Multiple effects of ryanodine on intracellular free Ca2+ in smooth muscle cells from bovine and porcine coronary artery: Modulation of sarcoplasmic reticulum function

C. Wagner-Mann, Q. Hu, Michael Sturek

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Abstract

1. The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+](i)) were studied by use of fura-2 microfluorometry in single smooth muscle cells freshly dispersed from bovine and porcine coronary artery. 2. Bovine and porcine cells demonstrated similar sensitivities to 10 min of exposure to ryanodine in physiological salt solution (PSS), as determined by comparable dose-dependent decreases in the subsequent [Ca2+](i) transient induced by 5 mM caffeine. 3. Ryanodine (10 μM) caused a significant increase in [Ca2+](i) to a plateau level 27 ± 3% and 38 ± 4% above baseline [Ca2+](i) (baseline [Ca2+](i) = [Ca2+](i) at 0 min) in porcine and bovine cells, respectively, when bathed in PSS. In bovine cells the time required to reach 1/2 the plateau level was only 3 min versus 6 min for porcine cells. 4. The ryanodine-induced plateau increase in [Ca2+](i) was 35 ± 5% above baseline for bovine cells bathed in 0 Ca PSS (PSS including 10 μM EGTA with no added Ca2+), but only 7 ± 3% baseline in porcine cells during 10 min exposure to 10 μM ryanodine. In bovine cells [Ca2+](i) showed proportional increases when extracellular Ca2+ was increased from the normal 2 mM Ca2+ PSS to 5 and 10 mM. 5. Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, showed no increase in [Ca2+](i) when challenged with 10 μM ryanodine. The ryanodine-associated increase in [Ca2+](i), which was sustained in 0 Ca PSS during the 10 min ryanodine exposure in cells not pretreated with caffeine, suggests that ryanodine releases Ca2+ from the sarcoplasmic reticulum, but also inhibits Ca2+ efflux. 6. Intracellular free Ba2+ ([Ba2+](i)) was measured with fura-2 microfluorometry to define further the Ca2+ efflux pathway inhibited by ryanodine; specifically, Ba2+ is not transported by the Ca2+ pump, but will substitute for Ca2+ in Na+-Ca2+ exchange. In porcine cells pretreated with caffeine in 0 Ca PSS to deplete the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, depolarization with 80 mM K+ in 2 mM external Ba2+ caused a 100 ± 6% increase in fura-2 fluorescence ([Ba2+](i)). During the 17.5 min 0 Ca PSS recovery from depolarization, exposure to 10 μM ryanodine inhibited the removal of [Ba2+](i) by 69 ± 3% when compared with control (0 Ca PSS without ryanodine). 7. It was concluded that in bovine and porcine smooth muscle cells; (a) ryanodine (≥ 10 μM) releases Ca2+ from the sarcoplasmic reticulum; (b) ryanodine (≥ 10 μM) decreases Ca2+ efflux, probably by inhibition of Na+-Ca2+ exchange; (c) the sarcoplasmic reticulum Ca2+ store may be larger in bovine than in porcine smooth muscle cells; thus, porcine cells have a relatively greater reliance on Ca2+ influx to increase [Ca2+](i).

Original languageEnglish (US)
Pages (from-to)903-911
Number of pages9
JournalBritish Journal of Pharmacology
Volume105
Issue number4
StatePublished - 1992
Externally publishedYes

Fingerprint

Ryanodine
Sarcoplasmic Reticulum
Smooth Muscle Myocytes
Coronary Vessels
Swine
Salts
Caffeine
Fura-2
Cytophotometry
Egtazic Acid

Keywords

  • Ba
  • Ca
  • Ca efflux
  • Caffeine
  • Na-Ca exchange
  • Ryanodine
  • Sarcoplasmic reticulum
  • Vascular smooth muscle

ASJC Scopus subject areas

  • Pharmacology

Cite this

@article{84bc8947dfca446cb005da903fb70a33,
title = "Multiple effects of ryanodine on intracellular free Ca2+ in smooth muscle cells from bovine and porcine coronary artery: Modulation of sarcoplasmic reticulum function",
abstract = "1. The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+](i)) were studied by use of fura-2 microfluorometry in single smooth muscle cells freshly dispersed from bovine and porcine coronary artery. 2. Bovine and porcine cells demonstrated similar sensitivities to 10 min of exposure to ryanodine in physiological salt solution (PSS), as determined by comparable dose-dependent decreases in the subsequent [Ca2+](i) transient induced by 5 mM caffeine. 3. Ryanodine (10 μM) caused a significant increase in [Ca2+](i) to a plateau level 27 ± 3{\%} and 38 ± 4{\%} above baseline [Ca2+](i) (baseline [Ca2+](i) = [Ca2+](i) at 0 min) in porcine and bovine cells, respectively, when bathed in PSS. In bovine cells the time required to reach 1/2 the plateau level was only 3 min versus 6 min for porcine cells. 4. The ryanodine-induced plateau increase in [Ca2+](i) was 35 ± 5{\%} above baseline for bovine cells bathed in 0 Ca PSS (PSS including 10 μM EGTA with no added Ca2+), but only 7 ± 3{\%} baseline in porcine cells during 10 min exposure to 10 μM ryanodine. In bovine cells [Ca2+](i) showed proportional increases when extracellular Ca2+ was increased from the normal 2 mM Ca2+ PSS to 5 and 10 mM. 5. Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, showed no increase in [Ca2+](i) when challenged with 10 μM ryanodine. The ryanodine-associated increase in [Ca2+](i), which was sustained in 0 Ca PSS during the 10 min ryanodine exposure in cells not pretreated with caffeine, suggests that ryanodine releases Ca2+ from the sarcoplasmic reticulum, but also inhibits Ca2+ efflux. 6. Intracellular free Ba2+ ([Ba2+](i)) was measured with fura-2 microfluorometry to define further the Ca2+ efflux pathway inhibited by ryanodine; specifically, Ba2+ is not transported by the Ca2+ pump, but will substitute for Ca2+ in Na+-Ca2+ exchange. In porcine cells pretreated with caffeine in 0 Ca PSS to deplete the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, depolarization with 80 mM K+ in 2 mM external Ba2+ caused a 100 ± 6{\%} increase in fura-2 fluorescence ([Ba2+](i)). During the 17.5 min 0 Ca PSS recovery from depolarization, exposure to 10 μM ryanodine inhibited the removal of [Ba2+](i) by 69 ± 3{\%} when compared with control (0 Ca PSS without ryanodine). 7. It was concluded that in bovine and porcine smooth muscle cells; (a) ryanodine (≥ 10 μM) releases Ca2+ from the sarcoplasmic reticulum; (b) ryanodine (≥ 10 μM) decreases Ca2+ efflux, probably by inhibition of Na+-Ca2+ exchange; (c) the sarcoplasmic reticulum Ca2+ store may be larger in bovine than in porcine smooth muscle cells; thus, porcine cells have a relatively greater reliance on Ca2+ influx to increase [Ca2+](i).",
keywords = "Ba, Ca, Ca efflux, Caffeine, Na-Ca exchange, Ryanodine, Sarcoplasmic reticulum, Vascular smooth muscle",
author = "C. Wagner-Mann and Q. Hu and Michael Sturek",
year = "1992",
language = "English (US)",
volume = "105",
pages = "903--911",
journal = "British Journal of Pharmacology",
issn = "0007-1188",
publisher = "Wiley-Blackwell",
number = "4",

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TY - JOUR

T1 - Multiple effects of ryanodine on intracellular free Ca2+ in smooth muscle cells from bovine and porcine coronary artery

T2 - Modulation of sarcoplasmic reticulum function

AU - Wagner-Mann, C.

AU - Hu, Q.

AU - Sturek, Michael

PY - 1992

Y1 - 1992

N2 - 1. The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+](i)) were studied by use of fura-2 microfluorometry in single smooth muscle cells freshly dispersed from bovine and porcine coronary artery. 2. Bovine and porcine cells demonstrated similar sensitivities to 10 min of exposure to ryanodine in physiological salt solution (PSS), as determined by comparable dose-dependent decreases in the subsequent [Ca2+](i) transient induced by 5 mM caffeine. 3. Ryanodine (10 μM) caused a significant increase in [Ca2+](i) to a plateau level 27 ± 3% and 38 ± 4% above baseline [Ca2+](i) (baseline [Ca2+](i) = [Ca2+](i) at 0 min) in porcine and bovine cells, respectively, when bathed in PSS. In bovine cells the time required to reach 1/2 the plateau level was only 3 min versus 6 min for porcine cells. 4. The ryanodine-induced plateau increase in [Ca2+](i) was 35 ± 5% above baseline for bovine cells bathed in 0 Ca PSS (PSS including 10 μM EGTA with no added Ca2+), but only 7 ± 3% baseline in porcine cells during 10 min exposure to 10 μM ryanodine. In bovine cells [Ca2+](i) showed proportional increases when extracellular Ca2+ was increased from the normal 2 mM Ca2+ PSS to 5 and 10 mM. 5. Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, showed no increase in [Ca2+](i) when challenged with 10 μM ryanodine. The ryanodine-associated increase in [Ca2+](i), which was sustained in 0 Ca PSS during the 10 min ryanodine exposure in cells not pretreated with caffeine, suggests that ryanodine releases Ca2+ from the sarcoplasmic reticulum, but also inhibits Ca2+ efflux. 6. Intracellular free Ba2+ ([Ba2+](i)) was measured with fura-2 microfluorometry to define further the Ca2+ efflux pathway inhibited by ryanodine; specifically, Ba2+ is not transported by the Ca2+ pump, but will substitute for Ca2+ in Na+-Ca2+ exchange. In porcine cells pretreated with caffeine in 0 Ca PSS to deplete the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, depolarization with 80 mM K+ in 2 mM external Ba2+ caused a 100 ± 6% increase in fura-2 fluorescence ([Ba2+](i)). During the 17.5 min 0 Ca PSS recovery from depolarization, exposure to 10 μM ryanodine inhibited the removal of [Ba2+](i) by 69 ± 3% when compared with control (0 Ca PSS without ryanodine). 7. It was concluded that in bovine and porcine smooth muscle cells; (a) ryanodine (≥ 10 μM) releases Ca2+ from the sarcoplasmic reticulum; (b) ryanodine (≥ 10 μM) decreases Ca2+ efflux, probably by inhibition of Na+-Ca2+ exchange; (c) the sarcoplasmic reticulum Ca2+ store may be larger in bovine than in porcine smooth muscle cells; thus, porcine cells have a relatively greater reliance on Ca2+ influx to increase [Ca2+](i).

AB - 1. The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+](i)) were studied by use of fura-2 microfluorometry in single smooth muscle cells freshly dispersed from bovine and porcine coronary artery. 2. Bovine and porcine cells demonstrated similar sensitivities to 10 min of exposure to ryanodine in physiological salt solution (PSS), as determined by comparable dose-dependent decreases in the subsequent [Ca2+](i) transient induced by 5 mM caffeine. 3. Ryanodine (10 μM) caused a significant increase in [Ca2+](i) to a plateau level 27 ± 3% and 38 ± 4% above baseline [Ca2+](i) (baseline [Ca2+](i) = [Ca2+](i) at 0 min) in porcine and bovine cells, respectively, when bathed in PSS. In bovine cells the time required to reach 1/2 the plateau level was only 3 min versus 6 min for porcine cells. 4. The ryanodine-induced plateau increase in [Ca2+](i) was 35 ± 5% above baseline for bovine cells bathed in 0 Ca PSS (PSS including 10 μM EGTA with no added Ca2+), but only 7 ± 3% baseline in porcine cells during 10 min exposure to 10 μM ryanodine. In bovine cells [Ca2+](i) showed proportional increases when extracellular Ca2+ was increased from the normal 2 mM Ca2+ PSS to 5 and 10 mM. 5. Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, showed no increase in [Ca2+](i) when challenged with 10 μM ryanodine. The ryanodine-associated increase in [Ca2+](i), which was sustained in 0 Ca PSS during the 10 min ryanodine exposure in cells not pretreated with caffeine, suggests that ryanodine releases Ca2+ from the sarcoplasmic reticulum, but also inhibits Ca2+ efflux. 6. Intracellular free Ba2+ ([Ba2+](i)) was measured with fura-2 microfluorometry to define further the Ca2+ efflux pathway inhibited by ryanodine; specifically, Ba2+ is not transported by the Ca2+ pump, but will substitute for Ca2+ in Na+-Ca2+ exchange. In porcine cells pretreated with caffeine in 0 Ca PSS to deplete the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, depolarization with 80 mM K+ in 2 mM external Ba2+ caused a 100 ± 6% increase in fura-2 fluorescence ([Ba2+](i)). During the 17.5 min 0 Ca PSS recovery from depolarization, exposure to 10 μM ryanodine inhibited the removal of [Ba2+](i) by 69 ± 3% when compared with control (0 Ca PSS without ryanodine). 7. It was concluded that in bovine and porcine smooth muscle cells; (a) ryanodine (≥ 10 μM) releases Ca2+ from the sarcoplasmic reticulum; (b) ryanodine (≥ 10 μM) decreases Ca2+ efflux, probably by inhibition of Na+-Ca2+ exchange; (c) the sarcoplasmic reticulum Ca2+ store may be larger in bovine than in porcine smooth muscle cells; thus, porcine cells have a relatively greater reliance on Ca2+ influx to increase [Ca2+](i).

KW - Ba

KW - Ca

KW - Ca efflux

KW - Caffeine

KW - Na-Ca exchange

KW - Ryanodine

KW - Sarcoplasmic reticulum

KW - Vascular smooth muscle

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