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J Y Cheung

Publications and source records attributed to J Y Cheung.

At least 73 records · Page 4Linked to original sources
PubMed1991

Nuclear calcium gradients in cultured rat hepatocytes.

Ca2+ concentrations ([Ca2+]) in cytosol and nucleus in fura-2-loaded cultured rat hepatocytes were determined by three-dimensional (3-D) optical-sectioning microscopy. After determining the empirical 3-D point spread function of the fluorescence microscope-coupled digital video imaging system, contaminating light arising from optical planes above and below the plane of interest was removed by deconvolution using the nearest-neighboring approach (NNA) algorithm. Although deconvolution resulted in substantial improvement in accuracy of fluorescence intensity determinations in single-wavelength excitation images as well as sharper delineation of boundaries between cellular compartments, the complicated mathematical process did not significantly enhance the precision of [Ca2+] values derived from ratiometric (ratio of dual-wavelength excitation) images. In resting hepatocytes, cytosolic Ca2+ (210 +/- 15 nM) was 1.6- to 2-fold higher than nuclear Ca2+ (128 +/- 12 nM). This difference in Ca2+ between the two compartments was detected both in raw ratiometric images and in those processed with NNA algorithm. Addition of arginine vasopressin or epidermal growth factor resulted in significant increases (2- to 3-fold) in both cytosolic and nuclear Ca2+; however, the nuclear-to-cytosolic Ca2+ gradient was preserved in hepatocytes stimulated with mitogens. We conclude that the hepatocyte nuclear membrane contains Ca2+ permeability barriers and Ca2+ transport mechanisms that may be hormonally sensitive. We postulate that the increase in nuclear Ca2+ may be important in regulation of cell proliferation induced by mitogens, possibly by activating Ca(2+)-dependent endonucleases, nuclear calmodulin, or nuclear protein kinase C.

Algorithms↗
PubMed1991

Modulation of cardiac contractility by myosin light chain phosphorylation.

Cytosolic free [Ca2+] ([Ca2+]c) mediates primary regulation of cardiac contractility. Both the magnitude and time-course of [Ca2+]c transient that is elicited by a sarcolemmal action potential play central roles in defining the characteristics of the mechanical response that occurs during a single excitation-contraction coupling cycle. Numerous modulators of cardiac contractility, both hormonal and autoregulatory, act to influence contractile function via direct effects on various cellular processes that govern [Ca2+]c dynamics. Cardiac contractility can also be influenced by mechanisms that alter the responsiveness of the contractile element to activation by Ca2+ (see preceding paper). There is growing interest in the possibility that the phosphorylation of the P-light chain subunit of cardiac myosin by a Ca(2+)-calmodulin-dependent myosin light chain kinase may modulate cardiac muscle contractility by increasing the sensitivity of the contractile element to activation by Ca2+. The types of experimental data that have led to the development of this hypothesis and the unique aspects of cardiac P-light chain phosphate content regulation will be briefly addressed in this paper. Furthermore, several unresolved issues regarding the functional significance of cardiac P-light chain phosphorylation in intact myocardium are identified.

Animals↗
PubMed1990

Three-dimensional intracellular calcium gradients in single human burst-forming units-erythroid-derived erythroblasts induced by erythropoietin.

We have previously shown that the intracellular free Ca2+ increase induced by erythropoietin is likely related to differentiation rather than proliferation in human BFU-E-derived erythroblasts (1989. Blood. 73:1188-1194). Since cell differentiation involves transcription of specific regions of the genome, and since nuclear endonucleases responsible for single strand DNA breaks observed in cells undergoing differentiation are Ca2+ dependent, we investigated whether the erythropoietin-induced calcium signal is transmitted from cytosol to nucleus in this study. To elucidate subcellular Ca2+ gradients, the technique of optical sectioning microscopy was used. After determining the empirical three-dimensional point spread function of the video imaging system, contaminating light signals from optical planes above and below the focal plane of interest were removed by deconvolution using the nearest neighboring approach. Processed images did not reveal any discernible subcellular Ca2+ gradients in unstimulated erythroblasts. By contrast, with erythropoietin stimulation, there was a two- to threefold higher Ca2+ concentration in the nucleus compared to the surrounding cytoplasm. We suggest that the rise in nuclear Ca2+ may activate Ca2(+)-dependent endonucleases and initiate differentiation. The approach described here offers the opportunity to follow subcellular Ca2+ changes in response to a wide range of stimuli, allowing new insights into the role of regional Ca2+ changes in regulation of cell function.

Calcium↗
PubMed1989

Regulation of citric acid cycle by calcium.

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.

Animals↗
PubMed1989

The simultaneous occurrence of renal artery stenosis and an aldosteronoma in a patient with hypertension.

The simultaneous occurrence of renovascular hypertension and an adrenocortical adenoma is a rare entity. The case of a 64-year-old woman who underwent an aortorenal bypass graft for renovascular hypertension requiring a multidrug antihypertensive regimen is presented. Persistently elevated blood pressures in the postoperative period prompted further workup for other causes of hypertension. Laboratory evaluation showed hyperaldosteronism and hyporeninemia despite enalapril administration. Abdominal computerized tomography (CT) revealed a left adrenal mass which, on surgical removal, was found to be a cortical adenoma. Subsequently, her antihypertensive therapy has been reduced to a single agent. Previous authors have described only four patients with malignant hypertension who had the rare clinical combination of renal artery stenosis and an aldosteronoma. This case reemphasizes the critical need for a thorough search for other surgically correctable lesions in those patients who remain severely hypertensive after the "definitive" operation.

Adenoma↗
PubMed1989

Cytosolic free calcium concentration in individual cardiac myocytes in primary culture.

Cytosolic free Ca2+ concentration, [Ca2+]i, of single isolated Ca2+-tolerant rat ventricular myocytes in primary culture was determined by digital video imaging of intracellular fura-2 fluorescence. In deenergized myocytes in which contractile elements were uncoupled by 2,3-butanedione monoxime, the maximum and minimum fluorescence intensity ratio values of fura-2 in the cell were similar when compared with those of fura-2 solutions observed in the microscope. Through the use of in vitro calibration, [Ca2+]i in quiescent, rod-shaped myocytes was 90 +/- 6 nM. There was no detectable spatial heterogeneity in [Ca2+]i in resting myocytes. Localized regions of [Ca2+]i elevation were observed in cells undergoing spontaneous rhythmic contractions or when subjected to mild depolarization by KCl. Additions of gramicidin or veratridine resulted in massive increases in [Ca2+]i (greater than 1 microM) and immediate cell hypercontracture. Ruthenium red elicited a modest increase in [Ca2+]i but extracellular ATP or epinephrine had no effect. We conclude the following: 1) digital video imaging of resting cardiac cells did not reveal any subcellular Ca2+ gradients; 2) the fluorescence properties of intracellular fura-2 were similar to that in free solution; and 3) subcellular heterogeneity of [Ca2+]i in isolated myocytes was observed in cells undergoing spontaneous rhythmic contraction.

Adenosine Triphosphate↗
PubMed1989

Erythropoietin stimulates a rise in intracellular-free calcium concentration in single BFU-E derived erythroblasts at specific stages of differentiation.

Human cord blood progenitor-derived erythroblasts have recently been shown to respond to erythropoietin (Epo) or granulocyte-macrophage colony-stimulating factor (GM-CSF) with a transient increase in intracellular free calcium concentration [Cac]. However, the importance of [Cac] changes in mediating cell proliferation and/or differentiation is undefined. In the present study, the response of erythroid precursors at different stages of differentiation to Epo was examined. Erythroblasts were derived from adult blood erythroid progenitors (BFU-E) at day 7 or day 10 of culture. [Cac] was measured in individual Fura-2 loaded cells with fluorescence microscopy coupled digital video imaging. The dynamic range (Rmax/Rmin) of intracellular Fura-2 was similar to that measured in free solution, suggesting insignificant amounts of intracellular Ca insensitive forms of Fura-2. Baseline [Cac] of erythroid cells calculated with an in vitro calibration method was 44 +/- 4 nmol/L and with an in vivo method was 46 +/- 4 nmol/L. Treatment of day 7 BFU-E derived erythroblasts with Epo resulted in no significant increase in [Cac]. In contrast, in more mature erythroblasts (day 10 of culture), Epo stimulated a large increase in [Cac] from 49 +/- 11 nmol/L at baseline to 279 +/- 47 nmol/L. This [Cac] increase occurred in phosphate buffered saline (PBS) containing no added calcium. The increase in [Cac] persisted for 18 minutes and was dose dependent. Day 7 and day 10 control cells treated with either insulin or media showed no significant change in [Cac] during 18 minutes of observation. Our data demonstrate that early (day 7) and late (day 10) erythroblasts display different responses to Epo, at least in terms of intracellular Ca++ fluxes. The differential [Cac] response observed in early and late erythroid precursors to growth factor stimulation suggests that [Cac] may be an important signal in cell differentiation.

Adult↗
PubMed1988

Erythropoietin stimulates a rise in intracellular free calcium concentration in single early human erythroid precursors.

Erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the differentiation and proliferation of erythroid cells. To determine the cellular mechanism of action of these growth factors, we measured changes in intracellular free calcium concentration [( Cac]) in single human erythroid precursors in response to recombinant erythropoietin and GM-CSF. [Cac] in immature erythroblasts derived from cultured human cord blood erythroid progenitors was measured with fluorescence microscopy digital video imaging. When stimulated with erythropoietin, [Cac] in the majority of erythroblasts increased within 3 min, peaked at 5 min, and returned toward baseline at 10 min. The percentage of cells that responded to erythropoietin stimulation increased in a dose-dependent manner. Additional stimulation with GM-CSF in cells previously exposed to erythropoietin resulted in a second [Cac] increase. Immature erythroblasts treated with GM-CSF followed by erythropoietin responded similarly to each factor with a rise in [Cac]. The source of transient calcium is intracellular since erythroblasts were incubated in medium devoid of extracellular calcium. Our observations suggest that changes in [Cac] may be an intracellular signal that mediates the proliferative/differentiating effect of hematopoietic growth factors.

Body Fluids↗
PubMed1987

Cytosolic free calcium concentration and glucose transport in isolated cardiac myocytes.

The role of cytosolic free Ca2+ concentration, [Cai], in mediating insulin's stimulatory effect on glucose transport was investigated in isolated Ca2+-tolerant rat ventricular cells. Approximately 98% of glucose transport in isolated myocytes was inhibited by phloretin. Insulin-accelerated glucose transport by 50-115% over basal transport rate. Removal of extracellular Ca2+ had no effect on either the basal transport rate or insulin's stimulatory action, indicating that extracellular Ca2+ was not necessary for insulin's effect to be manifest. Addition of A23187 had no effect on glucose transport rate. Under basal conditions, [Cai] was 167 +/- 12 nM as measured by fura-2 fluorescence and 239 +/- 22 nM by null-point titration with arsenazo III. Loading cells with fura-2 did not affect basal glucose transport rates. In addition, the stimulatory effect of insulin on glucose transport was preserved in fura-2 loaded cells. In paired experiments, insulin did not increase [Cai] as measured by fura-2 fluorescence or null-point titration despite acceleration of glucose transport. In contrast, addition of KCl (40 mM) increased [Cai] from 168 +/- 30 to 287 +/- 51 nM and resulted in 50% reduction in glucose transport rate. In other experiments designed to control for the hyperosmolar effects of KCl, NaCl (40 mM) caused no change in [Cai] but also inhibited glucose transport rate by 50%. We conclude that an elevation in [Cai] is unlikely to be the intracellular signal mediating insulin's effect on glucose transport since insulin's stimulatory effect was not reduced by Ca2+ -free media, insulin had no detectable effect on [Cai], and elevation of [Cai] by KCl did not result in stimulation of glucose transport.

Animals↗
PubMed1986

Mitochondrial function and intracellular calcium in anoxic cardiac myocytes.

Mitochondrial dysfunction has been implicated as the cause of irreversible injury in the ischemic heart. To circumvent artifacts associated with organelle isolation, mitochondrial function was studied in intact isolated, Ca2+-tolerant rat ventricular myocytes. After 30 min of anaerobic incubation, myocyte viability decreased from 76 +/- 1 to 33 +/- 4%. Basal O2 consumption rates (nanoatoms . mg cell protein-1 . min-1) were 17.1 +/- 1.3 in aerobic cells and 51.0 +/- 9.8 in anoxic cells. Carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP)-stimulated rates were 65.5 +/- 9.2 and 84.5 +/- 15.3 in aerobic and anoxic cells, respectively. Respiratory control ratio was lower in anoxic cells: 2.3 +/- 0.3 versus 4.2 +/- 0.4 in aerobic cells. These data suggest that early anoxic mitochondrial injury is due to increased permeability of the inner membrane. Addition of pyruvate, malate, and FCCP to cells made permeable by digitonin resulted in similar maximal O2 consumption rates: 276.5 +/- 31.8 in aerobic and 299.3 +/- 31.9 in anoxic cells, suggesting the electron transport chain is intact in anoxic cells. For purposes of investigating whether anoxic mitochondrial dysfunction is secondary to cellular or mitochondrial Ca2+ overload, total cell Ca2+, cytosolic free Ca2+ levels (measured by null-point titration), and mitochondrial Ca2+ contents (measured as FCCP-releasable Ca2+) were measured. There were no differences in these three parameters between aerobic and anoxic cells, suggesting that mitochondrial dysfunction and irreversible hypercontraction of isolated cardiac myocytes exposed to 30 min of anoxia are not related to Ca2+ overload.

Animals↗
PubMed1986

Cytosolic free calcium concentration in cultured renal epithelial cells.

Cytosolic free Ca2+ concentration ([Ca2+]i) was determined in isolated cultured renal LLC-PK1 cells by two independent techniques involving 1) measurement of the fluorescence of an intracellular Ca2+ probe (quin 2) or measurement of the change in extracellular Ca2+ with arsenazo III after making the cell membrane permeable with digitonin, a null-point titration determination. [Ca2+]i determined with the former technique was 98 +/- 5 nM (n = 81) and was 101 +/- 23 nM (n = 7) with the latter. The value of [Ca2+]i was independent of intracellular quin 2 concentration within the range of 0.7-3.0 mM. Increasing extracellular [Ca2+] from 0.5 to 2.0 mM had no effect on [Ca2+]i as determined with quin 2. Ouabain (10(-4) M) or replacement of 120 mM of the extracellular Na+ with choline or lithium resulted in increases in [Ca2+]i. When extracellular [Ca2+] was higher than [Ca2+]i and cell plasma membranes were made permeable with digitonin, Ca2+ was taken up by the intracellular organelles. Ca2+ taken up into the intracellular compartments could be released with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and Ca2+ uptake could be blocked by ruthenium red, suggesting that the mitochondria were the primary Ca2+-buffering sites under our experimental conditions in the absence of Mg2+ and ATP. The mitochondrial compartment was found to have a very large capacity for Ca2+ buffering. This study represents the first full report using two independent techniques (quin 2 and null-point titration) for determination of [Ca2+]i in epithelial cells and demonstrates excellent agreement between the techniques. The study also demonstrates the utility of these techniques in studying intracellular Ca2+ homeostasis in renal epithelial cells.

Aminoquinolines↗
PubMed1986

Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells.

Cytosolic free calcium concentration ([Ca2+]f) was determined in cultured rat glomerular mesangial cells under basal conditions and after exposure to arginine vasopressin (AVP) or angiotensin II (ANG II). [Ca2+]f was determined using quin 2 or fura-2, two intracellular fluorescent probes. [Ca2+]f was measured to be 102 +/- 3 nM (n = 154) using quin 2 and 82 +/- 4 (n = 34) using fura-2. AVP and ANG II increased [Ca2+]f. Maximal levels of [Ca2+]f were achieved in less than 10 s after addition of the hormone. This peak value was followed by a rapid fall toward the base line. With fura-2 as the intracellular Ca2+ indicator, [Ca2+]f increased from 74 +/- 7 to a peak of 578 +/- 39 nM (n = 17) with 100 nM AVP. At 115 s after addition of AVP, [Ca2+]f was 125 +/- 9 nM. Similar peak levels of [Ca2+]f were observed using quin 2. The increase in [Ca2+]f was due in large part to release of Ca2+ from intracellular stores, since reduction in extracellular free [Ca2+] with EGTA did not prevent the hormone-induced increase in [Ca2+]f, although it did result in a decreased peak level and a more rapid return to base line. The AVP-induced increase in [Ca2+]f was blocked by the V1 receptor antagonist (CH2)5Tyr(Me)VDAVP. Neither isoproterenol, which increased adenylate cyclase activity, nor dibutyryl cAMP had any affect on [Ca2+]f directly or on the AVP-induced increase in [Ca2+]f. In this report we present the first direct measurements of [Ca2+]f and hormone-induced changes in [Ca2+]f in glomerular mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1986

Regulation of cytosolic free calcium concentration in cultured renal epithelial cells.

The relative contribution by intracellular organelles to the regulation of cytosolic free Ca2+ concentration was investigated in isolated cultured renal LLC-PK1 cells. Exchangeable mitochondrial and nonmitochondrial Ca2+ represented 10 and 50% of total cell Ca2+ content, respectively. In the absence of Mg2+, Ca2+ added to cells made permeable with digitonin was rapidly sequestered by the mitochondria so that basal cytosolic free Ca2+ concentration (approximately 100 nM) was reestablished within minutes. Provision of Mg2+ and ATP resulted in biphasic Ca2+ buffering behavior. A slow, low-capacity system buffered exogenous Ca2+ to a steady-state level of 50-300 nM. Ca2+ buffering at these low Ca2+ concentrations was likely by the endoplasmic reticulum, since vanadate but not ruthenium red blocked the Ca2+ buffering ability. After the endoplasmic reticulum was saturated with Ca2+, exogenous Ca2+ was buffered by a fast, high-capacity organelle to a new steady-state level of 600-1,500 nM. The high-capacity system was identified as the mitochondria, since ruthenium red but not vanadate abolished Ca2+ buffering and resulted in release of previously sequestered Ca2+. Addition of inositol-1,4,5-trisphosphate (IP3) resulted in rapid release from and reuptake of Ca2+ by the endoplasmic reticulum in digitonin-treated cells. We conclude that, under physiological conditions, most of the cell Ca2+ is stored in endoplasmic reticulum which is the functionally important organelle in buffering small changes in Ca2+ at the resting cytosolic free Ca2+ level. The mitochondrial compartment represents a high-capacity Ca2+ buffering system that may serve important physiological functions when large loads of Ca2+ are presented to the cell. In addition, IP3 mobilizes Ca2+ stored in the endoplasmic reticulum and may serve as the intracellular messenger to raise cytosolic free Ca2+ concentration transiently in response to stimuli.

Calcium↗
PubMed1985

Determination of isolated myocyte viability: staining methods and functional criteria.

Single ventricular cells tolerant to physiological levels of calcium have been successfully isolated from adult rat heart. These cells exhibit morphological as well as functional characteristics of the intact myocardium. Under anaerobic incubations either in the absence of extracellular calcium or in the presence of 6% PEG, myocytes excluded Trypan blue despite severe derangements in biochemical functions. We suggest that Trypan blue entry may be a rather insensitive criterion of cell injury. We recommend the combination of rod-shape morphology, ATP levels and the ability to contract under external pacing as alternative simple tests of cell viability.

Adenosine Triphosphate↗
PubMed1985

Effect of electro-acupuncture on chronic painful conditions in general medical practice--a 4 years' study.

A total of 204 consecutive, recalcitrant painful conditions were treated by electroacupuncture and closely observed up to 4 years in a group medical practice in Australia. The majority (151 cases or 74%) had enjoyed significant pain reduction well beyond 3 months. The causes of failure in the remainders were analysed and discussed. The encouraging overall results led to a renewed call for this mode of therapy to be used more widely as an adjunct in family medicine.

Acupuncture Therapy↗