scholarly journals Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

1999 ◽  
Vol 144 (2) ◽  
pp. 241-254 ◽  
Author(s):  
Maria Teresa Alonso ◽  
Maria José Barrero ◽  
Pedro Michelena ◽  
Estela Carnicero ◽  
Inmaculada Cuchillo ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Physiological Role ◽  
Dependent Manner ◽  
Excitable Cells ◽  
Permeabilized Cells ◽  
Low Concentrations ◽  
Cyclic Adp Ribose ◽  
Cell Depolarization ◽  
Adrenal Chromaffin

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4,5-trisphosphate (InsP3)- producing agonists released only 60–80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells

1990 ◽  
Vol 48 (3) ◽  
pp. 952-953
Author(s):  
Gemma A.J. Kuijpers ◽  
Harvey B. Pollard
Keyword(s):  
Adrenal Medulla ◽  
Calcium Binding ◽  
Chromaffin Cells ◽  
Cell Activation ◽  
Structural Information ◽  
Dependent Manner ◽  
Chromaffin Granules ◽  
Immuno Electron Microscopy ◽  
Ultrathin Sections ◽  
Adrenal Chromaffin

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+ upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+ (1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.

scholarly journals Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5′-[γ-thio]triphosphate and guanosine 5′-[β γ-imido]triphosphate. Evidence for the involvement of various guanine nucleotide-binding proteins

1992 ◽  
Vol 284 (2) ◽  
pp. 321-326 ◽  
Author(s):  
G Ahnert-Hilger ◽  
U Wegenhorst ◽  
B Stecher ◽  
K Spicher ◽  
W Rosenthal ◽  
...  
Keyword(s):  
G Protein ◽  
Chromaffin Cells ◽  
Inhibitory Effect ◽  
Adrenal Chromaffin Cells ◽  
Prolonged Incubation ◽  
Permeabilized Cells ◽  
Alpha Subunits ◽  
Cytoplasmic Material ◽  
Streptolysin O ◽  
Adrenal Chromaffin

1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.

Abstract 489: Mechanisms of Carbon Monoxide Attenuation of Tubuloglomerular Feedback

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
YiLin Ren ◽  
Martin A D'Ambrosio ◽  
Hong Wang ◽  
Jeffrey L Garvin ◽  
Oscar A Carretero
Keyword(s):  
Carbon Monoxide ◽  
Physiological Role ◽  
Inhibitory Effect ◽  
Tubuloglomerular Feedback ◽  
Renal Microcirculation ◽  
Low Concentrations ◽  
Cgmp Analog ◽  
Cell Depolarization ◽  
Toxic Concentrations

Tubuloglomerular feedback (TGF) is an autoregulatory mechanism of the renal microcirculation in which the macula densa (MD) senses NaCl concentration in the lumen of the nephron and sends a signal that controls glomerular filtration rate by constricting the afferent arteriole (Af-Art). We have shown that MD depolarization is sufficient for inducing TGF. Carbon monoxide (CO), either endogenous or exogenous, is known to inhibit TGF, at least in part via cGMP. However, whether cGMP-independent mechanisms are involved, and where in the TGF cascade CO exerts its inhibitory effect, remain unknown. Thus we hypothesize that CO, acting via both cGMP-dependent and -independent mechanisms, attenuates TGF by acting downstream from MD cell depolarization. In vitro , microdissected rabbit Af-Arts and their attached MD were simultaneously perfused and TGF was measured as the decrease in Af-Art diameter. Depolarization of the MD was induced by switching luminal KCl from 4 to 50 mM in the presence of the potassium ionophore valinomycin, while adding the CO-releasing molecule CORM-3 to the MD perfusate at non-toxic concentrations. CORM-3 blunted depolarization-induced TGF at a concentration of 50 μM, from 3.6±0.4 to 2.5±0.4 μm (P<0.01), and completely abolished it at a concentration of 100 μM, to 0.1±0.1 μm (P<0.001, n=6). Similar results were found with 100 μM CORM-3 when depolarization was induced by nystatin (3.0±0.2 vs. 0.4±0.2 μm, P <0.001, n=6). This indicates that CO inhibits TGF acting downstream from depolarization. When cGMP generation was blocked with the guanylate cyclase inhibitor LY-83583 (1 μM) added to the MD, CORM-3 no longer had an effect on depolarization-induced TGF at 50 μM (2.9±0.4 vs. 3.0±0.4 μm), but retained partial inhibitory effect on TGF at 100 μM (1.3±0.2 μm, P =0.02, n=9). This suggests that CO acts via cGMP at low concentrations, but additional mechanisms of action may be involved at higher concentrations. Finally, we confirmed that cGMP inhibits TGF downstream from MD depolarization by adding the degradation-resistant cGMP analog dibutyryl-cGMP (500 μM), which attenuated depolarization-induced TGF (from 3.9±0.5 to 0.6±0.2 μm, P <0.01, n=6). Our results could help explain the physiological role of CO in controlling the renal microcirculation.

Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape

1992 ◽  
Vol 103 (4) ◽  
pp. 1215-1222 ◽  
Author(s):  
J.R. Sims ◽  
S. Karp ◽  
D.E. Ingber
Keyword(s):  
Shape Control ◽  
Nuclear Shape ◽  
Force Balance ◽  
Dependent Manner ◽  
Integrin Receptors ◽  
Permeabilized Cells ◽  
Capillary Endothelial Cells ◽  
Shape Determination ◽  
Dose Dependent

Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular ADP-ribose inhibits ATP-sensitive K+ channels in rat ventricular myocytes

1996 ◽  
Vol 271 (2) ◽  
pp. C464-C468 ◽  
Author(s):  
Y. G. Kwak ◽  
S. K. Park ◽  
U. H. Kim ◽  
M. K. Han ◽  
J. S. Eun ◽  
...  
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Physiological Role ◽  
Hill Coefficient ◽  
K Channel ◽  
Channel Activity ◽  
Rat Ventricular Myocytes ◽  
Cyclic Adp Ribose ◽  
The Hill

Cyclic ADP-ribose (cADPR), an NAD metabolite, has been shown to be a messenger for Ca2+ mobilization from intracellular Ca2+ stores. However, the physiological role of ADP-ribose (ADPR), another metabolite of NAD, is not known. We examined the effects of cADPR and ADPR on the ATP-sensitive K+ channel (KATP) activity in rat ventricular myocytes by use of the inside-out patch-clamp configuration. ADPR, but not cADPR, inhibited the channel activity at micromolar range with an inhibitor constant (Ki) of 38.4 microM. The Hill coefficient was 0.9. ATP inhibited the K+ channel with a Ki of 77.8 microM, and the Hill coefficient was 1.8. Single-channel conductance was not affected by ADPR. These findings strongly suggest that ADPR may act as a regulator of KATP channel activity.

scholarly journals Dominant Role of Mitochondria in Clearance of Large Ca2+ Loads from Rat Adrenal Chromaffin Cells

Neuron ◽  
1996 ◽  
Vol 16 (1) ◽  
pp. 219-228 ◽  
Author(s):  
James Herrington ◽  
Young Bae Park ◽  
Donner F Babcock ◽  
Bertil Hille
Keyword(s):  
Chromaffin Cells ◽  
Dominant Role ◽  
Adrenal Chromaffin Cells ◽  
Adrenal Chromaffin
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