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O Thastrup

Publications and source records attributed to O Thastrup.

At least 19 recordsLinked to original sources
PubMed2004

Functional identification and monitoring of individual alpha and beta cells in cultured mouse islets of Langerhans.

The aim of the present study was to evaluate, by use of fluorescence microscopy and immunofluorescence stainings, the use of a fluorescent membrane potential sensitive probe as a means to identify and monitor changes in membrane potential of individual cell types in whole islets of Langerhans over time. Our work supports the use of the fluorescent probe bis-(1,3 dibutylbarbituric acid) trimethine oxonol (diBAC(4)(3)), in identification of single alpha and beta cells in the periphery of mouse pancreatic islets cultured on extracellular matrix. At a low extracellular glucose concentration (3 mM), heterogeneous staining of the islets was observed. Approximately 97% of the peripheral cells that stained brightly with diBAC(4)(3) were glucagon positive. Additional diBAC(4)(3) studies, demonstrated that an increase in glucose concentration from 3 to 10 mM is paralleled by repolarization of alpha cells and depolarization of beta cells. This suggests that reciprocity of glucagon and insulin release upon glucose stimulation is coupled to divergent changes in membrane potential of these cell types and supports the use of diBAC(4)(3) as a means to detect changes in secretion in both cell types.

Animals↗
PubMed2001

Excitation wave propagation as a possible mechanism for signal transmission in pancreatic islets of Langerhans.

In response to glucose application, beta-cells forming pancreatic islets of Langerhans start bursting oscillations of the membrane potential and intracellular calcium concentration, inducing insulin secretion by the cells. Until recently, it has been assumed that the bursting activity of beta-cells in a single islet of Langerhans is synchronized across the whole islet due to coupling between the cells. However, time delays of several seconds in the activity of distant cells are usually observed in the islets of Langerhans, indicating that electrical/calcium wave propagation through the islets can occur. This work presents both experimental and theoretical evidence for wave propagation in the islets of Langerhans. Experiments with Fura-2 fluorescence monitoring of spatiotemporal calcium dynamics in the islets have clearly shown such wave propagation. Furthermore, numerical simulations of the model describing a cluster of electrically coupled beta-cells have supported our view that the experimentally observed calcium waves are due to electric pulses propagating through the cluster. This point of view is also supported by independent experimental results. Based on the model equations, an approximate analytical expression for the wave velocity is introduced, indicating which parameters can alter the velocity. We point to the possible role of the observed waves as signals controlling the insulin secretion inside the islets of Langerhans, in particular, in the regions that cannot be reached by any external stimuli such as high glucose concentration outside the islets.

Animals↗
PubMed2000

Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet beta cells.

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be approximately 30 microM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be approximately 7 microM. Likewise, the NAD(P)H increase of the mitochondrial domain is approximately 60 microM and is delayed with respect to the change in cytoplasmic NAD(P)H by approximately 20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.

Animals↗
PubMed2000

Exclusion of known protease-activated receptors in factor VIIa-induced signal transduction.

The protease activity is mandatory for intracellular activities induced by coagulation factor VIIa (FVIIa), and in this way it resembles signal transduction induced by thrombin and trypsin caused by specific, proteolytic cleavage of protease activated receptors (PARs). The mechanism for FVIIa-induced signal transduction is, however, not known although a mechanism involving PAR cleavage has been deduced from studies of cytosolic Ca2+ release and p44/p42 mitogen activated protein kinase (MAPK) activation. In the present work we have examined the possibilities that i) FVIIa-induced signal transduction involves the activation of one of the four known PARs, or ii) exposure of cells to FVIIa releases a soluble ligand that is responsible for MAPK activation. For this purpose, we evaluated the effects of FVIIa, thrombin, FXa, trypsin and PAR agonist peptides on the Ca2+ release and MAPK activation in tissue factor-(TF) transfected baby hamster kidney (BHK[+TF]) cells and Madin-Darby canine kidney (MDCK) cells. FVIIa induced a significant MAPK signal in BHK(+TF) cells and in MDCK-I and -II cells whereas no MAPK activation was observed with thrombin, FXa or PAR agonist peptides. Thrombin, trypsin, PAR-1 and PAR-2 agonist peptides induced a prominent Ca2+ response in both cell types. In contrast the cells did not respond with a detectable Ca2+ signal when treated with FVIIa. These results suggest that the intracellular activity induced by FVIIa is distinctly different from that induced by trypsin, thrombin and FXa not involving any of the known PARs. Conditioned medium from BHK(+TF) cells treated with FVIIa failed to induce a MAPK response in untreated BHK(+TF) cells when FVIIa was removed by immunoadsorption from the medium prior to its transfer to the untreated BHK(+TF) cells. Although it is not possible entirely to exclude a transient response close to the cell surface, the data suggest that the intracellular response was not induced by an autocrine release of a soluble mediator to the medium.

Animals↗
PubMed1999

Simultaneous visualization of the translocation of protein kinase Calpha-green fluorescent protein hybrids and intracellular calcium concentrations.

The alpha isoform of protein kinase C (PKCalpha) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKCalpha was tagged with a highly fluorescent derivative of green fluorescent protein and stably expressed in baby hamster kidney cells overexpressing the muscarinic type 1 receptor. Addition of the agonist carbamylcholine triggered the onset of translocation within 1 s. Half-maximal and maximal translocation occurred after about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within about 45 s. A high-resolution study showed an almost homogeneous cytoplasmic distribution of tagged PKCalpha in unstimulated cells and virtually complete translocation to the plasma membrane in response to the phorbol ester, PMA. Simultaneous visualization of intracellular calcium concentration ([Ca2+]i) and PKCalpha translocation in single cells showed a good correlation between these parameters at intermediate and high concentrations of agonist. At low agonist concentration, a small increase in [Ca2+]i was observed, without detectable translocation of PKCalpha. In contrast, PMA induced translocation of PKCalpha without any increase in [Ca2+]i. Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKCalpha, indicating that PKCalpha translocation may be independent of both actin filaments and microtubules. The time course of PKCalpha translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.

Animals↗
PubMed1998

Signal transduction via the mitogen-activated protein kinase pathway induced by binding of coagulation factor VIIa to tissue factor.

The putative role of tissue factor (TF) as a receptor involved in signal transduction is indicated by its sequence homology to cytokine receptors (Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938). Signal transduction induced by binding of FVIIa to cells expressing TF was studied with baby hamster kidney (BHK) cells stably transfected with TF and with a reporter gene construct encoding a luciferase gene under transcriptional control of tandem cassettes of signal transducer and activator of transcription (STAT) elements and one serum response element (SRE). FVIIa induced a significant luciferase response in cells expressing TF, BHK(+TF), but not in cells without TF. The BHK(+TF) cells responded to the addition of FVIIa in a dose-dependent manner, whereas no response was observed with active site-inhibited FVIIa, which also worked as an antagonist to FVIIa-induced signaling. Activation of the p44/42 MAPK pathway upon binding of FVIIa to TF was demonstrated by suppression of signaling with the specific kinase inhibitor PD98059 and demonstration of a transient p44/42 MAPK phosphorylation. No stimulation of p44/42 MAPK phosphorylation was observed with catalytically inactive FVIIa derivatives suggesting that the catalytic activity of FVIIa was obligatory for activation of the MAPK pathway. Signal transduction caused by a putative generation of FXa activity was excluded by experiments showing that FVIIa/TF-induced signaling was not quenched by tick anticoagulant protein, just as addition of FXa could not induce phosphorylation of p44/42 MAPK in BHK(+TF) cells. These results suggest a specific mechanism by which binding of FVIIa to cell surface TF independent of coagulation can modulate cellular functions and possibly play a role in angiogenesis and tumor metastasis as indicated by several recent observations.

Animals↗
PubMed1996

Flow injection microscopy: a novel tool for the study of cellular response and drug discovery.

Studying responses of live cells to agonists, antagonists and other physical stimuli offers insight into their complex membrane and internal biochemistry. An experimental technique has been developed in which responses of living cells in an inverted radial flow chamber are continuously monitored while being repeatedly stimulated using controlled pulses of a biologically active ligand. Precisely defined flow conditions result in reproducible peaks which can be numerically analysed by comparison with a tracer curve obtained by substituting a dye for the stimulus. Exploratory studies have demonstrated that the flow injection technique can provide a novel method for kinetics of receptor binding and cellular responses. Flow injection microscopy (FIM) allows identification of biologically active ligands and their ranking based on measurement of the cellular responses in a short time frame. The use of FIM for rapid drug screening, through monitoring of the initial kinetics of cellular responses, is demonstrated on a model system.

Cell Line↗
PubMed1995

Structure-activity relationships of analogues of thapsigargin modified at O-11 and O-12.

A number of analogues of thapsigargin have been synthesized by alkylating or acylating O-11 and O-12 in the lactol obtained by reducing thapsigargicin. Introduction of alpha-disposed substituents decreased the Ca(2+)-ATPase inhibitory potency of the analogue, whereas the enzyme was more tolerant toward beta-disposed substituents, indicating that the alpha-face of the lactone ring is in close contact with the binding site when the inhibitor is bound to the enzyme.

Animals↗
PubMed1994

Characteristics of stably expressed human dopamine D1a and D1b receptors: atypical behavior of the dopamine D1b receptor.

Human dopamine D1a and D1b receptors were stably expressed in Baby Hamster Kidney (BHK) or Chinese Hamster Ovary (CHO) cells. [3H]SCH23390 saturation experiments indicated the presence of only a single binding site in the D1a expressing cell line with a Kd of 0.5 nM. In D1b expressing cell lines, two binding sites were observed with Kd values of 0.5 and 5 nM in CHO cells and 0.05 and 1.6 nM in BHK cells, respectively. Neither of the receptors affected Ca2+ metabolism whereas they both were coupled in a stimulatory fashion to adenylyl cyclase. The pharmacological profile of both the D1a and D1b receptors as assessed from inhibition of specific [3H]SCH 23390 binding was classical D1-like. Thus, benzazepine derivatives as well as the atypical neuroleptics, clozapine and fluperlapine, exhibited high affinity whereas D2 selective compounds like sulpiride and spiperone had low affinity for these receptors. Besides SCH 23390, only NNC 112, fluphenazine and bulbocapnine were able to discriminate between the two states of the D1b receptor. In case of the D1a receptor, the Ki values obtained in binding experiments were very similar to Ki values obtained from inhibition of dopamine stimulated adenylyl cyclase. In the D1b expressing cell line, the Ki values obtained from inhibition of the dopamine stimulated adenylyl cyclase indicated a significantly better correlation with the state of the D1b receptor showing high affinity for antagonists. In agreement with observations from binding experiments, dopamine was around 20 fold more potent in stimulating adenylyl cyclase via the D1b receptor as compared to the D1a receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1992

In situ imaging of agonist-sensitive calcium pools in AR4-2J pancreatoma cells. Evidence for an agonist- and inositol 1,4,5-trisphosphate-sensitive calcium pool in or closely associated with the nuclear envelope.

The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells.

Animals↗
PubMed1992

Biochemical and ultrastructural studies suggest that the effects of thapsigargin on human platelets are mediated by changes in intracellular calcium but not by intracellular histamine.

The involvement of intracellular histamine in thapsigargin (Tg)-induced platelet aggregation was studied. Platelet aggregation induced by 0.25 and 0.5 microM Tg was not accompanied by a rise in intracellular histamine but a significant (p < 0.01) increase in the level of intracellular histamine was observed at 1 microM Tg. Preincubation of platelets with inhibitors of histamine metabolizing enzymes had little effect on intracellular histamine levels in platelets stimulated by 0.5 microM Tg. In addition, the inhibitors of histidine decarboxylase (HDC), alpha-methyl histidine (alpha-MH) and alpha-fluoromethyl histidine (alpha-FMH) failed to inhibit Tg-induced aggregation. The intracellular histamine receptor antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine. HCl (DPPE), inhibited Tg-induced aggregation but with IC50 values dependent on the concentration of agonist used. The inhibitory effects of DPPE on Tg-induced aggregation were not reversed by the addition of histamine to saponin-permeabilized platelets suggesting non-histamine mediated effects of DPPE on Tg-induced aggregation. Tg stimulated an increase in the cytosolic free calcium concentration which was unaffected by DPPE indicating that the effects of DPPE are also not due to the inhibition of mobilization of cytosolic calcium. The ultrastructural studies suggest that the major Tg-induced changes (pseudopod formation and granule centralization) are consistent with a primary role for Tg to mobilize calcium; DPPE had very little effect on these ultrastructural changes. The results indicate that the effects of Tg on human platelets are mediated by an increase in cytosolic calcium but not by intracellular histamine.

Blood Platelets↗
PubMed1992

Mechanisms of activated Ca2+ entry in the rat pancreatoma cell line, AR4-2J.

The characteristics of Ca2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K+ activated Ca2+ entry. Ca2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca(2+)-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca2+ pool and sustained Ca2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca2+ influx through voltage-sensitive Ca2+ channels. Instead, we conclude that agonist-activated Ca2+ mobilization is initiated by (1,4,5)IP3-mediated intracellular Ca2+ release and that Ca2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP3-sensitive intracellular Ca2+ pool.

Animals↗
PubMed1992

The molluscicidal activity of coumarins from Ethulia conyzoides and of dicumarol.

The molluscicidal principles of Ethulia conyzoides were identified as ethuliacoumarin A (1) and isoethuliacoumarin A (2). Ethuliacoumarin A possessed an LC90 between 19 and 23.5 ppm depending on the age of the snail against Biomphalaria glabrata, and between 12 and 15 ppm against Bulinus truncatus. In addition, ethuliacoumarin A was found to be cercaricidal at 25 ppm and ovicidal. Ethuliacoumarin has the structural requirements considered essential for anticoagulant activity. Consequently the anticoagulant dicumarol (4) was tested and found to be molluscicidal in the range from 2.5 to 10 ppm. In contrast, the coumarin anticoagulant warfarin (3) did not show molluscicidal activity.

Animals↗
PubMed1992

On the botanical distribution of chiral forms of gossypol.

Plants belonging to Malvaceae (about 90 species belonging to 33 genera) were analysed for the presence of gossypol by reversed-phase HPLC using absorbance detection at 365 nm, and the chirality and optical purity of gossypol were determined whenever the analysis was positive. Only plants belonging to the cotton tribe (Gossypieae) contained gossypol (detection limit better than 0.001%). In particular, no gossypol could be detected in ABELMOSCHUS ESCULENTUS (ocra), HIBISCUS TILIACEUS, H. SABDARIFFA, and HEREA BRASI-LIENSIS, earlier claimed to contain this compound. Gossypol-containing plants usually produced dextrorotatory gossypol of varying optical purity; an enantiomeric excess of (-)-gossypol was detected in only one plant, GOSSYPIUTN BARBADENSE.

Journal Article↗
PubMed1991

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines.

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.

Animals↗
PubMed1991

Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles.

In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100- and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes.

Antibodies↗
PubMed1991

Mobilization of calcium by inositol trisphosphates from permeabilized rat parotid acinar cells. Evidence for translocation of calcium from uptake to release sites within the inositol 1,4,5-trisphosphate- and thapsigargin-sensitive calcium pool.

D-myo-Inositol (1,4,5)-trisphosphate ((1,4,5)IP3)-induced Ca2+ release and subsequent Ca2+ reuptake were investigated in saponin-permeabilized rat parotid acinar cells. Following the rapid release of Ca2+ by (1,4,5)IP3, Ca2+ was resequestered. The sequential addition of submaximal concentrations of (1,4,5)IP3 resulted in sequential Ca2+ release. However, when the cells were challenged with the poorly metabolized (1,4,5)IP3 analogues, (1,4,5)IPS3 or (2,4,5)IP3, or under conditions where the metabolism of authentic (1,4,5)IP3 was reduced, Ca2+ reuptake again occurred, but sequestered Ca2+ was not released by subsequent additions of (1,4,5)IP3. The sequestered Ca2+ was, however, released by thapsigargin, an agent which inhibits active Ca2+ uptake into the (1,4,5)IP3-sensitive pool. Furthermore, the rate of thapsigargin-induced release was significantly increased in the continued presence of an (1,4,5)IP3 stimulus. Thus, Ca2+ reuptake apparently occurred into the (1,4,5)IP3- and thapsigargin-sensitive Ca2+ store and (1,4,5)IP3 continued to influence the permeability of this pool to Ca2+ during Ca2+ reuptake. In contrast to the findings in permeabilized cells, Ca2+ reuptake did not occur in the sustained presence of (1,4,5)IP3 in intact parotid cells. We conclude that cell permeabilization reveals a kinetic, and presumably structural, separation of Ca2+ uptake and release sites within the (1,4,5)IP3-regulated intracellular organelle.

Animals↗