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J B Parys

Publications and source records attributed to J B Parys.

At least 91 records · Page 5Linked to original sources
PubMed1994

Inhibition of inositol trisphosphate-induced calcium release by caffeine is prevented by ATP.

We have investigated the effect of various methylxanthines on the basal and InsP3-stimulated unidirectional 45Ca2+ efflux from permeabilized A7r5 cells under different experimental conditions. We report that caffeine and theophylline inhibit the InsP3-induced Ca2+ release, whereas the basal Ca2+ leak remained largely unaffected. The effect on InsP3-induced Ca2+ release was specific, since isocaffeine was not effective. The inhibition occurred similarly in the absence or presence of extravesicular Ca2+ and was not associated with a decrease in the [3H]InsP3 binding to the receptor. ATP and MgATP (5 mM) prevented the inhibition, suggesting that caffeine may interact with an ATP-binding site on the InsP3 receptor or some associated protein.

Adenosine Triphosphate↗
PubMed1994

Co-activation of inositol trisphosphate-induced Ca2+ release by cytosolic Ca2+ is loading-dependent.

Luminal and cytosolic Ca2+ control the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3). In this work, we have characterized how luminal Ca2+ interfered with the stimulation of the InsP3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. InsP3-induced Ca2+ release from stores containing 34 pmol of Ca2+/10(6) cells absolutely depended on a simultaneous rise in cytosolic Ca2+ concentration in the submicromolar range. This stimulation by cytosolic Ca2+ was more pronounced when the stores were preincubated with Ca2+. In contrast, fully loaded stores containing 3400 pmol of Ca2+/10(6) cells already responded to InsP3 in the absence of cytosolic Ca2+ and the release from these stores was much less stimulated by increasing the cytosolic Ca2+ concentration. The loading dependence of the release was not due to a higher cytosolic Ca2+ concentration around more loaded stores sensitizing the InsP3 receptor by binding at a cytoplasmic site. Luminal Ca2+ was therefore found to functionally substitute for cytosolic Ca2+ in triggering Ca2+ release in the presence of a constant InsP3 concentration. These findings are relevant for explaining base-line Ca2+ spiking in non-excitable cells.

Animals↗
PubMed1994

Presence of inositol 1,4,5-trisphosphate receptor, calreticulin, and calsequestrin in eggs of sea urchins and Xenopus laevis.

The presence of inositol 1,4,5-triphosphate receptor (InsP3R), calreticulin, and calsequestrin was demonstrated in eggs of sea urchins (Lytechinus pictus, Lytechinus variegatus, and Strongylocentroutus purpuratus) and Xenopus laevis. Binding of inositol 1,4,5-trisphosphate (InsP3) to microsomes of L. pictus eggs was inhibited by heparin and NaCl. An affinity-purified antibody against the C-terminal of the type I InsP3R, which recognizes InsP3R isoforms of rabbit brain (273 kDa) and Xenopus oocytes and eggs (256 kDa), reacted with a 373-kDa protein in sea urchin eggs. The 373-kDa protein was tentatively identified as the sea urchin egg InsP3R. Observations with fluorescence microscopy indicated that the InsP3R is present throughout the cytoplasm of sea urchin eggs in a pattern consistent with the distribution of endoplasmic reticulum. Small differences in the relative amount of reaction deposits in cortex vs subcortex were noted among the species of sea urchins examined. Reaction product was also localized to the periphery of female pronuclei in eggs of all three sea urchins. InsP3R reactivity was present in the perinuclear region, along the periphery of the germinal vesicle, and throughout the animal and vegetal hemispheres of Xenopus oocytes. A similar cytoplasmic staining pattern was also observed in eggs, although islands of reactivity, much larger than those in oocytes, were present in the animal hemisphere of eggs. Calreticulin and calsequestrin in sea urchin eggs had the same molecular mass as in rabbit brain (56 and 60 kDa, respectively), but differed from those present in Xenopus oocytes/eggs (61 and 57 kDa, respectively). The distribution of calreticulin and calsequestrin in both sea urchin and Xenopus oocytes and eggs was similar to that observed for the InsP3R. These results are discussed in relation to previous studies of Ca2+ regulation during egg development and fertilization and suggest that in the oocytes and eggs of the species examined, InsP3-sensitive Ca2+ stores play an important role in the regulation of cellular Ca2+.

Animals↗
PubMed1994

Vasopressin responses in electrically coupled A7r5 cells.

Changes in cytosolic Ca2+ concentration ([Ca2+]i) and in membrane potential were monitored in single A7r5 smooth-muscle cells during spontaneous spiking and after arginine vasopressin stimulation. Spontaneous Ca2+ oscillations, which were associated with the occurrence of action potentials, occurred in about 90% of the confluent monolayers investigated. This spontaneous activity was synchronized amongst all the cells of the monolayer, indicating that the cells were electrically coupled. Arginine vasopressin stimulation produced a [Ca2+]i rise that was about 5 times higher than the amplitude of the spontaneous Ca2+ oscillations and resulted in a subsequent cessation of spontaneous electrical activity and associated Ca2+ spiking, which persisted after [Ca2+]i returned to baseline. Individual cells in the monolayer responded to arginine vasopressin with a different latency. Agonist-induced Ca2+ waves within one cell propagated much more slowly than spontaneous [Ca2+]i rises. We conclude that agonist-induced [Ca2+]i increases in an electrically coupled cell monolayer can be asynchronous.

Animals↗
PubMed1994

Partial calcium release in response to submaximal inositol 1,4,5-trisphosphate receptor activation.

Even a prolonged application of a submaximal dose of Ins(1,4,5)P3 is unable to release the same amount of Ca2+ from the Ins(1,4,5)P3-sensitive store as a higher dose of Ins(1,4,5)P3. Low doses of Ins(1,4,5)P3 therefore only induce a partial release of the stored Ca2+. In this review, we will focus on the mechanisms that may contribute to this behaviour. Molecular heterogeneity of the Ins(1,4,5)P3 receptor can contribute to such behaviour if all the gene products and alternatively spliced isoforms would have different functional properties and be located in different store units. We will show that the control of the Ins(1,4,5)P3 receptor by by luminal Ca2+ also contributes to the partial release behaviour; it can set the sensitivity of the Ins(1,4,5)P3 receptor and the decreasing luminal Ca2+ concentration may inhibit further release while some Ca2+ is still left in the store. It is finally possible that the Ins(1,4,5)P3 receptor may adapt to a maintained stimulus.

Animals↗
PubMed1994

Second messenger specificity of the inositol trisphosphate receptor: reappraisal based on novel inositol phosphates.

To further understand how the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] interacts with its intracellular receptor, we injected 47 highly purified inositol phosphate (InsP) positional isomers in Xenopus oocytes and compared their potency in releasing intracellular Ca2+. The potency of the Ca(2+)-releasing InsPs spanned four orders of magnitude. Seven compounds, including the novel inositol 1,2,4,5-tetrakisphosphate [D/L-Ins (1,2,4,5)P4] and D/L-Ins(1,4,6)P3, had a very high potency. All of these highly active InsPs shared the following structure: two D-trans-equatorial phosphates (eq-P) and one equatorial hydroxyl (eq-OH) attached to ring carbons D-4, D-5, and D-6 (or to the structurally equivalent D-1, D-6, and D-5 carbons). This permissive structure was not sufficient for Ca2+ release, because it was also found in two inactive compounds, Ins(1,6)P2 and Ins(1,3,6)P3. To be active, InsPs also required the structural equivalent of a D-3 eq-OH and/or a D-1 eq-P. Together, our data reveal how the structure of the InsP molecule affects its ability to release Ca2+.

Animals↗
PubMed1993

Loading dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in the clonal cell line A7r5. Implications for the mechanism of quantal Ca2+ release.

The Ca2+ content of the intracellular Ca2+ stores controls the inositol 1,4,5-trisphosphate receptor (InsP3R) in the clonal cell line A7r5. This regulation was characterized with respect to the understanding of the "quantal" release phenomenon. Independent of the loading protocol used, increasing the Ca2+ content of the stores increased the sensitivity of the inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release until a Ca2+ content of about 30% of the steady-state value was reached. Loading of the stores to higher levels had only a marginal effect on the Ca2+ release. The effects of luminal Ca2+ were still observed in the presence of 10 mM BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), excluding the possibility that luminal Ca2+ acted indirectly via cytoplasmic binding sites. Conditions were developed to simultaneously measure [3H]InsP3 binding to the InsP3R and the 45Ca2+ content of the stores in the same cells. [3H]InsP3 binding to a high affinity binding site was potentiated by luminal Ca2+. Analysis at the molecular level revealed the simultaneous expression of different splice variants of InsP3R-I, as well as the expression of InsP3R-III, and of the putative InsP3R-IV. We conclude that the control of the InsP3R by luminal Ca2+ could account for quantal release and that the observed heterogeneity of the InsP3R may also contribute to this behavior, especially at high levels of store loading.

Animals↗
PubMed1993

Ins(1,4,5)P3 and glutathione increase the passive Ca2+ leak in permeabilized A7r5 cells.

Thapsigargin depletes intracellular Ca2+ stores by its inhibitory effect on the Ca2+ pumps, which unmasks an aspecific Ca2+ leak from the stores. This aspecific Ca2+ permeability of the stores was further investigated using 45Ca2+ fluxes on intact and permeabilized A7r5 smooth-muscle cells. Stores in intact cells were found to be more leaky for Ca2+ than those in saponin-permeabilized or Staphylococcus aureus alpha-toxin-permeabilized cells, which suggests that a cytosolic factor may be involved. Supplementing the medium bathing the permeabilized cells with a submaximal Ins(1,4,5)P3 concentration increased the leakiness of the stores. Glutathione also increased the aspecific Ca2+ leak. This effect occurred with both the reduced and the oxidized form but reduced glutathione was more effective. Our data show that basal Ins(1,4,5)P3 levels and glutathione can contribute to the relatively high Ca2+ leak in intact cells. The washing out of these substances during permeabilization can reduce the aspecific leakiness of the stores.

Bacterial Toxins↗
PubMed1993

Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2+ release by thimerosal in permeabilized A7r5 smooth-muscle cells.

There is no consensus about the different types of Ca2+ transport processes in the endoplasmic reticulum that are targeted by the sulphydryl reagent thimerosal. We have therefore investigated how thimerosal affects the various Ca2+ transport processes in permeabilized A7r5 smooth-muscle cells, using an unidirectional 45Ca2+ flux technique. Thimerosal up to a concentration of 32 microM did not have an effect on the passive 45Ca2+ leak from the stores, while higher concentrations increased this aspecific leak. Thimerosal inhibited the endoplasmic reticulum Ca2+ pump with an EC50 of 9 microM. Thimerosal exerted a biphasic effect on the Ca2+ release induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with a stimulation of the release at thimerosal concentrations below 10 microM, and an inhibitory effect at higher concentrations. Thimerosal (2.5-250 microM) did not exert an effect on the specific binding of [3H]Ins(1,4,5)P3 to its receptor, indicating that it probably did not act at the level of the binding site. This finding contrasts with the effect of the closely related sulphydryl reagent parachloromercuriphenylsulphonate, which, at high concentrations, inhibited [3H]Ins(1,4,5)P3 binding. The effects of thimerosal were largely prevented by the sulphydryl reducing agent dithiothreitol (3 mM). We conclude that thimerosal concentrations ranging from 0.32 to 1 microM can stimulate the Ins(1,4,5)P3-induced Ca2+ release without inhibiting the Ca2+ pumps or without increasing the passive Ca2+ permeability of the endoplasmic reticulum.

Animals↗
PubMed1993

Normal Ca2+ signalling in glutathione-depleted and dithiothreitol-treated HeLa cells.

We have investigated whether reducing agents and substances that interfere with glutathione metabolism would affect the histamine-induced rises in internal Ca2+ concentration ([Ca2+]i) in indo-1-loaded HeLa cells. Individual cells responded to 1 microM histamine with either baseline or sinusoidal Ca2+ oscillations, a single Ca2+ peak or a maintained elevation of the [Ca2+]i. Only a few cells did not respond. The sulphydryl reducing agent dithiothreitol (5 mM) did not affect these responses to histamine. A 24-h preincubation with 1 mM DL-buthionine (SR)-sulphoximine, which reduces the cellular glutathione content to less than 20% of its control value, affected neither these histamine responses, nor the [Ca2+]i rises after application of 2 microM thapsigargin. We conclude that oxidation of critical sulphydryl groups is not required for the normal response to histamine and also that glutathione plays no role in agonist-induced Ca2+ signalling in HeLa cells.

Antimetabolites↗
PubMed1992

Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.

Amino Acid Sequence↗
PubMed1991

Isoform switching of the sarco(endo)plasmic reticulum Ca2+ pump during differentiation of BC3H1 myoblasts.

We have studied the expression of the gene 2 for the sarco(endo)plasmic reticulum Ca2+ pump (SERCA2) in BC3H1 cells. Myogenic differentiation not only activated the SERCA2 expression but it also induced an isoform switch. Undifferentiated myoblasts only expressed the SERCA2b isoform (non-muscle) whereas differentiated myocytes predominantly contained the SERCA2a isoform (cardiac/slow skeletal muscle). The isoform switch was documented by immunoblot analysis with isoform-specific antibodies. This observation was confirmed at the mRNA level by using antisense RNA probes specific for class 1 (SERCA2a) or class 2 (SERCA2b) messengers. The expression of the SERCA2a isoform after differentiation was accompanied by a decreased sensitivity of the Ca2+ uptake in permeabilized cells to the Ca2+ pump inhibitor thapsigargin.

Animals↗
PubMed1991

Changes in the mechanism of Ca2(+) mobilization during the differentiation of BC3H1 muscle cells.

Ca2+ sequestration and release in BC3H1 muscle cells is strongly dependent on the stage of differentiation. In proliferating cells, more than 90% of the sequestered Ca2+ was Ins(1,4,5)P3-sensitive and 25% was caffeine-sensitive. In differentiated cells, the Ca2+ accumulation was 5-fold higher and was InsP3-insensitive, but about 60% of the sequestered Ca2+ was caffeine-sensitive. These changes were reversible upon addition of growth stimuli. Similarly, by measuring the intracellular Ca2+ concentration in single intact BC3H1 cells, it was found that the number of histamine-responsive cells decreased and the number of caffeine-responsive cells increased during muscle cell differentiation. These data indicate that the development of the muscle phenotype in BC3H1 myoblasts induces a major rearrangement of the mechanisms for Ca2+ mobilization.

Animals↗
PubMed1990

Transport systems for polyamines in the established renal cell line LLC-PK. Polarized expression of an Na(+)-dependent transporter.

We present evidence for the existence of an Na(+)-dependent transporter and an Na(+)-independent transporter for polyamines in LLC-PK1 cells. Both transporters could be discriminated by their sensitivity to inhibitors, particularly rho-chloromercuriphenyl sulphate and various polycationic molecules. By using cell monolayers grown on a permeable filter support, we have found that the Na(+)-dependent polyamine uptake occurred preferentially from the basolateral side. The Na(+)-independent uptake, on the other hand, occurred to the same extent from either the apical or the basolateral side.

4-Chloromercuribenzenesulfonate↗
PubMed1990

Regulation of the Na(+)-dependent and the Na(+)-independent polyamine transporters in renal epithelial cells (LLC-PK1).

We have studied the regulation of the Na(+)-dependent and Na(+)-independent polyamine transport pathways in the renal LLC-PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL-2-difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth-promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na(+)-dependent than for the Na(+)-independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth-promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na(+)-dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 less than 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non-metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na(+)-dependent pathway against the polyamine-induced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine-induced decay. These observations suggest that this polyamine-induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine-induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na(+)-dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na(+)-dependent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a physiological role for these transporters, such as an involvement in urinary polyamine disposal.

Animals↗
PubMed1987

Calmodulin and calmodulin-binding proteins in the renal brush border.

The calmodulin content of renal brush-border membrane vesicles, prepared by Mg2+-precipitation in EGTA-containing solutions, amounts to 1.8 micrograms per mg protein. The amount and the distribution of this EGTA-insensitive calmodulin was determined in membrane and cytoskeletal fractions prepared from the brush-border membrane vesicles by extraction with Triton X-100. The Triton X-100 insoluble pellet contains 21.2% of the protein and 52.2% of the EGTA-insensitive calmodulin, which amounts in this fraction to 4.4 micrograms per mg protein. Treatment of the Triton X-100 insoluble pellet, consisting of the microvillar core residue, with ATP and Mg2+ results in the solubilization of a relatively small number of proteins among which are actin, myosin, calmodulin and several calmodulin-binding proteins. The solubilization is partially reversible and a fraction of the proteins can be precipitated by centrifugation after the enzymatic hydrolysis of ATP. Readdition of ATP to the pellet results in the resolubilization of myosin, part of the actin, an 115-kDa calmodulin-binding protein and calmodulin. The calmodulin content of the final extract was 61.8 micrograms per mg protein. We have found roughly the same distribution pattern of calmodulin and ATP-solubilized, calmodulin-binding proteins in renal and intestinal brush-border preparations. The calmodulin content, however, as well as the relative amount of the calmodulin-binding proteins versus actin are about 4 to 5-times higher in intestinal than in renal microvillar core residues.

Adenosine Triphosphate↗
PubMed1986

Calcium transport systems in the LLC-PK1 renal epithelial established cell line.

ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na+-Ca2+ exchange mechanism is also present in LLC-PK1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK1 cells lowered the free calcium concentration in the medium to less than 0.4 microM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK1 cell line can be successfully used as model system for the study of renal calcium handling.

Animals↗