Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion.

The role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca2+, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca2+ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 mumol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 mumol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca2+. Increasing the Ca2+ concentration to 1.26 mmol/l in TCM 199 during the 22-24 h exposure to glucose (16.7 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca2+ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Regulation of the type III InsP3 receptor and its role in beta cell function

The type III inositol 1,4,5-trisphosphate receptor (InsP3R) is an important intracellular calcium (Ca2+) release channel in the pancreatic beta cell. Pancreatic beta cells secrete insulin following a characteristic change in membrane potential that leads to an increase in cytoplasmic Ca2+. Both extracellular Ca2+ and Ca2+ mobilized from InsP3-sensitive stores contribute to this increase. RIN-m5F cells, an insulin-secreting beta cell line, preferentially express the type III InsP3R. These cells have been useful in determining the regulatory properties of the type III InsP3R and the role of this isoform in an intact cell. The type III InsP3R is ideal for signal initiation because high cytoplasmic Ca2+ does not inhibit its activity. Altered insulin secretion, the result of changes in Ca2+ handling by the beta cell, has significant clinical consequences.     

Dysfunctional insulin secretion in type 2 diabetes: role of metabolic abnormalities

Insulin secretion is finely tuned to the requirements of tissues by tight coupling to prevailing blood glucose levels. The normal regulation of insulin secretion is coupled to glucose metabolism in the pancreatic B cell, a major but not exclusive signal for secretion being closure of K⁺ATP (adenosine triphosphate)-dependent channels in the cell membrane through an increase in cytosolic ATP/adenosine diphosphate. Insulin secretion in type 2 diabetes is abnormal in several respects due to genetic causes but also due to the metabolic environment of the pancreatic B cells. This environment may be particularly important for the deterioration of insulin secretion which occurs with increasing duration of diabetes. Factors in the environment with potential importance include overstimulation, a negative effect of hyperglycemia per se (‘glucotoxicity’) as well as adverse effects of elevated fatty acids (‘lipotoxicity’). Elucidating the mechanisms behind these factors as well as their clinical importance will pave the way for treatment which could preserve B-cell function in type 2 diabetic patients. Received 4 October 1999; received after revision 1 November 1999; accepted 3 December 1999     

Influence of ethanol on calcium, inositol phospholipids and intracellular signalling mechanisms

Studies have implicated Ca++ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca++ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca++/Mg++-ATPase, the high affinity membrane Ca++ pump. Current research suggests that a subtype of the voltage-dependent Ca++ channel, the dihydropyridine-sensitive Ca++ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases 45Ca++-influx and [3H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca++/Mg++-ATPase activity in neuronal membranes. Changes in Ca++ channel and Ca++/Mg++-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca++ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca++/Mg++-ATPase activity. The increased sensitivity of three Ca++-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion

The role of protein kinase C and Ca²⁺ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca²⁺, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca²⁺ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca²⁺. Increasing the Ca²⁺ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca²⁺ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Calcium and sodium distribution and movements in smooth muscle

Summary Electron probe microanalysis (EPMA) has been used to study the subcellular distribution of Ca, Na, K. Cl, and Mg in smooth muscle. The EPMA results indicate that the sarcoplasmic reticulum (SR) is the majorintracellular source and sink of activator Ca: norepinephrine decreases the Ca content of the junctional SR in portal vein smooth muscle. Mitochondria do not play a significant role in regulating cytoplasmic free Ca²⁺, but mitochondrial Ca content can be altered to a degree compatible with suggestions that fluctuations in matrix Ca contribute to the control of mitochondrial metabolism. The rise intotal cytoplasmic Ca during a maintained, maximal contraction is very much greater than the rise in free Ca²⁺, and is probably in excess of the known binding sites available on calmodulin and myosin. Cell Ca is not increased in normal cells that are Na-loaded. The non-Donnan distribution of Cl is not due to compartmentalization, but reflects high cytoplasmic Cl. Na-loading of smooth muscle in K-free solutions is temperature dependent, and may exhibit cellular heterogeneity undetected by conventional techniques. The total cell Mg is equivalent to approximately 12 mM, and less than 50% of it can be accounted for by binding to ATP and to actin. Mitochondrial monovalent cations in smooth muscle are relatively rapidly exchangeable.     

Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores

This review discusses multiple ways in which the endoplasmic reticulum participates in and is influenced by signal transduction pathways. The endoplasmic reticulum provides a Ca2+ store that can be mobilized either by calcium-induced calcium release or by the diffusible messenger inositol 1,4,5-trisphosphate. Depletion of endoplasmic reticulum Ca2+ stores provides a signal that activates surface membrane Ca2+ channels, a process known as capacitative calcium entry. Depletion of endoplasmic reticulum stores can also signal long-term cellular responses such as gene expression and programmed cell death or apoptosis. In addition to serving as a source of cellular signals, the endoplasmic reticulum is also functionally and structurally modified by the Ca2+ and protein kinase C pathways. Elevated cytoplasmic Ca2+ causes a rearrangement and fragmentation of endoplasmic reticulum membranes. Protein kinase C activation reduces the storage capacity of the endoplasmic reticulum Ca2+ pool. In some cell types, protein kinase C inhibits capacitative calcium entry. Protein kinase C activation also protects the endoplasmic reticulum from the structural effects of high cytoplasmic Ca2+. The emerging view is one of a complex network of pathways through which the endoplasmic reticulum and the Ca2+ and protein kinase C signaling pathways interact at various levels regulating cellular structure and function.     

Calcium signalling: a historical account, recent developments and future perspectives

Ca2+ is a uniquely important messenger that penetrates into cells through gated channels to transmit signals to a large number of enzymes. The evolutionary choice of Ca2+ was dictated by its unusual chemical properties, which permit its reversible complexation by specific proteins in the presence of much larger amounts of other potentially competing cations. The decoding of the Ca2+ signal consists in two conformational changes of the complexing proteins, of which calmodulin is the most important. The first occurs when Ca2+ is bound, the second (a collapse of the elongated protein) when interaction with the targeted enzymes occurs. Soluble proteins such as calmodulin contribute to the buffering of cell Ca2+, but membrane intrinsic transporting proteins are more important. Ca2+ is transported across the plasma membrane (channel, a pump, a Na+/Ca2+ exchanger) and across the membrane of the organelles. The endoplasmic reticulum is the most dynamic store: it accumulates Ca2+ by a pump, and releases it via channels gated by either inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPr). The mitochondrion is more sluggish, but it is closed-connected with the reticulum, and senses microdomains of high Ca2+ close to IP3 or cADPr release channels. The regulation of Ca2+ in the nucleus, where important Ca(2+)-sensitive processes reside, is a debated issue. Finally, if the control of cellular Ca2+ homeostasis somehow fails (excess penetration), mitochondria 'buy time' by precipitating inside Ca2+ and phosphate. If injury persists, Ca2(+)-death eventually ensues.     

Calcium and sodium distribution and movements in smooth muscle

Electron probe microanalysis (EPMA) has been used to study the subcellular distribution of Ca, Na, K, Cl, and Mg in smooth muscle. The EPMA results indicate that the sarcoplasmic reticulum (SR) is the major intracellular source and sink of activator Ca: norepinephrine decreases the Ca content of the junctional SR in portal vein smooth muscle. Mitochondria do not play a significant role in regulating cytoplasmic free Ca2+, but mitochondrial Ca content can be altered to a degree compatible with suggestions that fluctuations in matrix Ca contribute to the control of mitochondrial metabolism. The rise in total cytoplasmic Ca during a maintained, maximal contraction is very much greater than the rise in free Ca2+, and is probably in excess of the known binding sites available on calmodulin and myosin. Cell Ca is not increased in normal cells that are Na-loaded. The non-Donnan distribution of Cl is not due to compartmentalization, but reflects high cytoplasmic Cl. Na-loading of smooth muscle in K-free solutions is temperature dependent, and may exhibit cellular heterogeneity undetected by conventional techniques. The total cell Mg is equivalent to approximately 12 mM, and less than 50% of it can be accounted for by binding to ATP and to actin. Mitochondrial monovalent cations in smooth muscle are relatively rapidly exchangeable.     

Biochemical events associated with rapid cellular damage during the oxygen- and calcium-paradoxes of the mammalian heart

The O2- and Ca2(+)-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H+ and e- and also oxygen radicals or redox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca2+ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised conditions, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca2+ or by raising [Ca2+]i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca2(+)-paradox by the membrane perturbation associated with removal of extracellular Ca2+; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca2+]i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca2(+)-paradox which continues under anoxia. Massive entry of Ca2+ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

The role of Ca2+ in secretagogue-induced insulin release is documented not only by the measurements of 45Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca2+, [Ca2+]i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca2+]i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca2+ homeostasis by organelles from a rat insulinoma, was investigated with a Ca2+ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca2+ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca2+]i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca2+ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

α-Endosulfine, a new entity in the control of insulin secretion

ATP-dependent potassium (K_ATP) channels occupy a key position in the control of insulin release from the pancreatic β cell since they couple cell polarity to metabolism. These channels close when more ATP is produced via glucose metabolism. They are also controlled by sulfonylureas, a class of drugs used in type 2 diabetic patients for triggering insulin secretion from β cells that have lost part of their sensitivity to glucose. We have demonstrated the existence of endogenous counterparts to sulfonylureas which we have called ‘endosulfines.’ In this review, we describe the discovery, isolation, cloning, and biological features of the high-molecular-mass form, α-endosulfine, and discuss its possible role in the physiology of the β cell as well as in pathology. Received 1 February 1999; received after revision 26 March 1999; accepted 26 March 1999     

Neuronal calcium signaling: function and dysfunction

Calcium (Ca²⁺) is an universal second messenger that regulates the most important activities of all eukaryotic cells. It is of critical importance to neurons as it participates in the transmission of the depolarizing signal and contributes to synaptic activity. Neurons have thus developed extensive and intricate Ca²⁺ signaling pathways to couple the Ca²⁺ signal to their biochemical machinery. Ca²⁺ influx into neurons occurs through plasma membrane receptors and voltage-dependent ion channels. The release of Ca²⁺ from the intracellular stores, such as the endoplasmic reticulum, by intracellular channels also contributes to the elevation of cytosolic Ca²⁺. Inside the cell, Ca²⁺ is controlled by the buffering action of cytosolic Ca²⁺-binding proteins and by its uptake and release by mitochondria. The uptake of Ca²⁺ in the mitochondrial matrix stimulates the citric acid cycle, thus enhancing ATP production and the removal of Ca²⁺ from the cytosol by the ATP-driven pumps in the endoplasmic reticulum and the plasma membrane. A Na⁺/Ca²⁺ exchanger in the plasma membrane also participates in the control of neuronal Ca²⁺. The impaired ability of neurons to maintain an adequate energy level may impact Ca²⁺ signaling: this occurs during aging and in neurodegenerative disease processes. The focus of this review is on neuronal Ca²⁺ signaling and its involvement in synaptic signaling processes, neuronal energy metabolism, and neurotransmission. The contribution of altered Ca²⁺ signaling in the most important neurological disorders will then be considered.     

The signal transducing system coupled to serotonin-S2 receptors

The signal transducing system coupled to the serotonin-S2 receptor on platelets involves metabolism of inositol-containing phospholipid, elevation of intracellular free Ca2+ and activation of protein kinase C. Evidence for coupling of the serotonin-S2 receptor to the same signal transducing system in brain and smooth muscle tissue is reviewed.     

Effect of gentamycin on insulin release and45Ca net uptake by isolated islets

Summary In isolated islets, gentamycin reduced both the⁴⁵Ca net uptake and insulin release induced by glucose, but failed to inhibit the insulin secretion provoked by the combination of Ba²⁺ and theophylline. This indicates that the inhibitory effect of gentamycin is due to a reduction of Ca²⁺ entry into B-cells instead of to a harmful effect upon the integrity of the effector system, responsible for the extrusion of the insulin containing granules.Supported by a Grant (No. 79/1872) from the São Paulo State Research Foundation (FAPESP), Brazil.     

Regulation of mitochondrial oxidative phosphorylation by second messenger-mediated signal transduction mechanisms

The mitochondrial oxidative phosphorylation system is responsible for providing the bulk of cellular ATP molecules. There is a growing body of information regarding the regulation of this process by a number of second messenger-mediated signal transduction mechanisms, although direct studies aimed at elucidating this regulation are limited. The main second messengers affecting mitochondrial signal transduction are cAMP and calcium. Other second messengers include ceramide and reactive oxygen species as well as nitric oxide and reactive nitrogen species. This review focuses on available data on the regulation of the mitochondrial oxidative phosphorylation system by signal transduction mechanisms and is organised according to the second messengers involved, because of their pivotal role in mitochondrial function. Future perspectives for further investigations regarding these mechanisms in the regulation of the oxidative phosphorylation system are formulated. Received 11 December 2005; received after revision 14 January 2006; accepted 6 February 2006     

Platelet abnormalities and the pathophysiology of essential hypertension

The mechanisms whereby intracellular calcium concentration is controlled are briefly reviewed. With the current knowledge of both calcium homeostasis and the function and properties of cellular Ca2+-target proteins/signal transduction systems, a dysfunction of cellular calcium metabolism is considered in relation to the pathogenesis of hypertension. Although the enhanced peripheral vascular resistance characteristic of hypertension is ultimately a function of Ca2+ availability for smooth muscle cell contraction, the platelet possesses many parallel biochemical and physiological properties. Therefore, we have utilized the platelet as the cell-model for investigating the role of Ca2+ in hypertension disorders. An overview of Ca2+-linked platelet processes altered in essential hypertension is presented, and an attempt is made to integrate these multiple aberrations in a fundamental membrane lesion.