Rik Casteels

Exchange characteristics of the noradrenaline‐sensitive calcium store in vascular smooth muscle cells or rabbit ear artery.

1. The amplitude of the noradrenaline‐sensitive Ca stores has been estimated by measuring the amplitude of the transient contraction induced by the agonist in Ca‐free solution. or by measuring the amount of 45Ca released under these conditions. 2. The rate of filling of this store after depletion is much faster than the rate of depletion in Ca‐free solution, and depends on [Ca]o in the bathing solution. The degree of filling also depends on [Ca]o. 3. At the same [Ca]o the degree of filling is higher in K‐depolarized tissues than in control tissues. However at 10 mM‐[Ca]o and 5.9 mM‐K the amount of Ca taken up by the store is larger than that after loading in 0.2 mM‐Ca and 141.4 mM‐K, although the tissues remain relaxed during loading at 5.9 mM‐K and contracted at 141.4 mM‐K. 4. The Ca antagonists D600 and nicardipine selectively block the contraction induced by K depolarization, but do not affect appreciably the noradrenaline‐induced contraction. 5. The filling of the store is not significantly reduced by the presence of the Ca antagonists in solutions containing 5.9 mM‐K. However these antagonists reduce the degree of filling in K‐rich loading solution to a level which is lower than that observed in the control. 6. Mn blocks both the contraction induced by K‐rich solution and the tonic component of the noradrenaline‐induced contraction and its also inhibits filling of the store. 7. The results suggest that the filling of the store under physiological conditions occurs by a direct pathway between the store and the extracellular medium.

The action of lanthanum and D600 on the calcium exchange in the smooth muscle cells of the guinea-pig Taenia coli.

SummaryThe calcium distribution in smooth muscle cells of the taenia coli has been studied with a method based on the action of lanthanum ions. La3+-ions make it possible to separate the extracellular calcium from the cellular calcium by their high affinity for extracellular calcium binding sites and by their blocking action on the transmembrane calcium movements. Using this method the cellular calcium content and its exchangeable fraction have been determined. In spontaneously active tissues the exchange of the cellular calcium reaches a maximal value of 108 μM/kg wet wt or 1/6 of the total cellular calcium, while in quiescent tissues (treated with D600) this maximal value is 80 μM/kg wet wt.During K depolarization a large amount of extracellular calcium penetrates in the cellular compartment. This entry and the corresponding tension development are inhibited by D600.These results are interpreted by assuming that even during spike discharge the Ca-permeability of the membrane is very small and that this entering calcium could function as a trigger, releasing intracellularly localized calcium. This physiological mechanism should be distinguished from the important uptake of calcium occurring during K-depolarization.

Molecular and Functional Evidence for Multiple Ca2+-binding Domains in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+. To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria. The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures. Six new cytosolic Ca2+-binding regions were detected on the InsP3R in addition to the one described earlier (Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996)J. Biol. Chem. 271, 27005–27012). Strong45Ca2+ and ruthenium red binding domains were localized in the N-terminal region of the InsP3R as follows: two Ca2+-binding domains were located within the InsP3-binding domain, and three Ca2+ binding stretches were localized in a 500-amino acid region just downstream of the InsP3-binding domain. A sixth Ca2+-binding stretch was detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca2+-binding sites in the regulation of the InsP3R was obtained from functional studies on permeabilized A7r5 cells, in which we characterized the effects of Ca2+ and Sr2+ on the EC50 and cooperativity of the InsP3-induced Ca2+ release. The activation by cytosolic Ca2+was due to a shift in EC50 toward lower InsP3concentrations, and this effect was mimicked by Sr2+. The inhibition by cytosolic Ca2+ was caused by a decrease in cooperativity and by a shift in EC50 toward higher InsP3 concentrations. The effect on the cooperativity occurred at lower Ca2+ concentrations than the inhibitory effect on the EC50. In addition, Sr2+ mimicked the effect of Ca2+ on the cooperativity but not the inhibitory effect on the EC50. The different [Ca2+] and [Sr2+] dependencies suggest that three different cytosolic interaction sites were involved. Luminal Ca2+ stimulated the release without affecting the Hill coefficient or the EC50, excluding the involvement of one of the cytosolic Ca2+-binding sites. We conclude that multiple Ca2+-binding sites are localized on the InsP3R-1 and that at least four different Ca2+-interaction sites may be involved in the complex feedback regulation of the release by Ca2+.

Electrogenic sodium pump in arterial smooth muscle cells.

Summary1.The membrane potential, the tension development and the42K-efflux were studied in the ear artery of the rabbit under different experimental conditions.2.K-free solution depolarized the cells. Readmission of K in the external medium caused a transient hyperpolarization.3.Ouabain 10−5 M also depolarized the cells. On washing out the ouabain the membrane potential gradually returned to its control value.4.Tension development appeared simultaneously with the depolarization, but it subsided during prolonged depolarization.5.K-free solution and ouabain induced a small increase of the rate coefficient of K-efflux from the ear artery. This effect appeared later than the depolarization or the tension development. These changes of42K-efflux make a rapid change of (Na)i unlikely.6.It is concluded that the electrogenic Na-pump plays an important role in maintaining the membrane potential of arterial smooth muscle cells. All procedures which inhibit this pump, induce a depolarization of the cells and thereby a tension development. There is no experimental evidence in favour of the hypothesis that the inhibition of a Na−Ca exchange system would be the primary event in inducing contraction under our experimental conditions.

Calcium ion homeostasis in smooth muscle

Ca2+ plays an important role in the regulation of smooth-muscle contraction. In this review, we will focus on the various Ca(2+)-transport processes that contribute to the cytosolic Ca2+ concentration. Mainly the functional aspects will be covered. The smooth-muscle inositol 1,4,5-trisphosphate receptor and ryanodine receptor will be extensively discussed. Smooth-muscle contraction also depends on extracellular Ca2+ and both voltage- and Ca(2+)-release-activated plasma-membrane Ca2+ channels will be reviewed. We will finally discuss some functional properties of the Ca2+ pumps that remove Ca2+ from the cytoplasm and of the Ca2+ regulation of the nucleus.

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.

Agonist-dependent Ca2+ and Mn2+ entry dependent on state of filling of Ca2+ stores in aortic smooth muscle cells of the rat.

1. The properties of intracellular Ca2+ stores of intact‐ and of saponin‐skinned A7r5 (an established cell line from embryonic rat aorta) smooth muscle cells were studied by measuring 45Ca2+ and 54Mn2+ fluxes. 2. Application of 5 microM‐vasopressin to intact cells increased the fractional loss of 45Ca2+ in Ca2(+)‐free solution by a factor of 5.2. This effect was not influenced by a pre‐incubation with 10 microM‐ryanodine. Caffeine (25 mM) did not stimulate the fractional loss of 45Ca2+ from intact cells. 3. In skinned cells 10 microM‐IP3 (inositol 1,4,5‐trisphosphate) and 5 microM‐A23187 (a calcium ionophore) released the same amount of 45Ca2+. This release did not require GTP and was not affected by a pre‐incubation with 10 microM‐ryanodine. Caffeine (25 mM) did not release stored Ca2+. 4. NaF (1 mM) plus 10 microM‐AlCl3 inhibited by 72% the 45Ca2+ uptake by the IP3‐sensitive store of skinned cells at 0.15 microM‐Ca2+. Cyclic AMP‐dependent protein kinase did not stimulate this ATP‐dependent 45Ca2+ uptake, nor could the presence of phospholamban be demonstrated immunologically. 5. The 45Ca2+ uptake by cells which had been depleted of Ca2+ with 5 microM‐vasopressin was 69% higher than the uptake obtained without such proceeding depletion. This enhanced 45Ca2+ uptake did not occur through voltage‐operated Ca2+ channels, because blockade of these channels with verapamil, or depolarization of the plasma membrane by increasing [K+] from 5.9 to 59 mM in the presence of verapamil, did not modify this uptake. 6. A similar increase of the 54Mn2+ uptake occurred in intact cells with a depleted Ca2+ store. If, however, the cells were first skinned and subsequently exposed to 54Mn2+, the ATP‐dependent 54Mn2+ uptake amounted to less than 6% of the ATP‐dependent 45Ca2+ uptake. 7. If intact cells were first exposed to a 45Ca2(+)‐ or 54Mn2(+)‐containing solution, and subsequently skinned in a non‐radioactive intracellular solution, the addition of 10 microM‐A23187 to these cells released stored Ca2+ or Mn2+. The amount of released Ca2+ was only slightly larger than the amount of released Mn2+. If the intracellular store was depleted before loading, the amount of Ca2+ or Mn2+ released by the ionophore increased by 68 and 28%, respectively. 8. It is concluded that A7r5 smooth muscle cells do not express a Ca2(+)‐induced Ca2+ release mechanism, but do contain an IP3‐induced Ca2+ release mechanism which can release approximately all intracellularly accumulated 45Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat basophilic leukemia cells as model system for inositol 1,4,5-trisphosphate receptor IV, a receptor of the type II family: functional comparison and immunological detection.

This study concerns the detection and analysis of the highly homologous type II-like inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3R-II, -IV and -V). We have particularly investigated RBL-2H3 cells, which at the mRNA level predominantly expressed InsP3R-IV [De Smedt H. Missiaen L. Parys JB. et al. (1994) Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. J. Biol. Chem., 269, 21691-21698]. When measured in identical experimental conditions, microsomes from RBL-2H3 cells were characterized by a much higher InsP3 binding affinity (Kd 3.8 +/- 0.8 nM, Bmax 0.40 +/- 0.08 pmol/mg protein) than microsomes from A7r5 cells (Kd 65 +/- 7 nM, Bmax 0.65 +/- 0.08 pmol/mg protein) or from cerebellum (Kd 135 +/- 14 nM, Bmax 7.35 +/- 1.13 pmol/mg protein). An affinity-purified antibody against the C-terminus of type II-like InsP3Rs detected, after SDS-PAGE and immunoblotting, a 250 kD protein in RBL-2H3 and C3H10T1/2 cells, but not in other cell types. An isoform-specific antibody against the C-terminus of InsP3R-I was used to determine the presence of the various InsP3R-I splice isoforms at the protein level. The 273 kD (brain), 261 kD (peripheral tissues) and 256 kD (Xenopus oocytes) isoforms were recognized. Expression of InsP3R-I in RBL-2H3 cells was very low. Taken together, our results support the hypothesis that InsP3R isoforms may differ to a large extent in their affinity for InsP3 and suggest that RBL-2H3 cells are a useful model for the study of InsP3R-IV.

Ca2+ Homeostasis in Vascular Smooth Muscle

The free intracellular calcium concentration is an important link in the excitation-contraction coupling mechanism of vascular smooth muscle. In this review, some current topics about vascular smooth muscle as regards Ca2+ storage, Ca2+ release, Ca2+ extrusion and Ca2+ regulation are discussed. Particular attention is paid to Ca2+ mobilized from the sarcoplasmic reticulum, the physiologically important Ca2+ reservoir in vascular smooth muscle. This occurs through two Ca2+ release channels: the inositol 1,4,5-trisphosphate receptor and the ryanodine receptor; the characteristics, function and control of these two receptors are summarized. Emphasis is also placed on a role of the nucleus as a potential Ca2+ storage site.

Ca2+ extrusion across plasma membrane and Ca2+ uptake by intracellular stores

The aim of this review is to summarize the various systems that remove Ca2+ from the cytoplasm. We will initially focus on the Ca2+ pump and the Na(+)-Ca2+ exchanger of the plasma membrane. We will review the functional regulation of these systems and the recent progress obtained with molecular-biology techniques, which pointed to the existence of different isoforms of the Ca2+ pump. The Ca2+ pumps of the sarco(endo)plasmic reticulum will be discussed next, by summarizing the discoveries obtained with molecular-biology techniques, and by reviewing the physiological regulation of these proteins. We will finally briefly review the mitochondrial Ca(2+)-uptake mechanism.