ACTA PHYSIOLOGICA, Issue 1 2010
A. J. Kolnes
Abstract Aim:, Caffeine and theophylline inhibit phosphatidylinositol 3-kinase (PI3-kinase) activity and insulin-stimulated protein kinase B (PKB) phosphorylation. Insulin-stimulated glucose uptake involves PI3-kinase/PKB, and the aim of the present study was to test the hypothesis that caffeine and theophylline inhibit insulin-stimulated glucose uptake in skeletal muscles. Methods:, Rat epitrochlearis muscles and soleus strips were incubated with insulin and different concentrations of caffeine and theophylline for measurement of glucose uptake, force development and PKB phosphorylation. The effect of caffeine was also investigated in muscles stimulated electrically. Results:, Caffeine and theophylline completely blocked insulin-stimulated glucose uptake in both soleus and epitrochlearis muscles at 10 mm. Furthermore, insulin-stimulated PKB Ser473 and Thr308 and GSK-3, Ser9 phosphorylation were blocked by caffeine and theophylline. Caffeine reduced and theophylline blocked insulin-stimulated glycogen synthase activation. Caffeine stimulates Ca2+ release and force development increased rapidly to 10,20% of maximal tetanic contraction. Dantrolene (25 ,m), a well-known inhibitor of Ca2+ -release, prevented caffeine-induced force development, but caffeine inhibited insulin-stimulated glucose uptake in the presence of dantrolene. Contraction, like insulin, stimulates glucose uptake via translocation of glucose transporter-4 (GLUT4). Caffeine and theophylline reduced contraction-stimulated glucose uptake by about 50%, whereas contraction-stimulated glycogen breakdown was normal. Conclusion:, Caffeine and theophylline block insulin-stimulated glucose uptake independently of Ca2+ release, and the likely mechanism is via blockade of insulin-stimulated PI3-kinase/PKB activation. Caffeine and theophylline also reduced contraction-stimulated glucose uptake, which occurs independently of PI3-kinase/PKB, and we hypothesize that caffeine and theophylline also inhibit glucose uptake in skeletal muscles via an additional and hitherto unknown molecule involved in GLUT4 translocation. [source]

Cardiac basal metabolism: energetic cost of calcium withdrawal in the adult rat heart

ACTA PHYSIOLOGICA, Issue 3 2010
P. Bonazzola
Abstract Aim:, Cardiac basal metabolism upon extracellular calcium removal and its relationship with intracellular sodium and calcium homeostasis was evaluated. Methods:, A mechano-calorimetric technique was used that allowed the simultaneous and continuous measurement of both heat rate and resting pressure in arterially perfused quiescent adult rat hearts. Using pharmacological tools, the possible underlying mechanisms related to sodium and calcium movements were investigated. Results:, Resting heat rate (expressed in mW g,1dry wt) increased upon calcium withdrawal (+4.4 ± 0.2). This response was: (1) unaffected by the presence of tetrodotoxin (+4.3 ± 0.6), (2) fully blocked by both, the decrease in extracellular sodium concentration and the increase in extracellular magnesium concentration, (3) partially blocked by the presence of either nifedipine (+2.8 ± 0.4), KB-R7943 (KBR; +2.5 ± 0.2), clonazepam (CLO; +3.1 ± 0.3) or EGTA (+1.9 ± 0.3). The steady heat rate under Ca2+ -free conditions was partially reduced by the addition of Ru360 (,1.1 ± 0.2) but not CLO in the presence of EGTA, KBR or Ru360. Conclusion:, Energy expenditure for resting state maintenance upon calcium withdrawal depends on the intracellular rise in both sodium and calcium. Our data are consistent with a mitochondrial Ca2+ cycling, not detectable under normal calcium diastolic levels. The experimental condition here analysed, partially simulates findings reported under certain pathological situations including heart failure in which mildly increased levels of both diastolic sodium and calcium have also been found. Therefore, under such pathological conditions, hearts should distract chemical energy to fuel processes associated with sodium and calcium handling, making more expensive the maintenance of their functions. [source]

Effects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal rats

ACTA PHYSIOLOGICA, Issue 1 2010
P. Fang
Abstract Aim:, The aim of this study was to determine the effects of motilin on [Ca2+]i regulation and its underlying molecular mechanism in cultured antral smooth muscle cells (ASMCs). Methods:, Antral cells were isolated and cultured from neonatal rats, and then the [Ca2+]i in these cells was evaluated by calcium fluorescent probe Fluo-3/AM on a laser scanning confocal microscope. Results:, We show that motilin dose-dependently increased [Ca2+]i concentration in cultured ASMCs. Pre-incubation of cells with either the calcium antagonist verapamil (10,5 mol L,1) or the calcium chelator Egtazic (EGTA, 0.1 mmol L,1) significantly suppressed motilin (10,6 mol L,1) induced [Ca2+]i increase as indicated by fluorescent intensity. Interestingly, after mixing with the non-selective intracellular calcium release blocker TMB-8 (10,5 mol L,1), guanosine triphosphate regulatory protein antagonist NEM (10,5 mol L,1), phospholipase C (PLC) inhibitor compound 48/80 (1.2 ,g mL,1) and ryanodine at high concentration (10,5 mol L,1), the motilin-induced [Ca2+]i increase was only partially blocked. The protein kinase C inhibitor d -sphingosine (10,6 mol L,1), however, did not show any inhibitory effect on motilin-induced [Ca2+]i elevation. Conclusions:, Our study suggests that motilin-stimulated [Ca2+]i elevation in ASMCs is probably due to sustained extracellular Ca2+ influx and Ca2+ release from Ca2+ stores via inositol tris-phosphate receptors and ryanodine receptors. Specifically, motilin-induced [Ca2+]i release is accompanied with guanosine triphosphate-binding protein-coupled receptor,PLC,inositol tris-phosphate signalling cascades. [source]

Angiotensin II regulates endothelial cell migration through calcium influx via T-type calcium channel in human umbilical vein endothelial cells

ACTA PHYSIOLOGICA, Issue 4 2010
A. Martini
Abstract Aim:, The T-type calcium channel is expressed in vascular endothelial cells, but its role in endothelial cell function is yet to be elucidated. We analysed the endothelial functional role of T-type calcium channel-dependent calcium under angiotensin II (Ang II) stimulation. Methods:, Human umbilical vein endothelial cells were co-incubated with hormone at 10,7 m and either Efonidipine 10,5 m or Verapamil 10,5 m or Mibefradil 10,5 m or Wortmannin 10,6 m. The contribution of Ang II receptors was evaluated using PD123319 10,7 m and ZD 7155 10,7 m. The calcium ion concentration was observed using Fluo-3 acetossimetil ester. The cells were observed after 3, 6, 9 and 12 h. Results:, The microfluorescence method points out that Ang II induces intracellular calcium modulation in time by distinct mechanisms. AT2 receptor blockade is necessary to observe significant increase in [Ca2+]i levels. Pre-treatment with Mibefradil abolishes Ang II -induced cell migration. Conclusions:, Our data show that Ang II, via AT1 receptor, modulates calcium concentration involving T-type calcium channel and L-type calcium channel but only the calcium influx via T-type calcium channels regulates endothelial cell migration which is essential for angiogenesis. [source]

Contraction-induced changes in skeletal muscle Na+,K+ pump mRNA expression , importance of exercise intensity and Ca2+ -mediated signalling

ACTA PHYSIOLOGICA, Issue 4 2010
N. B. Nordsborg
Abstract Aim:, To investigate if exercise intensity and Ca2+ signalling regulate Na+,K+ pump mRNA expression in skeletal muscle. Methods:, The importance of exercise intensity was evaluated by having trained and untrained humans perform intense intermittent and prolonged exercise. The importance of Ca2+ signalling was investigated by electrical stimulation of rat soleus and extensor digitorum longus (EDL) muscles in combination with studies of cell cultures. Results:, Intermittent cycling exercise at ,85% of VO2peak increased (P < 0.05) ,1 and ,1 mRNA expression ,2-fold in untrained and trained subjects. In trained subjects, intermittent exercise at ,70% of VO2peak resulted in a less (P < 0.05) pronounced increase (,1.4-fold; P < 0.05) for ,1 and no change in ,1 mRNA. Prolonged low intensity exercise increased (P < 0.05) mRNA expression of ,1 ,3.0-fold and ,2 ,1.8-fold in untrained but not in trained subjects. Electrical stimulation of rat soleus, but not EDL, muscle increased (P < 0.05) ,1 mRNA expression, but not when combined with KN62 and cyclosporin A incubation. Ionomycin incubation of cultured primary rat skeletal muscle cells increased (P < 0.05) ,1 and reduced (P < 0.001) ,2 mRNA expression and these responses were abolished (P < 0.05) by co-incubation with cyclosporin A or KN62. Conclusion:, (1) Exercise-induced increases in Na+,K+ pump ,1 and ,1 mRNA expression in trained subjects are more pronounced after high- than after moderate- and low-intensity exercise. (2) Both prolonged low and short-duration high-intensity exercise increase ,1 mRNA expression in untrained subjects. (3) Ca2+i regulates ,1 mRNA expression in oxidative muscles via Ca2+/calmodulin-dependent protein kinase (CaMK) and calcineurin signalling pathways. [source]

Effects of short-term food deprivation on orexin-A-induced intestinal bicarbonate secretion in comparison with related secretagogues

ACTA PHYSIOLOGICA, Issue 3 2010
G. Flemström
Abstract Studies of gastrointestinal physiology in humans and intact animals are usually conducted after overnight fast. We compared the effects of orexin-A, vasoactive intestinal polypeptide (VIP), melatonin, serotonin, uroguanylin, ghrelin and prostaglandin E2 (PGE2) on duodenal bicarbonate secretion in fed and overnight fasted animals. This review is a summary of our findings. Secretagogues were administered by intra-arterial infusion or luminally (PGE2). Enterocyte intracellular calcium ([Ca2+]i) signalling was studied by fluorescence imaging. Total RNA was extracted, reverse transcripted to cDNA and expression of orexin receptors measured by quantitative real-time PCR. Orexin-A stimulates the duodenal secretion in continuously fed animals but not in food-deprived animals. Similarly, short-term fasting causes a 100-fold decrease in the amount of the muscarinic agonist bethanechol required for stimulation of secretion. In contrast, fasting does not affect secretory responses to intra-arterial VIP, melatonin, serotonin, uroguanylin and ghrelin, or that to luminal PGE2. Orexin-A induces [Ca2+]i signalling in enterocytes from fed rats but no significant [Ca2+]i responses occur in enterocytes from fasted animals. In addition, overnight fasting decreases the expression of mucosal orexin receptors. Short-term food deprivation thus decreases duodenal expression of orexin receptors and abolishes the secretory response to orexin-A as well as orexin-A-induced [Ca2+]i signalling. Fasting, furthermore, decreases mucosal sensitivity to bethanechol. The absence of declines in secretory responses to other secretagogues tested strongly suggests that short-term fasting does not affect the secretory capacity of the duodenal mucosa in general. Studies of intestinal secretion require particular evaluation with respect to feeding status. [source]

Potassium-transporting proteins in skeletal muscle: cellular location and fibre-type differences

ACTA PHYSIOLOGICA, Issue 2 2010
M. Kristensen
Abstract Potassium (K+) displacement in skeletal muscle may be an important factor in the development of muscle fatigue during intense exercise. It has been shown in vitro that an increase in the extracellular K+ concentration ([K+]e) to values higher than approx. 10 mm significantly reduce force development in unfatigued skeletal muscle. Several in vivo studies have shown that [K+]e increases progressively with increasing work intensity, reaching values higher than 10 mm. This increase in [K+]e is expected to be even higher in the transverse (T)-tubules than the concentration reached in the interstitium. Besides the voltage-sensitive K+ (Kv) channels that generate the action potential (AP) it is suggested that the big-conductance Ca2+ -dependent K+ (KCa1.1) channel contributes significantly to the K+ release into the T-tubules. Also the ATP-dependent K+ (KATP) channel participates, but is suggested primarily to participate in K+ release to the interstitium. Because there is restricted diffusion of K+ to the interstitium, K+ released to the T-tubules during AP propagation will be removed primarily by reuptake mediated by transport proteins located in the T-tubule membrane. The most important protein that mediates K+ reuptake in the T-tubules is the Na+,K+ -ATPase ,2 dimers, but a significant contribution of the strong inward rectifier K+ (Kir2.1) channel is also suggested. The Na+, K+, 2Cl, 1 (NKCC1) cotransporter also participates in K+ reuptake but probably mainly from the interstitium. The relative content of the different K+ -transporting proteins differs in oxidative and glycolytic muscles, and might explain the different [K+]e tolerance observed. [source]

Acute atrial arrhythmogenesis in murine hearts following enhanced extracellular Ca2+ entry depends on intracellular Ca2+ stores

ACTA PHYSIOLOGICA, Issue 2 2010
Y. Zhang
Abstract Aim:, To investigate the effect of increases in extracellular Ca2+ entry produced by the L-type Ca2+ channel agonist FPL-64176 (FPL) upon acute atrial arrhythmogenesis in intact Langendorff-perfused mouse hearts and its dependence upon diastolic Ca2+ release from sarcoplasmic reticular Ca2+ stores. Methods:, Confocal microscope studies of Fluo-3 fluorescence in isolated atrial myocytes were performed in parallel with electrophysiological examination of Langendorff-perfused mouse hearts. Results:, Atrial myocytes stimulated at 1 Hz and exposed to FPL (0.1 ,m) initially showed (<10 min) frequent, often multiple, diastolic peaks following the evoked Ca2+ transients whose amplitudes remained close to control values. With continued pacing (>10 min) this reverted to a regular pattern of evoked transients with increased amplitudes but in which diastolic peaks were absent. Higher FPL concentrations (1.0 ,m) produced sustained and irregular patterns of cytosolic Ca2+ activity, independent of pacing. Nifedipine (0.5 ,m), and caffeine (1.0 mm) and cyclopiazonic acid (CPA) (0.15 ,m) pre-treatments respectively produced immediate and gradual reductions in the F/F0 peaks. Such nifedipine and caffeine, or CPA pre-treatments, abolished, or reduced, the effects of 0.1 and 1.0 ,m FPL on cytosolic Ca2+ signals. FPL (1.0 ,m) increased the incidence of atrial tachycardia and fibrillation in intact Langendorff-perfused hearts without altering atrial effective refractory periods. These effects were inhibited by nifedipine and caffeine, and reduced by CPA. Conclusion:, Enhanced extracellular Ca2+ entry exerts acute atrial arrhythmogenic effects that is nevertheless dependent upon diastolic Ca2+ release. These findings complement reports that associate established, chronic, atrial arrhythmogenesis with decreased overall inward Ca2+ current. [source]

Released nucleotides amplify the cilium-dependent, flow-induced [Ca2+]i response in MDCK cells

ACTA PHYSIOLOGICA, Issue 3 2009
H. A. Praetorius
Abstract Aim:, Changes in perfusate flow produce increases in [Ca2+]i in renal epithelial cells. Cultured renal epithelia require primary cilia to sense subtle changes in flow. In perfused kidney tubules this flow response is caused by nucleotide signalling via P2Y2 receptors. It is, however, not known whether nucleotides are released by mechanical stress applied to renal primary cilia. Here we investigate whether nucleotides are released during the cilium-dependent flow response and contribute to the flow-induced, cilium-dependent [Ca2+]i signal. Methods:, MDCK cells loaded with Fluo-4-AM were observed at 37 °C in semi-open single or closed-double perfusion chambers. Results:, Our data suggest a purinergic component of the cilium-dependent flow-response: (1) ATP scavengers and P2 receptor antagonists reduced (55%) the cilium-dependent flow-response; (2) ATP added at subthreshold concentration sensitized the renal epithelia to flow changes; (3) increases in fluid flow transiently enhanced the ATP concentration in the superfusate (measured by biosensor-cells). To test if nucleotides were released in sufficient quantities to stimulate renal epithelia we used non-confluent MDCK cells without cilia as reporter cells. We confirmed that non-confluent cells do not respond to changes in fluid flow. Placing confluent, ciliated cells upstream in the in-flow path of the non-confluent cells made them responsive to fluid flow changes. This phenomenon was not observed if either non-confluent or de-ciliated confluent cells were placed upstream. The [Ca2+]i -response in the non-confluent cells with ciliated cells upstream was abolished by apyrase and suramin. Conclusion:, This suggests that subtle flow changes sensed by the primary cilium induces nucleotide release, which amplifies the epithelial [Ca2+]i -response. [source]

Calmodulin kinase II initiates arrhythmogenicity during metabolic acidification in murine hearts

ACTA PHYSIOLOGICA, Issue 1 2009
T. H. Pedersen
Abstract Aim:, The multifunctional signal molecule calmodulin kinase II (CaMKII) has been associated with cardiac arrhythmogenesis under conditions where its activity is chronically elevated. Recent studies report that its activity is also acutely elevated during acidosis. We test a hypothesis implicating CaMKII in the arrhythmogenesis accompanying metabolic acidification. Methods:, We obtained monophasic action potential recordings from Langendorff-perfused whole heart preparations and single cell action potentials (AP) using whole-cell patch-clamped ventricular myocytes. Spontaneous sarcoplasmic reticular (SR) Ca2+release events during metabolic acidification were investigated using confocal microscope imaging of Fluo-4-loaded ventricular myocytes. Results:, In Langendorff-perfused murine hearts, introduction of lactic acid into the Krebs-Henseleit perfusate resulted in abnormal electrical activity and ventricular tachycardia. The CaMKII inhibitor, KN-93 (2 ,m), reversibly suppressed this spontaneous arrhythmogenesis during intrinsic rhythm and regular 8 Hz pacing. However, it failed to suppress arrhythmia evoked by programmed electrical stimulation. These findings paralleled a CaMKII-independent reduction in the transmural repolarization gradients during acidosis, which previously has been associated with the re-entrant substrate under other conditions. Similar acidification produced spontaneous AP firing and membrane potential oscillations in patch-clamped isolated ventricular myocytes when pipette solutions permitted cytosolic Ca2+ to increase following acidification. However, these were abolished by both KN-93 and use of pipette solutions that held cytosolic Ca2+ constant during acidosis. Acidosis also induced spontaneous Ca2+ waves in isolated intact Fluo-4-loaded myocytes studied using confocal microscopy that were abolished by KN-93. Conclusion:, These findings together implicate CaMKII-dependent SR Ca2+ waves in spontaneous arrhythmic events during metabolic acidification. [source]

No relationship between enzyme activity and structure of nucleotide binding site in sarcoplasmic reticulum Ca2+ -ATPase from short-term stimulated rat muscle

ACTA PHYSIOLOGICA, Issue 4 2009
T. Mishima
Abstract Aim:, We examined whether structural alterations to the adenine nucleotide binding site (ANBS) within sarcoplasmic (endo) reticulum Ca2+ -ATPase (SERCA) would account for contraction-induced changes in the catalytic activity of the enzyme as assessed in vitro. Methods:, Repetitive contractions were induced in rat gastrocnemius by electrical nerve stimulation. Measurements of sarcoplasmic reticulum properties were performed on control and stimulated muscles immediately after or at 30 min after the cessation of 5-min stimulation. In order to examine the properties at the ANBS, the binding capacity of SERCA to fluorescence isothiocyanate (FITC), a competitive inhibitor at the ANBS, was analysed in microsomes. Results:, Short-term electrical stimulation evoked a 23.9% and 32.6% decrease (P < 0.05) in SERCA activity and in the FITC binding capacity, respectively, in the superficial region of the muscle. Whereas SERCA activity reverted to normal levels during 30-min recovery, a restoration of the FITC binding capacity did not occur. Conclusion:, The discordant changes between the enzyme activity and the FITC binding suggest that, at least during recovery after exercise, changes in SERCA activity may not correlate closely with structural alterations to the ANBS within the enzyme. [source]

The role of free fatty acids, pancreatic lipase and Ca2+ signalling in injury of isolated acinar cells and pancreatitis model in lipoprotein lipase-deficient mice

ACTA PHYSIOLOGICA, Issue 1 2009
F. Yang
Abstract Aim and methods:, Recurrent pancreatitis is a common complication of severe hypertriglyceridaemia (HTG) often seen in patients carrying various gene mutations in lipoprotein lipase (LPL). This study investigates a possible pathogenic mechanism of cell damage in isolated mouse pancreatic acinar cells and of pancreatitis in LPL-deficient and in wild type mice. Results:, Addition of free fatty acids (FFA) or of chylomicrons to isolated pancreatic acinar cells caused stimulation of amylase release, and at higher concentrations it also caused cell damage. This effect was decreased in the presence of the lipase inhibitor orlistat. Surprisingly, pancreatic lipase whether in its active or inactive state could act like an agonist by inducing amylase secretion, increasing cellular cGMP levels and converting cell damaging sustained elevations of [Ca2+]cyt to normal Ca2+ oscillations. Caerulein increases the levels of serum amylase and caused more severe inflammation in the pancreas of LPL-deficient mice than in wild type mice. Conclusion:, We conclude that high concentrations of FFA as present in the plasma of LPL-deficient mice and in patients with HTG lead to pancreatic cell damage and are high risk factors for the development of acute pancreatitis. In addition to its enzymatic effect which leads to the generation of cell-damaging FFA from triglycerides, pancreatic lipase also prevents Ca2+ overload in pancreatic acinar cells and, therefore, counteracts cell injury. [source]

Local Ca2+ influx through CRAC channels activates temporally and spatially distinct cellular responses

ACTA PHYSIOLOGICA, Issue 1 2009
A. B. Parekh
Abstract Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels controls a disparate array of key cellular responses. In this review, recent work will be described that shows local Ca2+ influx through CRAC channels has important spatial and temporal consequences on cell function. A localized Ca2+ rise below the plasma membrane activates, within tens of seconds, catabolic enzymes resulting in the generation of the intracellular messenger arachidonic acid and the paracrine pro-inflammatory molecule LTC4. In addition, local Ca2+ entry can activate gene expression, which develops over tens of minutes. Local Ca2+ influx through CRAC channels therefore has far-reaching consequences on intra- and intercellular communication. [source]

Modulation of calcium signalling by intracellular organelles seen with targeted aequorins

ACTA PHYSIOLOGICA, Issue 1 2009
M. T. Alonso
Abstract The cytosolic Ca2+ signals that trigger cell responses occur either as localized domains of high Ca2+ concentration or as propagating Ca2+ waves. Cytoplasmic organelles, taking up or releasing Ca2+ to the cytosol, shape the cytosolic signals. On the other hand, Ca2+ concentration inside organelles is also important in physiology and pathophysiology. Comprehensive study of these matters requires to measure [Ca2+] inside organelles and at the relevant cytosolic domains. Aequorins, the best-known chemiluminescent Ca2+ probes, are excellent for this end as they do not require stressing illumination, have a large dynamic range and a sharp Ca2+ -dependence, can be targeted to the appropriate location and engineered to have the proper Ca2+ affinity. Using this methodology, we have evidenced the existence in chromaffin cells of functional units composed by three closely interrelated elements: (1) plasma membrane Ca2+ channels, (2) subplasmalemmal endoplasmic reticulum and (3) mitochondria. These Ca2+ -signalling triads optimize Ca2+ microdomains for secretion and prevent propagation of the Ca2+ wave towards the cell core. Oscillatory cytosolic Ca2+ signals originate also oscillations of mitochondrial Ca2+ in several cell types. The nuclear envelope slows down the propagation of the Ca2+ wave to the nucleus and filters high frequencies. On the other hand, inositol-trisphosphate may produce direct release of Ca2+ to the nucleoplasm in GH3 pituitary cells, thus providing mechanisms for selective nuclear signalling. Aequorins emitting at different wavelengths, prepared by fusion either with green or red fluorescent protein, permit simultaneous and independent monitorization of the Ca2+ signals in different subcellular domains within the same cell. [source]

Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspects

ACTA PHYSIOLOGICA, Issue 1 2009
N. Fedirko
Abstract Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca2+ store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors [Ins(1,4,5)P3Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P3Rs by Ins(1,4,5)P3. We have also investigated the possible consequences of nuclear calcium signals: the role of Ca2+ in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca2+ -mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca2+: the IEG expression level was lowest in Ca2+ -free solution, increased at the physiological level of 1 mm [Ca2+]o and was maximal at 10 mm [Ca2+]o, i.e.: 102 ± 22% and 163 ± 15% for c-fos; c-myc ,73 ± 13% and 106 ± 24%; c-jun ,49 ± 8% and 59 ± 9% at 1 and 10 mm of extracellular Ca2+ respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca2+ together with nuclear Ca2+ signals induced by CCK play important roles in the induction of IEG expression. [source]

Roles of the actin-binding proteins in intracellular Ca2+ signalling

ACTA PHYSIOLOGICA, Issue 1 2009
J. T. Chun
Abstract Starfish oocytes undergo massive intracellular Ca2+ signalling during meiotic maturation and fertilization. Although the igniting stimulus of Ca2+ mobilization may differ in different cell contexts, its final leverage is usually the Ca2+ -releasing second messengers such as InsP3, cADPr and NAADP. The general scheme of intracellular Ca2+ release is that the corresponding receptors for these molecules serve as ion channels to release free Ca2+ from its internal stores such as the lumen of the endoplasmic reticulum. However, a growing body of evidence has suggested that intracellular Ca2+ release can be strongly modulated by the actin cytoskeleton. Although it is known that Ca2+ contributes to remodelling of the actin cytoskeleton, whether the actin cytoskeleton modulates Ca2+ signalling in return has not been much explored. An emerging candidate to answer to this reciprocal causality of Ca2+ and the actin cytoskeleton may be actin-binding proteins. In this review, we discuss how the actin cytoskeleton may fit into the known mechanisms of intracellular Ca2+ release, and propose two models to explain the experimental data. [source]

Mechanisms of metabotropic glutamate receptor-mediated synaptic signalling in cerebellar Purkinje cells

ACTA PHYSIOLOGICA, Issue 1 2009
J. Hartmann
Abstract The metabotropic glutamate receptors type 1 (mGluR1s) are required for a normal function of the mammalian cerebellum. These G-protein-coupled receptors are abundantly expressed in the principle cerebellar cells, namely the Purkinje neurones. Under physiological conditions, mGluR1s are activated during repetitive activity of both afferent glutamatergic synaptic inputs provided by the climbing and parallel fibres respectively. Unlike the common ionotropic glutamate receptors that underlie rapid synaptic excitation, mGluR1s produce a complex post-synaptic response consisting of a Ca2+ -release signal from intracellular stores and a slow excitatory post-synaptic potential. While it is well established that the mGluR1-dependent Ca2+ -release signal from intracellular stores involves the activation of inositol-trisphosphate receptors, the mechanisms underlying the slow synaptic excitation remained unclear. Here we will review recent evidence indicating an essential role of C-type transient receptor potential (TRPC) cation channels, especially that of the subunit TRPC3, for the generation of the mGluR1-dependent synaptic current. For the signalling pathways underlying both, Ca2+ -release from intracellular stores and the slow synaptic potential, we present current knowledge about the activators, downstream effectors and possible roles for mGluR1-dependent signalling in Purkinje neurones. [source]

When is high-Ca2+ microdomain required for mitochondrial Ca2+ uptake?,

ACTA PHYSIOLOGICA, Issue 1 2009
A. Spät
Abstract Ca2+ release from IP3 -sensitive stores in the endoplasmic reticulum (ER) induced by Ca2+ -mobilizing agonists generates high-Ca2+ microdomains between ER vesicles and neighbouring mitochondria. Here we present a model that describes when such microdomains are required and when submicromolar [Ca2+] is sufficient for mitochondrial Ca2+ uptake. Mitochondrial Ca2+ uptake rate in angiotensin II-stimulated H295R adrenocortical cells correlates with the proximity between ER vesicles and the mitochondrion, reflecting the uptake promoting effect of high-Ca2+ peri-mitochondrial microdomains. Silencing or inhibition of p38 mitogen-activated protein kinase (MAPK) or inhibition of the novel isoforms of protein kinase C enhances mitochondrial Ca2+ uptake and abolishes the positive correlation between Ca2+ uptake and ER-mitochondrion proximity. Inhibition of protein phosphatases attenuates mitochondrial Ca2+ uptake and also abolishes its positive correlation with ER-mitochondrion proximity. We postulate that during IP3 -induced Ca2+ release, Ca2+ uptake is confined to ER-close mitochondria, because of the simultaneous activation of the protein kinases. Attenuation of Ca2+ uptake prevents Ca2+ overload of mitochondria and thus protects the cell against apoptosis. On the other hand, all the mitochondria accumulate Ca2+ at a non-inhibited rate during physiological Ca2+ influx through the plasma membrane. Membrane potential is higher in ER-distant mitochondria, providing a bigger driving force for Ca2+ uptake. Our model explains why comparable mitochondrial Ca2+ signals are formed in response to K+ and angiotensin II (equipotent in respect to global cytosolic Ca2+ signals), although only the latter generates high-Ca2+ microdomains. [source]

Downstream from calcium signalling: mitochondria, vacuoles and pancreatic acinar cell damage

ACTA PHYSIOLOGICA, Issue 1 2009
S. Voronina
Abstract Ca2+ is one of the most ancient and ubiquitous second messengers. Highly polarized pancreatic acinar cells serve as an important cellular model for studies of Ca2+ signalling and homeostasis. Downstream effects of Ca2+ signalling have been and continue to be an important research avenue. The primary functions regulated by Ca2+ in pancreatic acinar cells , exocytotic secretion and fluid secretion , have been defined and extensively characterized in the second part of the last century. The role of cytosolic Ca2+ in cellular pathology and the related question of the interplay between Ca2+ signalling and bioenergetics are important current research lines in our and other laboratories. Recent findings in these interwoven research areas are discussed in the current review. [source]

Could chronic pain and spread of pain sensation be induced and maintained by glial activation?

ACTA PHYSIOLOGICA, Issue 1-2 2006
E. Hansson
Abstract An injury often starts with acute physiological pain, which becomes inflammatory or neuropathic, and may sometimes become chronic. It has been proposed recently that activated glial cells, astrocytes and microglia within the central nervous system could maintain the pain sensation even after the original injury or inflammation has healed, and convert it into chronic by altering neuronal excitability. Glial cell activation has also been proposed to be involved in the phenomenon of spread of pain sensation ipsilaterally or to the contralateral side (i.e. mirror image pain). Substance P and calcitonin gene-related peptide, released due to an inflammatory process, interact with the endothelial cells of the blood,spinal cord and blood,brain barriers. The barriers open partially and substances may influence adjacent glial cells. Such substances are also released from neurones carrying the ,pain message' all the way from the injury to the cerebral cortex. Pro-inflammatory cytokines may be released from the microglial cells, and astroglial Ca2+ -transients or oscillations may spread within the astroglial networks. One theory is that Ca2+ -oscillations could facilitate the formation of new synapses. These new synapses could establish neuronal contacts for maintaining and spreading the pain sensation. If this theory holds true, it is possible that Ca2+ waves, production of cytokines and growth factors could be modified by selective anti-inflammatory drugs to achieve a balance in the activities of the different intercellular and intracellular processes. This paper reviews current knowledge about glial mechanisms underlying the phenomena of chronic pain and spread of the pain sensation. [source]

Gating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+

ACTA PHYSIOLOGICA, Issue 4 2006
L. Lacinová
Abstract Aim:, We have investigated the influence of Ca2+ ions on the basic biophysical properties of T-type calcium channels. Methods:, The Cav3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca2+ or Na+ ions were used as charge carriers. The intracellular Ca2+ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 ,m Ca2+ into the non-buffered intracellular solution. Various combinations of extra- and intracellular solutions yielded high, intermediate or low intracellular Ca2+ levels. Results:, The amplitude of the calcium current was independent of intracellular Ca2+ concentrations. High levels of intracellular Ca2+ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca2+ by Na+ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca2+ was a charge carrier. The number of available channels saturated for membrane voltages more negative than ,100 mV for the Ca2+ current, but did not reach steady state even at ,150 mV for the Na+ current. Conclusions:, Ca2+ ions facilitate transitions of Cav3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor. [source]

Calcium handling in afferent arterioles

ACTA PHYSIOLOGICA, Issue 4 2004
M. Salomonsson
Abstract The cytosolic intracellular calcium concentration ([Ca2+]i) is a major determining factor in the vascular smooth muscle tone. In the afferent arteriole it has been shown that agonists utilizing G-protein coupled receptors recruit Ca2+ via release from intracellular stores and entry via pathways in the plasma membrane. The relative importances of entry vs. mobilization seem to differ between different agonists, species and preparations. The entry pathway might include different types of voltage sensitive Ca2+ channels located in the plasmalemma such as dihydropyridine sensitive L-type channels, T-type channels and P/Q channels. A role for non-voltage sensitive entry pathways has also been suggested. The importance of voltage sensitive Ca2+ channels in the control of the tone of the afferent arteriole (and thus in the control of renal function and whole body control of extracellular fluid volume and blood pressure) sheds light on the control of the membrane potential of afferent arteriolar smooth muscle cells. Thus, K+ and Cl, channels are of importance in their role as major determinants of membrane potential. Some studies suggest a role for calcium-activated chloride (ClCa) channels in the renal vasoconstriction elicited by agonists. Other investigators have found evidence for several types of K+ channels in the regulation of the afferent arteriolar tone. The available literature in this field regarding afferent arterioles is, however, relatively sparse and not conclusive. This review is an attempt to summarize the results obtained by others and ourselves in the field of agonist induced afferent arteriolar Ca2+ recruitment, with special emphasis on the control of voltage sensitive Ca2+ entry. Outline of the Manuscript: This manuscript is structured as follows: it begins with an introduction where the general role for [Ca2+]i as a key factor in the regulation of the tone of vascular smooth muscles (VSMC) is detailed. In this section there is an emphasis is on observations that could be attributed to afferent arteriolar function. We then investigate the literature and describe our results regarding the relative roles for Ca2+ entry and intracellular release in afferent arterioles in response to vasoactive agents, with the focus on noradrenalin (NA) and angiotensin II (Ang II). Finally, we examine the role of ion channels (i.e. K+ and Cl, channels) for the membrane potential, and thus activation of voltage sensitive Ca2+ channels. [source]

Reduced calcium tolerance in rat cardiomyocytes after myocardial infarction

ACTA PHYSIOLOGICA, Issue 4 2002
I. Sjaastad
ABSTRACT During ischaemia and reperfusion the intracellular Na+ concentration is elevated in the cardiomyocytes and the cells are depolarized, both favouring reverse mode Na,Ca-exchange loading of the cell with Ca2+. We examined whether cardiomyocytes from rats with congestive heart failure (CHF) and younger rats (HINCX) which both have a high expression of the Na,Ca-exchanger protein (NCX) showed reduced tolerance to extracellular Ca2+. The CHF was induced in Isofluran anaesthetized rats by left coronary artery ligation. Isolated cardiomyocytes were loaded with Fura-2AM and 140 mm Na+ and exposed to 0.05 mm Ca2+. Expression of the Na,Ca-exchanger protein was analysed. Fura-2 340/380 ratio rose more rapidly in HINCX and CHF than in SHAM, and the rise was abolished by Ni2+. Hypercontracture developed more frequently in HINCX and CHF than in SHAM cells. The amount of NCX was 54% higher in HINCX and 76% higher in CHF compared with SHAM. Na+ -loaded cardiomyocytes from CHF and HINCX rats are more susceptible to Ca2+ overload than SHAM cells because of the increased capacity for Na,Ca-exchange. [source]

Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumber

CYTOSKELETON, Issue 4 2009
Masaya Morita
Abstract The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (>7.8) solution without cAMP, and a phosphorylation assay using (,-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+ -binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+ -sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+ -sensitive flagellar protein phosphorylation. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source]

Anisotropic contraction in forisomes: Simple models won't fit

CYTOSKELETON, Issue 5 2008
Winfried S. Peters
Abstract Forisomes are ATP-independent, Ca2+ -driven contractile protein bodies acting as reversible valves in the phloem of plants of the legume family. Forisome contraction is anisotropic, as shrinkage in length is associated with radial expansion and vice versa. To test the hypothesis that changes in length and width are causally related, we monitored Ca2+ - and pH-dependent deformations in the exceptionally large forisomes of Canavalia gladiata by high-speed photography, and computed time-courses of derived geometric parameters (including volume and surface area). Soybean forisomes, which in the resting state resemble those of Canavalia geometrically but have less than 2% of the volume, were also studied to identify size effects. Calcium induced sixfold volume increases in forisomes of both species; in soybean, responses were completed in 0.15 s, compared to about 0.5 s required for a rapid response in Canavalia followed by slow swelling for several minutes. This size-dependent behavior supports the idea that forisome contractility might rest on similar mechanisms as those of polyelectrolyte gels, a class of artificial "smart" materials. In both species, time-courses of forisome length and diameter were variable and lacked correlation, arguing against a simple causal relationship between changes in length and width. Moreover, changes in the geometry of soybean forisomes differed qualitatively between Ca2+ - and pH-responses, suggesting that divalent cations and protons target different sites on the forisome proteins. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries

ACTA PHYSIOLOGICA, Issue 2 2001
S. Horiguchi
The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 ,M) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (,35 ± 2 mV at 143 mM [Na+]o, ,37 ± 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 ± 4 vs. 47 ± 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+ -extrusion. [source]

Platelet-derived growth factor receptors expressed in response to injury of differentiated vascular smooth muscle in vitro: effects on Ca2+ and growth signals

ACTA PHYSIOLOGICA, Issue 2 2001
A. Lindqvist
Vascular smooth muscle cells (VSMCs) in the intact vascular wall are differentiated for contraction, whereas the response to vascular injury involves transition towards a synthetic phenotype, with increased tendency for proliferation. Platelet-derived growth factor (PDGF) is thought to be important for this process. We investigated expression and functional coupling of PDGF receptors (PDGFRs) , and , in rat tail arterial rings kept in organ culture, in order to capture early events in the phenotypic transition. In freshly dissected rings no PDGFR immunoreactivity was found in medial VSMCs, whereas PDGFR , was detected in nerve fibres. After organ culture for 1,4 days PDGFR , and , as well as phospholipase C,2 (PLC,2), known to couple to PDGFR, were expressed in VSMCs within 100 ,m of the cut ends. Calponin, a marker for the contractile phenotype, was decreased near the injured area, suggesting that cells were in transition towards synthetic phenotype. In these cells, which showed functional Ca2+ -release from the sarcoplasmic reticulum, PDGF-AB (100 ng mL,1) had no effect on [Ca2+]i, whereas cultured VSMCs obtained from explants of rat tail arterial rings responded to PDGF-AB with an increase in [Ca2+]i. However, PDGFR within the cultured rings coupled to growth signalling pathways, as PDGF-AB caused a tyrphostin AG1295-sensitive activation of extracellular signal-regulated kinases 1 and 2 and of [3H]-thymidine incorporation. Thus, early expression of PDGFR in VSMC adjacent to sites of vascular injury coincides with signs of dedifferentiation. These receptors couple to growth signalling, but do not activate intracellular Ca2+ release. [source]

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation

CYTOSKELETON, Issue 8 2006
Masaya Morita
Abstract Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10,6 M Ca2+. Motility features changed when Ca2+ concentrations were increased from 10,6 to 10,5 M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca2+. Thus, it is possible that Ca2+ binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca2+ -binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by 45Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca2+ -binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca2+/CaM-dependent manner for regulating the dynein activity. A 32P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca2+/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10,5 M Ca2+. It is possible that an increase in Ca2+ induces Ca2+/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source]

Ca2+ -dependent in vitro contractility of a precipitate isolated from an extract of the heliozoon Actinophrys sol

CYTOSKELETON, Issue 2 2006
Mikihiko Arikawa
Abstract Contraction of axopodia in actinophrid heliozoons (protozoa) is induced by a unique contractile structure, the "contractile tubules structure (CTS)". We have previously shown that a cell homogenate of the heliozoon Actinophrys sol yields a precipitate on addition of Ca2+ that is mainly composed of filamentous structures morphologically identical to the CTS. In this study, to further characterize the nature of the CTS in vitro, biochemical and physiological properties of the precipitate were examined. SDS-PAGE analysis showed that the Ca2+ -induced precipitate was composed of many proteins, and that no proteins in the precipitate showed any detectable changes in electrophoretic mobility on addition of Ca2+. Addition of extraneous proteins such as bovine serum albumin to the cell homogenate resulted in cosedimentation of the proteins with the Ca2+ -induced precipitate, suggesting that the CTS has a high affinity for other proteins that are not related to precipitate formation. Appearance and disappearance of the precipitate were repeatedly induced by alternating addition of Ca2+ and EGTA, and its protein composition remained unchanged even after repeated cycles. When adhered to a glass surface, the precipitate showed Ca2+ -dependent contractility with a threshold of 10,100 nM, and this contractility was not inhibited by colchicine or cytochalasin B. The precipitate repeatedly contracted and relaxed with successive addition and removal of Ca2+, indicating that the contraction was controlled by Ca2+ alone with no need for any other energy supply. From our characterization of the precipitate, we concluded that its Ca2+ -dependent formation and contraction are associated with the unique contractile organelle, the "contractile tubules structure". Cell Motil. Cytoskeleton 2006. © 2005 Wiley-Liss, Inc. [source]

Role of the Na+/Ca2+ exchanger in calcium homeostasis and human sperm motility regulation

CYTOSKELETON, Issue 2 2006
Zoltán Krasznai
Abstract A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing ,1 ,M Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3,,4, -dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 ,M, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50 = 2.44 ,M). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role. Cell Motil. Cytoskeleton 2006. © 2005 Wiley-Liss, Inc. [source]