Measurement of SR free Ca2+ and Mg2+ in permeabilized smooth muscle cells with use of furaptra

1995 ◽  
Vol 269 (3) ◽  
pp. C698-C705 ◽  
Author(s):  
T. Sugiyama ◽  
W. F. Goldman
Keyword(s):  
Smooth Muscle ◽  
Dissociation Constants ◽  
Time Dependent ◽  
Isosbestic Point ◽  
Half Time ◽  
Permeabilized Cells ◽  
Aortic Smooth Muscle ◽  
A7r5 Cells ◽  
Inositol 1,4,5 Trisphosphate

The concentrations of intrasarcoplasmic reticulum (SR) free Ca2+ ([Ca2+]SR) and Mg2+ ([Mg2+]SR) were measured in furaptra-loaded saponin-permeabilized cultured aortic smooth muscle (A7r5) cells. Ca(2+)-independent fluorescence emitted by furaptra trapped within organelles, excited at 346 nm (isosbestic point), decreased with a half time of 30 min. All Ca2+ measurements appeared to be from SR, because the apparent Ca2+ distribution within permeabilized cells was uniform and therefore inconsistent with furaptra loading into mitochondria. Moreover, thapsigargin-induced SR Ca(2+)-adenosinetriphosphatase inhibition caused near-total depletion of Ca2+, and the metabolic poisons oligomycin and rotenone had no effect. Calibration curves relating 370 nm-to-346 nm ratios to [Ca2+] and to [Mg2+] were calculated in situ; dissociation constants for Ca2+ and Mg2+ binding were 49 microM and 6.8 mM, respectively. Resting [Ca2+]SR was 75-130 microM, with a mean of 97.2 +/- 2.2 microM (n = 376), whereas [Mg2+]SR, estimated in the absence of Ca2+, was 1.0 mM. Stimulation with inositol 1,4,5-trisphosphate resulted in time-dependent declines in [Ca2+]SR, and pretreatment with guanosine 5'-triphosphate caused a large increase in the rate of inositol 1,4,5-trisphosphate-evoked SR Ca2+ release, although guanosine 5'-triphosphate had no effect by itself. These observations indicate that furaptra will be a valuable tool with which to directly study [Ca2+]SR and SR function.

scholarly journals Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release

1997 ◽  
Vol 325 (3) ◽  
pp. 661-666 ◽  
Author(s):  
Ludwig MISSIAEN ◽  
Jan B. PARYS ◽  
Humbert DE SMEDT ◽  
Ilse SIENAERT ◽  
Henk SIPMA ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Site ◽  
Calcium Release ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Release Process ◽  
A7r5 Cells ◽  
Channel Opening ◽  
Inositol 1,4,5 Trisphosphate

The effects of a whole series of adenine nucleotides on Ins(1,4,5)P3-induced Ca2+ release were characterized in permeabilized A7r5 smooth-muscle cells. Several adenine nucleotides activated the Ins(1,4,5)P3 receptor. It was observed that 3′-phosphoadenosine 5′-phosphosulphate, CoA, di(adenosine-5′)tetraphosphate (Ap4A) and di(adenosine-5′)pentaphosphate (Ap5A) were more effective than ATP. Ap4A and Ap5A also interacted with a lower EC50 than ATP. In order to find out how these adenine nucleotides affected Ins(1,4,5)P3-induced Ca2+ release, we have measured their effect on the response of permeabilized A7r5 cells to a progressively increasing Ins(1,4,5)P3 concentration. Stimulatory ATP and Ap5A concentrations had no effect on the threshold Ins(1,4,5)P3 concentration for initiating Ca2+ release, but they stimulated Ca2+ release in the presence of supra-threshold Ins(1,4,5)P3 concentrations by increasing the co-operativity of the release process. Inhibition of the Ins(1,4,5)P3-induced Ca2+ release at higher ATP concentrations was associated with a further increase in co-operativity and also with a shift in threshold towards higher Ins(1,4,5)P3 concentrations. ATP had no effect on the non-specific Ca2+ leak in the absence of Ins(1,4,5)P3. We conclude that the adenine-nucleotide-binding site can be activated by many different adenine nucleotides. Binding of these compounds to the transducing domain of the Ins(1,4,5)P3 receptor increases the efficiency of transmitting Ins(1,4,5)P3 binding to channel opening. The inhibition by high ATP concentrations is exerted at a different site, related to Ins(1,4,5)P3 binding.

Dual regulation of L-type Ca2+ channels by serotonin 2 receptor stimulation in vascular smooth muscle cells

1995 ◽  
Vol 268 (2) ◽  
pp. H544-H549 ◽  
Author(s):  
Y. Hirakawa ◽  
T. Kuga ◽  
S. Kobayashi ◽  
H. Kanaide ◽  
A. Takeshita
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cell Voltage ◽  
Muscle Cells ◽  
Receptor Stimulation ◽  
Aortic Smooth Muscle ◽  
A 23187 ◽  
Inositol 1,4,5 Trisphosphate ◽  
Voltage Dependent ◽  
Ca2 Channels

The purpose of the present study was to investigate regulation of voltage-dependent Ca2+ channels by serotonin in rat aortic smooth muscle cells in primary culture. L- and T-type Ca2+ currents (ICa) were recorded using the whole cell voltage-clamp method. Without pretreatment, in 25 of 30 cells examined, 10 microM serotonin decreased L-type ICa to various extents (-14 to -72%). However, in the remaining five cells, serotonin increased L-type ICa 21 +/- 4%. Thus, in 30 cells, serotonin decreased L-type ICa an average of 22 +/- 5%. In the presence of intracellular heparin (100 micrograms/ml), a blocker of inositol 1,4,5-trisphosphate binding to its receptor, serotonin increased L-type ICa in all cells 29 +/- 3% (n = 6). When stored Ca2+ was depleted by pretreatment either with 20 microM ryanodine and 20 mM caffeine or with 100 nM A-23187, serotonin also increased L-type ICa in all cells 30 +/- 5 (n = 4) or 37 +/- 5% (n = 12), respectively. In the presence of heparin, the serotonin-induced increase of L-type ICa was prevented by 100 nM staurosporine (2 +/- 3%; n = 6, P < 0.01). The serotonin-induced decrease of L-type ICa was significantly augmented by 100 nM staurosporine (-43 +/- 10%; n = 5). Phorbol 12,13-dibutylate (PDBu; 1 microM) increased L-type ICa 29 +/- 3% (n = 6), and serotonin did not further increase L-type ICa after its potentiation by PDBu.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells

1999 ◽  
Vol 276 (1) ◽  
pp. L81-L89 ◽  
Author(s):  
Christina M. Pabelick ◽  
Y. S. Prakash ◽  
Mathur S. Kannan ◽  
Keith A. Jones ◽  
David O. Warner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ryanodine Receptor ◽  
Muscle Cells ◽  
Calcium Oscillations ◽  
Tracheal Smooth Muscle ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Trisphosphate Receptor

The effect of halothane on intracellular Ca2+ concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]iwhen Ca2+ influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+ leak and/or decreased reuptake. In β-escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]ioscillations (which represent SR Ca2+ release through ryanodine-receptor channels), abolishing oscillations in ∼20% of tracheal smooth muscle cells at 2 MAC. When Ca2+ influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]ioscillations, inhibiting oscillations in ∼70% of cells at 2 MAC. The fall time of [Ca2+]ioscillations and the rate of fall of the [Ca2+]iresponse to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]ioscillations by 1) depleting SR Ca2+ via increased Ca2+ leak through inositol 1,4,5-trisphosphate-receptor channels, 2) decreasing Ca2+ release through ryanodine-receptor channels, and 3) inhibiting reuptake.

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

2001 ◽  
Vol 281 (2) ◽  
pp. C555-C562 ◽  
Author(s):  
Madhumita Jena Mohanty ◽  
Maian Ye ◽  
Xingli Li ◽  
Noreen F. Rossi
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Nicotinic Acid ◽  
Vascular Smooth Muscle Cells ◽  
Ruthenium Red ◽  
A7r5 Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Hypotonic Swelling

Hypotonic swelling increases the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC). The source of this Ca2+ is not clear. To study the source of increase in [Ca2+]i in response to hypotonic swelling, we measured [Ca2+]i in fura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a 40.7-nM increase in [Ca2+]i that was not inhibited by EGTA but was inhibited by 1 μM thapsigargin. Prior depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores with vasopressin did not inhibit the increase in [Ca2+]i in response to hypotonic swelling. Exposure of 45Ca2+-loaded intracellular stores to hypotonic swelling in permeabilized VSMC produced an increase in45Ca2+ efflux, which was inhibited by 1 μM thapsigargin but not by 50 μg/ml heparin, 50 μM ruthenium red, or 25 μM thio-NADP. Thus hypotonic swelling of VSMC causes a release of Ca2+ from the intracellular stores from a novel site distinct from the IP3-, ryanodine-, and nicotinic acid adenine dinucleotide phosphate-sensitive stores.

scholarly journals Evidence against reciprocal regulation of Ca2+ entry by vasopressin in A7r5 rat aortic smooth-muscle cells

2005 ◽  
Vol 388 (1) ◽  
pp. 237-244 ◽  
Author(s):  
Lioubov I. BRUEGGEMANN ◽  
Daniel R. MARKUN ◽  
John A. BARAKAT ◽  
Haiyan CHEN ◽  
Kenneth L. BYRON
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Cyclopiazonic Acid ◽  
Reciprocal Regulation ◽  
Aortic Smooth Muscle ◽  
Fura 2 ◽  
Fluorescence Techniques ◽  
A7r5 Cells ◽  
Perforated Patch Clamp

Recent studies by Moneer and Taylor [(2002) Biochem. J. 362, 13–21] have proposed a reciprocal regulation of two Ca2+-entry pathways by AVP ([Arg8]-vasopressin) in A7r5 vascular smooth-amuscle cells. Their model proposes that AVP inhibits CCE (capacitative Ca2+ entry) and predicts a rebound of CCE after the removal of AVP. In the present study, we used whole-cell perforated patch-clamp techniques to measure ISOC (store-operated current) corresponding to CCE in A7r5 cells. When 100 nM AVP is present, it activates ISOC with no apparent rebound on removal of AVP. ISOC activated by thapsigargin or cyclopiazonic acid was not inhibited by 100 nM AVP. We also used fura 2 fluorescence techniques to re-examine the model of Moneer and Taylor, specifically focusing on the proposed inhibition of CCE by AVP. We find that 100 nM AVP activates capacitative Mn2+ entry and does not inhibit thapsigargin- or cyclopiazonic acid-activated Mn2+ entry. Moreover, Ca2+ entry after depletion of intracellular Ca2+ stores is enhanced by AVP and we detect no rebound of Ca2+ or Mn2+ entry after AVP removal. On the basis of these findings, we conclude that AVP does not inhibit CCE in A7r5 cells.

Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways

1992 ◽  
Vol 262 (5) ◽  
pp. C1258-C1265 ◽  
Author(s):  
Y. T. Xuan ◽  
O. L. Wang ◽  
A. R. Whorton
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate

We have investigated the role of the sarcoplasmic reticulum Ca2+ pool in regulating Ca2+ entry in vascular smooth muscle cells using a receptor-independent means of mobilizing the intracellular Ca2+ pool. Thapsigargin (TG) has been shown to inhibit the endoplasmic reticulum Ca(2+)-ATPase, mobilize intracellular Ca2+, and activate Ca2+ entry in nonmuscle tissues. When smooth muscle cells were treated with 0.2 microM TG, cytosolic Ca2+ concentrations rose gradually over 8 min to a peak value of 365 +/- 18 nM. Cytosolic Ca2+ remained elevated for at least 20 min and was supported by continued entry of extracellular Ca2+. TG also stimulated entry of Mn2+ and 45Ca2+ from outside the cell. Importantly, TG-induced Ca2+ entry and Mn2+ entry were found to occur through mechanisms that were independent of L-type Ca2+ channel activation because influx was not inhibited by concentrations of nicardipine that were found to block either endothelin- or 100 mM extracellular K(+)-induced cation influx. The mechanism through which TG activates cation entry appears to involve mobilization of the inositol 1,4,5-trisphosphate-responsive intracellular Ca2+ pool. In permeabilized cells, TG prevented ATP-stimulated Ca2+ uptake into the sarcoplasmic reticulum and slowly released sequestered Ca2+. The Ca2+ pool involved was responsive to inositol 1,4,5-trisphosphate. However, TG did not initiate the formation of inositol polyphosphates. Thus TG mobilizes the sarcoplasmic reticulum Ca2+ pool and activates Ca2+ entry through a nicardipine-insensitive Ca2+ channel in vascular smooth muscle. The mechanism is independent of inositol polyphosphate formation.

Effects of tonin on the response to norepinephrine by the aortic strip of the hypertensive rat

1981 ◽  
Vol 59 (8) ◽  
pp. 790-793 ◽  
Author(s):  
R. Garcia ◽  
E. L. Schiffrin ◽  
G. Thibault ◽  
R. Boucher ◽  
J. Genest
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Elevated Blood Pressure ◽  
Elevated Blood ◽  
Hypertensive Rats ◽  
Aortic Smooth Muscle ◽  
Hypertensive Group ◽  
Aortic Strip ◽  
The One

The response to norepinephrine (NE) of arterial smooth muscle from two types of experimental hypertensive rats was investigated. Aortic strips from one-kidney, one-clip hypertensive animals were less responsive to NE than those from their normotensive controls but strips from two-kidney, one-clip hypertensive animals showed no difference from their corresponding controls. The contractility in response to NE was the same in all groups. These results suggest that the mechanisms responsible for the lesser reactivity in the one-kidney hypertensive group are not a consequence of elevated blood pressure itself but may be related to changes in the intrinsic sensitivity of aortic smooth muscle.Tonin potentiated the contraction induced by NE in aortic strips from hypertensive and normotensive rats. This effect was more pronounced in the one-kidney, one-clip hypertensive animals, so that although the aortic smooth muscle from these animals is less reactive to NE, the decreased reactivity can be more than compensated by the presence of tonin. The mechanism of potentiation is not yet clear but the fact that Saralasin did not inhibit it suggests that angiotensin Il is not generated in situ.

Ca2+ stores in smooth muscle cells: Ca2+ buffering and coupling to AVP-evoked inositol phosphate synthesis

1994 ◽  
Vol 266 (1) ◽  
pp. C276-C283 ◽  
Author(s):  
D. M. Berman ◽  
T. Sugiyama ◽  
W. F. Goldman
Keyword(s):  
Smooth Muscle ◽  
Inverse Relationship ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Enzyme System ◽  
Similar Treatment ◽  
A7r5 Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Communication Pathway

Cytosolic Ca2+ concentrations ([Ca2+]cyt) and [3H]inositol phosphates ([3H]InsP) were correlated while decreasing the Ca2+ content of sarcoplasmic reticulum (SR) stores in cultured A7r5 cells at rest and after activation with 8-arginine vasopressin (AVP). Decreasing Ca2+ influx by reducing extracellular Ca2+ or by treatment with verapamil had no effect on resting [Ca2+]cyt but significantly inhibited the AVP-evoked Ca2+ transients (delta Ca2+). Neither treatment affected basal [3H]InsP, but both treatments increased AVP-evoked synthesis of [3H]InsP. Likewise, basal [3H]InsP were unaffected by brief (10-30 s) exposures to thapsigargin (TG), while AVP-induced [3H]InsP synthesis was significantly augmented. Similar treatment with TG rapidly increased resting [Ca2+]cyt and decreased SR Ca2+ by 9-25% as manifested by decreased delta Ca2+. By contrast, ryanodine induced slow increases in [Ca2+]cyt that stabilized within 30 min; subsequent AVP-induced delta Ca2+ were attenuated by 50%. Ryanodine had no effect on either basal or stimulated [3H]InsP levels. Agents that elevate adenosine 3',5'-cyclic monophosphate (cAMP) such as caffeine, 8-bromo-cAMP, and forskolin inhibited AVP-evoked [3H]InsP formation. These observations provide further characterization of a communication pathway between the AVP-sensitive Ca2+ stores in the SR and the plasmalemmal enzyme system involved in the synthesis of inositol 1,4,5-trisphosphate. This pathway is manifested by an inverse relationship between the Ca2+ content of an AVP-sensitive, ryanodine-insensitive SR Ca2+ store and evoked [3H]InsP synthesis and may represent an important component in the tonic regulation of resting [Ca2+]cyt and vasoconstrictor- and hormone-evoked SR Ca2+ release.

Sign in / Sign up

Export Citation Format

Share Document