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J G Iversen

Publications and source records attributed to J G Iversen.

At least 19 recordsLinked to original sources
PubMed2000

Ruled by waves? Intracellular and intercellular calcium signalling.

The field of calcium signalling has evolved rapidly the last 20 years. Physiologists had worked with cytosolic Ca2+ as the coupler of excitation and contraction of muscles and as a secretory signal in exocrine glands and in the synapses of the brain for several decades before the discovery of cellular calcium as a second messenger. Development of powerful techniques for measuring the concentration of cytosolic free calcium ions in cell suspensions and later in single cells and even in different cellular compartments, has resulted in an upsurge in the knowledge of the cellular machinery involved in intracellular calcium signalling. However, the focus on intracellular mechanisms might have led this field of study away from physiology. During the last few years there is an increasing evidence for an important role of calcium also as an intercellular signal. Via gap junctions calcium is able to co-ordinate cell populations and even organs like the liver. Here we will give an overview of the general mechanisms of intracellular calcium signalling, and then review the recent data on intercellular calcium signals. A functional coupling of cells in different tissues and organs by the way of calcium might be an important mechanism for controlling and synchronizing physiological responses

Animals↗
PubMed1999

Coagulation factors VIIa and Xa induce cell signaling leading to up-regulation of the egr-1 gene.

Intracellular signaling induced by the coagulation factors (F) VIIa and Xa is poorly understood. We report here studies on these processes in a human keratinocyte line (HaCaT), which is a constitutive producer of tissue factor (TF) and responds to both FVIIa and FXa with elevation of cytosolic Ca(2+), phosphorylation of extracellular signal-regulated kinase (Erk) 1/2, p38(MAPK), and c-Jun N-terminal kinase, and up-regulation of transcription of the early growth response gene-1 (egr-1). Using egr-1 as end point, we observed with both agonists that phosphatidylinositol-specific phospholipase C and the mitogen-activated protein kinase/Erk kinase/Erk pathway were mediators of the responses. The responses to FVIIa were TF-dependent and up-regulation of egr-1 mRNA did not require presence of the TF cytoplasmic domain. Antibodies to EPR-1 and factor V had no effect on the response to FXa. We have provided evidence that TF is not the sole component of the FVIIa receptor. The requirement for proteolytic activity of both FVIIa and FXa suggests that protease-activated receptors may be involved. We now report evidence suggesting that protease-activated receptor 2 or a close homologue may be a necessary but not sufficient component of this particular signal transduction pathway. The up-regulation of egr-1 describes one way by which the initiation of blood coagulation may influence gene transcription. The ability of these coagulation proteases to induce intracellular signals at concentrations at or below the plasma concentrations of their zymogen precursors suggests that these processes may occur also in vivo.

Animals↗
PubMed1999

Interferon-gamma elicits a G-protein-dependent Ca2+ signal in human neutrophils after depletion of intracellular Ca2+ stores.

Interferon-gamma (IFN-gamma) has multiple effects on Ca2+ signalling in polymorphonuclear neutrophils (PMNs), including evoked cytosolic Ca2+ transients, increased capacitative calcium influx and increased sequestration of Ca2+ in intracellular stores. The present study was conducted to elucidate the mechanism behind the Ca2+ transients. As observed before, the IFN-gamma-evoked Ca2+ signals were apparent when extracellular Ca2+ was removed. A new finding was that the proportion of responding cells and the extent of calcium release increased with increasing time in EGTA buffer. As assessed by N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated Ca2+ release, the intracellular stores were depleted during this incubation period, and the extent of depletion correlated well with the appearance of IFN-gamma-induced Ca2+ signals. This store dependence of the IFN-gamma-induced Ca2+ signals was confirmed by the appearance of IFN-gamma-evoked Ca2+ signals in the presence of extracellular Ca2+ after store depletion by thapsigargin. The appearance of IFN-gamma-mediated Ca2+-signals in the presence of EGTA indicates that IFN-gamma stimulates Ca2+ release from intracellular stores. This was confirmed by the inability of the calcium transportation blocker La3+ to abolish the IFN-gamma response and the total abrogation of the response by the phospholipase C inhibitor U73122. Although these latter results imply a role for inositol 1,4,5-trisphosphate(IP3) in IFN-gamma signalling, comparison of IFN-gamma-evoked responses with fMLP responses revealed clear differences that suggest different signal-transduction pathways. However, responses to fMLP and IFN-gamma were both depressed by pertussis toxin, and the IFN-gamma responses were, in addition, inhibited by the tyrosine kinase inhibitor genistein. Further evidence of the involvement of tyrosine kinase was a slight stimulatory effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate. The PI-3K activity was of minor importance. In conclusion, we present evidence of a novel signal-transduction mechanism for IFN-gamma in PMNs, dependent on tyrosine kinase activity, a pertussis toxin-sensitive G protein and phospholipase C activity.

Adult↗
PubMed1999

Mechanisms of the relaxant and contractile responses to bradykinin in rat duodenum.

The signal pathway for bradykinin-induced relaxation followed by contraction in the isolated rat duodenum was investigated by comparing the effect of blocking agents on the response to bradykinin and acetylcholine. The phospholipase C inhibitor U-73122 inhibited the relaxation induced by bradykinin, but had no effect on the contraction to either bradykinin or acetylcholine. The same response pattern was observed when the tissues were pre-treated with thapsigargin, a selective inhibitor of microsomal Ca2+ pumps. An inhibitor of non-voltage-dependent Ca2+ influx, SK&F 96365, inhibited the relaxant response to bradykinin and the contraction induced by acetylcholine, but not the contraction induced by bradykinin. In Ca2+-free Krebs-Henseleit buffer, the tissues failed to respond when they were exposed to either bradykinin or acetylcholine. When the tissues were partly depolarized (30 mM KCI), both bradykinin and acetylcholine induced contraction, while the relaxant response to bradykinin was almost completely abolished. Apamin (an antagonist of low-conductance calcium-activated K+ channel) together with charybdotoxin (CTX, an antagonist of large-conductance calcium-activated K+ channel) and CTX alone inhibited the relaxant but not the contractile response to bradykinin. We conclude that the biphasic response in isolated rat duodenum to bradykinin involves two distinct pathways. We propose that the relaxant component is induced indirectly via inositol-mediated increase in cytosolic Ca2+ in non-muscle cells with subsequent signals to the smooth muscle cells, whereas the contractile response is induced by direct effect on the smooth muscle cells.

Acetylcholine↗
PubMed1999

Bradykinin elevates cytosolic Ca2+ concentration in smooth muscle cells isolated from rat duodenum.

The effect of bradykinin on the cytosolic Ca2+ concentration were measured in single, Fura-2 loaded, smooth muscle cells isolated from rat duodenum. All cells responded with a Ca2+ signal when exposed to bradykinin. The bradykinin response consisted of an initial Ca2+ spike followed by a plateau. Pre-treatment of single muscle cells with either the phospholipase C blocker U-73122 or thapsigargin, which is a potent inhibitor of the endoplasmic reticulum Ca2+-ATPase, inhibited the response to bradykinin. Pre-treatment of the cells with EGTA or La3+ to inhibit the Ca2+ influx, abolished the response induced by bradykinin. We conclude that bradykinin applied to single smooth muscle cells from rat duodenum, increases cytosolic Ca2+ by emptying intracellular Ca2+ stores, and by contribution from extracellular Ca2+. In contrast to bradykinin-induced response in isolated rat duodenum (a relaxation followed by a contraction), we did not observe a biphasic effect of bradykinin on cytosolic Ca2+ in single muscle cells. Bradykinin may thus cause relaxation of duodenal smooth muscle indirectly through an effect on neighbouring cells as dilatation is brought about by this agent in blood vessels.

Animals↗
PubMed1998

Bradykinin causes contraction in rat uterus through the same signal pathway as oxytocin.

The signal pathway for bradykinin-induced contraction of the uterine smooth muscle was investigated by comparing the effect of blocking agents on bradykinin and oxytocin induced contractions of the isolated rat uterus in organ bath. The phospholipase C inhibitor U-73,122 abolished the effect of both bradykinin and oxytocin. Inhibition of non-voltage-dependent Ca2+ influx by SK & F 96,365 reduced the contraction induced by both agonists to about 20% of control. The tissues failed to contract when they were exposed to bradykinin or oxytocin in Ca(2+)-free Krebs-Henseleit buffer with 2 mM EDTA. Both bradykinin and oxytocin induced further contraction when the tissues were partially depolarized and partially contracted by 30 mM KCl. These observations suggest that bradykinin, like oxytocin, activates phospholipase C which generates IP3 with a subsequent release of Ca2+ from intracellular stores followed by store-operated Ca2+ influx. Thus, membrane potential independent steps appear to be important in bradykinin-induced contraction in the rat uterus.

Animals↗
PubMed1998

IFN-gamma induces calcium transients and increases the capacitative calcium entry in human neutrophils.

We have previously reported that long-term priming of human polymorphonuclear neutrophilic granulocytes (PMN) with interferon-gamma (IFN-gamma) increased the fMLP-stimulated calcium influx. We now show that also after short-term incubation with IFN-gamma, PMN calcium metabolism is modulated. Single adherent cells in three different calcium-containing buffers (high, normal, and low [Ca2+]) were stimulated with the bacterial peptide fMLP or the Ca-ATPase inhibitor thapsigargin (Tg) after about 5 min preincubation with IFN-gamma. The results of this protocol indicated that IFN-gamma increases both calcium influx and calcium sequestration. Store dependent Ca2+ influx, directly measured on readdition of calcium to Tg-treated cells incubated in EGTA buffer, was significantly enhanced in IFN-gamma-treated cells. This effect of IFN-gamma was enhanced by the tyrosine kinase inhibitor herbimycin A. Strikingly, in low extracellular calcium concentrations, IFN-gamma induced calcium transients in 20%-60% of the cells. The proportion of PMN responding with Ca2+ transients increased with decreasing extracellular calcium concentration. Average lagtime from addition of IFN-gamma to a response that could be measured was 7.3 sec, and average increase in [Ca2+] above the basal level was 790 nM. These IFN-gamma-induced transients could not be depressed by herbimycin A. Thus, IFN-gamma can increase capacitative calcium influx, induce calcium transients, and possibly affect calcium sequestration in human PMN.

Calcium↗
PubMed1997

Synchronized Ca2+ oscillations induced in Madin Darby canine kidney cells by bradykinin and thrombin but not by ATP.

In an earlier report, we described synchronous Ca2+ oscillations in globally stimulated, subconfluent MDCK cells [Røttingen J-A, Enden T., Camerer E., Iversen J-G., Prydz H. Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor. J Biol Chem 1995; 270: 4650-4660]. In order to elucidate the mechanisms behind these oscillations, we have analyzed the fluctuations in cytosolic Ca2+ in single, Fura-2 loaded, MDCK cells grown to subconfluence, after stimulation with bradykinin, thrombin and ATP. All three agonists gave rise to an initial Ca2+ spike followed by oscillations or transients. Both the initial and subsequent spikes appeared to be due mainly to release of Ca2+ from internal stores, since they remained after Ca2+ influx was impeded by either La3+ or by chelation of extracellular Ca2+ with EGTA. The secondary spikes were apparently synchronized when the cells were (permanently and globally) stimulated with bradykinin or thrombin, but each cell seemed to oscillate independently when stimulated in the same way with ATP. Synchronized secondary spikes arose with a constant frequency and amplitude, independent of agonist concentration in contrast to most Ca2+ oscillations observed. Pretreatment of the cells with octanol to block gap junctions, or with EGTA or La3+ to inhibit Ca2+ influx, abolished the synchronization induced by bradykinin or thrombin. We observed that in the MDCK cell layer there are some "pacemaker' cells and hypothesize that these have a higher sensitivity for the agonists than their neighboring cells. From these pacemakers, an intercellular Ca2+ wave can be seen to spread to adjacent cells in the presence of intact gap junctions, thereby initiating concurrent transients in all cells. The Ca2+ wave is amplified by release from internal stores, probably owing to the bell-shaped Ca2+ activation curve of the IP3 receptor and by subsequent Ca2+ influx through Ca2+ release activated channels.

Adenosine Triphosphate↗
PubMed1996

Coagulation factors VII and X induce Ca2+ oscillations in Madin-Darby canine kidney cells only when proteolytically active.

We have recently reported that the activated serine protease and blood coagulation Factor VII (FVIIa) can induce Ca2+ oscillations in Madin-Darby canine kidney cells. We now demonstrate a similar response by Madin-Darby canine kidney cells to the active coagulation Factor X (FXa), which is also a serine protease and a substrate of the tissue factor (TF).FVIIa complex in the initiation of the coagulation cascade. The phosphatidyl inositol-specific phospholipase C inhibitor U73122 inhibited the signals elicited by both FVIIa and FXa. Lack of sensibility to the tyrosine kinase inhibitors herbimycin A, genistein, and the tyrphostin AG18 and discordance between TF expression and FVIIa responsiveness argued against TF acting as a cytokine-like receptor, with tyrosine kinase-mediated activation by FVIIa. As demonstrated using the protease inhibitor benzamidine and by specific active site inhibition with 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone, both FVIIa and FXa lost their ability to elicit a calcium response when devoid of their proteolytic activity. Consistent with this, the native (zymogen) form of Factor X did not induce Ca2+ transients. Homologous but not heterologous inhibition of FVIIa- and FXa-evoked Ca2+ signals by 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated FVIIa and FXa suggested that each factor had its own specific cell surface anchoring receptor. The two coagulation factors did not show homologous desensitization as seen for thrombin stimulation. Studies with hirudin excluded involvement of the established activation pathway through thrombin itself. Lack of desensitization of the response to FVIIa or FXa by thrombin ruled out any involvement of proteinase activated receptor-1 (PAR-1), the thrombin receptor. We speculate that FXa and FVIIa may work via a receptor (possibly common) analogous to PAR-1 or its functional homologue PAR-2. Although TF is essential for the FVIIa-induced signaling event, its role in the phosphatidyl inositol-specific phospholipase C-mediated Ca2+ signal may be in anchoring FVIIa to the cell surface rather than in transmembrane signal mediation.

Animals↗
PubMed1996

Growth-promoting effects of Ca(2+)-mobilizing agents in hepatocytes: lack of correlation between the acute activation of phosphoinositide-specific phospholipase C and the stimulation of DNA synthesis by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2 alpha.

Although several hormones that promote hepatocyte proliferation also activate phosphoinositide-specific phospholipase C (PI-PLC) and mobilize Ca2+, the role of PI-PLC in the growth-stimulating effect of these agents is not clear. We have investigated this issue further, by exposing freshly isolated adult rat hepatocytes to vasopressin, angiotensin II, norepinephrine (in the presence of the beta-adrenoceptor blocker timolol) or PGF2 alpha, and examined both acute responses and the subsequent DNA synthesis when the cells were grown in monolayer culture. All the agonists elevated the level of inositol 1,4,5-trisphosphate (InsP3) and enhanced the DNA synthesis, amplifying the response to epidermal growth factor (EGF), and this comitogenic effect could be exerted by a single exposure of the cells 24 h prior to the addition of EGF. The acute activation of PI-PLC, measured as the early rise (peak 15-60 s) in InsP3, was 8-10-fold with vasopressin or angiotensin II, 3-4-fold with norepinephrine, and approximately 2-fold with PGF2 alpha. For all the agonists, a rise in cytosolic free Ca2+ in 100% of the cells and a maximal increase in glycogen phosphorylase activity were evoked at concentrations that approximately doubled the level of InsP3. However, the growth-stimulatory effects of these agonists showed a different order of efficacy as compared to the activation of PI-PLC; in terms of the maximal stimulation of DNA synthesis, the effects were: norepinephrine approximately PGF2 alpha > angiotensin II > vasopressin. Also, norepinephrine, PGF2 alpha, and angiotensin II, but not vasopressin, further enhanced the DNA synthesis when their concentrations were increased above those yielding maximal elevation of InsP3. In experiments where vasopressin and angiotensin II were combined, their effects on the DNA synthesis were additive while the InsP3 responses were not. The results show that the extent of the initial activation of PI-PLC is not the determinant for the magnitude of the growth effects of Ca(2+)-mobilizing hormones in hepatocytes. This suggests either (a) that the proliferative response to these agents is determined by the activity of PI-PLC at a later time, or its integral over an extended part of the prereplicative period, rather than by the acute activation, or (b) that additional, PI-PLC-independent, mechanisms are required.

Angiotensin II↗
PubMed1995

Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor.

Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions resulting in thrombin production. The cloning of TF revealed its structural characteristics to be those of a receptor related to the class 2 cytokine receptor superfamily, but until now no intracellular signal has been discovered related to binding of the ligand (FVIIa) to the putative receptor. We have studied possible intracellular signaling effects of the FVIIa-TF interaction by measuring cytosolic free Ca2+ in single fura-2-loaded cells and found that 200 nM FVIIa caused Ca2+ transients in about 30% of human umbilical vein endothelial cells treated with interleukin-1 beta to express TF, compared to below 5% in uninduced cells. A gradual increase of the basal Ca2+ level was also caused by binding of FVIIa. In the human bladder carcinoma cell line J82, which has a high constitutive TF activity, similar results were found. An antibody neutralizing TF activity decreased the response rate to control levels. COS-1 cells which do not make TF did not respond to FVIIa as opposed to COS-1 cells expressing TF after transfection with a human TF cDNA construct. The canine kidney cell line MDCK, a constitutive TF producer, responded especially well; up to 100% of the cells examined showed Ca2+ oscillations which were dose dependent with regard to frequency, latency, maximal amplitude, and recruitment of responding cells. The frequency was reduced by inhibition of Ca2+ influx with 100 microM LaCl3. In confluent MDCK cells the Ca2+ oscillations were synchronous, constituting the first evidence of a synchronous cytosolic Ca2+ oscillator generated by global application of agonist. Thus, TF mediates a cytosolic Ca2+ signal upon interaction with its ligand FVIIa, thereby suggesting a more complex biological role for TF.

Animals↗