Acetylcholine Increases the Free Intracellular Calcium Concentration in Podocytes in Intact Rat Glomeruli via Muscarinic M5 Receptors

2001 ◽  
Vol 12 (4) ◽  
pp. 678-687
Author(s):  
ROLAND NITSCHKE ◽  
ANNA HENGER ◽  
SIGRID RICKEN ◽  
VICTORIA MÜLLER ◽  
MICHAEL KÖTTGEN ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Receptor Expression ◽  
Intracellular Calcium Concentration ◽  
Acetoxymethyl Ester ◽  
Two Photon ◽  
Photon Excitation ◽  
Free Intracellular Calcium ◽  
Immunohistochemical Studies

Abstract. The effects of acetylcholine (ACh) on the free intracellular calcium concentration ([Ca2+]i) of microdissected glomeruli were investigated using fura-2 fluorescence digital imaging and two-photon confocal microscopy. ACh caused a concentration-dependent [Ca2+]i increases with an initial peak followed by a sustained plateau, which was suppressed by reduced extracellular Ca2+ concentrations. The [Ca2+]i plateau was not affected by the L-type Ca2+ channel blocker nicardipine, whereas gadolinium and lanthanum (both at 1 μM) blocked the plateau. Diphenylacetoxy-N-methylpiperidine methiodide (100 nM), an M3/M5 receptor antagonist, and pirenzepine (1 μM), an M1 receptor antagonist, completely inhibited the effect of ACh. [Ca2+]i measurements using two-photon excitation of fluo-3 and staining of the cells with calcein/acetoxymethyl ester, for observation of the capillary network together with the glomerular cells, showed that [Ca2+]i was increased in single podocytes. Immunohistochemical studies did not demonstrate M3 receptor expression in glomerular cells. M1 receptors could be detected only in the parietal sheet of Bowman's capsule, whereas M5 receptors were found only in podocytes. The data show that ACh increases [Ca2+]i in podocytes of intact glomeruli, most likely via muscarinic M5 receptors.

Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range

1995 ◽  
Vol 74 (6) ◽  
pp. 2625-2637 ◽  
Author(s):  
N. E. Ziv ◽  
M. E. Spira
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Intracellular Calcium Concentration ◽  
Concentration Gradients ◽  
Fura 2 ◽  
Direct Demonstration ◽  
Free Intracellular Calcium ◽  
Axonal Transection ◽  
Transient Elevation

1. Axonal transection triggers a cascade of pathological processes that frequently lead to the degeneration of the injured neuron. It is generally believed that the degenerative process is triggered by an overwhelming influx of calcium through the cut end of the axon. 2. Theoretical considerations and indirect observations suggest that axotomy is followed by an increase in the free intracellular calcium concentration ([Ca2+]i) to the millimolar level. In contrast, only relatively modest and transient elevation in [Ca2+]i to the micromolar level was revealed by recent fura-2 studies. 3. In the current study we used the low-affinity Ca2+ indicator mag-fura-2 to reexamine the spatiotemporal distribution pattern of Ca2+ after axotomy and to map the free intracellular Mg2+ concentration gradients. 4. We report that axotomy elevates [Ca2+]i well beyond the "physiological" range of calcium concentrations, to levels > 1 mM near the tip of the cut axon and to hundreds of micromolars along the axon further away from the cut end. Nevertheless, [Ca2+]i recovers to the control levels within 2-3 min after the resealing of the cut end. 5. A comparison of the behavior of fura-2 and mag-fura-2 in the cytosol of the axotomized neurons reveals that the determination of [Ca2+]i by fura-2 largely underestimates the actual intracellular Ca2+ concentrations. 6. Experiments in which one branch of a bifurcated axon was transected revealed that the elevation in [Ca2+]i is confined to the transected axonal branch and does not spread beyond the bifurcation point. 7. After axotomy, the intracellular Mg2+ concentration equilibrates rapidly with the external concentration and then recovers at a rate somewhat slower than that of [Ca2+]i. 8. To the best of our knowledge, this study is the first direct demonstration that axotomy elevates [Ca2+]i to the millimolar range and that neurons are able to recover from these extreme calcium concentrations.

scholarly journals Simultaneous arterial calcium dynamics and diameter measurements: application to myoendothelial communication

2001 ◽  
Vol 280 (3) ◽  
pp. H1088-H1096 ◽  
Author(s):  
Alexander Schuster ◽  
Hirotaka Oishi ◽  
Jean-Louis Bény ◽  
Nikolaos Stergiopulos ◽  
Jean-Jacques Meister
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Palmitoleic Acid ◽  
Intracellular Calcium Concentration ◽  
Calcium Flux ◽  
Arterial Segment ◽  
Free Intracellular Calcium ◽  
Image Acquisition System ◽  
Stimulation Of

The goal of the present study was to analyze the intercellular calcium communication between smooth muscle cells (SMCs) and endothelial cells (ECs) by simultaneously monitoring artery diameter and intracellular calcium concentration in a rat mesenteric arterial segment in vitro under physiological pressure (50 mmHg) and flow (50 μl/min) in a specially developed system. Intracellular calcium was expressed as the fura 2 ratio. The diameter was measured using a digital image acquisition system. Stimulation of SMCs with the α1-agonist phenylephrine (PE) caused not only an increase in the free intracellular calcium concentration of the SMCs as expected but also in the ECs, suggesting a calcium flux from the SMCs to the ECs. The gap junction uncoupler palmitoleic acid greatly reduced this increase in calcium in the ECs on stimulation of the SMCs with PE. This indicates that the signaling pathway passes through the gap junctions. Similarly, although vasomotion originates in the SMCs, calcium oscillates in both SMCs and ECs during vasomotion, suggesting again a calcium flux from the SMCs to the ECs.

Reactive oxygen species and calcium homeostasis in cultured human intestinal smooth muscle cells

1997 ◽  
Vol 272 (6) ◽  
pp. G1439-G1450 ◽  
Author(s):  
K. Bielefeldt ◽  
C. A. Whiteis ◽  
R. V. Sharma ◽  
F. M. Abboud ◽  
J. L. Conklin
Keyword(s):  
Reactive Oxygen Species ◽  
Intracellular Calcium ◽  
Calcium Homeostasis ◽  
Calcium Concentration ◽  
Calcium Release ◽  
Reactive Oxygen ◽  
Muscle Cells ◽  
Intracellular Calcium Concentration ◽  
Oxygen Species ◽  
Acetoxymethyl Ester

Reactive oxygen species (ROS) significantly alter cell function. We examined the effects of hydrogen peroxide (H2O2) and xanthine/xanthine oxidase (X/XO) on isolated intestinal muscle cells. We assessed cell viability with the exclusion dye trypan blue and assayed the effects of H2O2 and X/XO on the intracellular redox state with the fluorescent probe 2',7'-dichlorofluorescein. Intracellular calcium concentration was measured in cells loaded with fura 2-acetoxymethyl ester, and we recorded whole membrane currents with conventional patch-clamp methods. Cells remained viable after a 5-min exposure to H2O2 and X/XO. H2O2 and X/XO led to a significant rise of the intracellular concentration of ROS. H2O2 (270 microM to 2.7 mM) as well as X/XO (0.25-16 mU; 0.5 mM xanthine) significantly increased intracellular calcium concentrations. Depletion of intracellular calcium with ryanodine or thapsigargin did not abolish the effect of ROS on the intracellular calcium concentration. In the absence of external calcium or in the presence of the calcium channel blocker nifedipine, H2O2 and X/XO still increased the intracellular calcium level. Thus calcium influx and calcium release from internal stores contributed to this rise in cytosolic calcium. Catalase and superoxide dismutase blunted or completely abolished the changes in calcium concentration elicited by H2O2 and X/XO. Exposure to ROS resulted in a rapid decline of the membrane resistance without significant changes in voltage-sensitive ion currents. We conclude that ROS disrupt the calcium homeostasis of cells at concentrations that do not lead to immediate cell death. The resulting elevation in cytosolic free calcium will activate a variety of biochemical reactions and may thus contribute to the cytotoxicity of reactive oxygen molecules.

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