Skip Navigation

Cardiovascular Research 2007 76(1):61-70; doi:10.1016/j.cardiores.2007.05.020
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Sampson, L. J.
Right arrow Articles by Dart, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sampson, L. J.
Right arrow Articles by Dart, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2007, European Society of Cardiology

Angiotensin II-activated protein kinase C targets caveolae to inhibit aortic ATP-sensitive potassium channels

Laura J. Sampsonb, Lowri M. Daviesa, Richard Barrett-Jolleyc, Nick B. Standenb and Caroline Darta,*

aBiosciences Building, School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom
bDepartment of Cell Physiology ... Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, United Kingdom
cPreclinical Veterinary Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZJ, United Kingdom

*Corresponding author. Tel.: +44 151 795 4462; fax: +44 151 795 4404. c.dart{at}liv.ac.uk

Received 27 July 2006; revised 27 April 2007; accepted 15 May 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective The vasoconstrictor angiotensin II (Ang II) acts at Gq/11-coupled receptors to suppress ATP-sensitive potassium (KATP) channel activity via activation of protein kinase C (PKC). The aim of this study was to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic KATP channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels.

Methods and results We show that the inhibitory effect of Ang II on pinacidil-evoked whole-cell rat aortic KATP currents persists in the presence of Gö6976, an inhibitor of the conventional PKC isoforms, but is abolished by intracellular dialysis of a selective PKC{eta} translocation inhibitor peptide. This suggests that PKC-dependent inhibition of aortic KATP channels by Ang II arises exclusively from the activation and translocation of PKC{eta}. Using discontinuous sucrose density gradients and Western blot analysis, we show that Ang II induces the translocation of PKC{eta} to cholesterol-enriched rat aortic smooth muscle membrane fractions containing both caveolin, a protein found exclusively in caveolae, and Kir6.1, the pore-forming subunit of the vascular KATP channel. Immunogold electron microscopy of rat aortic smooth muscle plasma membrane sheets confirms both the presence of Kir6.1 in morphologically identifiable regions of the membrane rich in caveolin and Ang II-evoked migration of PKC{eta} to these membrane compartments.

Conclusions Ang II induces the recruitment of the novel PKC isoform, PKC{eta}, to arterial smooth muscle caveolae. This translocation allows PKC{eta} access to KATP channels compartmentalized within these specialized membrane microdomains and highlights a potential role for caveolae in targeting PKC isozymes to an ion channel effector.

KEYWORDS Angiotensin; K-ATP channel; Protein kinase C; Smooth muscle; Arteries; Caveolae; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Angiotensin II (Ang II) is a potent endogenous vasoconstrictor that elicits contraction of vascular smooth muscle via multiple pathways including enhanced Ca2+ influx across the sarcolemma, release of Ca2+ from intracellular stores and sensitization of the contractile machinery to Ca2+ [1]. Enhanced Ca2+ influx occurs via direct activation of Ca2+-permeable channels as well as indirect voltage-dependent elevation of Ca2+ channel activity through membrane depolarization [2–4]. Ang II has been shown to depolarize vascular smooth muscle via the activation of non-selective inward cation currents [5], or the suppression of outward currents mediated by large conductance Ca2+-activated K+ channels [6,7], voltage-dependent K+ channels [8,9] or ATP-sensitive K+(KATP) channels [10,11].

Arterial KATP channels are known to be important regulators of membrane potential and thus vascular tone at rest and in response to vasoactive transmitters [12–14]. Functional studies in animals and man highlight the role of KATP channels in regulating blood flow, with blockade of KATP channels increasing vascular resistance in the systemic and coronary circulations, and KATP channel activity contributing substantially to changes in vasomotor tone and blood flow in response to exercise [15–17]. Ang II, endothelin, noradrenaline, histamine and serotonin (5HT2) act at Gq/11-coupled receptors to inhibit KATP channels via activation of protein kinase C (PKC) [10,18–20]. While this is believed to be an important regulatory mechanism, the PKC isoforms involved and the mechanism/s by which PKC targets KATP channels are poorly understood.

The 12 closely related isoenzymes that make up the PKC family share extensive structural homology and show limited substrate specificity in vitro [21]. Within the cell however, PKC isozymes mediate unique cellular functions by phosphorylating specific subsets of target proteins. Such substrate specificity is most likely conferred by differential targeting of activated PKC isoforms to distinct subcellular locations governed by the distribution of their preferred substrate [22–24]. We have shown recently that vascular KATP channels localize primarily to small vesicular invaginations of the plasma membrane termed caveolae [25]. These specialized lipid microdomains comprise approximately 20% of the smooth muscle cell's total surface area and may act to generate subcellular signalling compartments by recruiting interacting signalling proteins [26–29]. Indeed, caveolae have been identified as potential focal points for PKC signalling, with receptor-driven loss [29] or recruitment [30] of PKC isoforms to these distinct membrane regions. Here we use a combination of electrophysiological, biochemical and electron microscopy techniques to investigate whether Ang II induces the recruitment of specific PKC isoforms to smooth muscle caveolae to modulate KATP channel activity.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Thoracic aorta was obtained from adult male Wistar rats (~200 g) killed by stunning and rapid cervical dislocation. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Antibodies, polyacrylamide gel electrophoresis and immunoblotting
The following primary antibodies were used: anti-PKC{alpha}, β, {gamma}, {delta}, {eta}, {iota}/{lambda}, anti-caveolin (BD Transduction Laboratories), anti-PKC{theta} (sc-212), anti-Kir6.1 (R-14; sc-11224) (Santa Cruz Biotechnology Inc), anti-transferrin receptor (Zymed Laboratories Inc), anti-smooth muscle {alpha}-actin (Sigma-Aldrich). Horseradish peroxidise (HRP)-conjugated secondary antibodies were all from Jackson Immunochemical Laboratories with the exception of HRP-conjugated anti-goat which was from Sigma-Aldrich. Gold-conjugated secondary antibodies for use in immunogold electron microscopy were from British Biocell International. Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide–Tris gels and transferred electrophoretically onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia Biotech). Immunoblotting was carried out as described previously [31].

2.3 Membrane fractionation
Rat thoracic aortae were incubated in the presence and absence of phorbol 12-myristate-13-acetate (PMA; 300 nM; 30 min, room temperature). Stimulated tissues were transferred to ice-cold lysis buffer (mM: 25 Tris, 250 NaCl, 3 EDTA, 1% Triton X-100) and disrupted rapidly in a hand-held homogenizer. Lysed samples were spun in a Beckman bench-top ultracentrifuge (30,000 rpm, 30 min) and samples from soluble and particulate fractions analysed by SDS-polyacrylamide gel electrophoresis and immunoblotting.

2.4 Fractionation of caveolin-enriched membrane and assay for cholesterol
Buoyant caveolae-enriched membrane fractions were isolated under detergent-free conditions from rat aortic smooth muscle cell homogenates by ultracentrifugation on discontinuous sucrose gradients using a method adapted from [32] as previously described [25]. Cholesterol levels in each fraction collected from the gradient were determined by analysis with the Amplex Red cholesterol assay kit (Molecular Probes) according to the manufacturer's protocol.

2.5 Electron microscopy of immunogold-labelled smooth muscle plasma membrane sheets
Plasma membrane sheets from cultured aortic smooth muscle cells (2–4 days in primary culture) were prepared, fixed and labelled with primary and gold-conjugated secondary antibodies as previously described [33]. Membrane sheets were subsequently imaged using a FEI 120 kV Tecnai G 2 Spirit BioTWIN transmission electron microscope. NIH ImageJ software was used to measure distances between gold particles. Macros within ImageJ were also used to simulate random particle distributions on EM images.

2.6 Electrophysiology
Whole-cell K+ currents were recorded from freshly dissociated rat aortic smooth muscle cells using an Axopatch 200B amplifier (Axon Instruments) as previously described [25]. The pipette-filling solution contained (mM): 110 KCl, 30 KOH, 10 HEPES, 10 EGTA, 1 MgCl2, 1 CaCl2 adjusted to pH 7.2. In conventional whole-cell experiments 1 Na2ATP, 0.1 ADP, 0.5 GTP was additionally added to the pipette-filling solution. For perforated patch experiments, amphotericin B (Sigma-Aldrich; stock concentration 30 mg/mL in dimethylsulphoxide (DMSO)) was diluted to a working concentration of 210 µg/mL in pipette-filling solution. The 6 mM K+ extracellular solution contained (mM): 134 NaCl, 6 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 10 glucose; adjusted to pH 7.4. 140 mM K+ extracellular solution contained (mM): 140 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 10 glucose; pH 7.4. Pinacidil, angiotensin II and glibenclamide were from Sigma-Aldrich. Rp-cAMPS (the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt); Gö6976, PKC inhibitor peptide 20-28 (previously named 19-27) and PKC{eta} translocation inhibitor peptide were from Calbiochem. Experiments were conducted in a temperature-controlled bath at 25 °C. Results are expressed as means±S.E.M. Statistical significance was evaluated using ANOVA followed by Tukey–Kramer multicomparison test (StatsDirect, Cheshire, UK).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
We have shown previously that Ang II inhibits KATP channels in rat mesenteric artery via a dual mechanism involving the inhibition of cAMP-dependent protein kinase (PKA) and the activation of PKC [11]. To establish that PKC is similarly involved in the modulation of KATP currents in rat aorta (a preparation more amenable to biochemical analysis due to the relatively large amounts of tissue it yields), we assessed the effect of Ang II on pinacidil-evoked whole-cell KATP current in the presence of specific and non-specific PKC inhibitors. These experiments were conducted in the presence of 100 µM of the PKA inhibitor Rp-cAMPS to remove any complicating effects of vasoconstrictor inhibition of PKA. Under control conditions, the addition of 100 nM Ang II caused a 30.6±3.3% inhibition (mean±SEM; n=6) of whole-cell current induced by the KATP channel opener pinacidil in cells isolated from rat aortic artery (Fig. 1A, D). The inhibitory effect of Ang II was abolished in the presence of the non-selective PKC inhibitor peptide 20-28 (Ang II inhibition 1.2±1.0%, n=5, p<0.0001, Fig. 1B, D), indicating that in the absence of PKA activity Ang II-induced inhibition of the channel occurs via the activation of PKC. Inclusion in the pipette-filling solution of 50 µM of a selective PKC{eta} translocation inhibitor peptide similarly rendered the pinacidil-evoked KATP current insensitive to inhibition by Ang II (Ang II inhibition 1.4±1.1%, n=6, p<0.0001, Fig. 1C, D). This suggests that the novel PKC isoform, PKC{eta}, is primarily involved in Ang II-induced inhibition of aortic KATP channels. To allow dialysis of the PKC{eta} inhibitor peptide into cells these experiments were conducted in the conventional whole-cell recording configuration with intracellular Ca2+ buffered by the presence of 10 mM EGTA. These recording conditions ([Ca2+]i<20 nM) could potentially inhibit the activity of the Ca2+-sensitive PKC isoforms ({alpha}, β and {gamma}) leading to an underestimation of their involvement in channel inhibition [34]. We therefore undertook perforated patch experiments (which allow receptor-driven changes in intracellular Ca2+) to assess the involvement of these conventional isoforms in Ang II-induced channel modulation. In perforated patch recordings, the presence of 1 µM of the conventional PKC isoform inhibitor Gö6976 had no effect upon the ability of Ang II to inhibit whole-cell pinacidil-induced currents (control Ang II inhibition 34.8±5.1%; Ang II inhibition in the presence of Gö6976 36.0±2.9%, n=6, 6, Fig. 2A, B, C). These findings indicate that PKC-dependent inhibition of aortic KATP channels by Ang II arises exclusively from the activation and translocation of PKC{eta}.


Figure 1
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Representative whole-cell currents recorded from single isolated rat aortic smooth muscle cells held at –60 mV under control conditions (A), in the presence of 100 µM of the non-specific PKC inhibitor peptide 20-28 (B), or following intracellular dialysis with 50 µM of PKC{eta} translocation inhibitor peptide (C). Experiments were conducted in the presence of 100 µM of the PKA inhibitor Rp-cAMPS to block any PKA-dependent effects on channel activity. Myristoylated PKC inhibitor peptide 20-28 was applied in the extracellular solution for 10 min prior to application of Ang II. In all recordings, the cells were dialysed with a 140 mM K+/1 mM ATP intracellular solution. At a point indicated by the vertical arrow, the extracellular solution was changed from 6 mM K+ to 140 mM K+ to increase the inward driving force for K+. Pinacidil (10 µM), Ang II (100 nM) and the KATP channel blocker, glibenclamide (10 µM) were applied as indicated. D, Mean inhibition by Ang II in experiments like those of A–C. (n=6, 5, 6 cells, **p<0.0001, ANOVA followed by Tukey–Kramer multicomparison test).

 

Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Perforated patch recordings of whole-cell currents from single isolated rat aortic smooth muscle cells held at –60 mV under control conditions (A) or in the presence of 1 µM of the conventional PKC isoform inhibitor Gö6976 (B). C, Mean inhibition by Ang II in experiments like those of A–B. (n=6, 6 cells, no significant difference).

 
To confirm our functional data, we investigated the expression and translocation properties of PKC isoforms in aortic smooth muscle. Typically the PKC isoenzymes {alpha}, β, {delta}, {eta} and {zeta} coexist in systemic vascular smooth muscle, although expression patterns vary depending upon species and vascular bed [35]. Using Western blot analysis of tissue homogenates we were able to detect all of these isoforms in rat aorta with the exception of PKC{zeta}, which we were unable to identify conclusively with our range of antibodies (Fig. 3A). Of the conventional, Ca2+- and DAG-sensitive isoforms, PKC{alpha} and low levels of PKCβ were present. Of the DAG-sensitive but Ca2+-insensitive novel isoforms we detected robust signals for PKC{delta} and {eta} and also the skeletal muscle isoform PKC{theta}, which has been reported in airways [36,37] and cultured porcine aortic smooth muscle [38]. Additionally, we saw a strong expression of the atypical Ca2+- and DAG-insensitive PKC{iota}/{lambda} isoform, which has also previously been identified in pulmonary [39], colonic circular [40] and cultured porcine aortic smooth muscle [38].


Figure 3
View larger version (52K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 A. Western blot analysis of rat aortic tissue homogenates showing expression of PKC{alpha}, β, {delta}, {eta}, {theta}, {iota}/{lambda}. B. Exposure to 300 nM PMA induces a redistribution of PKC{alpha}, {delta} and {eta} from cytosolic (C) and membrane (M) fractions. {alpha}-actin was used as a loading control.

 
Translocation to different cellular compartments enables PKC to colocalize with both activators and substrates. We thus assessed the ability of the strongly expressed aortic PKC isoforms to migrate to bulk membrane fractions in response to activation by the tumour-promoting phorbol ester, phorbal-12-myristate-13-acetate (PMA). Western blot analysis of cytosolic and membrane fractions separated by high-speed centrifugation of rat aortic smooth muscle cells showed a marked redistribution of PKC{alpha}, {delta} and {eta} from cytosol to membrane following stimulation by PMA (300 nM; Fig. 3B). The cellular distribution of DAG (and hence phorbol ester)-insensitive isoforms PKC{iota}/{lambda} was unchanged by exposure to PMA.

Since vascular KATP channels localize primarily to caveolae [25], we would predict that isoforms that modulate channel activity, such as PKC{eta}, would specifically target these membrane compartments. The cholesterol and sphingolipid-enriched composition of caveolae gives them distinct biochemical properties– namely a highly reduced ‘buoyant’ density – that allows them to be separated from the bulk of the plasma membrane. We isolated buoyant membrane fractions under detergent-free conditions from rat aortic smooth muscle cell homogenates by ultracentrifugation on discontinuous sucrose gradients [25]. To assess the purity of caveolar and non-caveolar fractions, we used Western blot analysis to determine the distribution of specific caveolar and non-caveolar marker proteins and an assay to determine the level of cholesterol within each fraction. Western blot analysis of five 2 ml fractions collected from top to bottom of the sucrose density gradient showed the caveolae-specific protein caveolin-1 predominantly in fraction 2 (Fig. 4A), suggesting this represents the buoyant caveolae-containing layer. Consistent with this, cholesterol was high in upper fractions 1 and 2 (Fig. 4B). In contrast, transferrin receptor, a plasma membrane protein that is excluded from caveolae, localized to the low-cholesterol layers 3 and 4 of the gradient (Fig. 4A). Under non-stimulated control conditions, the majority of PKC was confined to the lower layers of the gradient, which correspond to both non-caveolar membrane and cytosolic fractions (Fig. 4C). A small proportion of PKC{alpha} and {eta} was associated with the caveolar layer at rest, while PKC{delta} and {iota}/{lambda} were largely excluded from these buoyant upper fractions. Exposure to 300 nM PMA induced a significant shift in the distribution of both PKC{alpha} and {eta} from the lower layers of the sucrose gradient to the caveolin-enriched membrane fractions. We observed a smaller shift in the distribution of PKC{delta}, but saw essentially no change in the distribution of the phorbol-insensitive {iota}/{lambda} isoforms (Fig. 4C, lower panels). Treatment with 4{alpha}-PMA, an inactive isoform of PMA, had no effect on the PKC distribution within the sucrose gradient.


Figure 4
View larger version (45K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 A, Western blot analysis of each of five 2 ml fractions collected from the top to the bottom of a discontinuous sucrose density gradient to determine the distribution of the caveolae-marker, caveolin-1 (top panel) and the non-caveolar transferrin receptor (lower panel). B, Relative cholesterol levels in each of five 2 ml fractions shown in A above. C, Exposure to 300 nM PMA induces a marked shift in the distribution of PKC{alpha}, {delta} and {eta} from the lower layers of the sucrose gradient to the buoyant caveolin-enriched fractions. Treatment with 4{alpha}-PMA, an inactive isoform of PMA, had no effect on the distribution of PKC isoforms within the sucrose gradient.

 
To assess the effects of a physiological vasoconstrictor we repeated the above experiments in the presence and absence of Ang II. Western blot analysis revealed a significant shift in the distribution of both PKC{alpha} and PKC{eta} from the lower layers of the sucrose gradient to the buoyant caveolin-enriched fractions following exposure to 300 nM Ang II (Fig. 5A, B). These upper layers also contain the pore-forming subunit of the KATP channel, which we have previously shown coimmunoprecipitates with the caveolae-specific protein caveolin [25]. In contrast, there was no statistically significant Ang II-induced movement of either PKC{delta} or PKC{iota}/{lambda} to caveolae.


Figure 5
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 A, Western blot analysis reveals a significant shift in the distribution of PKC{alpha} and PKC{eta} from the lower layers of the sucrose gradient to buoyant upper fractions containing caveolin and Kir6.1 following exposure to 300 nM Ang II. B, Mean ratios of PKC isoforms in caveolae/non-caveolae layers (layer 2/layer 4) under control conditions (open bars) or following stimulation with 300 nM Ang II (closed bars). Ratio of PKC{alpha} in caveolae/non-caveolae layers: unstimulated=0.04±0.01; Ang II-treated=0.42±0.07, n=3, *p<0.05. Ratio of PKC{eta} in caveolae/non-caveolae layers: unstimulated=0.20±0.03; Ang II-treated=0.49±0.09, n=3, *p<0.05.

 
To verify the location of KATP channels within caveolae and the Ang II-induced migration of PKC{eta} to these membrane microdomains we undertook immunogold electron microscopy. Aortic smooth muscle cells were plated onto poly-L-lysine-coated coverslips and allowed to adhere and grow to approximately 70% confluence. Plasma membrane sheets were ripped off these adherent cells directly onto EM grids as previously described [33]. The membrane sheets were fixed, labelled with primary antibodies (against Kir6.1 and/or caveolin, or PKC{eta} and caveolin) and the location of these proteins visualized by the addition of appropriate secondary antibodies conjugated with gold particles of either 5 or 10 nm. Fig. 6A shows a transmission electron microscope (TEM) image of an aortic smooth muscle cell membrane sheet. Caveolae are identifiable as ‘donut-shaped— circular structures clustered in a line running horizontally across the centre of the image. Labelling of the membrane sheet with antibodies against the caveolae-marker caveolin confirms these structures as caveolae (Fig. 6B). By co-staining the membrane sheets with antibodies against caveolin and Kir6.1, the pore-forming subunit of vascular KATP channels, we were able to visualize KATP channels within these membrane compartments (Fig. 6C). Measurement of the distance between gold particles corresponding to Kir6.1 (5 nm) and gold particles corresponding to caveolin (10 nm) was used to quantify the association of these proteins. Our results indicate that approximately 70% of Kir6.1 particles are found within 50 nm of caveolin particles (Fig. 6D; 67.8±3.1%; 1065 measurements made over 14 separate images). To assess the significance of this distribution we looked at what would be expected if Kir6.1 particles were randomly distributed. In simulated random distributions of approximately the same number of particles over the same images only 6.7±2.4% of particles were found to be within 50 nm of caveolin (933 measurements of randomly generated particles over 14 separate images, p<0.0001). Importantly, in control experiments no labelling was observed if the secondary antibodies were applied alone, and no cross-reactivity was seen with any of the secondary antibodies. These data therefore support our previous findings that vascular KATP channels localize to smooth muscle caveolae [25]. To confirm that Ang II induces the recruitment PKC{eta} to these membrane microdomains we labelled membrane sheets with antibodies against both PKC{eta} and caveolin under control conditions and following stimulation with Ang II. In unstimulated control experiments we saw limited co-localization of PKC{eta} and caveolin particles, with only 11.0±3.0% of PKC within 50 nm of caveolin (Fig. 6E i; 223 measurements made over 9 separate images). Membrane sheets ripped from cells that had been preincubated with 300 nM Ang II however exhibited a marked change in PKC{eta} distribution with 34.8±3.7% of PKC{eta} particles found within 50 nm (Fig. 6E ii; 210 measurements made over 11 separate images, p<0.0005), and clear labelling of morphologically identifiable caveolae with PKC{eta} (Fig. 6E ii inset).


Figure 6
View larger version (69K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 A, TEM image of an aortic smooth muscle cell membrane sheet. Caveolae appear as circular structures. A network of membrane-associated cytoskeleton can also been seen in the image. B, Areas of aortic smooth muscle cell membrane sheet showing morphologically identifiable caveolae labelled with rabbit anti-caveolin and 10 nm gold-conjugated anti-rabbit secondary antibodies. C, Morphologically identifiable caveolae co-labelled with rabbit anti-caveolin (with 10 nm gold-conjugated secondary) and goat anti-Kir6.1 (5 nm gold-conjugated secondary). D, Histogram showing the distribution of distances measured between caveolin and Kir6.1 particles (see text for details). E, Histograms showing distribution of distances between caveolin and PKC{eta} particles under unstimulated control conditions (i), and following stimulation with 300 nM Ang II (ii). Inset shows a membrane sheet from an Ang II-stimulated cell labelled with rabbit anti-caveolin (10 nm gold-conjugated secondary) and mouse anti-PKC{eta} (5 nm gold-conjugated secondary).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study we set out to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic KATP channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels. We show that multiple PKC isoenzymes ({alpha}, β, {delta}, {eta}, {theta} and {iota}/{lambda}) coexist in rat aortic smooth muscle. These isozymes have been shown to differ in their cofactor requirement for activation, their subcellular localization and the intracellular proteins they phosphorylate [41,42]. Such substrate specificity is apparent even amongst the family of K+ channel proteins. This and previous studies [9,11] indicate that PKC{eta} is involved in Ang II-induced inhibition of both KATP and voltage-dependent K+(Kv) channels, while other reports show that PKC{alpha} mediates Ang II inhibition of inwardly rectifying K+(Kir) channels in coronary arterial cells [43] and PKC{zeta} thromboxane-A2 suppression of Kv currents in the pulmonary artery [44]. Since PKC isozymes share considerable structural homology and demonstrate only poor substrate discrimination in in vitro assays it has been suggested that this in vivo specificity arises from differential targeting of activated PKC isoforms to distinct intracellular locations determined by the distribution of their substrate [22–24]. We have shown previously that vascular KATP channels localize primarily to cholesterol-enriched invaginations of the plasma membrane termed caveolae [25] that are believed to form subcellular signalling ‘pockets’ by recruiting interacting signalling molecules [26–28]. These findings are supported by the present study where immunogold electron microscopy of aortic smooth muscle plasma membrane sheets confirms the presence of Kir6.1 in regions of the membrane morphologically identifiable as caveolae and which label with the caveolae-marker caveolin. To test the idea that PKC isoforms responsible for the modulation of KATP channel activity are recruited to these membrane compartments, we isolated buoyant caveolar membranes from rat aortic smooth muscle cells under unstimulated control conditions and following exposure to Ang II. Western blot analysis revealed a significant shift in the distribution of PKC{eta} to cholesterol-enriched fractions containing caveolin following exposure to Ang II. This Ang II-evoked migration of PKC{eta} to caveolae was supported by immunogold TEM of Ang II-stimulated rat aortic smooth muscle cell membranes dual labelled with caveolin and PKC{eta}. Thus, our findings indicate that PKC-dependent modulation of aortic KATP channels by Ang II arises from the activation and translocation of PKC{eta} to smooth muscle caveolae to allow PKC{eta} access to KATP channels compartmentalized within these specialized membrane domains.

Interestingly, our biochemical data suggest that Ang II also induces the translocation of PKC{alpha} to caveolae. Functionally, we find that only PKC{eta} modulates aortic KATP channel activity since the inhibitory effect of Ang II on pinacidil-evoked whole-cell KATP currents persists in the presence of Gö6976, an inhibitor of the conventional PKC isoforms ({alpha}, β, {gamma}), but is abolished by the selective PKC{eta} translocation inhibitor peptide. One possible explanation for this is that PKC{alpha} and {eta} target separate populations of caveolae each containing a different compliment of signalling proteins. Only a proportion of caveolae would be expected to contain KATP channel proteins, for example, since there are only a few hundred KATP channels within the cell [45,46] and an estimated 170000 caveolae [47]. Selective targeting of a particular subset of caveolae by different PKC isozymes is an intriguing idea and could potentially work by different caveolae sequestering specific PKC targeting proteins. Proteins implicated in PKC targeting within cells include (i) substrates that interact with PKC (STICKs) such as annexins and multivalent kinase anchoring proteins (AKAP5/AKAP79, AKAP12/gravin); (ii) non-substrate targeting proteins (RACKs), and (iii) cytoskeletal or structural proteins such as caveolin [23,24]. We have demonstrated previously the involvement of an unidentified AKAP in PKA-dependent activation of vascular KATP channels [48], indicating that these anchoring proteins may be responsible for the localization of kinases in the vicinity of the channel. Annexins are another targeting candidate, particularly since annexin II has recently been shown to associate in a Ca2+-dependent manner with cholesterol and sphingolipid-enriched regions of the membrane [49]. RACKs seem a less likely means by which PKC would target caveolae since we (unpublished observations) and others [30,50] have shown that both RACK1 and RACK2 are largely excluded from caveolar membranes. Finally, structural proteins such as caveolins themselves may play a role in PKC targeting since caveolin isoforms have been shown to interact with many caveolae-localized signalling molecules, including selected PKC isozymes [51–53] and the pore-forming subunit of the KATP channel [25].

In conclusion, our data highlight a potential role for caveolae in vasoconstrictor-mediated targeting of specific PKC isoforms. Elevated plasma triglycerides and cholesterol, an essential component of caveolae, often accompany hypertension and alterations in membrane composition, fluidity and caveolar morphology have been reported in hypertensive humans and several animal models of hypertension [54,55]. A more complete understanding of the physiological roles of these cholesterol-enriched microdomains may help explain some of the alterations in smooth muscle signalling and contractility seen in conditions such as hypercholesterolaemia [56,57].

Time for primary review 25 days


   Acknowledgements
 
We thank the British Heart Foundation and The Royal Society for their support and Dr Ian Prior, Connie Muncke and Alison Beckett of the EM Unit, University of Liverpool for help and advice with TEM and plasma membrane rip-offs.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Touyz R.M., Schiffrin E.L. Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells. Pharmacol Rev (2000) 52:639–672.[Abstract/Free Full Text]
  2. Arnaudeau S., MacrezLepretre N., Mironneau J. Activation of calcium sparks by angiotensin II in vascular myocytes. Biochem Biophys Res Commun (1996) 222:809–815.[CrossRef][Web of Science][Medline]
  3. Lu H.K., Fern R.J., Luthin D., Linden J., Liu L.P., Cohen C.J., et al. Angiotensin II stimulates T-type Ca2+ channel currents via activation of a G protein, G(i). Am J Physiol Cell Physiol (1996) 40:C1340–C1349.
  4. Nelson M.T., Patlak J.B., Worley J.F., Standen N.B. Calcium channels, potassium channels, and voltage dependence of arterial smooth-muscle tone. Am J Physiol (1990) 259:C3–C18.[Web of Science][Medline]
  5. Hughes A.D., Bolton T.B. Action of angiotensin-II, 5-hydroxytryptamine and adenosine-triphosphate on ionic currents in single ear artery cells of the rabbit. Br J Pharmacol (1995) 116:2148–2154.[Web of Science][Medline]
  6. Minami K., Hirata Y., Tokumura A., Nagaya Y., Fukuzawa K. Protein kinase C-independent inhibition of the Ca2+-activated K+ channel by angiotensin-II and endothelin-1. Biochem Pharmacol (1995) 49:1051–1056.[CrossRef][Web of Science][Medline]
  7. Toro L., Amador M., Stefani E. Ang-II inhibits calcium-activated potassium channels from coronary smooth-muscle in lipid bilayers. Am J Physiol (1990) 258:H912–H915.[Web of Science][Medline]
  8. ClementChomienne O., Walsh M.P., Cole W.C. Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes. J Physiol (Lond) (1996) 495:689–700.[Abstract/Free Full Text]
  9. Hayabuchi Y., Standen N.B., Davies N.W. Angiotensin II inhibits and alters kinetics of voltage-gated K+ channels of rat arterial smooth muscle. Am J Physiol Heart Circ Physiol (2001) 281:H2480–H2489.[Abstract/Free Full Text]
  10. Kubo M., Quayle J.M., Standen N.B. Angiotensin II inhibition of ATP-sensitive K+ currents in rat arterial smooth muscle cells through protein kinase C. J Physiol (Lond) (1997) 503:489–496.[Abstract/Free Full Text]
  11. Hayabuchi Y., Davies N.W., Standen N.B. Angiotensin II inhibits rat arterial K-ATP channels by inhibiting steady-state protein kinase A activity and activating protein kinase C{eta}. J Physiol (Lond) (2001) 530:193–205.[Abstract/Free Full Text]
  12. Quayle J.M., Nelson M.T., Standen N.B. ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol Rev (1997) 77:1165–1232.[Abstract/Free Full Text]
  13. Clapp L.H., Tinker A. Potassium channels in the vasculature. Curr Opin Nephrol Hypertens (1998) 7:91–98.[Web of Science][Medline]
  14. Yokoshiki H., Sunagawa M., Seki T., Sperelakis N. ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. Am J Physiol Cell Physiol (1998) 43:C25–C37.
  15. Samaha F.F., Heineman F.W., Ince C., Fleming J., Balaban R.S. ATP-sensitive potassium channel is essential to maintain basal coronary vascular tone in vivo. Am J Physiol (1992) 262:C1220–C1227.[Web of Science][Medline]
  16. Goto K., Fujii K., Abe I., Fujishima M. Sympathetic control of arterial membrane potential by ATP-sensitive K+-channels. Hypertension (2000) 35:379–384.[Abstract/Free Full Text]
  17. Duncker D.J., Oei H.H., Hu F., Stubenitsky R., Verdouw P.D. Role of K-ATP(+) channels in regulation of systemic, pulmonary, and coronary vasomotor tone in exercising swine. Am J Physiol Heart Circ Physiol (2001) 280:H22–H33.[Abstract/Free Full Text]
  18. Kleppisch T., Nelson M.T. ATP-sensitive K+ currents in cerebral arterial smooth-muscle- pharmacological and hormonal modulation. Am J Physiol Heart Circ Physiol (1995) 38:H1634–H1640.
  19. Bonev A.D., Nelson M.T. Vasoconstrictors inhibit ATP-sensitive K+ channels in arterial smooth muscle through protein kinase C. J Gen Physiol (1996) 108:315–323.[Abstract/Free Full Text]
  20. Park W.S., Ko E.A., Han J., Kim N., Earm Y.E. Endothelin-1 acts via protein kinase C to block K-ATP channels in rabbit coronary and pulmonary arterial smooth muscle cells. J Cardiovasc Pharmacol (2005) 45:99–108.[CrossRef][Web of Science][Medline]
  21. Parker P.J., Kour G., Marais R.M., Mitchell F., Pears C., Schaap D., et al. Protein Kinase-C- A Family Affair. Mol Cell Endocrinol (1989) 65:1–11.[CrossRef][Web of Science][Medline]
  22. Schechtman D., Mochly-Rosen D. Adaptor proteins in protein kinase C-mediated signal transduction. Oncogene (2001) 20:6339–6347.[CrossRef][Web of Science][Medline]
  23. Jaken S., Parker P.J. Protein kinase C binding partners. BioEssays (2000) 22:245–254.[CrossRef][Web of Science][Medline]
  24. Poole A.A.W., Pwa G., Hers M., Crosby D., Jones M.L. PKC-interacting proteins: from function to pharmacology. Trends Pharmacol Sci (2004) 25:528–535.[CrossRef][Medline]
  25. Sampson L.J., Hayabuchi Y., Standen N.B., Dart C. Caveolae localize protein kinase A signaling to arterial ATP-sensitive potassium channels. Circ Res (2004) 95:1012–1018.[Abstract/Free Full Text]
  26. Lisanti M.P., Scherer P.E., Vidugiriene J., Tang Z.L., Hermanowskivosatka A., Tu Y.H., et al. Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source — implications for human-disease. J Cell Biol (1994) 126:111–126.[Abstract/Free Full Text]
  27. Razani B., Woodman S.E., Lisanti M.P. Caveolae: from cell biology to animal physiology. Pharmacol Rev (2002) 54:431–467.[Abstract/Free Full Text]
  28. Foster L.J., de Hoog C.L., Mann M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A (2003) 100:5813–5818.[Abstract/Free Full Text]
  29. Smart E.J., Ying Y.S., Anderson R.G.W. Hormonal-regulation of caveolae internalization. J Cell Biol (1995) 131:929–938.[Abstract/Free Full Text]
  30. Rybin V.O., Xu X.H., Steinberg S.F. Activated protein kinase C isoforms target to cardiomyocyte caveolae — stimulation of local protein phosphorylation. Circ Res (1999) 84:980–988.[Abstract/Free Full Text]
  31. Sampson L.J., Leyland M.L., Dart C. Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel, Kir2.1. J Biol Chem (2003) 278:41988–41997.[Abstract/Free Full Text]
  32. Song K.S., Li S.W., Okamoto T., Quilliam L.A., Sargiacomo M., Lisanti M.P. Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains — detergent-free purification of caveolae membranes. J Biol Chem (1996) 27:9690–9697.
  33. Prior IA, Parton RG, Hancock JF. Observing cell surface signalling domains using electron microscopy. Science STKE 2003, 2003: pI9.
  34. Maasch C., Wagner S., Lindschau C., Alexander G., Buchner K., Gollasch M., et al. Protein kinase C alpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+](i). FASEB J (2000) 14:1653–1663.[Abstract/Free Full Text]
  35. Salamanca D.A., Khalil R.A. Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension. Biochem Pharmacol (2005) 70:1537–1547.[CrossRef][Web of Science][Medline]
  36. Donnelly R., Yang K., Omary M.B., Azhar S., Black J.L. Expression of multiple isoenzymes of protein-kinase-C in airway smooth-muscle. Am J Resir Cell Mol Biol (1995) 13:253–256.
  37. Webb B.L.J., Lindsay M.A., Seybold J., Brand N.J., Yacoub M.H., Haddad E.B., et al. Identification of the protein kinase C isoenzymes in human lung and airways smooth muscle at the protein and mRNA level. Biochem Pharmacol (1997) 54:199–205.[CrossRef][Web of Science][Medline]
  38. Yano K.J., Bauchat J.R., Liimatta M.B., Clemmons D.R., Duan C.M. Down-regulation of protein kinase C inhibits insulin-like growth factor I-induced vascular smooth muscle cell proliferation, migration, and gene expression. Endocrinology (1999) 140:4622–4632.[Abstract/Free Full Text]
  39. Damron D.S., Nadim H.S., Hong S.J., Darvish A., Murray P.A. Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II. Am J Physiol Lung Cell Mol Physiol (1998) 18:L278–L288.
  40. Ali I., Sarna S.K. Selective modulation of PKC isozymes by inflammation in canine colonic circular muscle cells. Gastroenterology (2002) 122:483–494.[CrossRef][Web of Science][Medline]
  41. Newton A.C. Protein kinase C: Structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev (2001) 101:2353–2364.[CrossRef][Web of Science][Medline]
  42. Parker P.J., Murray-Rust J. PKC at a glance. J Cell Sci (2004) 11:131–132.
  43. Park W.S., Kim N., Youm J.B., Warda M., Ko J.H., Kim S.J., et al. Angiotensin II inhibits inward rectifier K+ channels in rabbit coronary arterial smooth muscle cells through protein kinase C alpha. Biochem Biophys Res Commun (2006) 341:728–735.[CrossRef][Web of Science][Medline]
  44. Cogolludo A., Moreno L., Bosca L., Tamargo J., Perez-Vizcaino F. Thromboxane A(2)-induced inhibition of voltage-gated K+ channels and pulmonary vasoconstriction — role of protein kinase C zeta. Circ Res (2003) 93:656–663.[Abstract/Free Full Text]
  45. Bychkov R., Gollasch M., Ried C., Luft F.C., Haller H. Effects of pinacidil on K+ channels in human coronary artery vascular smooth muscle cells. Am J Physiol Cell Physiol (1997) 42:C161–C171.
  46. Bonev A.D., Nelson M.T. ATP-sensitive potassium channels in smooth-muscle cells from guinea-pig urinary-bladder. Am J Physiol (1993) 264:C1190–C1200.[Web of Science][Medline]
  47. Gabella G. Quantitative morphological-study of smooth-muscle cells of guinea-pig taenia-coli. Cell Tissue Res (1976) 170:161–186.[CrossRef][Web of Science][Medline]
  48. Hayabuchi Y., Dart C., Standen N.B. Evidence for involvement of A-kinase anchoring protein in activation of rat arterial K-ATP channels by protein kinase A. J Physiol (Lond) (2001) 536:421–427.[Abstract/Free Full Text]
  49. Draeger A., Wray S., Babiychuk E.B. Domain architecture of the smooth-muscle plasma membrane: regulation by annexins. Biochem J (2005) 387:309–314.[CrossRef][Web of Science][Medline]
  50. Mineo C., Ying Y.S., Chapline C., Jaken S., Anderson R.G.W. Targeting of protein kinase C alpha to caveolae. J Cell Biol (1998) 141:601–610.[Abstract/Free Full Text]
  51. Li S.W., Couet J., Lisanti M.P. Src tyrosine kinases, G(alpha) subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin — caveolin binding negatively regulates the auto-activation of Src tyrosine kinases. J Biol Chem (1996) 271:29182–29190.[Abstract/Free Full Text]
  52. Oka N., Yamamoto M., Schwencke C., Kawabe J., Ebina T., Ohno S., et al. Caveolin interaction with protein kinase C-isoenzyme-dependent regulation of kinase activity by the caveolin scaffolding domain peptide. J Biol Chem (1997) 272:33416–33421.[Abstract/Free Full Text]
  53. Taggart M.J., Leavis P., Feron O., Morgan K.G. Inhibition of PKC alpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res (2000) 258:72–81.[CrossRef][Web of Science][Medline]
  54. Darblade B., Caillaud D., Poirot M., Fouques M.J., Thiers J.C., Rami J., et al. (2001) Alteration of plasmalemmal caveolae mimics endothelial dysfunction observed in hypercholesterolemic rabbit aorta. J Hypertens (2001) 19:A14–A15.
  55. Zicha J., Kunes J., Devynck M.A. Abnormalities of membrane function and lipid metabolism in hypertension— a review. Am J Hypertens (1999) 12:315–331.[CrossRef][Web of Science][Medline]
  56. Mathew V., Cannan C.R., Miller V.M., Barber D.A., Hasdai D., Schwartz R.S., et al. Enhanced endothelin-mediated coronary vasoconstriction and attenuated basal nitric oxide activity in experimental hypercholesterolemia. Circulation (1997) 96:1930–1936.[Abstract/Free Full Text]
  57. Merkel L.A., Bilder G.E. Modulation of vascular reactivity by vasoactive peptides in aortic rings from hypercholesterolemic rabbits. Eur J Pharmacol (1992) 222:175–179.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
R. D. Rainbow, R. I. Norman, D. E. Everitt, J. L. Brignell, N. W. Davies, and N. B. Standen
Endothelin-I and angiotensin II inhibit arterial voltage-gated K+ channels through different protein kinase C isoenzymes
Cardiovasc Res, June 5, 2009; (2009) cvp143v2.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
V. Garg, J. Jiao, and K. Hu
Regulation of ATP-sensitive K+ channels by caveolin-enriched microdomains in cardiac myocytes
Cardiovasc Res, April 1, 2009; 82(1): 51 - 58.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
S. Li, P. Duan, and G. You
Regulation of human organic anion transporter 1 by ANG II: involvement of protein kinase C{alpha}
Am J Physiol Endocrinol Metab, February 1, 2009; 296(2): E378 - E383.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
C. P. Nelson, J. M. Willets, N. W. Davies, R. A. J. Challiss, and N. B. Standen
Visualizing the temporal effects of vasoconstrictors on PKC translocation and Ca2+ signaling in single resistance arterial smooth muscle cells
Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1590 - C1601.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
J. Jiao, V. Garg, B. Yang, T. S. Elton, and K. Hu
Protein Kinase C-{epsilon} Induces Caveolin-Dependent Internalization of Vascular Adenosine 5'-Triphosphate-Sensitive K+ Channels
Hypertension, September 1, 2008; 52(3): 499 - 506.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
W. F. Jackson
Vanishing Act: Protein Kinase C-Dependent Internalization of Adenosine 5'-Triphosphate-Sensitive K+ Channels
Hypertension, September 1, 2008; 52(3): 470 - 472.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S.-K. Cha, T. Wu, and C.-L. Huang
Protein kinase C inhibits caveolae-mediated endocytosis of TRPV5
Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1212 - F1221.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Sampson, L. J.
Right arrow Articles by Dart, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sampson, L. J.
Right arrow Articles by Dart, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?