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Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry.

Gordienko DV, Harhun MI, Kustov MV, Pucovský V, Bolton TB - Cell Calcium (2007)

Bottom Line: The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine).Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%.Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Medical Sciences, Ion Channels and Cell Signalling Centre, St. George's University of London, UK. gordienk@sgul.ac.uk

ABSTRACT
Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.

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Spatio-temporal patterns of CCh-induced [Ca2+]i transients. The x–y confocal Ca2+ imaging was performed at 32 Hz (A and B), 40 Hz (C), 44 Hz (D) and 30 Hz (E). For each cell, the time course plot of the normalized fluo-4 fluorescence intensity was averaged (red trace) within sub-PM regions of interest where Ca2+ waves (induced by 10 μM CCh) were initiated (insets), and (green trace) within the total confocal optical slice of the SMC. The galleries below the plots show sequential confocal images (after rotation by 90°) captured during the periods highlighted by grey background in the plots. Magenta arrowheads in the galleries indicate sub-PM [Ca2+]i upstrokes.
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fig2: Spatio-temporal patterns of CCh-induced [Ca2+]i transients. The x–y confocal Ca2+ imaging was performed at 32 Hz (A and B), 40 Hz (C), 44 Hz (D) and 30 Hz (E). For each cell, the time course plot of the normalized fluo-4 fluorescence intensity was averaged (red trace) within sub-PM regions of interest where Ca2+ waves (induced by 10 μM CCh) were initiated (insets), and (green trace) within the total confocal optical slice of the SMC. The galleries below the plots show sequential confocal images (after rotation by 90°) captured during the periods highlighted by grey background in the plots. Magenta arrowheads in the galleries indicate sub-PM [Ca2+]i upstrokes.

Mentions: Spatio-temporal patterns of CCh-induced [Ca2+]i mobilization in non-patched SMCs are illustrated by Fig. 2 showing the results obtained in 5 different cells. Muscarinic receptors were activated by 10 μM CCh either superfused through the experimental bath (Fig. 2A) or applied to the cell as a 600-ms pulse through a glass micropipette (Fig. 2B–E). In all plots, the green traces show the temporal profile of the global [Ca2+]i changes, while red traces show the dynamics of [Ca2+]i changes averaged at multiple sub-PM regions (outlined in the images; insets on the plots) where CCh-induced [Ca2+]i transients were initiated. The galleries show sequential confocal images of fluo-4 fluorescence acquired during the periods marked by a grey background in the plots, respectively. In all cases CCh-induced [Ca2+]i transients were initiated by a SPCU (depicted by the arrowheads on the galleries). Muscarinic receptor activation evoked a SPCU independently of the extent of myocyte contraction (n = 147), suggesting that this phenomenon is not a result of the increase in the local density of Ca2+-release units, which could be caused by change in SMC geometry. In the vast majority of cases (97%) two phases can be clearly distinguished in the Ca2+ responses to CCh: (1) an initial high-amplitude [Ca2+]i transient and (2) a delayed increase of global [Ca2+]i characterized by a smaller amplitude and a tendency to oscillation.


Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry.

Gordienko DV, Harhun MI, Kustov MV, Pucovský V, Bolton TB - Cell Calcium (2007)

Spatio-temporal patterns of CCh-induced [Ca2+]i transients. The x–y confocal Ca2+ imaging was performed at 32 Hz (A and B), 40 Hz (C), 44 Hz (D) and 30 Hz (E). For each cell, the time course plot of the normalized fluo-4 fluorescence intensity was averaged (red trace) within sub-PM regions of interest where Ca2+ waves (induced by 10 μM CCh) were initiated (insets), and (green trace) within the total confocal optical slice of the SMC. The galleries below the plots show sequential confocal images (after rotation by 90°) captured during the periods highlighted by grey background in the plots. Magenta arrowheads in the galleries indicate sub-PM [Ca2+]i upstrokes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268754&req=5

fig2: Spatio-temporal patterns of CCh-induced [Ca2+]i transients. The x–y confocal Ca2+ imaging was performed at 32 Hz (A and B), 40 Hz (C), 44 Hz (D) and 30 Hz (E). For each cell, the time course plot of the normalized fluo-4 fluorescence intensity was averaged (red trace) within sub-PM regions of interest where Ca2+ waves (induced by 10 μM CCh) were initiated (insets), and (green trace) within the total confocal optical slice of the SMC. The galleries below the plots show sequential confocal images (after rotation by 90°) captured during the periods highlighted by grey background in the plots. Magenta arrowheads in the galleries indicate sub-PM [Ca2+]i upstrokes.
Mentions: Spatio-temporal patterns of CCh-induced [Ca2+]i mobilization in non-patched SMCs are illustrated by Fig. 2 showing the results obtained in 5 different cells. Muscarinic receptors were activated by 10 μM CCh either superfused through the experimental bath (Fig. 2A) or applied to the cell as a 600-ms pulse through a glass micropipette (Fig. 2B–E). In all plots, the green traces show the temporal profile of the global [Ca2+]i changes, while red traces show the dynamics of [Ca2+]i changes averaged at multiple sub-PM regions (outlined in the images; insets on the plots) where CCh-induced [Ca2+]i transients were initiated. The galleries show sequential confocal images of fluo-4 fluorescence acquired during the periods marked by a grey background in the plots, respectively. In all cases CCh-induced [Ca2+]i transients were initiated by a SPCU (depicted by the arrowheads on the galleries). Muscarinic receptor activation evoked a SPCU independently of the extent of myocyte contraction (n = 147), suggesting that this phenomenon is not a result of the increase in the local density of Ca2+-release units, which could be caused by change in SMC geometry. In the vast majority of cases (97%) two phases can be clearly distinguished in the Ca2+ responses to CCh: (1) an initial high-amplitude [Ca2+]i transient and (2) a delayed increase of global [Ca2+]i characterized by a smaller amplitude and a tendency to oscillation.

Bottom Line: The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine).Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%.Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Medical Sciences, Ion Channels and Cell Signalling Centre, St. George's University of London, UK. gordienk@sgul.ac.uk

ABSTRACT
Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.

Show MeSH
Related in: MedlinePlus