger Generation and Secretion in PC12 Cells. Role of Ca++ and Phosphoinositide Hydrolysis

PC12 is a line of neurosecretory cells originally developed by Greene and Tischler (1976) from a rat pheochromocytoma. This line has been extensively used in a number of laboratories around the world. Morphologically, growing PC12 cells resemble chromaffin cells of the adrenal medulla, although their secretion granules are smaller and less numerous (Greene and Tischler, 1976, 1982; Tischler and Greene, 1978; Watanabe et al., 1983). Because of this and other differences with respect to chromaffin cells, PC12 are now considered to be undifferentiated sympatoblasts. Their major catecholamine is dopamine, accumulated within secretory granules together with noradrenaline (Greene and Tischler, 1976, 1982; Rebois et al., 1980). Other neurotransmitters are synthesized in PC12 cells. Among these, acetylcholine is stored in organelles (not yet identified unambiguously), which are heavier than regular synaptic vesicles and lighter than the bulk of dopamine containing granules (Schubert et al., 1977). Peptides (enkephalins; neurotensin; Tischler et al., 1983; Panerai and Meldolesi, in preparation), as well as proteins typical of chromaffin and other secretory organelles (chromogranin A; secretogranin; Lee and Huttner, 1983) are present in PC12 cells, but their possible colocalization with dopamine has not been established with certainty yet.

Studies on Voltage-Gated Calcium Channels by Means of Fluorescent [Ca2+]i Indicators

Most excitable cells respond to depolarization with a rapid increase of their Ca2+ permeability, due to the opening of voltage-gated Ca2+ channels (Ca2+ VOCS) in the plasma membrane (Hagiwara and Byerly 1981; Reuter 1984). Opening of these channels causes the cytosolic concentration of Ca2+, [Ca2+]i, to rise quickly, beginning in the cytosolic region(s) immediately adjacent to the plasma membrane. The [Ca2+]i rise is opposed by processes that expell Ca2+ from the cell (active pumping by a Ca2+ ATPase and Na+/Ca2+ exchange) or cause its segregation within intracellular, membrane-bounded campartments (a high affinity microsomal store as well as the mitochondria). Up to now, studies on Ca2+ VOCs were carried out primarily by electro-physiology (classical techniques as well as patch clamping) and by Ca2+ influx experiments. By the use of these techniques, a number of important achievements were obtained (Hagiwara and Byerly 1981; Reuter 1984), including the recent demonstration of the heterogeneous nature of Ca2+ VOCs. Following the simple nomenclature proposed by Nowicky et al. (1985), the various types of Ca2+ VOCs so far identified will be referred to here as the T (transient, or low-threshold) VOCs, probably involved in pacemaking; the L (long-lasting, or high-threshold), the classical Ca2+ VOCs that are affected by dehydropyridine drugs; and the N (neither transient, nor long-lasting) VOCs, present in neurons, that might be preferentially located in the presynaptic membrane (Nowicky et al. 1985).

Stimulus Activation Coupling in Neutrophils

Evidence in favour of a primary role of two products of phosphatidylinositol (PI) metabolism, inositoltrisphosphate (InsP3) and diacylglycerol (DG) in triggering neutrophil functions are discussed. a) InsP3 is formed rapidly upon receptor stimulation. b) InsP3 is capable of releasing Ca2+ from membrane enclosed intracellular stores. c) InsP3 formation dogs not require a rise of intracellular free Ca2+ concentration, [Ca2+]i, but many actually precede and cause it. DG formation was not directly measured in this study but indirect evidence supports the hypothesis that DG formation is critical for full expression of metabolic responses: a) Synergistic effects on granule exocytosis and 02− production are observed between Ca2+ ionophores and Phorbol Miristate Acetate (PMA); PMA substitutes for endogenously formed diacylglycerol in activating protein kinase C. b) [Ca2+]i rises induced by receptor triggering result in a stimulation of neutrophil functions much larger than obtained when equivalent [Ca2+]i rises are induced by Ca2+ ionophores.