Immunocytochemistry of calciosomes in liver and pancreas.

Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K.

Generation of inositol phosphates, cytosolic Ca2+, and ionic fluxes in PC12 cells treated with bradykinin.

Accumulation of inositol phosphates (Ins-Ps, revealed by high performance liquid chromatography), changes of the cytosolic free Ca2+ [( Ca2+]i, revealed by fura-2), membrane potential and ionic currents (revealed by bis-oxonol and patch clamping) were investigated in PC12 cells treated with bradykinin (BK). The phenomena observed were (a) due to the activation of a B2 receptor (inhibitor studies) and (b) unaffected by pertussis toxin, cAMP analogs, and inhibitors of either cyclooxygenase or voltage-gated Ca2+ channels. During the initial tens of s, three interconnected events predominated: accumulation of Ins-1,4,5-P3, Ca2+ release from intracellular stores and hyperpolarization due to the opening of Ca2+-activated K+ channels.

Second-messenger control of catecholamine release from PC12 cells. Role of muscarinic receptors and nerve-growth-factor-induced cell differentiation.

The role of various intracellular signals and of their possible interactions in the control of neurotransmitter release was investigated in PC12 cells. To this purpose, agents that affect primarily the cytosolic concentration of Ca2+, [Ca2+]i (ionomycin, high K+), agents that affect cyclic AMP concentrations (forskolin; the adenosine analogue phenylisopropyladenosine; clonidine) and activators of protein kinase C (phorbol esters) were applied alone or in combination to either growing chromaffin-like PC12-cells, or to neuron-like PC12+ cells differentiated by treatment with NGF (nerve growth factor). In addition, the release effects of muscarinic-receptor stimulation (which causes increase in [Ca2+]i, activation of protein kinase C and decrease in cyclic AMP) were investigated.

Second-messenger generation in PC12 cells. Interactions between cyclic AMP and Ca2+ signals.

Changes in cyclic AMP concentrations were studied in intact PC12 pheochromocytoma cells exposed to a variety of treatments. A marked increase was triggered by N-(L-2-phenylisopropyl)adenosine, the activator of an adenosine receptor, whereas a decrease (observed even after phosphodiesterase blockade) was induced by carbachol, working through a muscarinic receptor inhibited by the selective muscarinic blocker pirenzepine, only at high concentration (Ki 450 nM). A decrease in cyclic AMP was also induced by clonidine, an alpha 2-adrenergic-receptor agonist.

The Ins(1,4,5)P3-sensitive Ca2+ store of non-muscle cells: endoplasmic reticulum or calciosomes?

The binding of a number of extracellular ligands (hormones, growth factors, neurotransmitters etc.) to their plasma membrane receptors causes hydrolysis of phosphatidylinositol bisphosphate to initiate the formation of two second messengers, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol, DAG. DAG has been shown to activate protein kinase C, whereas Ins(1,4,5)P3 induces the release of Ca2+ from an intracellular pool.

D-myo-inositol 1,4,5-trisphosphate phosphatase in skeletal muscle.

The presence and subcellular distribution of D-myo-inositol 1,4,5-trisphosphate phosphatase (InsP3ase) in rabbit fast-twitch skeletal muscle were investigated. A specific InsP3ase was found in both sarcotubular-membrane and soluble fractions. Membrane-bound InsP3ase accounted for 60-65% of total activity.

Activators of protein kinase C depolarize insulin-secreting cells by closing K+ channels.

Carbohydrate stimuli of insulin secretion depolarize the pancreatic B cell and the B-cell line RINm5F by inhibiting ATP-sensitive K+ channels. We examined the possibility that this effect is mediated by activation of protein kinase C. In RINm5F cells, the triose D-glyceraldehyde evoked a rapid increase of the mass of 1,2-diacylglycerol, the endogenous activator of protein kinase C.

"Calciosome," a cytoplasmic organelle: the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of nonmuscle cells?

Calsequestrin (CS) is the protein responsible for the high-capacity, moderate affinity binding of Ca2+ within the terminal cisternae of the sarcoplasmic reticulum, believed up to now to be specific for striated muscle. The cells of two nonmuscle lines (HL-60 and PC12) and of two rat tissues (liver and pancreas) are shown here to express a protein that resembles CS in many respects (apparent mass and pH-dependent migration in NaDodSO4/PAGE; blue staining with StainsAll dye; Ca2+ binding ability) and is specifically recognized by affinity-purified antibodies against skeletal muscle CS. In these cells, the CS-like protein is shown by immunofluorescence and immunogold procedures to be localized within peculiar, heretofore unrecognized structures distributed throughout the cytoplasm.

Immunocytological identification of the microsomal calcium store of nonmuscle cells.

The biochemical and functional similarities between skeletal muscle sarcoplasmic reticulum and the microsomal Ca2+ store of nonmuscle cells are discussed. It is shown that antibodies raised against two characteristic proteins of sarcoplasmic reticulum, Ca2+ ATPase and calsequestrin, recognize similar proteins in nonmuscle cells. The subcellular distribution of these two antigens was studied at the subcellular levels in ultrathin cryosections.

Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells.

The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e.

Dopamine inhibits cytosolic Ca2+ increases in rat lactotroph cells. Evidence of a dual mechanism of action.

Single rat lactotroph cells were studied after loading with the cytosolic free Ca2+ concentration ([Ca2+]i) indicator fura-2 either 1 or 3 days after cell dispersion. Under unstimulated conditions, two groups of lactotrophs were observed, the first (predominant at day 1) with large [Ca2+]i fluctuations (peaks up to 300 nM) probably due to spontaneous action potentials and the second (predominant at 3 days) with stable [Ca2+]i (values variable between 65 and 200 nM). The effect of dopamine on the resting [Ca2+]i was different in the two groups.

Antibody-induced modulation of the CD3/T cell receptor complex causes T cell refractoriness by inhibiting the early metabolic steps involved in T cell activation.

We investigated the mechanism involved in T cell unresponsiveness that follows the monoclonal antibody-induced surface modulation of the CD3-TCR complex. We determined whether modulation of CD3-TCR affected the early metabolic steps such as [Ca2+]i rise and InsP3 formation. A strong inhibition of the increase on [Ca2+]i mediated by either anti-TCR or anti-CD2 mAbs was detected.

Voltage-dependent activation and inactivation of calcium channels in PC12 cells. Correlation with neurotransmitter release.

The existence and mechanisms of inactivation of voltage-gated Ca2+ channels are important, but still debatable, physiological problems. By using the Ca2+ indicators quin2 and fura-2, we demonstrate that in PC12 cells voltage-gated Ca2+ channels undergo inactivation dependent on both voltage and [Ca2+]i. Inactivation, however, is never complete and a small number of channels remains open during prolonged depolarization, explaining the steady state elevation of [Ca2+]i observed in cells depolarized with high KCl.

Calcium and inositolphosphates in the activation of T cell-mediated cytotoxicity.

Reports from a number of laboratories have shown that mAbs against the T3-Ti receptor complex cause an increase in cytosolic-free Ca2+ [( Ca2+]i) and the hydrolysis of phosphatidylinositolbisphosphate (PIP2) in CTLs. In the present report we show that activation of CTLs by their specific targets causes: (a) release of Ca2+ from intracellular stores; (b) transient formation of inositol trisphosphate (InsP3); and (c) an increased permeability to Ca2+ of CTL plasma membrane. Killing of unrelated targets could be induced by cocentrifugation of the unrelated targets with CTLs in the presence of A23187 or PMA.

Activation of a muscarinic receptor selectively inhibits a rapidly inactivated Ca2+ current in rat sympathetic neurons.

Sympathetic neurons dissociated from the superior cervical ganglion of 2-day-old rats were studied by whole-cell patch clamp and by fura-2 measurements of the cytosolic free Ca2+ concentration, [Ca2+]i. Step depolarizations in the presence of tetrodotoxin and hexamethonium triggered two Ca2+ currents that differed in the voltage dependence of activation and kinetics of inactivation. These currents resemble the L and N currents previously described in chicken sensory neurons [Nowycky, M.

Plasma membrane potential modulates chemotactic peptide-stimulated cytosolic free Ca2+ changes in human neutrophils.

The relationship between fMet-Leu-Phe-induced changes in the cytosolic free Ca2+ concentration [( Ca2+]i), plasma membrane potential depolarization, and metabolic responses was studied in human neutrophils. Receptor-activated depolarization occurred both at high and resting [Ca2+]i, but was inhibited at very low [Ca2+]i. Phorbol 12-myristate 13-acetate-induced plasma membrane depolarization, on the contrary, was independent of [Ca2+]i.

Effect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes.

Cytochalasins are routinely used to stimulate a variety of functions in eukaryotic cells even though their precise mode of action remains to be elucidated. In the present work we used the fluorescent Ca2+ indicator quin2 to study the effect of various cytochalasins, cytochalasins A, B, C, D, E (CA, CB, CC, CD, CE) and dihydrocytochalasin B (dhCB) on the intracellular Ca2+ concentration ([Ca2+]i) in various types of leukocytes, viz, neutrophils and lymphocytes. In human neutrophils, cytochalasins increase [Ca2+]i mainly by releasing Ca2+ from membrane-bound, intracellular stores.

Role of cytosolic free calcium and phospholipase C in leukotriene-B4-stimulated secretion in human neutrophils. Comparison with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine.

Various leukotriene analogues were tested for their capacity to raise the cytosolic free calcium concentration, [Ca2+]i, and to stimulate exocytosis in human neutrophils. Their order of potency for both parameters was LTB4 greater than the stereochemical isomer of LTB4, (5S, 12S)-LTB4 much much greater than the sulphidopeptides LTD4, LTC4. The correlation between [Ca2+]i and secretion indicates that an increase of [Ca2+]i above a threshold level of about 300 nM is necessary for stimulating secretion with LTB4.

Characterization of fMet-Leu-Phe receptor-mediated Ca2+ influx across the plasma membrane of human neutrophils.

N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) stimulation of human neutrophils leads to a rapid increase of the cytosolic free Ca2+ concentration, [Ca2+]i, which is significantly reduced by removal of extracellular calcium. In the present study we show that fMet-Leu-Phe-induced [Ca2+]i increases are, in part, mediated by an increase of the plasma membrane permeability to Ca2+. This conclusion is based on the following evidence.