Spatial and temporal aspects of ACh-induced [Ca2+]i oscillations in porcine tracheal smooth muscle

This study evaluated the relationship between regional elevation in intracellular calcium concentration ([Ca2+]i) induced by acetylcholine (ACh) and the global cellular responses in porcine tracheal smooth muscle (TSM) cells. Regional (approximately 1.5 microm3) and global (whole cell) changes in [Ca2+]i were measured in fluo-3 loaded TSM cells using real-time confocal microscopy. Regional responses appeared as propagating [Ca2+]i oscillations whereas global responses reflected the spatiotemporal integration of these regional responses. Within a region, [Ca2+]i oscillations were 'biphasic' with initial higher frequencies, followed by slower steady-state oscillations. With increasing ACh concentration, the peak (maximum value relative to 0 nM) of regional [Ca2+]i oscillations remained relatively constant, whereas both frequency and propagation velocity increased. In contrast, the global spatiotemporal integration of the regional oscillatory responses appeared as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i. We conclude that the significance of ACh-induced [Ca2+]i oscillations lies in the establishment of mean [Ca2+]i level for slower Ca2+-dependent physiological processes via modulation of oscillation frequency and propagation velocity.     

Spatial distribution and temporal change of cytoplasmic free calcium in human platelets

Our digital imaging microscope equipped with a microspectrofluorometer revealed in single resting human platelets the existence of continuous Ca2+ gradient increasing towards the plasma membrane (frequency; 100%) and discontinuous ones (Ca2+ plateaus) in the endoplasmic regions (frequency: 70%). An average cytoplasmic free Ca2+ concentration ([ Ca2+]i) in a whole cytoplasm was 72 +/- 7 nM, ranging from 30 nM in the lowest to 150 nM in the highest region just beneath the plasma membrane. When stimulated with thrombin, [Ca2+]i uniformly increased to the average [Ca2+]i of 300 nM and these gradients disappeared. This [Ca2+]i transient was followed by the sustained increase in [Ca2+]i in both single cells and cell suspension.     

Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative.

Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 10(5) cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 +/- 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 +/- 4.4 nM (mean +/- SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653-656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K(+)-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 microM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 microM. The data suggest that different isoforms of the alpha-subunit of Na+/K(+)-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart.     

Regulation of airway ciliary activity by Ca2+: simultaneous measurement of beat frequency and intracellular Ca2+.

Airway ciliary activity is influenced by [Ca2+]i, but this mechanism is not fully understood. To investigate this relationship, ciliary activity and [Ca2+]i were measured simultaneously from airway epithelial ciliated cells. Ciliary beat frequency was determined, for each beat cycle, with phase-contrast optics and high-speed video imaging (at 240 images s-1) and correlated with [Ca2+]i determined, at the ciliary base, by fast imaging (30 images s-1) of fura-2 fluorescence. As a mechanically induced intercellular Ca2+ wave propagated through adjacent cells, [Ca2+]i was elevated from a baseline concentration of 45 to 100 nM, to a peak level of up to 650 nM. When the Ca2+ wave reached the ciliary base, the beat frequency rapidly increased, within a few beat cycles, from a basal rate of 6.4 to 11.6 Hz at 20-23 degrees C, and from 17.2 to 26.7 Hz at 37 degrees C. Changes in [Ca2+]i, above 350 nM, had no effect on the maximum beat frequency. We suggest that airway ciliary beat frequency is 1) controlled by a low range of [Ca2+]i acting directly at an axonemal site at the ciliary base and 2) that a maximum frequency is induced by a change in [Ca2+]i of approximately 250-300 nM.     

Intracellular calcium of longitudinal muscles isolated from pregnant rat myometrium.

Longitudinal muscle cells were successfully isolated from pregnant rat myometrium (21 days of gestation) with more than a 95% survival rate. The approximate size of relaxed cells was 232.2 +/- 74 microns in length and 16.2 +/- 7.0 microns in width. Using the fluorescent indicator Fura-2, the concentration of intracellular free calcium ([Ca2+]i) in resting state cells was calculated to be 116 +/- 18.5 nM. The isolated cells responded well to K+, acetylcholine and oxytocin in terms of contraction as well as the increase in [Ca2+]i. The increase in [Ca2+]i induced by acetylcholine and K+ appeared to be mainly due to an influx of extracellular Ca2+. On the other hand, the oxytocin-induced increase in [Ca2+]i was mainly due to a release of Ca2+ from intracellular storage sites in the isolated cells. Isolated longitudinal muscle cells can serve as a useful tool in establishing the relationship between [Ca2+]i and regulation of the uterine contraction at the final stage of pregnancy.     

How far does phospholipase C activity depend on the cell calcium concentration? A study in intact cells.

The dependence of phospholipase C activity on the cytosolic Ca2+ concentration ([Ca2+]i) was studied in intact liver cells treated with the Ca2+-mobilizing hormone vasopressin, or not so treated. Phospholipase C (PLC) activity was estimated from the formation of [3H]inositol trisphosphate (InsP3) and the degradation of [3H]phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The [Ca2+]i of the cells was clamped from 29 to 1130 nM by quin2 loading. This wide concentration range was obtained by loading the hepatocytes with a high concentration of the Ca2+ indicator in low-Ca2+ medium or by using the Ca2+ ionophore ionomycin in medium containing Ca2+. In resting cells, in which [Ca2+]i was 193 nM, treatment with 0.1 microM-vasopressin which stimulates liver PLC maximally, tripled InsP3 content and raised [Ca2+]i to 2 microM within 15 s. Lowering [Ca2+]i partially decreased cell InsP3 content as well as the ability of vasopressin to stimulate InsP3 formation maximally. At 29 nM, the lowest Ca2+ concentration obtained in isolated liver cells, basal InsP3 content was 64% of that measured in control cells. Addition of vasopressin no longer affected [Ca2+]i, but significantly increased InsP3 by 200%, although less than in the controls (300%). The maintenance of the greater part of the PLC response at constant [Ca2+]i indicated that, in the liver, InsP3 formation does not result from an increase in [Ca2+]i. The effects of lowering [Ca2+]i were reversible. When low cell [Ca2+]i was restored to a normal value, resting InsP3 content and the ability of vasopressin to stimulate InsP3 formation maximally by 300% were also restored. Raising [Ca2+]i from 193 to 1130 nM had little effect on the InsP3 content or the vasopressin-mediated increase in InsP3. In agreement with the stimulation of PLC activity by vasopressin, cell [3H]PtdInsP2 and total PtdInsP2 were degraded by application of this hormone for 15 s. In contrast, when [Ca2+]i was lowered to 29 nM, basal [3H]PtdInsP2 and total PtdInsP2 were increased by about 30%, [3H]PtdInsP2 was further increased by vasopressin, but total PtdInsP2 was not changed. These results show that, in intact hepatocytes, PLC is little affected by [Ca2+]i concentrations above 193 nM, but is partially dependent on Ca2+ below that value. They suggest that, in addition to activating PLC activity, vasopressin might stimulate PtdInsP2 synthesis, presumably via phosphatidylinositol-phosphate kinase, and that this pathway might predominate in cells with low [Ca2+]i.     

The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP.

The requirements of purified rat Leydig cells for intra- and extra-cellular Ca2+ during steroidogenesis stimulated by LH (lutropin), cyclic AMP analogues and LHRH (luliberin) agonist were investigated. The intracellular Ca2+ concentrations ([Ca2+]i) were measured by using the fluorescent Ca2+ chelator quin-2. The basal [Ca2+]i was found to be 89.4 +/- 16.6 nM (mean +/- S.D., n = 25). LH, 8-bromo cyclic AMP and dibutyryl cyclic AMP increased [Ca2+]i, by 300-500 nM at the highest concentrations of each stimulator, whereas LHRH agonist only increased [Ca2+]i by a maximum of approx. 60 nM. Low concentrations of LH (less than 1 pg/ml) and all concentrations of LHRH agonist increased testosterone without detectable changes in cyclic AMP. With amounts of LH greater than 1 pg/ml, parallel increases in cyclic AMP and [Ca2+]i occurred. The steroidogenic effect of the LHRH agonist was highly dependent on extracellular Ca2+ concentration ([Ca2+]e), whereas LH effects were only decreased by 35% when [Ca2+]e was lowered from 2.5 nM to 1.1 microM. No increase in [Ca2+]i occurred with the LHRH agonist in the low-[Ca2+]e medium, whereas LH (100 ng/ml) gave an increase of 52 nM. It is concluded that [Ca2+]i can be modulated in rat Leydig cells by LH via mechanisms that are both independent of and dependent on cyclic AMP, whereas LHRH-agonist action on [Ca2+]i is independent of cyclic AMP. The evidence obtained suggests that, at sub-maximal rates of testosterone production, Ca2+, rather than cyclic AMP, is the second messenger, whereas for maximum steroidogenesis both Ca2+- and cyclic-AMP-dependent pathways may be involved.     

Role of calcium and cAMP in the action of adrenocorticotropin on aldosterone secretion

When the dose-response curve of adrenocorticotropin (ACTH)-induced aldosterone secretion is compared to that of ACTH-induced intracellular cAMP, the ED50 for intracellular cAMP is more than 10 times as high as that for aldosterone production. In contrast, the dose-response curve of forskolin-induced aldosterone secretion correlates well with that for forskolin-induced intracellular cAMP. ACTH, but not forskolin, increases calcium influx into glomerulosa cells without inducing the mobilization of calcium from an intracellular pool. The effect of ACTH on calcium influx is dose-dependent and ED50 is 3.5 X 10(-11) M. In a perifusion system, the effect of 1 nM ACTH on aldosterone secretion is much greater than that of 1 microM forskolin, even though these two stimulators induce identical increases in the intracellular cAMP. Perifusion with combined A23187 (50 nM) and forskolin (1 microM) stimulates aldosterone secretion to a value comparable to that induced by 1 nM ACTH. Likewise, BAY K 8644 (1 nM), which induces a comparable increase in calcium influx, potentiates the effect of 1 microM forskolin. When the intracellular [Ca2+] is fixed at either 100 or 300 nM, forskolin-stimulated intracellular cAMP content is identical, but ACTH-stimulated intracellular cAMP content at 100 nM [Ca2+]i is 60% of that at 300 nM [Ca2+]i. Both the ACTH- and forskolin-induced aldosterone secretion rate is higher at 300 nM than at 100 nM [Ca2+]i. These results indicate that ACTH stimulates calcium influx, that calcium potentiates ACTH-induced but not forskolin-induced cAMP generation, and that Ca2+ and cAMP act as synarchic messengers in ACTH-mediated aldosterone secretion.     

Ethanol-induced increases in [Ca2+]i and inositol (1,4,5) triphosphate in rat hepatocytes

Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.     

A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte.

We construct a minimal model of cytosolic free Ca2+ oscillations based on Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channel (IP3R) of a single intracellular Ca2+ pool. The model relies on experimental evidence that the cytosolic free calcium concentration ([Ca2+]c) modulates the IP3R in a biphasic manner, with Ca2+ release inhibited by low and high [Ca2+]c and facilitated by intermediate [Ca2+]c, and that channel inactivation occurs on a slower time scale than activation. The model produces [Ca2+]c oscillations at constant [IP3] and reproduces a number of crucial experiments. The two-dimensional spatial model with IP3 dynamics, cytosolic diffusion of IP3 (Dp = 300 microns 2 s-1), and cytosolic diffusion of Ca2+ (Dc = 20 microns 2 s-1) produces circular, planar, and spiral waves of Ca2+ with speeds of 7-15 microns.s-1, which annihilate upon collision. Increasing extracellular [Ca2+] influx increases wave speed and baseline [Ca2+]c. A [Ca2+]c-dependent Ca2+ diffusion coefficient does not alter the qualitative behavior of the model. An important model prediction is that channel inactivation must occur on a slower time scale than activation in order for waves to propagate. The model serves to capture the essential macroscopic mechanisms that are involved in the production of intracellular Ca2+ oscillations and traveling waves in the Xenopus laevis oocyte.     

Ca2+-agonistic effect of a T-type Ca-channel blocker mibefradil (Ro 40-5967)

Here we report that a Ca2+ antagonist mibefradil (Ro 40-5967) which has been shown to be a selective inhibitor of T-type calcium channels increases free calcium concentration ([Ca2+]i) in the cytoplasm of cultured smooth muscle cells isolated from porcine coronary artery. Smooth muscle cells were loaded with Fura 2 and a videoimage system was used to follow the [Ca2+]i responses. It was shown that at a concentration of 1 nM mibefradil induced a transient [Ca2+]i elevation in individual cells and at a concentration of 100 nM this compound stimulated almost all the cells in monolayer. The [Ca2+]i response did not change with the further increase of the mibefradil concentration up to 10 microM. The half-maximal effect was observed at 10 nM. The increase in [Ca2+]i strongly depended on the presence of Ca in the extracellular medium. Calcium antagonists belonging to three different classes--verapamil (phenylalkylamines), diltiazem (benzothiazepines) and amlodipin (dihydropyridines) neither suppressed the mibefradil effect nor mimicked it. These data indicate that mibefradil increased [Ca2+]i acting via a distinct receptor site. We suggest that these receptors are coupled to calcium channels of plasma membrane.     

Ca2+]i in single isolated cardiac cells: a review of recent results obtained with digital imaging microscopy and fura-2

The use of fluorescent Ca2+ indicators to observe [Ca2+]i transients in voltage-clamped single cells has many advantages over previous methods, such as the use of aequorin in multicellular preparations, for studying excitation-contraction coupling. In the studies reviewed in this article, [Ca2+]i in single isolated mammalian ventricular myocytes was observed through the use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were generally studied with the use of inverted microscopes equipped with ultraviolet epifluorescence illumination, intensified silicon intensifier target cameras (ISIT), and (or) a photomultiplier tube. Analysis of subcellular patterns of fura-2 fluorescence was performed by digital analysis of the images obtained with the ISIT camera. Variation of membrane voltage and exposure of cells to ryanodine (which was assumed to selectively block the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The main results of these studies are the following. (1) In any population of enzymatically isolated heart cells, there are (i) mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; (ii) spontaneously contracting cells, which have a relatively elevated [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous, propagating waves of high [Ca2+]i; and (iii) cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.     

Influx of extracellular calcium is required for the membrane translocation of 5-lipoxygenase and leukotriene synthesis.

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Low concentrations of the stable prostaglandin endoperoxide U44069 stimulate shape change in quin2-loaded platelets without a measurable increase in [Ca2+]i

Dose-response relationships for raised cytoplasmic free calcium concentration, [Ca2+]i, and shape change were measured simultaneously in quin2-loaded human platelets. With the calcium ionophore ionomycin the threshold [Ca2+]i for shape change was 300 nM with a maximal response at 800 nM. With 1 mM external Ca2+ the U44069 concentrations required to stimulate half-maximal shape change and an increase in [Ca2+]i were 2 and 41 nM, respectively. For PAF these values were 8.7 and 164 pg/ml, respectively. Low concentrations of U44069 and PAF evoked substantial shape change without any rise in [Ca2+]i. In the absence of external Ca2+, U44069 stimulated half-maximal shape change at 2 nM, and half-maximal elevation of [Ca2+]i at 69 nM: here, increased [Ca2+]i never reached the threshold [Ca2+]i for shape-change derived with ionomycin. These results suggest that some transduction mechanism other than elevated [Ca2+]i, as yet unidentified, can cause shape change.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Calcium channel blocker and calmodulin antagonists affect the gradient of free calcium ions in lily pollen tubes.

The distribution of intracellular free calcium ions ([Ca2+]i) was measured in pollen tubes of Lilium longiflorum using video imaging microscopy and the calcium sensitive indicators fura-2 and quin-2. The mean [Ca2+]i in growing pollen tubes measured with fura-2 shows a maximum of 1.7 to 2.6 microM in the tube tip and decreases almost exponentially to 60 to 100 nM at 100 microns behind the tip. Using quin-2, the maximum [Ca2+]i was also found in the tube tip but with a lower Ca2+ concentration, namely 1 microM. Addition of the calcium channel blocker La3+ caused a decrease of the [Ca2+]i maximum in the tube tip, indicating a heterogeneous distribution of Ca2+ channels along the plasma membrane of pollen tubes. The [Ca2+]i increased after addition of vanadate or compound 48/80. This suggests an involvement of a calmodulin-dependent Ca2+ pump in generation of the Ca2+ gradient in lily pollen tubes. The high [Ca2+]i found in the tube tip with fura-2 seems to indicate the real Ca2+ concentration and is probably responsible for vesicle fusion, fragmentation of actin filaments, and inhibition of cytoplasmic streaming.     

Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist.

Calcium-mobilizing agonists induce intracellular Ca2+ concentration ([Ca2+]i) changes thought to trigger cellular responses. In connected cells, rises in [Ca2+]i can propagate from cell to cell as intercellular Ca2+ waves, the mechanisms of which are not elucidated. Using fura2-loaded rat hepatocytes, we studied the mechanisms controlling coordination and intercellular propagation of noradrenaline-induced Ca2+ signals. Gap junction blockade with 18 alpha-glycyrrhetinic acid resulted in a loss of coordination between connected cells. We found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of intercellular Ca2+ waves. In addition, our experiments revealed functional differences between noradrenaline-induced Ca2+ signals in connected hepatocytes. These results demonstrate that intercellular Ca2+ signals in multicellular systems of rat hepatocytes are propagated and highly organized through complex mechanisms involving at least three factors. First, gap junction coupling ensures coordination of [Ca2+]i oscillations between the different cells; second, the presence of hormone at each hepatocyte is required for cell-cell Ca2+ signal propagation; and third, functional differences between adjacent connected hepatocytes could allow a 'pacemaker-like' intercellular spread of Ca2+ waves.     

Positive correlation between cytosolic free calcium and surfactant secretion in cultured rat alveolar type II cells

To determine whether increases in the cytosolic free Ca2+ concentration ([Ca2+]i) accompany agonist-stimulated surfactant secretion by cultured alveolar type II cells, we measured the [Ca2+]i of quin2-loaded cells isolated from adult rats before and after cells were stimulated with ionomycin, terbutaline or tetradecanoylphorbol acetate (TPA). To determine whether increases in [Ca2+]i are necessary for stimulated surfactant secretion to occur, we measured secretion in cells after [Ca2+]i had been reduced by loading cells with quin2 in medium containing low [Ca2+]. Ionomycin increased [Ca2+]i and stimulated surfactant secretion in a dose-dependent manner. Reductions in [Ca2+]i correlated with reductions in secretion stimulated by ionomycin, terbutaline or TPA. Ionomycin-stimulated secretion was most sensitive to reductions in [Ca2+]i; terbutaline-stimulated secretion was more sensitive than TPA-stimulated secretion. When [Ca2+]i was less than 65 nM, all stimulated secretion was blocked. Restoration of [Ca2+]i to greater than 100 nM restored ionomycin-stimulated secretion. We conclude that ionomycin increases [Ca2+]i and stimulates surfactant secretion in cultured alveolar type II cells, and that increased [Ca2+]i appears to be necessary for ionomycin-stimulated secretion to occur. Terbutaline-stimulated surfactant secretion seems to be more easily inhibited by a reduction in [Ca2+]i than does TPA-stimulated secretion.