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.

scholarly journals Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells

2007 ◽  
Vol 292 (3) ◽  
pp. H1352-H1363 ◽  
Author(s):  
Lioubov I. Brueggemann ◽  
Christopher J. Moran ◽  
John A. Barakat ◽  
Jay Z. Yeh ◽  
Leanne L. Cribbs ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Action Potential ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Kinase C ◽  
A7r5 Cells ◽  
Dependent Inhibition

[Arg8]-vasopressin (AVP), at low concentrations (10–500 pM), stimulates oscillations in intracellular Ca2+ concentration (Ca2+ spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K+ (Kv) channels are crucial steps in this process. In the present study, Kv currents ( IKv) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of IKv. This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive IKv was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 μM) and XE-991 (10 μM) and enhanced by the KCNQ channel activator flupirtine (10 μM). BaCl2 (100 μM) or linopirdine (5 μM) mimicked the effects of AVP on K+ currents, AP generation, and Ca2+ spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in IKv in A7r5 cells. IKv was also inhibited in response to the PKC activator 4β-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of IKv by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca2+ spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.

scholarly journals Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists

2013 ◽  
Vol 305 (6) ◽  
pp. C609-C622 ◽  
Author(s):  
Barry D. Kyle ◽  
Eamonn Bradley ◽  
Roddy Large ◽  
Gerard P. Sergeant ◽  
Noel G. McHale ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Action Potentials ◽  
Muscle Cells ◽  
Current Clamp ◽  
Patch Clamp Technique ◽  
Intracellular Ca ◽  
Urethral Smooth Muscle ◽  
Perforated Patch Clamp

We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K+ (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca2+, resulting from ryanodine receptor (RyR) activation via Ca2+-induced Ca2+ release, triggered by Ca2+ influx through L-type Ca2+ (CaV) channels. Carbachol inhibited tBK current by reducing Ca2+ influx and Ca2+ release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca2+ was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd2+ (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca2+ release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca2+ release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy- N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone.

scholarly journals Ca2+ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells

2010 ◽  
Vol 299 (2) ◽  
pp. C279-C288 ◽  
Author(s):  
Albert L. Gonzales ◽  
Gregory C. Amberg ◽  
Scott Earley
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Cerebral Artery ◽  
Perforated Patch ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Trisphosphate Receptor ◽  
Perforated Patch Clamp

The melastatin transient receptor potential (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. Activation of the channel is Ca2+-dependent, but prolonged exposure to high (>1 μM) levels of intracellular Ca2+ causes rapid (within ∼2 min) desensitization of TRPM4 currents under conventional whole cell and inside-out patch-clamp conditions. The goal of the present study was to establish a novel method to record sustained TRPM4 currents in smooth muscle cells under near-physiological conditions. Using the amphotericin B-perforated patch-clamp technique, we recorded and characterized sustained (up to 30 min) transient inward cation currents (TICCs) in freshly isolated cerebral artery myocytes. In symmetrical cation solutions, TICCs reversed at 0 mV and had an apparent unitary conductance of 25 pS. Replacement of extracellular Na+ with the nonpermeable cation N-methyl-d-glucamine abolished the current. TICC activity was attenuated by the TRPM4 blockers fluflenamic acid and 9-phenanthrol. Selective silencing of TRPM4 expression using small interfering RNA diminished TICC activity, suggesting that the molecular identity of the responsible ion channel is TRPM4. We used the perforated patch-clamp method to test the hypothesis that TRPM4 is activated by intracellular Ca2+ signaling events. We found that TICC activity is independent of Ca2+ influx and ryanodine receptor activity but is attenuated by sarco(endo)plasmic reticulum Ca2+-ATPase inhibition and blockade of inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the sarcoplasmic reticulum. Our findings suggest that TRPM4 channels in cerebral artery myocytes are regulated by Ca2+ release from inositol 1,4,5-trisphosphate receptor on the sarcoplasmic reticulum.

Clearance of store-released Ca2+ by the Na+-Ca2+ exchanger is diminished in aortic smooth muscle from Na+-K+-ATPase α2-isoform gene-ablated mice

2008 ◽  
Vol 294 (3) ◽  
pp. H1407-H1416 ◽  
Author(s):  
Ronald M. Lynch ◽  
Craig S. Weber ◽  
Kevin D. Nullmeyer ◽  
Edwin D. W. Moore ◽  
Richard J. Paul
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Cyclopiazonic Acid ◽  
Selective Inhibition ◽  
Wild Type ◽  
Aortic Smooth Muscle ◽  
Plasmic Reticulum ◽  
Free Media

Two α-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The α1-isoform is proposed to serve a cytosolic housekeeping role, whereas the α2-isoform modulates Ca2+ storage via coupling to the Na+-Ca2+ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+ transporters to Ca2+ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase α2-isoform gene-ablated, homozygous null knockout (α2-KO) mice. Ca2+ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+ influx. Ca2+ clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+ efflux. In α2-KO VSMCs, preferential clearance of store-released Ca2+ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the α2-isoform (0.5 μM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not α2-KO mice. A subsequent analysis of capacitative Ca2+ entry (CCE) indicated that the magnitude of Ca2+ influx was significantly greater in α2-KO cells. Our findings support the concept of a subsarcolemmal space where the α2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Sorghum Fermented by Aspergillus oryzae NK Enhances Inhibition of Vascular Inflammation in TNF-α-stimulated Human Aortic Smooth Muscle Cells

2018 ◽  
Vol 88 (5-6) ◽  
pp. 309-318
Author(s):  
Hae Seong Song ◽  
Jung-Eun Kwon ◽  
Hyun Jin Baek ◽  
Chang Won Kim ◽  
Hyelin Jeon ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Inflammatory Response ◽  
Smooth Muscle Cells ◽  
Adhesion Molecule ◽  
Vascular Inflammation ◽  
Muscle Cells ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Tnf Α ◽  
Cox 2

Abstract. Sorghum bicolor L. Moench is widely grown all over the world for food and feed. The effects of sorghum extracts on general inflammation have been previously studied, but its anti-vascular inflammatory effects are unknown. Therefore, this study investigated the anti-vascular inflammation effects of sorghum extract (SBE) and fermented extract of sorghum (fSBE) on human aortic smooth muscle cells (HASMCs). After the cytotoxicity test of the sorghum extract, a series of experiments were conducted. The inhibition effects of SBE and fSBE on the inflammatory response and adhesion molecule expression were measured using treatment with tumor necrosis factor-α (TNF-α), a crucial promoter for the development of atherosclerotic lesions, on HASMCs. After TNF-α (10 ng/mL) treatment for 2 h, then SBE and fSBE (100 and 200 μg/mL) were applied for 12h. Western blotting analysis showed that the expression of vascular cell adhesion molecule-1 (VCAM-1) (2.4-fold) and cyclooxygenase-2 (COX-2) (6.7-fold) decreased, and heme oxygenase-1 (HO-1) (3.5-fold) increased compared to the TNF-α control when treated with 200 μg/mL fSBE (P<0.05). In addition, the fSBE significantly increased the expression of HO-1 and significantly decreased the expression of VCAM-1 and COX-2 compared to the TNF-α control in mRNA level (P<0.05). These reasons of results might be due to the increased concentrations of procyanidin B1 (about 6-fold) and C1 (about 30-fold) produced through fermentation with Aspergillus oryzae NK for 48 h, at 37 °C. Overall, the results demonstrated that fSBE enhanced the inhibition of the inflammatory response and adherent molecule expression in HASMCs.

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