Protein kinase C has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. When cells are stimulated, protein kinase C is transiently activated by diacylglycerol which is produced in the membrane during the signal-induced turnover of inositol phospholipids. Tumour-promoting phorbol esters, when intercalated into the cell membrane, may substitute for diacylglycerol and permanently activate protein kinase C. The enzyme probably serves as a receptor for the tumour promoters. Further exploration of the roles of this enzyme may provide clues for understanding the mechanism of cell growth and differentiation.

The role of protein kinase C in cell surface signal transduction and tumour promotion

Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

Inositol trisphosphate and calcium signalling

There has recently been rapid progress in understanding receptors that generate intracellular signals from inositol lipids. One of these lipids, phosphatidylinositol 4,5-bisphosphate, is hydrolysed to diacylglycerol and inositol trisphosphate as part of a signal transduction mechanism for controlling a variety of cellular processes including secretion, metabolism, phototransduction and cell proliferation. Diacylglycerol operates within the plane of the membrane to activate protein kinase C, whereas inositol trisphosphate is released into the cytoplasm to function as a second messenger for mobilizing intracellular calcium.

Inositol trisphosphate, a novel second messenger in cellular signal transduction.

Protein kinase C, an enzyme that is activated by the receptor-mediated hydrolysis of inositol phospholipids, relays information in the form of a variety of extracellular signals across the membrane to regulate many Ca2+-dependent processes. At an early phase of cellular responses, the enzyme appears to have a dual effect, providing positive forward as well as negative feedback controls over various steps of its own and other signaling pathways, such as the receptors that are coupled to inositol phospholipid hydrolysis and those of some growth factors. In biological systems, a positive signal is frequently followed by immediate negative feedback regulation. Such a novel role of this protein kinase system seems to give a logical basis for clarifying the biochemical mechanism of signal transduction, and to add a new dimension essential to our understanding of cell-to-cell communication.

https://science.sciencemag.org/content/sci/233/4761/305.full.pdf

Studies and perspectives of protein kinase C

Inositol 1,4,5-trisphosphate is a second messenger which regulates intracellular calcium both by mobilizing calcium from internal stores and, perhaps indirectly, by stimulating calcium entry. In these actions it may function with its phosphorylated metabolite, inositol 1,3,4,5-tetrakisphosphate. The subtlety of calcium regulation by inositol phosphates is emphasized by recent studies that have revealed oscillations in calcium concentration which are perhaps part of a frequency-encoded second-messenger system.

Inositol phosphates and cell signalling

Activation of receptors for a wide variety of hormones and neurotransmitters leads to an increase in the intracellular level of calcium. Much of this calcium is released from intracellular stores but the link between surface receptors and this internal calcium reservoir is unknown. Hydrolysis of the phosphoinositides, which is another characteristic feature of these receptors, has been implicated in calcium mobilization. The primary lipid substrates for the receptor mechanism seem to be two polyphosphoinositides, phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2), which are rapidly hydrolysed following receptor activation in various cells and tissues. The action of phospholipase C on these polyphosphoinositides results in the rapid formation of the water-soluble products inositol 1,4-bisphosphate (Ins1,4P2) and inositol 1,4,5-trisphosphate (Ins1,4,5P3). In the insect salivary gland, where changes in Ins1,4P2 and Ins1,4,5P2 have been studied at early time periods, increases in these inositol phosphates are sufficiently rapid to suggest that they might mobilize internal calcium. We report here that micromolar concentrations of Ins1,4,5P3 release Ca2+ from a nonmitochondrial intracellular Ca2+ store in pancreatic acinar cells. Our results strongly suggest that this is the same Ca2+ store that is released by acetylcholine.

Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate.

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells.

Ca2+ uptake into isolated exocrine pancreatic cells with highly permeable plasma membrane was determined by measuring the decrease in free Ca2+ concentration of the surrounding incubation medium with a Ca2+-specific electrode. In the presence of Mg-ATP and respiratory substrates the free Ca2+ concentration of the incubation medium decreased rapidly after addition of leaky cells until a stable medium free Ca2+ concentration of 4.2 +/- 0.1 X 10(-7) mol/l was obtained. Changes in the medium free Ca2+ concentration at steady state by addition of Ca2+ or EGTA were buffered by cellular uptake or release, respectively, until the steady-state free Ca2+ concentration was reestablished. When nonmitochondrial Ca2+ uptake was determined in the presence of a combination of mitochondrial inhibitors (10(-5) mol/l antimycin, 5 X 10(-6) mol/l oligomycin, and 10(-2) mol/l azide), the rate of uptake was considerably reduced, while the steady-state concentration was unaltered. In contrast, mitochondrial uptake that could be observed in the presence of the ATPase inhibitor vanadate (2 X 10(-3) mol/l) proceeded at the same rate as the control, but the minimal medium free Ca2+ concentration reached was 2.4 +/- 0.1 X 10(-7) mol/l higher than the control. Addition of secretagogues at steady-state free Ca2+ concentration resulted in a Ca2+ release of 0.73 +/- 0.08 nmol/mg protein. The increase in medium free Ca2+ concentration was entirely transient and followed by reuptake to the prestimulation level. The data indicate that a cytosolic free Ca2+ concentration of 4 X 10(-7) mol/l can be regulated in pancreatic acinar cells by a nonmitochondrial Mg2+-dependent Ca2+ pool.

Regulation of cytosolic free Ca2+ concentration in acinar cells of rat pancreas.

ATP-dependent Ca2+ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca2+-specific electrode and45Ca2+. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na+,K+(Mg2+)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared,45Ca2+ uptake correlated with the RNA content and not with the Na+,K+(Mg2+)ATPase activity. The characteristics of nonmitochondrial Ca2+ uptake into leaky isolated cells and45Ca2+ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg2+, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca2+ concentration of 2 and 2 μmol/liter, respectively. Calcium uptake decreased at higher free Ca2+ concentration.45Ca2+ uptake was dependent on monovalent cations (Rb+>K+>Na+>Li+>choline+) and different anions (Cl−>Br−>SO
 4
 2−
 >NO
 3
 −
 >I−>cyclamate−>SCN−) in both preparations. Twenty mmol/liter oxalate enhanced45Ca2+ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca2+ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca2+ concentration to 0.4 μmol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca2+ concentration in pancreatic acinar cells.

Hanspeter Streb

Papers

Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate.

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells.

Regulation of cytosolic free Ca2+ concentration in acinar cells of rat pancreas.

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas