scholarly journals Calcium Regulates the Association between Mitochondria and a Smooth Subdomain of the Endoplasmic Reticulum

2000 ◽  
Vol 150 (6) ◽  
pp. 1489-1498 ◽  
Author(s):  
Hui-Jun Wang ◽  
Ginette Guay ◽  
Liviu Pogan ◽  
Remy Sauvé ◽  
Ivan R. Nabi
Keyword(s):  
Confocal Microscopy ◽  
Mdck Cells ◽  
Close Association ◽  
Cytosolic Calcium ◽  
Free Calcium ◽  
Autocrine Motility Factor ◽  
Cytosolic Free Calcium ◽  
Motility Factor ◽  
Rat Liver Cytosol ◽  
Close Contacts

Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca2+ concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 μM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.

The AMF-R tubule is a smooth ilimaquinone-sensitive subdomain of the endoplasmic reticulum

1997 ◽  
Vol 110 (24) ◽  
pp. 3043-3053 ◽  
Author(s):  
H.J. Wang ◽  
N. Benlimame ◽  
I. Nabi
Keyword(s):  
Electron Microscopy ◽  
Confocal Microscopy ◽  
Immunofluorescence Microscopy ◽  
Mdck Cells ◽  
Autocrine Motility Factor ◽  
Motility Factor ◽  
Membranous Tubule ◽  
Intermediate Compartment ◽  
Rough Er ◽  
Golgi Network

Autocrine motility factor receptor (AMF-R) is a marker for a distinct smooth membranous tubule. Ilimaquinone (IQ) is a sea sponge metabolite which induces the complete vesiculation of the Golgi apparatus and we show here that the addition of IQ to MDCK cells also results in the disruption of the AMF-R tubule. By immunofluorescence microscopy, the resultant punctate AMF-R label resembles the products of IQ-mediated vesiculation of the trans-Golgi network, however, the two labels can be distinguished by confocal microscopy. AMF-R tubule fragmentation occurs after nocodazole or taxol treatment of the cells demonstrating that the action of IQ on AMF-R tubules is not related to the ability of IQ to depolymerize microtubules. IQ activity is therefore not Golgi-specific. Electron microscopy of IQ-treated cells reveals that AMF-R is distributed to fenestrated networks of narrow interconnected tubules which are distinguishable from the uniform Golgi-derived vesicles and morphologically equivalent to smooth ER. Distinct fenestrations are visible in incompletely fragmented tubules which may represent intermediates in the fragmentation process. Smooth AMF-R labeled tubules exhibit continuity with rough ER cisternae and IQ selectively targets smooth and not rough ER. AMF-R tubules can be distinguished from the intermediate compartment labeled for ERGIC-53 by confocal microscopy and thus constitute a distinct IQ-sensitive subdomain of the smooth ER.

Interaction of the smooth endoplasmic reticulum and mitochondria

2006 ◽  
Vol 34 (3) ◽  
pp. 370-373 ◽  
Author(s):  
J.G. Goetz ◽  
I.R. Nabi
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Close Association ◽  
Cytosolic Calcium ◽  
Functional Roles ◽  
Calcium Dependent ◽  
Motility Factor ◽  
Calcium Sequestration ◽  
Multiple Domains ◽  
The Relationship

The ER (endoplasmic reticulum) is composed of multiple domains including the nuclear envelope, ribosome-studded rough ER and the SER (smooth ER). The SER can also be functionally segregated into domains that regulate ER–Golgi traffic (transitional ER), ERAD (ER-associated degradation), sterol and lipid biosynthesis and calcium sequestration. The last two, as well as apoptosis, are critically regulated by the close association of the SER with mitochondria. Studies with AMFR (autocrine motility factor receptor) have defined an SER domain whose integrity and mitochondrial association can be modulated by ilimaquinone as well as by free cytosolic calcium levels in the normal physiological range. AMFR is an E3 ubiquitin ligase that targets its ligand directly to the SER via a caveolae/raft-dependent pathway. In the present review, we will address the relationship between the calcium-dependent morphology and mitochondrial association of the SER and its various functional roles in the cell.

Angiotensin II causes sustained elevations in cytosolic calcium in glomerulosa cells

1988 ◽  
Vol 255 (3) ◽  
pp. E338-E346 ◽  
Author(s):  
R. E. Kramer
Keyword(s):  
Angiotensin Ii ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Calcium Signal ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Cytosolic Calcium Concentration ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Glomerulosa Cells

Studies were conducted to examine the effects of angiotensin II on cytosolic free calcium concentration in bovine adrenal glomerulosa cells maintained in primary culture. The calcium indicator, fura-2, and discontinuous dual-wavelength fluorescence spectroscopy were used to measure cytosolic free calcium in superfused adherent cell monolayers. Basal cytosolic free calcium concentration was 63.7 +/- 3.3 nM. The threshold concentration for angiotensin II-stimulated increases in cytosolic calcium was 10(-14)-10(-13) M, and maximal elevation of cytosolic calcium was produced by 10(-9) M angiotensin II. Angiotensin II (10(-13) M) produced a gradual increase in cytosolic calcium concentration that plateaued after 3-5 min of superfusion at a level approximately 1.2 times that of control cells. The calcium signal invoked by a maximal concentration (10(-9) M) of angiotensin II, in contrast, was characterized by an immediate, intense (approximately 8-fold) increase in cytosolic calcium concentration that decayed within 5 min to a lower, but sustained, level 2.5-3 times that of control cells. The calcium signals invoked by intermediate concentrations (10(-12)-10(-10) M) of angiotensin II exhibited dose-dependent increases in magnitude and a gradual transition in nature between those invoked by threshold and maximal concentrations of the peptide. The effect of angiotensin II to increase cytosolic calcium concentration was accompanied by an increase in aldosterone output. The increase in steroidogenesis was most closely correlated with the magnitude of the initial calcium signal. At high concentrations (10(-10) and 10(-9) M) of angiotensin II, there was a clear dissociation between aldosterone output and the magnitude of the sustained calcium signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin increases cytosolic free calcium in LLC-PK1 cells through a V1-receptor

1987 ◽  
Vol 253 (2) ◽  
pp. F328-F332 ◽  
Author(s):  
M. A. Burnatowska-Hledin ◽  
W. S. Spielman
Keyword(s):  
Parathyroid Hormone ◽  
Arginine Vasopressin ◽  
Calcium Concentration ◽  
Mdck Cells ◽  
Free Calcium ◽  
Base Line ◽  
Cytosolic Free Calcium ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Dose Dependent

We examined the effects of arginine vasopressin (AVP), parathyroid hormone (PTH), and bradykinin (BK) on the cytosolic free calcium concentration ([Ca]i) in cultured LLC-PK1 and MDCK kidney cell lines by use of the fluorescent Ca chelator fura-2. In LLC-PK1 cells, the addition of AVP but not [1-desamino-8-D-arginine]vasopressin (dDAVP, V2 agonist), PTH, or BK (10(-6) M) caused a significant increase in [Ca]i. The AVP-induced increase in [Ca]i from 61 +/- 6 to 225 +/- 44 nM (n = 7, P less than 0.01) was rapid and transient, returning to base line in 2 to 3 min. The effect of AVP was dose dependent and was present at 1 (61% increase) but not 5 min after extracellular Ca was removed. The effect of 10(-6) M AVP could be blocked with the pressor (V1) antagonist, d(CH2)5Tyr(Me)AVP, but not dDAVP. In MDCK cells, BK, but not AVP and PTH, increased [Ca]i from 146 +/- 11 to 281 +/- 31 nM (n = 9, P less than 0.001). The removal of extracellular Ca (5 min), reduced but did not abolish this effect. These results indicate that [Ca]i mobilized by activation of V1-receptors may mediate AVP-regulated function in some transporting epithelia.

scholarly journals Hypertension Secondary to PHPT: Cause or Coincidence?

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Helmut Schiffl ◽  
Susanne M. Lang
Keyword(s):  
Cardiovascular Reactivity ◽  
Vascular Reactivity ◽  
Cytosolic Calcium ◽  
Free Calcium ◽  
Normal Blood Pressure ◽  
Cytosolic Free Calcium ◽  
Hypertensive Patients ◽  
Before And After ◽  
Underlying Mechanisms ◽  
Normal Controls

Primary hyperparathyroidism (PHPT) may be associated with arterial hypertension. The underlying mechanisms are not fully understood and reversibility by parathyroid surgery is controversial. This study aimed to characterize pressor hormones, vascular reactivity to norepinephrine, and cytosolic-free calcium in platelets in 15 hypertensive patients with hypercalcaemic PHPT before and after successful parathyroidectomy and to compare them with 5 pre-hypertensive patients with normocalcaemic PHPT, 8 normotensive patients with hypercalcaemic PHPT and 15 normal controls. Hypertensive patients with hypercalcaemic PHPT had slightly higher levels of pressor hormones (), enhanced cardiovascular reactivity to norepinephrine () and increased cytosolic calcium in platelets () than controls. Pre-hypertensive patients with normocalcaemic PHPT had intermediate values of increased cardiovascular reactivity and cytosolic calcium. Normotensive patients with hypercalcaemic PHPT and normotensive controls had comparable pressor hormone concentrations and intracellular calcium levels. Successful parathyroidectomy was associated with normal blood pressure values and normalisation of pressor hormone concentrations, cardiovascular pressor reactivity and cytosolic free calcium. Our results suggest that parathyroid hypertension is mediated/maintained, at least in part, by functional alterations of vascular smooth muscle cells and can be cured by parathyroidectomy in those patients who do not have primary hypertension.

scholarly journals AMF-R Tubules Concentrate in a Pericentriolar Microtubule Domain After MSV Transformation of Epithelial MDCK Cells

1997 ◽  
Vol 45 (10) ◽  
pp. 1351-1363 ◽  
Author(s):  
Ivan R. Nabi ◽  
Ginette Guay ◽  
Danièle Simard
Keyword(s):  
Mdck Cells ◽  
Cell Periphery ◽  
Perinuclear Region ◽  
Microtubule Organizing Center ◽  
Autocrine Motility Factor ◽  
Microtubule Cytoskeleton ◽  
Motility Factor ◽  
Organizing Center ◽  
Sarcoma Virus ◽  
Moloney Sarcoma Virus

Autocrine motility factor receptor (AMF-R) is localized to an intracellular microtubule-associated membranous organelle, the AMF-R tubule. In well-spread untrans-formed MDCK epithelial cells, the microtubules originate from a broad perinuclear region and AMF-R tubules extend throughout the cytoplasm of the cells. In Moloney sarcoma virus (mos)-transformed MDCK (MSV-MDCK) cells, microtubules accumulate around the centrosome, forming a microtubule domain rich in stabilized detyrosinated microtubules. AMF-R tubules are quantitatively associated with this pericentriolar microtubule domain and the rough endoplasmic reticulum and lysosomes also co-distribute with the pericentriolar mass of microtubules. The Golgi apparatus is closely associated with the microtubule organizing center (MTOC) within the juxtanuclear mass of AMF-R tubules, and no co-localization of AMF-R tubules with the Golgi marker β-COP could be detected by confocal microscopy. After nocodazole treatment and washout, microtubule nucleation occurs exclusively at the centrosome of MSV-MDCK cells, and only after microtubule extension to the cell periphery does the microtubule cytoskeleton reorganize to generate the pericentriolar microtubule domain after 30–60 min. AMF-R tubules dispersed by nocodazole treatment concentrate in the pericentriolar region in parallel with the reorganization of the microtubule cytoskeleton. MSV transformation of epithelial MDCK cells results in the stabilization of a pericentriolar microtubule domain responsible for the concentration and polarized distribution of AMF-R tubules.

scholarly journals Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle.

1995 ◽  
Vol 129 (2) ◽  
pp. 459-471 ◽  
Author(s):  
N Benlimame ◽  
D Simard ◽  
I R Nabi
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Mdck Cells ◽  
Electron Microscopic ◽  
Autocrine Motility Factor ◽  
Lysosomal Fraction ◽  
Motility Factor ◽  
Variable Diameter ◽  
Triton X

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule-associated tubular organelles.

Effect of 17 β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

1990 ◽  
Vol 10 (1) ◽  
pp. 73-78 ◽  
Author(s):  
Tiziana Bellini ◽  
Diana Degani ◽  
Maurizio Matteuzzi ◽  
Franco Dallocchio
Keyword(s):  
Cell Surface ◽  
Steroid Hormones ◽  
Human Lymphocytes ◽  
Cytosolic Calcium ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Calcium Response ◽  
Pre Treatment ◽  
Preincubation Time ◽  
Estradiol Concentration

Pre-treatment of human lymphocytes with 17 β-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17 β-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17 β-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17 β-estradiol, since the α isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5α-androstan), have no effect. Since the effect of the 17 β-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.

scholarly journals Localization of Autocrine Motility Factor Receptor to Caveolae and Clathrin-independent Internalization of Its Ligand to Smooth Endoplasmic Reticulum

1998 ◽  
Vol 9 (7) ◽  
pp. 1773-1786 ◽  
Author(s):  
Naciba Benlimame ◽  
Phuong U. Le ◽  
Ivan R. Nabi
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Cell Surface ◽  
Smooth Endoplasmic Reticulum ◽  
Fluid Phase ◽  
Cell Surface Receptor ◽  
Tubular Structures ◽  
Autocrine Motility Factor ◽  
Motility Factor ◽  
3T3 Fibroblasts

Autocrine motility factor receptor (AMF-R) is a cell surface receptor that is also localized to a smooth subdomain of the endoplasmic reticulum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4°C exhibits partial colocalization with caveolin, confirming the localization of cell surface AMF-R to caveolae. Labeling of cell surface AMF-R by either anti-AMF-R antibody or biotinylated AMF (bAMF) exhibits extensive colocalization and after a pulse of 1–2 h at 37°C, bAMF accumulates in densely labeled perinuclear structures as well as fainter tubular structures that colocalize with AMF-R tubules. After a subsequent 2- to 4-h chase, bAMF is localized predominantly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-mediated endocytosis, results in the essentially exclusive distribution of internalized bAMF to AMF-R tubules. By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules. bAMF internalized in the presence of a 10-fold excess of unlabeled AMF labels perinuclear punctate structures, which are therefore the product of fluid phase endocytosis, but does not label AMF-R tubules, demonstrating that bAMF targeting to AMF-R tubules occurs via a receptor-mediated pathway. By electron microscopy, bAMF internalized for 10 min is located to cell surface caveolae and after 30 min is present within smooth and rough endoplasmic reticulum tubules. AMF-R is therefore internalized via a receptor-mediated clathrin-independent pathway to smooth ER. The steady state localization of AMF-R to caveolae implicates these cell surface invaginations in AMF-R endocytosis.

Cytosolic free calcium regulation in response to acute changes in intracellular pH in vascular smooth muscle

1993 ◽  
Vol 264 (4) ◽  
pp. C932-C943 ◽  
Author(s):  
D. C. Batlle ◽  
R. Peces ◽  
M. S. LaPointe ◽  
M. Ye ◽  
J. T. Daugirdas
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Intracellular Ph ◽  
Cytosolic Calcium ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Intracellular Store ◽  
Acute Changes ◽  
Free Media ◽  
Intracellular Alkalinization

This study examined the mechanisms whereby alterations of intracellular pH (pHi) impact on free cytosolic calcium (Cai2+) in cultured rat aortic vascular smooth muscle cells (VSMC) assayed in the presence of HCO3/CO2. Rapid cell alkalinization, effected by the exposure to NH4Cl or removal of CO2 from the superfusate, produced a rapid increase in Cai2+. The rise in Cai2+ was markedly diminished when sarcoplasmic reticulum (SR) Ca2+ stores had been depleted by prior exposure to arginine vasopressin (AVP) in Ca(2+)-free media or when SR release and reuptake of Ca2+ were blocked by the addition of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), but was unaffected by the removal of external Ca2+ or inhibition of Ca2+ entry using NiCl2. Cell acidification also resulted in a rapid increase in Cai2+. This Cai2+ increase was most apparent when pHi was very low (< 6.6) and was unaffected by removal of external Ca2+ or NiCl2 addition. Unlike the effect of cell alkalinization, the increase in Cai2+ associated with cell acidification was not prevented by pretreatment with AVP or TMB-8. We conclude that, in cultured VSMC, acute intracellular alkalinization and, to a lesser extent, acidification result in release of Ca2+ from internal stores. Alkalinization increases Cai2+ by promoting its release from a store which is AVP and TMB-8 sensitive, most likely the SR. Cell acidification increases Cai2+ from an intracellular store(s) that is neither AVP nor TMB-8 sensitive. The increase in Cai2+ produced by cell acidification may be explained on the basis of cell buffering such that, as cytosolic H+ increases, it displaces Cai2+ from internal buffers with similar affinities for Ca2+ and H+.

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