Biomedical subjects

B Himpens

Publications and source records attributed to B Himpens.

At least 37 records · Page 2Linked to original sources

PubMed1997

Effect of increasing glucose concentrations and protein phosphorylation on intercellular communication in cultured rat retinal pigment epithelial cells.

PURPOSE: The intercellular communication between cultured rat retinal pigment epithelial (RPE) cells grown in increasing glucose concentrations or after modulation of the protein kinase C-induced protein phosphorylation was investigated by studying the conduction of the [Ca2+]i wave elicited by mechanical stimulation and by analyzing the fluorescence recovery after photobleaching (FRAP). METHODS: Subconfluent monolayers of RPE cells isolated from neonatal Long Evans rats were cultured in growth medium with various glucose levels and analyzed using the fluorescent dye fluo-3 for measurements of intracellular Ca2+ after mechanical stimulation and using 6-carboxyfluorescein diacetate to investigate the intercellular communication with FRAP. RESULTS: Mechanical stimulation in 5 or 12 mM glucose resulted in a Ca2+ wave that spread centrifugally through the neighboring cells. An inhibition of the propagation of this wave, similar to that induced by halothane, could be observed in cells grown for 72 hours in 14-mM or higher concentrations of glucose. This inhibitory effect was not caused by a hyperosmotic effect, in that results of experiments on cells cultured in growth medium supplemented with mannitol instead of glucose did not differ from those of experiments in the control medium. Activation of protein kinase C by incubation of the cells for 30 minutes with phorbol 12-myristate 13-acetate (PMA) resulted in a strong inhibition of [Ca2+]i-wave propagation. This inhibition did not depend on the oxidizing effects of PMA because the addition of glaucine, a known antioxidant, did not prevent the inhibition. Cells grown for 72 hours in glucose-rich medium (25 or 50 mM) and in which all protein kinase C activity was downregulated by a previous 72-hour exposure to 1 microM PMA, did not display the inhibitory effect on the propagation of the Ca2+ wave that is normally induced by this elevated glucose level. Stimulation or inhibition of protein kinase A activity by incubating RPE cells with Sp-cyclic adenosine monophosphate or Rp-cyclic adenosine monophosphate respectively, or inhibition of tyrosine kinase activity with herbimycin A did not alter the intercellular communication after mechanical stimulation. To determine whether the observed changes were caused by alterations in gap junction conductance (GJC), FRAP experiments were performed in control conditions, after a 30-minute incubation with PMA, and in cells cultured in 50 mM glucose in the presence and in the absence of 1 microM PMA. The measured GJC was consistent with the inhibitory effect on propagation of an intercellular Ca2+ wave in all tested conditions. CONCLUSIONS: In RPE cells, a glucose concentration of 14 mM (224 mg/dl) or higher inhibits Ca(2+)-wave propagation and intercellular GJC. This effect may be mediated by protein kinase C activity.

Animals↗

PubMed1995

Site-dependent pathological differences in smooth muscles and skeletal muscles of the adult mdx mouse.

This study presents a survey of the morphometric characteristics, the regeneration rate, and the extent of muscle dystrophy in several smooth and skeletal muscles from adult mdx mice, an animal model of the Duchenne muscular dystrophy (DMD). Smooth muscles from adult mdx mice showed neither cell necrosis nor fibrosis. As compared to control C57 mice, the thickness of the mdx smooth muscle was normal in the vascular and urogenital layers but significantly reduced in the digestive layers, a finding relevant to clinical reports of gastrointestinal dilatation in DMD patients, and suggesting that gastrointestinal dysfunctions should be systemically searched for in DMD patients. Adult mdx skeletal muscles, however, presented different patterns of muscle suffering: either absent (esophagus); very mild (trunk and limb muscles); or severe (diaphragm). In these three conditions we studied the fiber diameters, the nuclei locations, and the regeneration rate. From this comparative study, it seems that severe dystrophy occurs in muscle tissues showing large fiber diameter and peripheral location of the nuclei. We showed that this combination occurs in the mouse diaphragm which is thus a realistic model for human DMD muscles.

Animals↗

PubMed1995

Human cytomegalovirus modulates the Ca2+ response to vasopressin and ATP in fibroblast cultures.

The free calcium concentrations in the nucleus ([Ca2+]n) and in cytosol ([Ca2+]c) of cultured human embryonic lung (HEL) fibroblasts were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. In resting HEL cells, The free cellular Ca(2+)-concentration significantly increased upon human cytomegalovirus (HCMV) infection. The ratio between [Ca2+]n and [Ca2+]c was not affected. Following stimulation by ATP or [Arg8] vasopressin (AVP), a differential Ca2+ response of the HCMV-infected HEL cells was observed. While uninfected cells were highly sensitive to AVP and only poorly sensitive to ATP, infected cells showed a high responsiveness to ATP but not to AVP. This switch in sensitivity to the agonists first observed at 24 h post infection. The Ca(2+)-rise following ATP or AVP stimulation was derived from intracellular Ca2+ stores. The magnitude of the ATP-induced Ca(2+)-rise increased upon infection. In contrast to non-infected cells where [Ca2+]n > [Ca2+]c during stimulation with AVP or ATP, no nucleo-cytosolic Ca(2+)-gradient was observed in infected cells. Furthermore, the magnitude of the Ca2+ rise in the two compartments was higher in ATP-stimulated cells. It is concluded that HCMV infection significantly interferes with Ca(2+)-homeostasis in HEL cells which could be related to the pathogenesis of the disease.

Adenosine Triphosphate↗

PubMed1995

Mechanism of theophylline-induced inotropic effects on foreshortened canine diaphragm.

The mechanisms of theophylline-induced inotropic effects at shorter diaphragm length have not yet been explored. We wondered whether the greater inotropic effects of the drug at shorter diaphragm length might result from an effect on intracellular calcium level. Forty pairs of diaphragm bundles were stimulated at 70% of optimal length in the presence of either verapamil (10(-5)M), calcium-free Krebs solution (buffered or not with 2 mM ethylene glycol tetra-acetic acid (EGTA)) or ryanodine (10(-6) M). Theophylline (1 mM) was subsequently added to one muscle bundle and, after 15 min, twitches were repeated. The twitch potentiation induced by theophylline (37 +/- 21%) was unaffected by verapamil (43 +/- 26%), or zero calcium (39 +/- 18%) and virtually unchanged when the latter was buffered with EGTA. By contrast, theophylline failed to increase twitch tension after pretreatment with ryanodine, a blocker of the calcium release by the sarcoplasmic reticulum. This decreased twitch tension in control (-5 +/- 11%) and experimental (-14 +/- 12%) bundles and prolonged half-relaxation time as a result of impaired sarcoplasmic reticulum calcium reuptake. We conclude that the inotropic effects of theophylline on twitch tension in foreshortened canine diaphragm bundles were not related to sarcoplasmic reticulum. This is consistent with an action of theophylline on the sarcoplasmic reticulum.

Analysis of Variance↗

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

Relationship between [Ca2+] changes in nucleus and cytosol.

The free calcium concentration in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of single cells were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. It is shown that in various cell types a nucleo-cytosolic Ca(2+)-gradient is present at rest and during stimulation. The direction and the extent of the nucleo-cytosolic Ca(2+)-gradient may vary with the cell type, differentiation status, phosphorylation conditions and also with the type of agonist. Evidence is given for the role of extra- and intranuclear storage sites as well as for Ca(2+)-influx. Finally potential artefactual interference with the measurements is discussed.

Animals↗

PubMed1994

Subcellular Ca(2+)-gradients in A7r5 vascular smooth muscle.

The free calcium concentrations in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of cultured A7r5 smooth muscle were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. Upon stimulation with 5 microM vasopressin (AVP) a cytosolic Ca2+ gradient was observed whereby the highest increase was observed in the subplasmalemmal region. The maximal nuclear Ca2+ concentration ([Ca2+]n) attained a lower level than that in the cytoplasm ([Ca2+]c > [Ca2+]n). After the initial rise, a second sustained change of the Ca2+ level was found and the initial gradient ([Ca2+]c > [Ca2+]n) was preserved. In Ca(2+)-free solution containing 2 mM EGTA the maximal [Ca2+]c value after AVP stimulation was significantly lower than in the Ca(2+)-containing solution, but it remained higher than [Ca2+]n which was the same in both conditions. The initial Ca2+ rise was followed by a monoexponential decline. When the influx of Ca2+ through voltage-sensitive Ca2+ channels was blocked, the maximal and steady state values of [Ca2+]c but not of [Ca2+]n were lower as compared to the values in non-treated cells. Preincubation with 10 microM verapamil and 2 mM Ni2+ resulted in initial [Ca2+]c and [Ca2+]n rises which were not significantly different from the levels found in the absence of Ni2+, but the sustained phase was absent in both compartments. The differential effect with [Ca2+]c > [Ca2+]n was not observed if 1 nM AVP was applied instead of 5 microM AVP. The results indicate that cytosolic and nuclear Ca2+ stores behave differently with respect to their dependence on the agonist concentration and also with respect to the effect of Ca(2+)-entry mechanisms.

Animals↗

PubMed1994

Modulation of nucleocytosolic [Ca2+] gradient in smooth muscle by protein phosphorylation.

In resting DDT1MF-2 smooth muscle cells, the cytosolic free Ca2+ concentration ([Ca2+]c) was higher than the free Ca2+ concentration in the nucleus ([Ca2+]n). However, this nucleocytosolic [Ca2+] gradient was reversed by Ca2+ agonists like ATP or, as is shown here, by the epidermal growth factor (EGF). The ATP-induced reversal of the nucleocytosolic [Ca2+] gradient was blocked by stimulation of protein kinase C with phorbol 12-myristate 13-acetate or with the diacylglycerol kinase inhibitor R59949, or by inhibition of the Ser/Thr-specific protein phosphatases-1 and -2A with okadaic acid or calyculin A. Moreover, the magnitude of the ATP-induced reversal of the [Ca2+] gradient diminished during prolonged culture of the cells. The EGF-induced [Ca2+] rise in the cytosol and nucleus was blocked by okadaic acid and by the tyrosine kinase inhibitors herbimycin A and psi-tectorigenin. Our data suggest that the nucleocytosolic [Ca2+] gradient is modulated by (de)phosphorylation processes catalyzed by tyrosine protein kinases, by protein kinase C, and by Ser/Thr protein phosphatases-1 and -2A.

Adenosine Triphosphate↗

PubMed1994

Selective increase in the binding of the alpha 1 beta 1 integrin for collagen type IV during neurite outgrowth of human neuroblastoma TR 14 cells.

Regulation of beta 1 integrins in neurite outgrowth following N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dBcAMP) treatment was investigated using the human neuroblastoma cell line TR 14. Three beta 1 integrins were identified: the alpha 1 beta 1 receptor bound collagen type I, collagen type IV and probably laminin; the alpha 2 beta 1 integrin bound collagen type I; and the alpha v beta i receptor bound fibronectin. Neurite extension was detectable as early as 30 minutes following dBcAMP treatment, was maximal after 24 hours and remained constant during treatment for 4 days. Adhesion-perturbing beta 1 subunit-specific antibodies, added together with dBcAMP, prevented the outgrowth of new neurites. During the first 24 hours of neurite outgrowth, no change was observed in the amount of beta 1 integrins nor in their topographic distribution. However, dBcAMP treatment increased the binding of alpha 1 beta 1 receptors to collagen type IV-Sepharose by a factor 2.3 +/- 0.6 (P < 0.02), while no alteration in the binding to collagen type I was detected. Moreover, neurites and growth cones were immunoreactive for collagen type IV but not for collagen type I. Consistently dBcAMP-induced neurite outgrowth was inhibited by adhesion-perturbing alpha 1 subunit-specific antibodies. Following maximal neurite outgrowth, the amount of beta 1 integrins determined by immunoprecipitation and by confocal microscopy decreased to 58.3 +/- 11.2% (P < 0.001) and to 55.4 +/- 17.5% (P < 0.001) of untreated levels, respectively, without any change in the level of beta 1 mRNA or de novo synthesized beta 1 precursor. However, pulse-chase experiments showed an increased turnover of the beta 1 subunit: the amount of beta 1 precursor that was degraded after 1 hour chase was 50.5 +/- 8.4% in cells treated for 4 days and 34.2 +/- 3.9% in untreated cells (P < 0.02); the amount of mature beta 1 after 24 hours chase was smaller in cells treated for 4 days compared to untreated cells. In conclusion, during neurite outgrowth, alpha 1 beta 1 integrins are required and acquire an enhanced binding activity for collagen type IV; but following maximal neurite outgrowth, expression of beta 1 integrins is reduced.

Antigens, CD↗

PubMed1994

In vitro evidence that LHRH stimulates the recruitment of prolactin mRNA-expressing cells during the postnatal period in the rat.

We have studied the effect of LHRH and neuropeptide Y (NPY) on prolactin (PRL) mRNA levels in pituitary reaggregate cell cultures from 14-day-old female rats, by means of in situ hybridization and Northern blot analysis. As estimated by computer-image analysis, addition of LHRH on day 5 in culture for 40 h resulted in a 37% increase in the total cytoplasmic areas of cells containing PRL mRNA, visualized using a digoxigenin-labelled PRL cRNA. The size of individual PRL-expressing cells was not influenced, nor was the content of PRL mRNA per cell. A similar effect of LHRH was found by dot blot hybridization of extracted RNA. PRL mRNA levels were not affected by NPY. LHRH induced a 29% increase in the number of PRL mRNA-expressing cells processing through the S phase of the cell cycle, visualized by the incorporation of [3H]thymidine ([3H]T) into DNA over 16 h. The fraction of [3H]T-labelled cells was 10-12% of the total cell population. NPY did not influence the number of [3H]T-positive cells expressing PRL mRNA, but completely blocked the effect of LHRH on the latter population. The present data suggest that LHRH, probably via a paracrine action of gonadotrophs, stimulates the recruitment of new lactotrophs, an action which is negatively modulated by NPY. Since the magnitude of this effect was the same in the total pituitary cell population as in cells processing through the S phase of the cell cycle and presumably mitosis, recruitment of lactotrophs seems to be based on differentiation of progenitor or immature cells into PRL-expressing cells, rather than on a mitogenic action on pre-existing lactotrophs alone.

Analysis of Variance↗

PubMed1993

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells.

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release in renal epithelial LLC-PK1 cells. AVP-induced changes in the intracellular free calcium concentration ([Ca2+]i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca2+ mobilization was also observed in the absence of extracellular Ca2+, but was completely abolished after depletion of the intracellular Ca2+ stores by 2 microM thapsigargin. Using 45Ca2+ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP3 affected the Ca2+ content of non-mitochondrial Ca2+ pools in different loading and release conditions. Less than 10% of the Ca2+ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 microM Ca2+. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 microM Ca2+, but Ca2+ sequestered in this pool could not be released by InsP3. The thapsigargin-sensitive Ca2+ pool, in contrast, was nearly completely InsP3 sensitive. A submaximal [InsP3], however, released only a fraction of the sequestered Ca2+. This fraction was dependent on the cytosolic as well as on the luminal [Ca2+]. The cytosolic free [Ca2+] affected the InsP3-induced Ca2+ release in a biphasic way. Maximal sensitivity toward InsP3 was found at a free cytosolic [Ca2+] between 0.1 and 0.5 microM, whereas higher cytosolic [Ca2+] decreased the InsP3 sensitivity. Other divalent cations or La3+ did not provoke similar inhibitory effects on InsP3-induced Ca2+ release. The luminal free [Ca2+] was manipulated by varying the time of incubation of Ca(2+)-loaded cells in an EGTA-containing medium. Reduction of the Ca2+ content to one-third of its initial value resulted in a fivefold decrease in the InsP3 sensitivity of the Ca2+ release.

Arginine Vasopressin↗

PubMed1993

Lack of dystrophin but normal calcium homeostasis in smooth muscle from dystrophic mdx mice.

The free cytosolic Ca2+ concentration ([Ca2+]i) in the dystrophin-lacking smooth muscle from mdx mice was studied to gain new insights into the relation between dystrophin and cytoplasmic Ca2+ homeostasis, which was reported to be impaired in the mdx skeletal muscle. We observed that [Ca2+]i, as measured with the fluorescent Ca2+ indicator fura-2, was not elevated in resting smooth muscle of the vas deferens from mdx mice, in comparison with control C57 mice. Changes of the external Ca2+ concentration evoked similar changes of [Ca2+]i in mdx and control vas deferens. During contraction, cytosolic Ca2+ transients were identical, both in amplitude and in kinetics, whether or not dystrophin was present. Stretches evoked similar Ca2+ increases in muscles from both strains. Intracellular Ca2+ homeostasis appears to be unimpaired in mdx smooth muscle. Thus, the lack of dystrophin per se does not automatically induce a perturbation of Ca metabolism in muscle cells.

Animals↗

PubMed1993

Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice.

The fluorescent probe Fura-2/AM was used to determine cytosolic free calcium concentration in soleus muscle and in isolated flexor digitorum brevis fibres. This required a precise calibration; therefore, each calibration parameter was studied in situ. The influence of the dye concentration on calcium measurements was also examined. This precise calibration technique was used to compare absolute free calcium concentration in resting preparations from dystrophic (mdx) and control (C57) mice. We showed that the behavior of the dye was not similar in C57 and in mdx muscles. For this reason, we did not confirm the previous results that cytosolic free calcium concentration is increased in mdx muscles.

Animals↗

PubMed1993

ATP induced-relaxation in the mouse bladder smooth muscle.

1. The effect of adenosine 5'-triphosphate (ATP) on the free cytosolic Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca(2+)-indicator fura-2, and on force was investigated in the intact smooth muscle strips of the mouse urinary bladder. 2. ATP elicited, when exogenously applied, a large increase of [Ca2+]i with limited force development resulting in a marked Ca(2+)-force dissociation. 3. Release of endogenous neurotransmitters by transmural electrical stimulation (TES) for 30 s induced a steady increase of [Ca2+]i and a peak contraction, followed within 15 s by a relaxation. 4. In carbachol-prestimulated preparations, ATP elicited an initial rise of [Ca2+]i followed by a return to the initial precontraction Ca(2+)-level. Force in contrast presented a biphasic pattern, i.e. an initial contraction was followed by a sustained relaxation. 5. In the K(+)-depolarized precontracted preparation, ATP elicited a slight initial rise of [Ca2+]i. The partial relaxation of the force during depolarization was not preceded by a transient contraction. 6. The ATP-induced relaxation of the K(+)-prestimulated preparations was not inhibited by 8-phenyltheophylline, a potent P1-purinoceptor antagonist. 7. The order of potency for relaxation of the ATP analogues was 2-MeSATP > ATP > beta gamma Me-ATP, which is characteristic for P2y-purinoceptors. 8. These results indicate that, besides its activating effect, ATP also relaxes the mouse urinary bladder. It is suggested that the relaxant effect, mediated through P2y-purinoceptors, is mainly responsible for the low contractile potency of ATP in the bladder.

Adenosine Triphosphate↗

PubMed1993

Post-receptor pathway of the ATP-induced relaxation in smooth muscle of the mouse vas deferens.

1. The post-receptor pathway of the ATP relaxant effect in K(+)-precontracted vas deferens smooth muscle (VD) was examined. 2. The relaxation to ATP was not antagonized either by 10 microM methylene blue, a cyclic GMP inhibitor, by 10 microM indomethacin, an inhibitor of prostaglandin synthesis or by 100 microM NG-nitro-L-arginine, an inhibitor of NO production. 3. The Rp-diastereomer of adenosine 3':5'-cyclic monophosphorothioate (Rp-cAMPS) 200 microM, a competitive inhibitor of cyclic AMP significantly diminished the relaxant response to ATP. 4. Isoprenaline 10 microM, a beta-adrenoceptor agonist, produced a sustained relaxation, inhibited by Rp-cAMPS, without a significant change in [Ca2+]i, thereby mimicking the ATP-induced relaxant effect. 5. The level of the phosphorylated myosin light chain in the precontracted VD was significantly lowered by 1000 microM ATP. 6. ATP (1000 microM) and isoprenaline (10 microM) produced the same increase (+ 50%) of [cyclic AMP] when applied to a resting VD. 7. The effect of simultaneous increases of [Ca2+]i and of [cyclic AMP] produced by externally applied ATP are discussed. 8. These results suggest that ATP-induced relaxation in K(+)-precontracted VD is mediated by the activation of adenylyl cyclase.

Adenosine Triphosphate↗

PubMed1993

Staurosporine induced Ca2+ increase in DDT1MF-2 smooth muscle cells.

The free calcium concentrations in nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured DDT1MF-2 smooth muscle cells were estimated using the fluorescent dye indo-1 and laser confocal microscopy. The alkaloid staurosporine mainly increased [Ca2+]c during the initial minutes of stimulation and the nucleocytoplasmic gradient was maintained. Thereafter [Ca2+]n increased further while [Ca2+]c decreased, resulting after 10 min in a reversion of the nucleocytoplasmic gradient. Staurosporine increased the Ca2+ influx but also released intracellular Ca2+. In Ca(2+)-free solution no inversion of the nucleocytoplasmic gradient was seen in most cells. Ca2+ increased more rapidly in the area of the perinuclear sarcoplasmic reticulum (SR) than in the central cytoplasm. This is suggestive of the release of Ca2+ from this region. Ca2+ concentration remained also enhanced near the plasmalemma during this time period. This might indicate that the efflux mechanism is also affected. It is unlikely that these changes are due to inhibition of protein kinase C, since phorbol esters are ineffective and since downregulation by prolonged incubation with phorbol 12-myristate 13-acetate only affected the time periods required to attain the described [Ca2+]c and [Ca2+]n peaks. The slow time course of the staurosporine effects allowed demonstration of the spatiotemporal Ca2+ gradients between the nucleus and the cytoplasm and also inside the cytosol. The different time course of the increases in [Ca2+]c and [Ca2+]n suggests that two different Ca2+ mobilization events are present. The occurrence of an inversion of the nucleocytoplasmic gradient whereby [Ca2+]n increased while [Ca2+]c values decreased indicates that the increase in [Ca2+]n might result partially from release sites inside the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMed1993

Intracellular Ca2+ signaling induced by vasopressin, ATP, and epidermal growth factor in epithelial LLC-PK1 cells.

The free calcium concentrations in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of cultured renal LLC-PK1 epithelial cells were estimated by confocal laser microscopy. No difference between the resting mean [Ca2+]n and [Ca2+]c was found. During stimulation with maximal effective concentrations of arginine vasopressin (AVP) or the purinergic agonist ATP, the transient Ca2+ rise was followed mostly by a decline to basal levels. A differential rise could be observed when the increase in [Ca2+]n attained higher values than [Ca2+]c. In 50-60% of the cells, epidermal growth factor (EGF) also induced a transient Ca2+ rise, and a differential increase ([Ca2+]n > [Ca2+]c) was found. The G protein-linked stimuli AVP and ATP were however quantitatively much more efficacious at stimulating the [Ca2+]n and [Ca2+]c increases than was EGF. To investigate whether AVP, ATP, and EGF released Ca2+ from distinct or overlapping stores, the agonists were sequentially added. AVP and ATP applied after EGF in Ca(2+)-free medium elicited an increase in [Ca2+]n and [Ca2+]c that was not significantly lower than the release of Ca2+ in control cells without EGF prestimulation. Similarly, the amplitude of the Ca2+ responses attained by EGF in cells prestimulated by ATP or AVP was comparable to the response in naive cells. Neither EGF, ATP, nor AVP evoked a Ca2+ signal after thapsigargin treatment, indicating that the intracellular Ca2+ pools stimulated by all these agonists are part of the thapsigargin-sensitive Ca2+ pools. In contrast, when ATP was applied after AVP in Ca(2+)-containing as well as in Ca(2+)-free solutions, the Ca2+ transients were lower as compared with the response without preincubation. No differential rise could be found in Ca(2+)-free conditions. An explanation could be the use of different phospholipase C isozymes by the different receptor types, which possibly gives rise to the mobilization of different Ca2+ pools.

Adenosine Triphosphate↗

PubMed1993

[Ca2+ homeostasis in mammalian cells].

The cytoplasmic free calcium concentration ([Ca2+]i) is one of the most important factors in the regulation of the cellular activity. We therefore studied in this work the changes of [Ca2+]i during muscle excitation with optical techniques in intact smooth muscle preparations. Also the relation between [Ca2+]i, the phosphorylation of the myosin light chain and the force development in phasic and tonic smooth muscle during excitation-contraction coupling was investigated. We concluded that the changes of [Ca2+]i and force are not invariably tightly coupled during stimulation in spite of the fact that the increase of [Ca2+]i is the primary regulator of smooth muscle contraction. Agonists may, besides their Ca2+ mobilizing effect, also modulate the sensitivity of the regulatory-contractile apparatus for Ca2+. Ca(2+)-release from the internal stores during activation may modulate its further depletion. We demonstrated that the Ins(1,4,5)P3 receptor is controlled by luminal Ca2+. These observations allowed us to develop a model whereby Ca2+ oscillations during submaximal stimulation could be explained. This model of control by luminal Ca2+ also explained why the release in response to a low Ins(1,4,5)P3 concentration suddenly stops at a time when there is still plenty of Ca2+ in the internal stores. We furthermore showed that these Ca2+ channels in the SR are controlled by the redox-status of the cell. The study of Ca2+ extrusion was mainly focused on the Ca2+ pump of the plasma-membrane and its regulation by negatively charged phospholipids. Our main finding was that the plasma-membrane Ca2+ pump was stimulated by negatively charged phospholipids. Finally, we started the investigation of the existence of Ca2+ gradients between nucleus and cytoplasm in intact cells with confocal laser microscopy. We demonstrated in different cell types the existence of spatio-temporal differences in the Ca2+ distribution between nucleus and cytosol in basal and stimulated conditions. These observations indicate that a Ca2+ barrier must be present at the nuclear envelope, that different Ca2+ mobilizing phenomena are present and are in favour of the existence of a specific regulation of nuclear Ca2+.

Animals↗