Intracellular Calcium Distribution Research Articles

Subcellular compartmentation of elements in non‐mycorrhizal and mycorrhizal roots of Pinus sylvestris: an X‐ray microanalytical study. II. The distribution of calcium, potassium and sodium

The inter‐ and intracellular distribution of the elements calcium, potassium and sodium in non‐mycorrhizal and mycorrhizal roots of Pinus sylvestris dependent on different external nutrient supply conditions was detected by energy‐dispersive X‐ray microanalysis after cryofixation, freeze‐drying and pressure infiltration of the material. In non‐mycorrhizal and mycorrhizal roots, calcium was mainly detectable in the apoplastic regions. The levels in vacuoles and cytoplasm were near the limits of detection by X‐ray microanalysis. Incubation with high concentrations of potassium and sodium, or mycorrhizal infection with Suillus bovinus and Pisolithus tinctorius reduced the amounts of calcium detectable in the roots, especially in the apoplast of cortical cells. The studies revealed that: potassium is mainly localized in cytoplasm and cell walls; the cytoplasmic content is regulated over a wide range of external potassium concentrations; potassium levels in the inner parts of roots are higher than in the outer parts. Mycorrhizal infection with Suillus bovinus had no effect on the inter‐ and intracellular distribution of potassium in roots but, if the external supply was low, the potassium content in shoots was reduced. In non‐mycorrhizal pine roots and those infected with Paxillus involutus an increase in the sodium content of all cell compartments was observed after treatment with high external concentrations of NaH2PO4. However, an increase in sodium content in mycorrhizas of S. bovinus was not detected. The X‐ray microanalytical results are discussed in relation to the apoplastic movement of nutrients in non‐mycorrhizal and mycorrhizal fine roots of pine and to the demand for these nutrients in different intracellular compartments.

Role of calcium in apoptosis of HL-60 cells induced by harringtonine

The effect of Ca(2+) on HT-induced apoptosis in HL-60 cells was examined. Characteristics of apoptosis induced by harringtonine (HT) or campothecin (CAM) could not be abolished by extracellular calcium chelator EGTA; however, an intracellular Ca(2+) chelator BAPTA-AM could block HT or CAM-induced HL-60 cell apoptosis. Requirement of intracellular calcium for HT-induced apoptosis is further supported by the fact that intracellular Ca(2+) -depleted HL-60 cells could not undergo HT-induced apoptosis. No significant increase of intracellular Ca(2+) was found after HT treatment. By using video enhancement contrast microscopy (VEC), the dynamic changes of intracellular calcium distribution over the whole period of apoptosis in the same individual cell were detected. The results demonstrated the movement of Ca(2+) from cytosol to nucleus after initiation of apoptosis by treatment with HT.

Intracellular calcium distribution in apoptosis of HL-60 cells induced by harringtonine: intranuclear accumulation and regionalization

Harringtonine (HT), an anticancer drug with high chemotherapeutic efficiency to human chronic granulocytic/myelomonocytic leukemia, has been reported to rapidly induce apoptosis in HL-60 cells in a wide scope/range of dosage by investigators from our lab and others. In the present studies, by using video enhancement contrast (VEC) microscopy, we dynamically analyzed changes in intracellular calcium distribution in a single HL-60 cell over the period from the initiation of apoptosis to the obvious appearance of chromatin condensation. The results from this paper demonstrated the striking distinction of intracellular calcium distribution at different time points after treatment with HT. Before treatment in normal HL-60 cells the highest [Ca 2+] i accumulation was observed in the peri-nuclear area and the lowest was observed in the nucleus; after treatment with 1 μg/ml HT for 30 min intracellular calcium diffused all over the cell compartments, while intranuclear calcium increased comparatively and significantly. The phenomenon of intranuclear calcium accumulation was further confirmed by using laser scanning confocal microscopy (LSCM). In addition, co-localization of the highest calcium region with condensed chromatin in apoptotic HL-60 cells was also observed by LSCM. Our results suggest that two sequential alterations of intracellular calcium distribution occurred in apoptotic HL-60 cells induced by HT, i.e. (a) accumulation of calcium in the nucleus and (b) regionalization in a specific nuclear region.

Dedifferentiated cardiomyocytes from chronic hibernating myocardium are ischemia-tolerant.

Left ventricular biopsies from 21 patients with clinically diagnosed chronic hibernating myocardium (CHM) were examined by light- and electron microscopy. A mean of 27% of cardiomyocytes were structurally altered and were characterized as hibernating, because of reduced amount of myofibrils and increased glycogen content. Electron microscopy of these cells showed reduction of T-tubules and numerous small mitochondria, but few changes associated with degeneration, acute ischemia or apoptosis. The structural changes found in CHM are reminiscent of dedifferentiation rather than degeneration. The expression patterns of some structural proteins show resemblance with those in embryonic cardiomyocytes. Histochemically, mitochondrial NADH-oxidase and proton translocating ATPase activities were absent, whereas cytochrome c activity was present. Intracellular calcium distribution indicated normally bound sarcolemmal calcium and absence of excess mitochondrial calcium accumulation. Nuclear chromatin ranged from normal to dispersed with only a few nuclei that were clumped. These results suggest that cardiomyocytes from human CHM hearts are structurally altered, but viable, and lack morphologic and cytochemical characteristics suggestive of apoptosis or acute ischemia.

Actin modifications and calcium homoeostasis in neurotoxicity. The case of organotin salts

The cytoskeleton is a major constituent of the neuronal cytoplasm; it controls cell shape and plays important roles in regulating various physiological processes. In neurons, actin filaments are involved in the growth of the neurite and the neurotransmitter release. Recent findings suggest that actin filaments play a role in modulating [Ca 2+] i responses to neurotoxic insults. The physiological functioning of the neural cell is critically dependent on the intracellular distribution of calcium. An increase of cytosolic free calcium can activate a number of intracellular reactions, including neurotransmitter release, protein phosphorylation, protease activity, and, eventually, cell death. Many neurotoxic agents with diverse mechanisms have been reported to affect mechanisms associated with calcium. Among these are organotin compounds: they can both raise the cytosolic and synaptosomal [Ca 2+] i concentrations and interfere with the [Ca 2+] i response evoked by different agonists. Furthermore, some of these compounds cause actin depolymerization. The interference of triethyltin (TET)—a compound inducing myelin vacuolization and brain oedema—with Ca 2+ homoeostasis and actin polymerization results in an adverse effect on neurotransmitter release in different neural cell lines. However, another neurotoxic organotin compound (trimethyltin, TMT) induces apoptosis in neural cells through the activation of a Ca 2+-dependent pathway. In conclusion, the identification of the key changes in actin and Ca 2+ homoeostasis could give early information on neural cell perturbation resulting in altered functionality or even cell death.

Pulsed Laser Imaging Techniques: Calcium and Exocytosis in Excitable Cells.

Abstract A rapid Ca++ signal is known to be the main trigger for exocytosis in excitable cells. However, its mode of action is unknown. Recently, it has become clear that the spatial distribution of a Ca++ stimulus is important for exocytosis. To investigate this question we have developed a novel instrument capable of imaging Ca++ gradients in patch clamped cells. We have equipped a standard fluorescence microscope with a CCD camera and an image processing station. This combination can generate a thin section view of the fluorescence of a single cell. We have equipped this microscope with a pulsed laser illumination system. The distribution of intracellular calcium can be obtained by exciting the Ca++ indicator dye (e.g., rhod-2) with a brief laser pulse [300 ns long at 525 nm ], then an image can be formed with the light emitted by the dye. by synchronizing the laser pulse with a depolarizing stimulus in a patch-clamped chromaffin cell loaded with the fluorescent Ca++ indicator rhod-2, we could easily obtain snapshots of the Ca++ distribution at known times after a stimulus.

Distribution of sarcomere length and intracellular calcium in mouse skeletal muscle following stretch-induced injury.

1. The effect on sarcomere organization of stretching intact single skeletal muscle fibres by 50% of their optimum length (Lo) during ten consecutive short tetani was investigated. Stretch reduced tetanic force to 36 +/- 4% of the pre-stretch condition. Sarcomere organization was analysed using both electron and confocal microscopy. For confocal microscopy the striation pattern was examined by fluorescently staining F-actin with rhodamine-phalloidin. 2. Electron microscopy revealed that fibres which had been stretched during contraction contained areas of severe sarcomere disorganization, as well as adjacent sarcomeres of normal appearance. 3. Confocal images of stretched fibres, which had been fixed and stained with rhodamine-phalloidin, showed focal regions of overstretched sarcomeres and regions where sarcomeres of adjacent myofibrils were out of alignment with each other. Analysis of all sarcomeres along the length of fibres showed regions of sarcomere inhomogeneity were distributed throughout the fibre length and cross-section. 4. Fibres were microinjected with the fluorescent [Ca2+]i indicator fura-2 before being stretched. Conventional wide-field fluorescence imaging microscopy showed that the tetanic [Ca2+]i was reduced after stretching but remained uniformly distributed. 5. This study confirms the finding that stretch-induced muscle injury has components caused by disorganization of the myofibrillar array and by failure of tetanic Ca2+ release. The structural damage is spatially heterogeneous whereas the changes in Ca2+ release appear to be spatially homogeneous.

Nuclear and cytosolic calcium levels in NIH 3T3 fibroblasts

The spatial distribution of intracellular calcium in resting NIH 3T3 fibroblasts loaded with Fura-2 has been studied by digital image analysis. Calibration parameters were determined separately for the nucleus and the cytosol to take into account possible differences in the physico-chemical properties of the two compartments and were found not to differ significantly. The apparent resting calcium concentration in these cells was found to be significantly lower in the nucleus than in the cytoplasm; however, this difference appears to be an artefact arising from the presence in the cytoplasm of regions with higher calcium levels. Application of thapsigargin, to block active uptake of calcium into these compartments, substantially eliminated the differences between nuclear and cytosolic calcium concentrations. These observations indicate that nuclear and cytosolic calcium are in equilibrium in the resting fibroblasts and argue against the existence of diffusional barriers between these two compartments.

From signal to form: aspects of the cytoskeleton-plasma membrane—cell wall continuuum in root hair tips

Root hairs are excellent cells for the study of the exocytotic process that leads to growth in higher plants, because the exocytotic event takes place locally and because the cells are directly accessible for signals, drugs, fixatives, microinjection, and microscopic observation. Well—characterized lipochito—oligosaccharides, signal molecules excreted by Rhizobium bacteria, induce root hair growth which can be recorded microscopically in a root hair deformation assay developed for Vicia sativa L. Root hair deformation is a morphogenetic process involving swelling of the hair tip and subsequent new hair outgrowth from that swelling. This response to the signal occurs at a specific developmental stage, namely when hairs are terminating growth. Thus, since polar growth can be triggered intentionally, the system allows the study of growth phenomena in higher plants at the cellular level. Furthermore, important advances are being made with molecular genetics that will allow the unravelling of the signal transduction pathways in root hair morphogenesis leading to growth. This paper first discusses cytological phenomena involved in the process of polar growth, such as cytoplasmic polarity, cyto—plasmic streaming and the organization of actin filaments, the location of a spectrin—like antigen, the distribution of intracellular calcium, cortical microtubules and cell wall texture, endocytosis by means of coated pits, and physical aspects of the incorporation of exocytotic vesicles into the plasma membrane. In the second part, changes are discussed that occur in some of these phenomena when growth is influenced by growth regulators and mutations.

Effects of aluminum on the growth and distribution of calcium in roots of an aluminum-sensitive cultivar of barley (Hordeum vulgare)

Aluminum-induced inhibition of root growth in the Al-sensitive cultivar Kearney of barley (Hordeum vulgare L.) is the result of disruption of both cell division in the meristematic region and cell expansion in the zone of elongation of the roots. In seedlings directly germinated in 50 μM Al, inhibition of root growth is detected 48 h after initiation of germination and it results primarily from the disruption of cell elongation. In seedlings germinated for 2 days under Al-free conditions, inhibition of root growth is apparent 8 h after transfer to 50 μM Al. In this instance, root growth inhibition is mainly the result of disruption of cell division in the meristematic region of the root. The calcium indicator dyes chlorotetracycline and Fluo-3 are used to study the distribution of intracellular calcium and its relationship to aluminum phototoxicity. Aluminum increases both chlorotetracycline and Fluo-3 fluorescence intensities. Fluorescence of the cytosolic calcium indicator dye Fluo-3 increases primarily in the zone of elongation of the roots of seedlings directly germinated in 50 μM aluminum. The increase in Fluo-3 fluorescence occurs concomitantly with major changes in both the length and width of the cells in the zone of elongation. The evidence suggests that changes in calcium homeostasis occurring in cells of the zone of elongation may be a major factor in the disruption of cell expansion and consequently root growth in seedlings directly germinated in 50 μM aluminum. Key words: aluminum, calcium, barley, chlorotetracycline, Fluo-3.

Distribution of intracellular calcium in isolated mouse skeletal muscle fibres

Distribution of intracellular calcium in isolated mouse skeletal muscle fibres

Intracellular calcium distribution regulates protein synthesis at translational initiation or elongation in the exocrine pancreas: [formula omitted] and Stephen Pandol. Department of Medicine, University of California and VA Medical Center, San Diego, CA

Intracellular calcium distribution regulates protein synthesis at translational initiation or elongation in the exocrine pancreas: [formula omitted] and Stephen Pandol. Department of Medicine, University of California and VA Medical Center, San Diego, CA

Investigative Urology: An Analysis of the Maxi-K sup + (k sub Ca) Channel in Cultured Human Corporal Smooth Muscle Cells

Previous studies have demonstrated that cultured corporal smooth muscle cells have prominent outward K currents composed of several different K channel subtypes. The goals of the present investigation were (1) to assert the nature of these channels and to evaluate the characteristics of the most predominant of these channel subtypes, the Maxi-K+ (KCa) channel, and (2) to compare KCa channel behavior in cultured corporal smooth muscle cells derived from the human corpus cavernosum of two distinct patient populations. The patient population was subdivided into two broad diagnostic categories: Group 1: 4 patients without evidence of organic disease of the corpus cavernosum, 3 of whom had documented erections; and Group 2: 4 patients with organic erectile dysfunction. Consistent with previous observations, 3 different K channel subtypes were detected in both patient populations, with corresponding conductances of 180, 100 and 40 pS, respectively. The approximately 183 pS channel was identified as the KCa channel based on its selective permeability to K+ and the fact that its open probability was modulated by both membrane potential and intracellular calcium levels. Specifically, the relative permeability of the 183 pS KCa channel to K+, Rb+, and NH4+ was 1.00:0.64:0.46. The channel was virtually impermeable to Na+ and Li+ (relative permeability < 0.02). In addition, the KCa channel was responsible for more than 90% of the outward K+ current passed through the cell membrane when depolarized. Furthermore, pharmacological studies using the K channel blocker tetraethylammonium ion (TEA) revealed that the sensitivity of KCa channels to TEA inhibition (as judged by the [TEA] required to block one-half of the outward whole cell current induced by a 90 mV depolarizing pulse) in cells from Group 1 patients was 1.05 +/- 0.22 mM. (n = 10 cells), while in sharp contrast the observed value for cells from Group 2 patients was 12.7 +/- 3.8 (n = 9 cells). The difference between the two groups was highly significant. These observations confirm and extend our previous studies to suggest that the KCa channel plays an important role in corporal smooth muscle physiology and, moreover, that alterations in the function/regulation of KCa channels may be an important feature of organic erectile dysfunction. As such, altered KCa channel behavior may contribute to an impaired hyperpolarizing ability of corporal smooth muscle, possibly altering intracellular calcium homeostasis and, perhaps, corporal smooth muscle reactivity and tone.

Intracellular calcium distribution in bone cells by eels and ESI: Optimization of background-fitting by symplex algorithm and by white line subtraction

Intracellular calcium distribution in bone cells by eels and ESI: Optimization of background-fitting by symplex algorithm and by white line subtraction

Effect of pretreatment with diltiazem on left ventricular function and intracellular calcium distribution in postischemic reperfused guinea-pig hearts

It has been previously demonstrated that pretreatment with diltiazem preserves mitochondrial function during postischemic reperfusion. The purpose of this study was to perform cytochemical and hemodynamical assessment to confirm this demonstration. Isolated Langendorff-perfused guinea-pig hearts received 10 min of diltiazem (7.5 microM) treatment, were subjected to 10 min of global ischemia and to 20 min of reperfusion. Left ventricular function was monitored by connecting a balloon to a pressure transducer. Intracellular calcium was precipitated with potassium pyroantimonate and examined with a transmission electron microscope. Compared with the control and the ischemic hearts, the diltiazem-pretreated hearts showed a significant increase in the left ventricular developed pressure (LVDP), dP/dtmax (P < 0.01), and recovery rates of the LVDP (P < 0.01 versus ischemic hearts) and dP/dtmax (P < 0.05), and a decrease in the heart rate (P < 0.01). The left ventricular end-diastolic pressure (LVEDP) and leakage of creatine kinase were not significantly different. Calcium deposits were seen along the inner aspects of the sarcolemma and t-tubule membranes, and in the mitochondria of the control hearts. The number of these deposits was considerably reduced after ischemia. They reappeared principally in the mitochondria by reperfusion. In contrast, the calcium deposits reappeared along the sarcolemma, t-tubule membranes, and cell junctions, and in the mitochondria in the diltiazem-pretreated hearts. These results suggest that pretreatment with diltiazem may improve cardiac function during postischemic reperfusion, probably in part by maintenance of sarcolemmal integrity rather than by mitochondrial buffering function.

Different calcium storage pools in vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar???Kyoto rats

To evaluate whether the distribution of intracellular free calcium may be impaired in primary hypertension. Cytosolic free calcium and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR). The concentrations of intracellular and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats aged 6 months from the Münster strain (SHR) and from age-matched normotensive Wistar-Kyoto (WKY) rats. Vascular smooth muscle cells were grown on coverslips, and fluorescence measurements of the intracellular calcium concentration were performed using fura-2. The different effects of thapsigargin, a selective Ca-ATPase inhibitor, and of angiotensin II (Ang II) on the calcium storage pools were investigated. In the absence of external calcium thapsigargin produced a dose-dependent transient increase in the concentration of intracellular calcium in vascular smooth muscle cells. The thapsigargin-induced maximum peak increase in the concentration of intracellular calcium was not significantly different in SHR and WKY rats. After depletion of the thapsigargin-sensitive calcium pools the addition of 100 nmol/l Ang II produced a rise in the concentration of intracellular calcium in vascular smooth muscle cells from SHR and WKY rats. Using vascular smooth muscle cells from the SHR the Ang II-induced increase in the concentration of intracellular calcium was not significantly different in the presence and absence of thapsigargin, indicating that the calcium pools depleted by thapsigargin and Ang II do not overlap significantly in vascular smooth muscle cells from SHR. In contrast, in the WKY rats the response to Ang II was significantly diminished after depletion of the thapsigargin-sensitive pool. When Ang II and thapsigargin were administered in the reverse order, i.e. Ang II before thapsigargin, the thapsigargin response was diminished in the WKY rats but not in the SHR. SHR differ from WKY rats in having vascular smooth muscle cells that contain thapsigargin-sensitive calcium storage pools that are distinct from the Ang II-sensitive calcium pools.

Standing calcium gradients in olfactory receptor neurons can be abolished by amiloride or ruthenium red.

Digital imaging and the patch clamp technique were used to investigate the intracellular calcium concentration in olfactory receptor neurons using the Ca2+ indicator dyes fura-2 and fura-2/AM. The spatial distribution of Cai2+ as well as its modification by the drugs Amiloride and Ruthenium Red were studied. Resting calcium concentrations in cells loaded with fura-2/AM were between 10 and 200 nM. In cells that were loaded with the pentapotassium salt of fura-2 through the patch pipette, calcium concentrations were in the same range if ATP was added to the pipette solution. Otherwise, Ca2+ reached concentrations of approximately 500 nM. Most of the observed cells showed a standing gradient of calcium, the calcium concentrations in the distal dendritic end of the cell being higher than in the soma. In some cells, the gradient was markedly reduced or abolished by adding either Amiloride or Ruthenium Red to the bath solution. In a few cells, neither drug had any effect upon the gradient. It is suggested that the inhomogenous spatial distribution of intracellular calcium in olfactory cells of Xenopus laevis is brought about by an influx of calcium ions through two different calcium permeable conductances in the peripheral compartments of the cells. The fact that only either Ruthenium Red or Amiloride abolished the standing calcium gradient further suggested that the two conductances blocked were presumably not coexpressed in the same cells.

Ultrastructural calcium distribution and myocardial calcium content in human idiopathic dilated cardiomyopathy

Myocardial calcium overload in chronic heart failure is still a debatable issue. The aim of this study was to investigate the myocardial calcium content and intracellular calcium distribution in end-stage dilated cardiomyopathy. The explanted hearts of 13 patients (9 male, 4 female, mean age 49 ± 12 years) undergoing heart transplantation because of end-stage dilated cardiomyopathy were examined. Samples were obtained from the right and left ventricular free wall and from the septum. Calcium and magnesium content were measured by atomic absorption spectrophotometry. Ultrastructural calcium distribution was examined in dilated cardiomyopathy using the phosphate-pyroantimonate method. Ultrastructural calcium distribution was also examined in left ventricular biopsies obtained from 3 patients (male, mean age 47 ± 3.6 years) with nonfailing hearts. The number of mitochondrial calcium precipitates was estimated morphometrically by a point counting method. Myocardial calcium and magnesium content in dilated cardiomyopathy did not differ significantly among the right and left ventricles and septum ranging from 8.5 to 10.8 mmol/kg dry weight. The phosphate-pyroantimonate method visualized calcium precipitates being confined to the sarcolemma, T-tubules, intercalated disks, and mitochondria in both nonfailing myocardium and dilated cardiomyopathy. Because mitochondria may act as buffers of cytoplasmic calcium, mitochondrial calcium precipitates served as a criterion for a possible cellular calcium overload. No differences in the amount of mitochondrial calcium deposits were observed between dilated cardiomyopathy and nonfailing hearts. The data suggest that there is no global myocardial calcium overload in human eng-stage dilated cardiomyopathy.

Regulation of protein synthesis by modulation of intracellular calcium in rat liver.

The rate of protein synthesis can be modulated in intact cells by varying the concentration and subcellular distribution of intracellular calcium. Because the biochemical reactions required for the pathway of protein synthesis occur in the cytosol of the cell, it might be expected that protein synthesis would be controlled by free cytosolic calcium rather than the sequestered cation. However, a recent report proposed that maintenance of optimal rates of protein synthesis depends on the amount of calcium sequestered in the endoplasmic reticulum rather than free cytosolic calcium (C.O. Brostrom and M. A. Brostrom, Annu. Rev. Physiol. 52: 577-590, 1990). In the present study, rat livers were perfused with buffer containing various compounds previously shown to alter intracellular calcium concentration and distribution in isolated cells. It was found that conditions designed to cause a rise in free cytosolic calcium had no effect on protein synthesis. In contrast, conditions designed to cause depletion of sequestered calcium resulted in an inhibition of protein synthesis characterized by a reduction in peptide-chain initiation relative to elongation. The inhibition of protein synthesis was further localized to a decrease in the activity of eukaryotic initiation factor (eIF) 2B as measured in extracts from perfused livers. The inhibition of eIF-2B activity was associated with a 2.4-fold increase in the proportion of the alpha-subunit of eIF-2 in the phosphorylated form. In summary, the results of the present study support a model whereby mobilization of calcium sequestered in the endoplasmic reticulum results in an inhibition of protein synthesis in rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Fast digital imaging of intracellular free calcium distribution in cultured single ventricular myocyte

Fast digital imaging of intracellular free calcium distribution in cultured single ventricular myocyte