Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC

2002 ◽  
Vol 283 (6) ◽  
pp. C1627-C1636 ◽  
Author(s):  
Juming Zhong ◽  
Ge-Xin Wang ◽  
William J. Hatton ◽  
Ilia A. Yamboliev ◽  
Michael P. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Swelling ◽  
Detectable Effect ◽  
Anion Channels ◽  
Inhibitory Peptide ◽  
Endogenous Protein ◽  
Membrane Bound ◽  
Hypotonic Cell Swelling

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl− currents ( I Cl.vol) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.1 μM) or by hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 μM) or Ro-31–8425 (0.1 μM), inhibitors of both conventional and novel PKC isozymes, significantly activated I Cl.vol and prevented further modulation by subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 μM), a selective conventional PKC inhibitor, was without effect. Dialyzing acutely dispersed and cultured PASMC with εV1–2 (10 μM), a translocation inhibitory peptide derived from the V1 region of εPKC, activated I Cl.vol under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with βC2–2 (10 μM), a translocation inhibitory peptide derived from the C2 region of βPKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of εPKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound εPKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.

Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells

2003 ◽  
Vol 285 (4) ◽  
pp. H1453-H1463 ◽  
Author(s):  
Ge-Xin Wang ◽  
William J. Hatton ◽  
Grace L. Wang ◽  
Juming Zhong ◽  
Ilia Yamboliev ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Molecular Mass ◽  
Cardiac Myocytes ◽  
Muscle Cells ◽  
Cell Swelling ◽  
Atrial Myocytes ◽  
Anion Channels ◽  
Immunoreactive Band

Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxy terminal antibody (Alm C592–661 antibody), although recent studies have raised questions related to the specificity of Alm C592–661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea pig cardiac myocytes. These new antibodies produced a common prominent immunoreactive band with an apparent molecular mass of 90–92 kDa in the guinea pig heart and PASMCs, and a similar molecular mass immunoreactive band was observed in the brain from homozgygous Clcn3+/+ mice but not from homozygous Clcn3–/– mice. VSOACs elicited by hypotonic cell swelling in PASMCs and guinea pig atrial myocytes were nearly completely abolished by intracellular dialysis with two new anti-ClC-3 antibodies specifically targeting the ClC-3 carboxy (C670–687 antibody) and amino terminus (A1–14 antibody). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, because 1) preabsorption of the antibodies with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new antibody raised against an extracellular epitope (Ex133–148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an antibody targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea pig atrial myocytes, and 4) anti-ClC-3 C670–687 antibody had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current in guinea pig ventricular myocytes, although VSOACs in the same cells were inhibited by the antibody. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea pig cardiac myocytes.

Abstract 507: G Proteins are Required for Lipoxygenase EDHF Activity of Rabbit Arterial Smooth Muscle Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Kathryn M Gauthier ◽  
J. R Falck ◽  
William B Campbell
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
G Proteins ◽  
Muscle Cells ◽  
Specific Structure ◽  
Inhibitory Peptide ◽  
Receptor Binding Site ◽  
Channel Activation ◽  
Sk Channel ◽  
Vascular Activity

Arachidonic acid 15-lipoxygenase (15-LO) metabolites function as endothelium-derived hyperpolarizing factors in rabbit and human arteries. In rabbit arteries, LO metabolites mediate nitric-oxide and prostaglandin-independent relaxations to acetylcholine and AA. Previously, we characterized 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) as a major vasoactive 15-LO metabolite in rabbit arteries. 11,12,15-THETA requires a specific structure for vascular activity. 11(R),12(S),15(S)-THETA causes concentration-related relaxation whereas 11(R),12(R),15(S)-THETA is without activity. The specific structure requirement suggests a role for a receptor. Therefore, we examined the role of G proteins in 11(R),12(S),15(S)-THETA vascular activity. Western immunoblot verified protein expression of Gαs, Gαi and a Gαo in rabbit endothelial and smooth muscle cells. 11(R),12(S),15(S)-THETA increased GTPγ35S binding to rabbit arterial membranes 280±25% while 11(R),12(S),15(S)-THETA was without effect. In cell-attached patches of rabbit smooth muscle, 11(R),12(S),15(S)-THETA (100 nM) increased mean open time of apamin-sensitive, calcium-activated, small conductance potassium (SK) channels from 0.0001±0.0001 to 0.0015±0.0006. In inside-out patches, 11(R),12(S),15(S)-THETA did not increase channel opening (0.0001±0.0001) unless GTP was present (0.0051±0.0023). In the presence of GTP, an antibody against Gαs and a Gαs inhibitory peptide inhibited 11(R),12(S),15(S)-THETA SK channel activation (0.0007±0.0005, 0.0013±0.0012, respectively) whereas an antibody against Gαi was without effect (0.0042±0.0018). A cell-permeant, penetratin-linked Gαs inhibitory peptide also inhibited 11(R),12(S),15(S)-THETA SK channel activation in cell-attached patches (0.0005±0.0002) and blocked 11(R),12(S),15(S)-THETA relaxations in rabbit aorta (max relaxations = 74±6%, 23±7% for control and permeant peptide, respectively). These studies indicate that 11,12,15-THETA-induced SK channel activation and vascular relaxation are mediated by a Gs-coupled mechanism and that 11,12,15-THETA acts via a stereo-specific G protein coupled receptor/binding site.

scholarly journals Caveolin-1 and caveolin-3 regulate Ca2+ homeostasis of single smooth muscle cells from rat cerebral resistance arteries

2007 ◽  
Vol 293 (1) ◽  
pp. H204-H214 ◽  
Author(s):  
T. Kamishima ◽  
T. Burdyga ◽  
J. A. Gallagher ◽  
J. M. Quayle
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Removal Rate ◽  
Muscle Cells ◽  
Amino Acid Residues ◽  
Resistance Arteries ◽  
Inhibitory Peptide ◽  
Caveolin 1 ◽  
Domain Peptide ◽  
Cerebral Resistance

The role of caveolins, signature proteins of caveolae, in arterial Ca2+ regulation is unknown. We investigated modulation of Ca2+ homeostasis by caveolin-1 and caveolin-3 using smooth muscle cells from rat cerebral resistance arteries. Membrane current and Ca2+ transients were simultaneously measured with voltage-clamped single cells. Membrane depolarization triggered Ca2+ current and increased intracellular Ca2+ concentration ([Ca2+]i). After repolarization, elevated [Ca2+]i returned to the resting level. Ca2+ removal rate was determined from the declining phase of the Ca2+ transient. Application of caveolin-1 antibody or caveolin-1 scaffolding domain peptide, corresponding to amino acid residues 82–101 of caveolin-1, significantly slowed Ca2+ removal rate at a measured [Ca2+]i of 250 nM, with little effect at a measured [Ca2+]i of 600 nM. Application of caveolin-3 antibody or caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55–74 of caveolin-3, also significantly slowed Ca2+ removal rate at a measured [Ca2+]i of 250 nM, with little effect at a measured [Ca2+]i of 600 nM. Likewise, application of calmodulin inhibitory peptide, autocamtide-2-related inhibitory peptide, and cyclosporine A, inhibitors for calmodulin, Ca2+/calmodulin-dependent protein kinase II, and calcineurin, also significantly inhibited Ca2+ removal rate at a measured [Ca2+]i of 250 nM but not at 600 nM. Application of cyclopiazonic acid, a sarcoplasmic reticulum Ca2+ ATPase inhibitor, also significantly inhibited Ca2+ removal rate at a measured [Ca2+]i of 250 nM but not at 600 nM. Our results suggest that caveolin-1 and caveolin-3 are important in Ca2+ removal of resistance artery smooth muscle cells.

Bradykinin and angiotensin II: activation of protein kinase C in arterial smooth muscle

1994 ◽  
Vol 266 (5) ◽  
pp. C1406-C1420 ◽  
Author(s):  
B. S. Dixon ◽  
R. V. Sharma ◽  
T. Dickerson ◽  
J. Fortune
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Ang Ii ◽  
Kinase C ◽  
Membrane Bound

The effects of bradykinin (BK) and angiotensin II (ANG II) were compared in cultured rat mesenteric arterial smooth muscle cells. BK and ANG II activated a phosphoinositide-specific phospholipase C, leading to the rapid release of [3H]inositol phosphates, an increase in intracellular calcium, and formation of sn-1,2-diacylglycerol (DAG). DAG formation was biphasic with a transient peak at 5 s followed by a sustained increase from 60 to 600 s. The BK-mediated increases in inositol triphosphate and DAG were dose dependent with half-maximal increases at concentrations of 5 and 2 nM, respectively. Both hormones were found to activate protein kinase C (PKC) as assessed by phosphorylation of the 68- to 72-kDa intracellular PKC substrate myristoylated alanine-rich C kinase substrate. However, despite similar phosphorylation of this substrate, only ANG II produced a significant increase in membrane-bound PKC activity. The mechanism accounting for the inability of BK to increase membrane-bound PKC activity is unclear. Our studies excluded differential translocation of PKC to the nuclear membrane, production of an inhibitor of membrane-bound PKC activity, and expression of BK and ANG II receptors on different cells as the mechanism. Vascular smooth muscle cells were found to express at least four different PKC isozymes: alpha, delta, zeta, and a faint band for epsilon. All of the isozymes except zeta-PKC were translocated by treatment with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. However, neither ANG II nor BK produced significant translocation of any measured isozyme; therefore, we could not exclude the possibility that ANG II and BK activate different isozymes of PKC. Both hormones were found to have a similar small and inconsistent effect in stimulating [3H]thymidine incorporation. These observations demonstrate that BK and ANG II have similar biochemical effects on vascular smooth muscle cells and imply that, in selected vessels, the vasodilatory effects of BK mediated by the endothelium may be partially counterbalanced by a vasoconstrictor effect on the underlying vascular smooth muscle cells.

Properties of a Cl−current activated by cell swelling in rabbit portal vein vascular smooth muscle cells

1998 ◽  
Vol 275 (5) ◽  
pp. H1524-H1532 ◽  
Author(s):  
I. A. Greenwood ◽  
W. A. Large
Keyword(s):  
Smooth Muscle ◽  
Portal Vein ◽  
Smooth Muscle Cells ◽  
Physiological Role ◽  
External Solution ◽  
Muscle Cells ◽  
Cell Swelling ◽  
Equilibrium Potential ◽  
Dependent Manner ◽  
Rabbit Portal Vein

In rabbit portal vein smooth muscle cells, application of a hypotonic external solution caused cell swelling and evoked an outwardly rectifying Cl− current. The hypotonicity-activated current was markedly reduced by the anti-estrogen tamoxifen (10 μM) and was inhibited by DIDS in a voltage-dependent manner [the concentration required to inhibit the current by 50% (IC50) at −50 and +100 mV was 21 and 5 μM DIDS, respectively]. Indanyloxyacetic acid 94 (IAA-94) and niflumic acid also inhibited the hypotonicity-activated current, with 50% inhibition produced at concentrations of ∼200 and 100 μM, respectively. In isotonic conditions, application of tamoxifen and DIDS to cells decreased the holding current due to the inhibition of a resting conductance that was outwardly rectifying and reversed at the Cl− equilibrium potential. These data show that rabbit portal vein myocytes have a resting Cl− conductance that is enhanced by cell swelling; its possible physiological role is discussed.

scholarly journals Agonist-induced calcium regulation in freshly isolated renal microvascular smooth muscle cells.

1997 ◽  
Vol 8 (4) ◽  
pp. 569-579
Author(s):  
E W Inscho ◽  
M J Mason ◽  
A C Schroeder ◽  
P C Deichmann ◽  
K D Stiegler ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Control Level ◽  
Muscle Cells ◽  
Detectable Effect ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Transduction Mechanisms ◽  
K Concentration

The studies presented here were performed to determine the effect of agonist stimulation on the cytosolic free Ca2+ concentration ([Ca2+]i) in single smooth muscle cells, freshly isolated from afferent arterioles and interlobular arteries averaging between 10 to 40 microns in diameter. Microvessels were obtained from male Sprague-Dawley rats using an iron oxide collection technique followed by collagenase digestion. Freshly isolated microvascular smooth muscle cells (MVSMC) were loaded with fura 2 and studied using fluorescence photometry techniques. The resting [Ca2+]i averaged 67 +/- 3 nM (N = 82 cells). Increasing the extracellular K+ concentration significantly increased [Ca2+]i dose-dependently (P < 0.05). Involvement of extracellular Ca2+ in the response to KCl-induced depolarization was also evaluated. Resting [Ca2+]i increased approximately 132% from 40 +/- 5 nM to 93 +/- 26 nM in response to 90 mM extracellular KCl. This change was abolished in nominally Ca(2+)-free conditions and markedly attenuated by diltiazem. Inhibition of K+ channels with charybdotoxin or tetraethylammonium chloride produced a modest transient increase in [Ca2+]i during the response to 30 mM K+ and had no detectable effect on responses to 90 mM K+. Studies were also performed to establish whether freshly isolated renal MVSMC exhibit appropriate responses to receptor-dependent physiological agonists. Angiotensin II (100 nM) increased cell Ca2+ from 97 +/- 10 nM to 265 +/- 47 nM (N = 12 cells). Similarly, 100 microM ATP increased MVSMC [Ca2+]i from a control level of 71 +/- 14 nM to 251 +/- 47 nM (N = 11 cells). Norepinephrine administration caused [Ca2+]i to increase from 63 +/- 4 nM to 212 +/- 47 nM (N = six cells), and vasopressin increased [Ca2+]i from 86 +/- 10 nM to 352 +/- 79 nM (N = five cells). These data demonstrate that receptor-dependent and -independent vasoconstrictor agonists increase [Ca2+]i in MVSMC, freshly isolated from rat preglomerular vessels. Furthermore, the ability to measure [Ca2+]i in responses to physiological stimuli in these single cells permits investigation of signal transduction mechanisms involved in regulating renal microvascular resistance.

scholarly journals Role of phosphodiesterase isoenzymes in regulating intracellular cyclic AMP in adenosine-stimulated smooth muscle cells

1995 ◽  
Vol 305 (2) ◽  
pp. 627-633 ◽  
Author(s):  
Y Xiong ◽  
E W Westhead ◽  
L L Slakey
Keyword(s):  
Smooth Muscle ◽  
Cyclic Amp ◽  
Smooth Muscle Cells ◽  
Soluble Fraction ◽  
Muscle Cells ◽  
Total Activity ◽  
Cytosolic Fraction ◽  
Rapid Fall ◽  
Membrane Bound ◽  
The Individual

Three phosphodiesterase (PDE) isoenzymes were separated by Mono Q h.p.l.c. column chromatography from the soluble fraction of a homogenate of pig aortic smooth muscle cells. The first peak of PDE activity was stimulated by calmodulin in the presence of calcium. The second broad peak contained at least two activities, which were sensitive to inhibition by CI-930 or rolipram respectively. The distribution of total cellular enzyme activity in different subcellular fractions was also determined. The majority (78%) of the total activity was present in the cytosolic fraction, 18% of activity was in a membrane-bound form and 4% of activity was associated with the cytoskeleton. Rolipram-sensitive PDE was present predominantly in the cytosolic fraction, whereas cyclic GMP-inhibited, CI-930-sensitive PDE was evenly distributed between the cytosolic and particulate fractions. All of the calmodulin-dependent PDE activity was found in the soluble fraction. CI-930 and rolipram enhanced, by 2-fold and 3-4-fold respectively, the adenosine-stimulated rise in cellular cyclic AMP level. The increase in cyclic AMP levels due to CI-930 or rolipram was dose-dependent. Removal of adenosine once cyclic AMP had risen resulted in a rapid fall in cyclic AMP levels even in the presence of rolipram and CI-930. M&B 22,948, the calmodulin-dependent PDE inhibitor, caused less than a 25% increase of the adenosine-stimulated cyclic AMP levels by itself, but it contributed substantially to controlling the cyclic AMP levels after the removal of adenosine when used together with CI-930 and rolipram. These phenomena suggested that all three PDE isoenzymes participated in modulating cellular cyclic AMP levels after adenosine stimulation, and that differential importance of the individual isoenzymes depends on cellular cyclic AMP levels.

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