HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 292/5/H2248    most recent 01170.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Clarke, C. J. Articles by Ohanian, J. Search for Related Content PubMed PubMed Citation Articles by Clarke, C. J. Articles by Ohanian, J.

Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase

The Cardiovascular Research Group, Division of Cardiovascular and Endocrine Sciences, Core Technology Facility, University of Manchester, Manchester, United Kingdom

Submitted 25 October 2006 ; accepted in final form 29 December 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP₂), PA, and DGK- are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [³³P]PIP₂ hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [³³P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK- in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [³³P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction.

signal transduction; vascular smooth muscle; lipid second messengers; phosphatidylinositol 3-kinase

Diacylglycerol kinases (DGKs) phosphorylate DG to produce phosphatidic acid (PA). In nonstimulated cells, their activity is low, allowing DG to be used for glycerolipid biosynthesis, but on activation of the PI pathway, DGK activity increases. This drives the conversion of DG generated by PI-PLC to PA, presumably inactivating PKC, although it could also sustain signaling by increasing PIP₂ formation (reviewed in Ref. 48). PA is also a bioactive lipid with several putative targets, such as sphingosine kinase 1 (11), Raf-1 (18, 41), phosphatases (24), and phosphoinositide 4-phosphate 5-kinase (23). Aside from interaction with protein effectors, PA may also play a role in signal transduction as a precursor of phosphoinositide lipids, necessary for replenishment of PI pools after agonist-stimulation of PLC (48).

It is well known that subcellular localization and targeting of signaling molecules are important for regulating signal transduction in biological systems, and a major mechanism occurs through interaction with lipids at specific membrane sites (reviewed in Ref. 46). However, there is relatively sparse information on the generation of lipids at specific membrane sites. Lipid rafts are plasma membrane microdomains enriched in cholesterol and sphingolipids. Although biochemically similar, caveolae are a subset of lipid rafts identified by their characteristic flask-shaped morphology and the presence of caveolins. Caveolae/lipid rafts are implicated in signal transduction, owing to their high content of signaling proteins such as G protein-coupled receptors (GPCRs), heterotrimeric and small G proteins, and PKC (40). However, differences in morphology and associated proteins suggest that these domains may serve distinct or indeed complementary functions. Caveolae/lipid rafts are abundant in smooth muscle and endothelial cells and have been implicated in VSM contraction (2, 5, 25). In nonmuscle cells, PI lipids have been identified in lipid rafts (20, 38) and in A431 cells, and PIP₂ hydrolysis in response to GPCR agonists and growth factors occurs solely at these sites (38). Furthermore, a recent study in platelets reported the presence of DGK activity in lipid rafts and showed a thrombin-stimulated increase in PA levels in these domains (8), demonstrating agonist-dependent PA generation within lipid rafts. These studies suggest that caveolae/rafts may be important sites for signaling through the PI pathway.

The aim of this study was to investigate whether vasoconstrictor agonist activation of PI signaling was spatially restricted within plasma membrane microdomains in vascular tissue. We show that norepinephrine (NE) and endothelin-1 (ET-1) induce PIP₂ hydrolysis solely within caveolae/rafts of rat mesenteric small arteries (RMSAs), leading to activation of DGK and generation of PA in these domains. Furthermore, inhibition of DGK activation and reduction of PA production within these domains correlated with reduced sensitivity of contraction, suggesting that localization of PI signaling to caveolae/rafts is linked to the regulation of VSM contractility.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The investigation was carried out in accordance with The University of Manchester Animal Experimentation Guidelines and the UK Animals (Scientific Procedures) Act 1986. Experiments were performed with the approval of the Review Board of the University of Manchester and the Home Office.

Animals and incubation conditions. Adult female Sprague-Dawley rats (8–10 wk of age, 180–220 g body wt) were used for all experiments. The mesentery artery was excised and placed in ice-cold physiological salt solution. Mesenteric small arteries (internal diameter <400 µm) were cleaned of adherent fat and connective tissue and dissected from the mesenteric bed. Unless stated otherwise, arteries were equilibrated in 1 ml of tissue culture medium M199 (GIBCO) for 1 h at 37°C before stimulation with NE (15 µM) or ET-1 (100 nM) for various time points.

Isolation of caveolae/raft microdomains. Caveolae/raft-enriched microdomains were purified from RMSAs according to the method of Song et al. (45) with the following modifications. Arteries were homogenized in 500 µl of 0.5 M Na₂CO₃ (pH 11) in ground glass homogenizers and further homogenized sequentially with an Omni homogenizer (3 x 10 s, 20,000 rpm at 10-s intervals) and sonication (3 x 20 s at 20-s intervals); all steps were carried out on ice. Aliquots of homogenate were removed for protein estimation by the Bradford Assay (9). A 450-µl aliquot of homogenate, protein concentration 1 mg/ml, was mixed with 450 µl 80% sucrose in MES-buffered saline [MBS; 25 mM MES (pH 6.5) and 0.15 M NaCl] and placed at the bottom of a polyallomer centrifuge tube. A discontinuous sucrose gradient was formed by layering 700 µl of 35% sucrose in MBS + 0.25 M Na₂CO₃ (pH 11) on top, followed by 625 µl of 5% sucrose in MBS + 0.25 M Na₂CO₃ (pH 11), and centrifuging at 55,000 rpm (160,000 g) for 16 h at 4°C (TLS-55 rotor, Beckman TL100 centrifuge). From the top of the gradient, 13 fractions of 175 µl were removed. The noncaveolae/raft pellet was removed and resuspended in fraction 13.

Immunoblotting. Proteins in the individual fractions were precipitated with 5% trichloroacetic acid and processed for SDS-PAGE and Western blot analysis with the appropriate primary antibody. Signals were developed by horseradish peroxidase-conjugated secondary antibody and chemiluminescence (Pierce). Signal intensity was quantified by densitometry (Bio-Rad GS800 Densitometer), and images showing saturation were not used in the analysis.

Ganglioside-GM₁ detection. The ganglioside GM₁ was detected using horseradish peroxidase-conjugated -cholera toxin as described previously (47).

Measurement of endogenously labeled phospholipids. RMSA phospholipids were labeled with ³³P_i as previously described (37), and tissue was processed for caveolae/raft separation.

Lipid extraction. Lipids were extracted according to the method of Bligh and Dyer (7). To each gradient fraction, 1 ml of chloroform-methanol (1:1 vol/vol) was added, and samples were agitated and left on ice for 10 min. Distilled H₂O (dH₂O; 200 µl) was added, and the mixture was agitated and centrifuged at 13,000 rpm for 5 min to render the extract biphasic. Organic layers were transferred to glass vials, dried with O₂-free N₂ gas, and reconstituted in 15 µl chloroform-methanol (1:1 vol/vol).

Thin-layer chromatography. Lipids were separated by one-dimensional TLC on 0.25-mm-thick Silica Gel 60 plates. For cholesterol, plates were developed in chloroform-methanol-acetic acid-formic acid-dH₂O (70:30:12:4:2 vol/vol/vol/vol/vol) followed by hexane-diisopropylether-acetic acid (130:70:4 vol/vol) (28). To visualize cholesterol, plates were stained with 8% cupric acetate in 3% phosphoric acid and heated to 100°C for 30 min. [³³P]PIP₂ was separated from other ³³P-labeled phospholipids on oxalate-coated TLC plates (37) and developed in chloroform-methanol-ammonium hydroxide-dH₂O (17:13.2:2.8:1 vol/vol/vol/vol) (19). PA was separated as described previously (37). Radiolabeled lipids were visualized by electronic autoradiography (InstantImager, Perkin Elmer) and identified by use of cochromatographed standards stained with I₂ vapour.

Assay of DGK activity. DGK activity at the membrane was extracted and assayed using the n-octyl--D-glucopyranoside (OBG)-mixed micelle assay as previously described for rat subcutaneous small arteries (35). Bradford assays (17) were performed on extracted samples to ensure that equal protein amounts were used in each assay. Following sucrose density centrifugation, fractions were pooled as caveolae/rafts (fractions 2–5), noncaveolae/rafts 1 (fractions 6–9), and noncaveolae/rafts 2 (fractions 10–13). Membranes were recovered by diluting pooled fractions threefold in 0.2 M MBS [pH 6.5; 25 mM MES (pH 6.5) and 0.15 M NaCl] and centrifuged at 100,000 g for 2 h. Concentrated membrane pellets were washed in 0.2 M MES (pH 6.0) and resuspended in DGK homogenization buffer, and DGK activity was extracted and assayed using the OBG-mixed micelle assay as previously described (35). Since protein levels were too low to measure in individual caveolae/raft fractions, samples were normalized by loading equal protein amounts onto sucrose gradients before centrifugation.

Measurement of contractile responses. Contractile responses of small arteries to NE and ET-1 were measured by using pressure myography. Briefly, segments of small artery (<350 µm ID) were cannulated and mounted in a Living Systems pressure myograph as described previously (34). Following equilibration for 45 min in physiological salt solution (pH 7.4, gassed with 5% CO₂-95% O₂) at 37°C and 20 mmHg intraluminal pressure, the intraluminal pressure was raised to 70 mmHg and the vessel was left to stabilize for a minimum of 15 min before construction of cumulative concentration response curves to ET-1 (0.03–300 nM) and NE (0.1–15 µM). Lumen diameter was measured 2 min following the addition of agonist, immediately before the addition of the next concentration. To study the effect of phosphatidylinositol 3-kinase (PI3-kinase) inhibition, arteries were incubated in 10 µM LY-294002 or 0.1% DMSO (vehicle) for 1 h before cumulative addition of NE or ET-1. NE concentration response curves in the presence of vehicle and inhibitor were obtained from a single vessel segment, for ET-1 individual segments were used for each treatment.

Materials. LY-294002 and ET-1 were purchased from Calbiochem, and TLC plates (Merck 5721) and "HiPerSolv" grade solvents were from VWR International (Leics, UK). [³³P]PO₄ (specific activity, 370 MBq/ml) was from Amersham International (Amersham, Bucks, UK). -[³²P]ATP was from MP Biomedicals. Monoclonal anti-caveolin-1 clone 2297 was from Transduction Laboratories. Polyclonal anti-PKB was purchased from Upstate, and polyclonal anti-DGK- was a gift from W. van Blitterswijk (Netherlands Cancer Institute, Amsterdam, The Netherlands). NE, PA standard, -Cholera toxin-horseradish peroxidase conjugate, and all other chemicals were supplied by Sigma Chemicals.

Statistical analysis. Comparisons between two groups were analyzed by Student's t-test. Repeated-measures ANOVA was used for comparison between multiple groups. P < 0.05 was considered statistically significant with n indicating the number of experiments.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Caveolae/raft isolation from RMSAs. Caveolae/lipid rafts are characterized by their buoyancy due to high-lipid content and enrichment with cholesterol (rafts/caveolae), caveolin-1 (caveolae), and the ganglioside GM₁ (rafts) (15, 26, 40). Accordingly, we used these markers to identify caveolae/raft fractions.

Caveolin-1, cholesterol, and GM₁ were predominantly localized to fractions 2–5 at the 5–35% sucrose interface (66 ± 5%, 44 ± 2%, and 54 ± 4% of total, respectively), identifying these fractions as enriched in caveolae/rafts (Fig. 1) in agreement with published data (45).

View larger version (30K): [in this window] [in a new window]   Fig. 1. Isolation of caveolae/rafts from rat mesenteric small arteries. Rat mesenteric small arteries (RMSAs) were processed for caveolae/raft isolation, and fraction content was analyzed for caveolin-1 (Cav-1), ganglioside GM1, and cholesterol as described in MATERIALS AND METHODS. A: Cav-1 immunoblot, GM1 dot-blot, and cholesterol TLC plate. B: densitometric data expressed as means ± SE %total marker. Results are representative of 4 separate experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Caveolae/rafts are enriched in [33P]phosphatidylinositol 4,5-bisphosphate (PIP2) and are sites of norepinephrine (NE) and endothelin 1 (ET or ET-1)-stimulated [33P]PIP2 hydrolysis. RMSAs were labeled with 33Pi, stimulated with NE (15 µM), ET-1 (100 nM), or distilled water (dH2O), and caveolae/rafts were isolated. Lipids were extracted from fractions, pooled as fractions 2–5 caveolae/raft, fractions 6–9 noncaveolae/raft-1, and fractions 10–13 noncaveolae/raft-2. [33P]PIP2 content was analyzed as described in MATERIALS AND METHODS. A: basal distribution of [33P]PIP2. Data are expressed as means ± SE %total [33P]PIP2 (*P < 0.05, n = 5 experiments). B: effect of NE on [33P]PIP2 levels. Data are expressed as means ± SE %basal PIP2 level in each of the individual fractions, where basal PIP2 = 100% (*P < 0.05 compared with basal, from a minimum of 4 separate experiments). C: effect of ET-1 on [33P]PIP2 levels. Data are expressed as means ± SE %basal PIP2 level in each of the individual fractions, where basal PIP2 = 100% (*P < 0.05 compared with basal, from a minimum of 5 separate experiments).

View larger version (12K): [in this window] [in a new window]   Fig. 3. Time course of NE and ET-1 stimulation of [33P]phosphatidic acid (PA). A: [33P]-labeled RMSAs were stimulated with 15 µM NE (n = 3 experiments) or 100 nM ET-1 (n = 5 experiments) for time points from 0–20 min, lipids were extracted, and [33P]PA was quantified as described in MATERIALS AND METHODS. Data are means ± SE [in counts/min (cpm)] normalized for total protein. B and C: [33P]-labeled RMSAs were stimulated with NE (15 µM; B) or ET-1 (100 nM; C) membrane fractions isolated and pooled as fractions 2–5 caveolae/raft, fractions 6–9 noncaveolae/raft-1, and fractions 10–13 noncaveolae/raft-2, and [33P]PA content was analyzed as described in MATERIALS AND METHODS. Data are means ± SE (in cpm), normalized for total protein loaded on the gradient (NE, n = 5 experiments; ET-1: n = 5 experiments, nonstimulated; n = 6 experiments, 20 s; and n = 4 experiments, 10 min). *P < 0.05.

View larger version (23K): [in this window] [in a new window]   Fig. 4. NE- and ET-1-induced [33P]PA in RMSAs is mediated by 1-adrenoceptors and ETA receptors, respectively. [33P]-labeled RMSAs were stimulated with phenylephrine (PE, 15 µM) for 5 min (n = 4 experiments), NE (15 µM) for 5 min (n = 5 experiments), incubated with prazosin (PZ, 10 µM) for 60 min before stimulation with NE (15 µM) for 5 min (n = 4 experiments), ET-1 (100 nM) for 10 min (n = 4 experiments), or incubated with BQ-123 (BQ, 10 µM) for 60 min before stimulation with ET-1 (100 nM, n = 4 experiments). Lipids were extracted; fractions were pooled as fractions 2–5 caveolae/raft, fractions 6–9 noncaveolae/raft-1, and fractions 10–13 noncaveolae/raft-2; and [33P]PA content was analyzed as described in MATERIALS AND METHODS. Data are means ± SE (in cpm), normalized for total protein loaded on the gradient. *P < 0.05.

View larger version (7K): [in this window] [in a new window]   Fig. 5. The effect of ET-1 on membrane-associated diacylglycerol (DG) kinase (DGK) activity. RMSAs were stimulated with ET-1 (100 nM) for time points from 0–10 min, and membrane-associated DGK activity was extracted and assayed as described in MATERIALS AND METHODS. Data are means ± SE DGK activity (in pmol PA·min–1·mg protein–1) *P < 0.05 compared with 0 min; n = 5 experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 6. The effect of phosphatidylinositol 3-kinase (PI3K) inhibition on NE and ET-1 stimulated DGK activity and [33P]PA production in caveolae/rafts and noncaveolae/rafts. A: immunoblot of DGK- distribution in sucrose density fractions 1–13, representative of 3 independent experiments. B: RMSAs were stimulated with vehicle (dH2O, 5 min, n = 5 experiments), ET-1 (100 nM, 5 min, n = 5 experiments), or NE (15 µM, 1 min, n = 5 experiments) or incubated for 1 h with LY-294002 (LY, 10 µM) before stimulation with NE (15 µM, 1 min, n = 4 experiments). Caveolae/rafts and noncaveolae/rafts were prepared and pooled as fractions 2–5 caveolae/raft, fractions 6–9 noncaveolae/raft-1, and fractions 10–13 noncaveolae/raft-2, and DGK activity was extracted and assayed as described in MATERIALS AND METHODS. Data are means ± SE (in pmol PA·min–1·10 µl extract–1). *P < 0.05 compared with vehicle. C: [33P]-labeled RMSAs were incubated in the absence or presence of 10 µM L7294002 for 60 min before stimulation with vehicle (dH2O, 5 min), NE (15 µM, 1 min), or ET-1 (100 nM, 5 min); membrane fractions were isolated; and [33P]PA content was analyzed as described in MATERIALS AND METHODS. Data are expressed as means ± SE (in cpm), normalized for total protein, from a minimum of 3 experiments. *P < 0.05, NE compared with LY + NE.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Distribution of PKB in membrane fractions. A: RMSAs were stimulated with vehicle (dH2O, 5 min), NE (15 µM, 1 min), or ET-1 (100 nM, 5 min), and PKB content in caveolae/rafts (fractions 2–5) was analyzed by immunoblot as described in MATERIALS AND METHODS. Numbers shown are densitometry data normalized to control. B: densitometry data of the PKB signal in fractions 2–5 caveolae/raft, fractions 6–9 noncaveolae/raft-1, and fractions 10–13 noncaveolae/raft-2 were expressed as means ± SE %total PKB from a minimum of 3 experiments. *P < 0.05 compared with control. C: RMSAs were incubated in the presence of 10 µM LY for 60 min before stimulation with NE (15 µM, 1 min), and PKB content in caveolae/rafts (fractions 2–5) was analyzed by immunoblot as described in MATERIALS AND METHODS. Numbers shown are densitometry data normalized to control. D: densitometry data of the PKB signal in fractions 2–5 caveolae/raft, fractions 6–9 noncaveolae/raft-1, and fractions 10–13 noncaveolae/raft-2 are expressed as means ± SE %total PKB from a minimum of 3 experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 8. The effect of PI3K inhibition on NE and ET-1-induced contraction. Segments of small artery were mounted in a pressure myograph, and lumen diameter was monitored continuously as described in MATERIALS AND METHODS. Vessels were treated with vehicle (0.01% DMSO) or 10 µM LY for 60 min before construction of a cumulative concentration response to NE (n = 8 experiments; A) or ET-1 (n = 4 experiments; B). Data are means ± SE. *P < 0.05 by ANOVA and Bonferroni posttest.

Mechanisms involved in NE- and ET-1-stimulated [³³P]PA formation in caveolae/rafts. We have previously shown that NE stimulation increases DGK activity in rat small arteries, resulting in the formation of PA (35, 37), and here we show that ET-1 also stimulates membrane-associated DGK activity (Fig. 5). Accordingly, we investigated whether DGK was involved in the production of [³³P]PA in caveolae/rafts following NE or ET-1 stimulation. In nonstimulated RMSAs, 24 ± 2% of DGK activity was localized to caveolae/rafts with 30 ± 2% and 47 ± 4% in noncaveolae/rafts-1 and -2, respectively. NE increased DGK activity 1.59 ± 0.28-fold in caveolae/rafts with no significant effect observed in noncaveolae/rafts, whereas ET-1 increased DGK activity 1.40 ± 0.18-fold in caveolae/rafts and 1.37 ± 0.12-fold in noncaveolae/rafts-1 with no significant effect observed in noncaveolae/rafts-2 (Fig. 6). Membrane-associated DGK activity measured after high-pH Na₂CO₃ extraction confirmed that the caveolae/raft extraction protocol had no effect on the magnitude of DGK activation (not shown).

DGKs exist as multiple isoforms, and in RMSAs, NE activates DGK- in a PI3-kinase-dependent manner (49). In this study we confirmed the presence of DGK- in caveolae/rafts (Fig. 6A) and pretreatment of RMSAs with 10 µM LY-294002, a concentration that blocks PI3-kinase activity in NE-stimulated RMSAs (49), inhibited NE-stimulated membrane-associated DGK activity (Table 1) that was localized to caveolae/rafts (Fig. 6B). Furthermore, PI3-kinase inhibition had no effect on basal [³³P]PA levels in any fraction but reduced NE-stimulated [³³P]PA levels in caveolae/rafts and noncaveolae/rafts-2 (Fig. 6C). However, LY-294002 had no effect on ET-1-stimulated DGK activity (Table 1) or [³³P]PA levels in any of the fractions (Fig. 6C).

Effect of PI3-kinase inhibition on contractile responses. To investigate the functional significance of PI3-kinase inhibition, the effects of LY-294002 on RMSA tension development in response to NE and ET-1 were studied. Cumulative concentration-response curves to ET-1 and NE were obtained in the presence and absence of LY-294002 (10 µM; Fig. 8). PI3-kinase inhibition decreased the contractile response to NE at submaximal concentrations (Fig. 8A) and increased the EC₅₀ from 2.12 ± 0.28 to 4.57 ± 0.47 µM (P < 0.05), demonstrating altered sensitivity. The effects of LY-294002 on maximal contraction (27% inhibition) were comparable with the effects of this inhibitor on [³³P]PA in caveolae/rafts (23% inhibition; Figs. 8A and 6B), showing a good agreement between PA levels and the contractile response. In contrast, PI3-kinase inhibition had no effect on ET-1-stimulated contraction (Fig. 8B), again agreeing with the lack of effect of LY-294002 on [³³P]PA levels with this agonist (Fig. 6B).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Caveolae/rafts are plasma membrane microdomains, abundant in smooth muscle and endothelial cells and implicated in signal transduction, owing to their enrichment with signaling proteins (26, 40). In this study, we have identified PIP₂ and PA in caveolae/rafts isolated from RMSAs and evaluated alterations in the levels of these lipids after NE and ET-1 stimulation. Additionally, we have demonstrated agonist-specific activation of DGK- and redistribution of PKB to these sites by NE but not ET-1. Our data suggest that, in vascular tissue, caveolae/rafts are sites of vasoconstrictor-induced activation of the PI signaling cascade and that the generation of lipid second messengers at these sites is necessary for modulating the contractile response.

Using a detergent-free method (45), we prepared buoyant membranes from RMSAs enriched in three caveolae/lipid rafts markers: cholesterol, caveolin-1, and GM₁. These fractions contained 52% of the total [³³P]PIP₂, in agreement with another study where [³H]inositol was used to label PIP₂ in A431 cells (20). However, we cannot say whether PIP₂ is preferentially localized in rafts or caveolae, because our preparation is a heterogeneous mix of these domains. However, a recent study reported that the PIP₂ present in caveolae represents <10% of the total plasma membrane PIP₂ (50), suggesting that, if concentrated in plasma membrane microdomains, PIP₂ is likely to be within lipid rafts.

We also found [³³P]PA (40 ± 2.4% of total) and DGK activity (24 ± 2% of total) present in caveolae/rafts. Given that the protein content of caveolae/rafts is markedly lower than noncaveolae/raft fractions (26, 45) and, indeed, was below detection levels in our preparation, this suggests that caveolae/rafts contain relatively high DGK activity in nonstimulated RMSAs. This is in good agreement with a study in platelets where DGK activity and PA were found in lipid rafts (8) but in contrast to a study in MDCK cells where DGK activity was absent from these domains (20). These differences could reflect our use of intact tissue versus cultured cells, DGK assay conditions, or conditions used for preparation of lipid rafts, because the composition of these fractions is critically dependent on the detergents and their concentrations used for extraction (1). However, given that PA is metabolized by lipid phosphate phosphohydrolases, the activity of which has been detected in caveolae from rat lung (31), this is indicative of caveolae/rafts being sites of PA production and metabolism in tissues, further strengthening the view that caveolae/rafts are sites of phospholipid signaling.

Agonist stimulation of PIP₂ hydrolysis and PA production in caveolae/rafts. There is considerable evidence that cells contain agonist-responsive and -unresponsive pools of PIs that are compartmentalized at the plasma membrane (reviewed in Ref. 29). This is considered to be an important regulatory mechanism, because although the most documented function of PIP₂ is that of a precursor for Ins(1,4,5)P₃ and DG, PIP₂ can also directly interact with specific domains in proteins to regulate their activity, and it can also act as a substrate for PI3-kinase to generate additional second messengers. A consequence of this is that PIP₂ has a pivotal role in regulating many cellular functions such as actin cytoskeleton reorganization, cell migration, vesicle trafficking, and ion channel activity in addition to the classical regulation of intracellular calcium and PKC activity (reviewed in Refs. 3, 12, 21). Therefore, compartmentalisation of PIP₂ into specific cellular pools is essential to lend selectivity to its diverse functions. Our observation that vasoconstrictors stimulate PIP₂ hydrolysis solely within caveolae/rafts shows that agonist responsive and unresponsive pools of PIP₂ do exist in vascular tissue. This agrees with a study in A431 cells where bradykinin-induced PIP₂ hydrolysis was confined to lipid rafts (38) and is also consistent with the reported caveolae localization of the ₁-adrenoreceptor and ET_A receptor (10, 16). NE and ET-1 stimulation also increased [³³P]PA levels in caveolae/rafts within the same time frame as PIP₂ hydrolysis. However, [³³P]PA production was not restricted to caveolae/rafts, suggesting that either DG or PA is capable of diffusing away from the initial site of production. Alternatively, there may be a generation of DG and [³³P]PA in noncaveolae/raft fractions by the action of other DGK isoforms or by hydrolysis of phospholipids other than PIP₂. To resolve this issue, a detailed analysis of PA species produced at different sites in the plasma membrane is being undertaken.

PI3-kinase-dependent activation of DGK in caveolae/rafts. The initial transient increase in [³³P]PA in caveolae/rafts induced by NE was consistent with the transient activation of DGK- by this agonist (49). Recently, we showed that NE activates PI3-kinase/PKB signaling in RMSAs and that this pathway is essential for activation of DGK- (49). Here we show that caveolae/rafts are sites where PI3-kinase/PKB regulates DGK activity and provides evidence that DGK- is involved in the response. Moreover, we also show that PI3-kinase is involved in the contractile response to NE, suggesting a link between PI3-kinase/PKB activation of DGK in caveolae/rafts and contraction. Another study in VSM cells has shown that mechanosensitive activation of PI3-kinase/PKB-dependent signaling requires intact caveolae (42), suggesting these membrane domains are important for this signaling pathway in smooth muscle. However, although PI3-kinase inhibition completely inhibited NE-stimulated DGK activity in caveolae/rafts, there was only a partial reduction of [³³P]PA levels. This probably reflects the presence of other constitutively active DGK isoforms such as DGK- (48). In contrast, ET-1 induced a sustained increase in DGK activity and [³³P]PA production in caveolae/rafts that was independent of PI3-kinase activity. This suggests that ET-1 activates different DGK isoforms to NE, demonstrating differential regulation of DGK and DG metabolism by vasoconstrictor hormones in vascular tissues. However, this is unlikely to be a type 1 DGK isoform (, , and ) since calcium removal or the DGK inhibitor R-59949 had no effect on ET-1-stimulated DGK activity (data not shown).

Caveolae/rafts and the contractile response. There is growing evidence that caveolae and caveolins are important for VSM contractility. For instance, caveolin knockout models are reported to have aberrations in endothelial-dependent relaxation, contractility, and maintenance of myogenic tone (reviewed in Ref. 25), whereas disruption of caveolae by cholesterol sequestration inhibits smooth muscle contraction by many agonists (reviewed in Ref. 5). Cholesterol depletion of rat-tail artery was reported to disrupt ET-1-stimulated contraction (14), and this was subsequently reported to be through a disruption of calcium influx (4). However, there is little consensus in the data concerning the role of caveolae in ₁-adrenergic contractile responses. In caveolin-1 null mice, normal (13) or blunted (39) contraction to ₁-adrenergic stimulation was reported, although the latter was due to enhanced endothelial relaxation rather than an alteration of smooth muscle response. Cholesterol depletion also suggests that ₁-adrenergic contraction is not dependent on intact caveolae (5), but two recent reports challenge this view, demonstrating blunted contractile responses to ₁-adrenergic stimulation in ferret aorta (22) and RMSAs (43) after cholesterol depletion. This suggests tissue-specific differences.

Here we observed NE- and ET-1-stimulated activation of the PI signaling cascade, important for contraction, in caveolae/rafts (schematically represented in Fig. 9). Furthermore, with both agonists, there was a direct correlation with the effects of LY-294002 on contraction and [³³P]PA levels in caveolae/rafts. With ET-1, both contraction and [³³P]PA were unaffected by PI3-kinase inhibition, whereas LY-294002 attenuated NE-stimulated maximal contraction (27% inhibition) and comparably reduced [³³P]PA levels in caveolae/rafts (23% inhibition; Figs. 6C and 8A). These data suggest a link between signaling events within caveolae/rafts and contraction. This also suggests that generation of PA within caveolae/rafts by GPCR agonists may be a common component of the signaling pathway leading to VSM contraction. Finally, although our tissue contains endothelial cells in addition to smooth muscle, the latter are the predominant cell type in our tissue, and because both ₁-adrenoceptors and ET_A receptors are mainly expressed on smooth muscle cells, it is most probable that the changes we observed are of smooth muscle origin.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Proposed schematic of NE- and ET-1-induced phosphatidylinositol (PI) signaling in caveolae/rafts. NE and ET-1 stimulate PLC-mediated hydrolysis of PIP2 within caveolae/rafts leading to contraction through increased intracellular calcium and PKC activation. In addition, activation of DGKs generates PA within caveolae/rafts. Inhibition of NE-induced DGK activity reduces PA levels in these domains as well as contraction, implicating the DGK/PA pathway in the contractile response. SR sarcoplasmic reticulum; IP3, D-myo-inositol 1,4,5-trisphosphate.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was funded by the British Heart Foundation.

	ACKNOWLEDGMENTS

 
Present address of C. J. Clarke: Dept. of Biochemistry and Molecular Biology, Medical Univ. of South Carolina, 173 Ashley Ave., Charleston, SC 29425.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. Ohanian, Cardiovascular Research Group, Div. of Cardiovascular and Endocrine Sciences, Core Technology Facility, 3rd Fl., Univ. of Manchester, 46 Grafton St., Manchester M13 9NT, UK (e-mail: johanian{at}manchester.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Babiychuk EB, Draeger A. Biochemical characterization of detergent-resistant membranes: a systematic approach. Biochem J 397: 407–416, 2006.[CrossRef][ISI][Medline]
2. Babiychuk EB, Monastyrskaya K, Burkhard FC, Wray S, Draeger A. Modulating signaling events in smooth muscle: cleavage of annexin 2 abolishes its binding to lipid rafts. FASEB J 16: 1177–1184, 2002.[Abstract/Free Full Text]
3. Balla T. Inositol-lipid binding motifs: signal integrators through protein-lipid and protein-protein interactions. J Cell Sci 118: 2093–2104, 2005.[Abstract/Free Full Text]
4. Bergdahl A, Gomez MF, Dreja K, Xu SZ, Adner M, Beech DJ, Broman J, Hellstrand P, Sward K. Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca²⁺ entry dependent on TRPC1. Circ Res 93: 839–847, 2003.[Abstract/Free Full Text]
5. Bergdahl A, Sward K. Caveolae-associated signalling in smooth muscle. Can J Physiol Pharmacol 82: 289–299, 2004.[CrossRef][ISI][Medline]
6. Billah MM, Anthes JC. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J 269: 281–291, 1990.[ISI][Medline]
7. Bligh EG and Dyer WJ. A Rapid Method of Total Lipid Extraction and Purification. Can J Biochem Physiol 37: 911–917, 1959.[Medline]
8. Bodin S, Giuriato S, Ragab J, Humbel BM, Viala C, Vieu C, Chap H, Payrastre B. Production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidic acid in platelet rafts: evidence for a critical role of cholesterol-enriched domains in human platelet activation. Biochemistry 40: 15290–15299, 2001.[CrossRef][Medline]
9. Bradford MM. A rapid and sensitive method for the quantitation of protein utilising the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
10. Chun M, Liyanage UK, Lisanti MP, Lodish HF. Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci USA 91: 11728–11732, 1994.[Abstract/Free Full Text]
11. Delon C, Manifava M, Wood E, Thompson D, Krugmann S, Pyne S, Ktistakis NT. Sphingosine kinase 1 is an intracellular effector of phosphatidic acid. J Biol Chem 279: 44763–44774, 2004.[Abstract/Free Full Text]
12. Downes CP, Gray A, Lucocq JM. Probing phosphoinositide functions in signaling and membrane trafficking. Trends Cell Biol 15: 259–268, 2005.[CrossRef][ISI][Medline]
13. Drab M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, Menne J, Lindschau C, Mende F, Luft FC, Schedl A, Haller H, Kurzchalia TV. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293: 2449–2452, 2001.[Abstract/Free Full Text]
14. Dreja K, Voldstedlund M, Vinten J, Tranum-Jensen J, Hellstrand P, Sward K. Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction. Arterioscler Thromb Vasc Biol 22: 1267–1272, 2002.[Abstract/Free Full Text]
15. Friedrichson T, Kurzchalia TV. Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature 394: 802–805, 1998.[CrossRef][Medline]
16. Fujita T, Toya Y, Iwatsubo K, Onda T, Kimura K, Umemura S, Ishikawa Y. Accumulation of molecules involved in ₁-adrenergic signal within caveolae: caveolin expression and the development of cardiac hypertrophy. Cardiovasc Res 51: 709–716, 2001.[Abstract/Free Full Text]
17. Ganitkevich V, Hasse V, Pfitzer G. Ca²⁺-dependent and Ca²⁺-independent regulation of smooth muscle contraction. J Muscle Res Cell Motil 23: 47–52, 2002.[CrossRef][ISI][Medline]
18. Ghosh S, Strum JC, Sciorra VA, Daniel L, Bell RM. Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic-acid-phosphatidic-acid regulates the translocation of raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated madin-darby canine kidney-cells. J Biol Chem 271: 8472–8480, 1996.[Abstract/Free Full Text]
19. Gonzalez-Sastre F, Folch-Pi J. Thin-layer chromatography of the phosphoinositides. J Lipid Res 9: 532–533, 1968.[Abstract]
20. Hope HR, Pike LJ. Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains. Mol Biol Cell 7: 843–851, 1996.[Abstract]
21. Janmey PA, Lindberg U. Cytoskeletal regulation: rich in lipids. Nat Rev Mol Cell Biol 5: 658–666, 2004.[CrossRef][ISI][Medline]
22. Je HD, Gallant C, Leavis PC, Morgan KG. Caveolin-1 regulates contractility in differentiated vascular smooth muscle. Am J Physiol Heart Circ Physiol 286: H91–H98, 2004.[Abstract/Free Full Text]
23. Jenkins GH, Fisette PL, Anderson RA. Type I phosphatidylinositol 4-phosphate 5-kinase isoforms are specifically stimulated by phosphatidic acid. J Biol Chem 269: 11547–11554, 1994.[Abstract/Free Full Text]
24. Jones JA, Hannun YA. Tight binding inhibition of protein phosphatase-1 by phosphatidic acid: specificity of inhibition by the phospholipid. J Biol Chem 277: 15530–15538, 2002.[Abstract/Free Full Text]
25. Li XA, Everson WV, Smart EJ. Caveolae, lipid rafts, and vascular disease. Trends Cardiovasc Med 15: 92–96, 2005.[CrossRef][ISI][Medline]
26. Lisanti MP, Scherer PE, Vidugiriene J, Tang Z, Hermanowski-Vosatka A, Tu YH, Cook RF, Sargiacomo M. Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J Cell Biol 126: 111–126, 1994.[Abstract/Free Full Text]
27. Liu G, Shaw L, Heagerty AM, Ohanian V, Ohanian J. Endothelin-1 stimulates hydrolysis of phosphatidylcholine by phospholipase C and D in intact rat mesenteric small arteries. J Vasc Res 36: 35–46, 1999.[CrossRef][ISI][Medline]
28. Macala LJ, Yu RK, Ando S. Analysis of brain lipids by high performance thin-layer chromatography and densitometry. J Lipid Res 24, 1243–1250, 1983.
29. Monaco ME, Gershengorn MC. Subcellular organization of receptor-mediated phosphoinositide turnover. Endocr Rev 13: 707–718, 1992.[CrossRef][ISI][Medline]
30. Morris AJ, Frohman MA, Engebrecht J. Measurement of phospholipase D activity. Anal Biochem 252: 1–9, 1997.[CrossRef][ISI][Medline]
31. Nanjundan M, Possmayer F. Pulmonary lipid phosphate phosphohydrolase in plasma membrane signalling platforms. Biochem J 358: 637–646, 2001.[CrossRef][ISI][Medline]
32. Northcott CA, Poy MN, Najjar SM, Watts SW. Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats. Circ Res 91: 360–369, 2002.[Abstract/Free Full Text]
33. Northcott CA, Watts SW. Low [Mg²⁺]_e enhances arterial spontaneous tone via phosphatidylinositol 3-kinase in DOCA-salt hypertension. Hypertension 43: 125–129, 2004.[Abstract/Free Full Text]
34. Ohanian J, Gatfield KM, Ward DT, Ohanian V. Evidence for a functional calcium-sensing receptor that modulates myogenic tone in rat subcutaneous small arteries. Am J Physiol Heart Circ Physiol 288: H1756–H1762, 2005.[Abstract/Free Full Text]
35. Ohanian J, Heagerty AM. Membrane-associated diacylglycerol kinase activity is increased by noradrenaline, but not by angiotensin-II, in arterial smooth muscle. Biochem J 300: 51–56, 1994.[ISI][Medline]
36. Ohanian J, Ohanian V, Shaw L, Bruce C, Heagerty AM. Involvement of tyrosine phosphorylation in endothelin-1-induced calcium-sensitization in rat small mesenteric arteries. Br J Pharmacol 120: 653–661, 1997.[ISI][Medline]
37. Ohanian J, Ollerenshaw J, Collins P, Heagerty A. Agonist-induced production of 1,2-diacylglycerol and phosphatidic acid in intact resistance arteries. Evidence that accumulation of diacylglycerol is not a prerequisite for contraction. J Biol Chem 265: 8921–8928, 1990.[Abstract/Free Full Text]
38. Pike LJ, Casey L. Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains. J Biol Chem 271: 26453–26456, 1996.[Abstract/Free Full Text]
39. Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, Marks CB, Macaluso F, Russell RG, Li M, Pestell RG, Di Vizio D, Hou H Jr, Kneitz B, Lagaud G, Christ GJ, Edelmann W, Lisanti MP. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J Biol Chem 276: 38121–38138, 2001.[Abstract/Free Full Text]
40. Razani B, Woodman SE, Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev 54: 431–467, 2002.[Abstract/Free Full Text]
41. Rizzo MA, Shome K, Watkins SC, Romero G. The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras. J Biol Chem 275: 23911–23918, 2000.[Abstract/Free Full Text]
42. Sedding DG, Hermsen J, Seay U, Eickelberg O, Kummer W, Schwencke C, Strasser RH, Tillmanns H, Braun-Dullaeus RC. Caveolin-1 facilitates mechanosensitive protein kinase B (Akt) signaling in vitro and in vivo. Circ Res 96: 635–642, 2005.[Abstract/Free Full Text]
43. Shaw L, Sweeney MA, O'Neill SC, Jones CJ, Austin C, Taggart MJ. Caveolae and sarcoplasmic reticular coupling in smooth muscle cells of pressurised arteries: the relevance for Ca²⁺ oscillations and tone. Cardiovasc Res 69: 825–835, 2006.[Abstract/Free Full Text]
44. Somlyo AP, Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177–185, 2000.[Abstract/Free Full Text]
45. Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP. Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. J Biol Chem 271: 9690–9697, 1996.[Abstract/Free Full Text]
46. Sprong H, van der Sluijs P, van Meer G. How proteins move lipids and lipids move proteins. Nat Rev Mol Cell Biol 2: 504–513, 2001.[CrossRef][ISI][Medline]
47. Tkachenko E, Simons M. Clustering induces redistribution of syndecan-4 core protein into raft membrane domains. J Biol Chem 277: 19946–19951, 2002.[Abstract/Free Full Text]
48. Van Blitterswijk WJ, Houssa B. Properties and functions of diacylglycerol kinases. Cell Signal 12: 595–605, 2000.[CrossRef][ISI][Medline]
49. Walker AJ, Draeger A, Houssa B, van Blitterswijk WJ, Ohanian V, Ohanian J. Diacylglycerol kinase theta is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries. Biochem J 353: 129–137, 2001.[CrossRef][ISI][Medline]
50. Watt SA, Kular G, Fleming IN, Downes CP, Lucocq JM. Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C-₁. Biochem J 363: 657–666, 2002.[CrossRef][ISI][Medline]

This article has been cited by other articles:

				C. J. Clarke, S. Forman, J. Pritchett, V. Ohanian, and J. Ohanian Phospholipase C-{delta}1 modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1 Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H826 - H834. [Abstract] [Full Text] [PDF]

				R. Gosens, G. L. Stelmack, G. Dueck, M. M. Mutawe, M. Hinton, K. D. McNeill, A. Paulson, S. Dakshinamurti, W. T. Gerthoffer, J. A. Thliveris, et al. Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle Am J Physiol Lung Cell Mol Physiol, December 1, 2007; 293(6): L1406 - L1418. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 292/5/H2248    most recent 01170.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Clarke, C. J. Articles by Ohanian, J. Search for Related Content