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Am J Physiol Heart Circ Physiol 290: H2059-H2065, 2006. First published December 22, 2005; doi:10.1152/ajpheart.01210.2005
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Inositol phospholipids localized to caveolae in rat heart are regulated by {alpha}1-adrenergic receptors and by ischemia-reperfusion

Alfred A. Lanzafame, Lynne Turnbull, Fatemeh Amiramahdi, Jane F. Arthur, Huy Huynh, and Elizabeth A. Woodcock

Cellular Biochemistry Laboratory, Baker Heart Research Institute, Melbourne, Victoria, Australia

Submitted 16 November 2005 ; accepted in final form 20 December 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Postischemic reperfusion of rat or mouse hearts causes generation of inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] and the initiation of arrhythmias. In the current study we investigated the possibility that the enhanced Ins(1,4,5)P3 generation in postischemic reperfusion was associated with an increased availability of the precursor lipid phosphatidylinositol(4,5)bisphosphate (PIP2) for {alpha}1-adrenergic receptor-activated phospholipase C (PLC). Isolated-perfused rat hearts were labeled with [3H]inositol and subjected to ischemia-reperfusion or stimulation with norepinephrine under normoxic conditions. Caveolar fractions were prepared by buoyant density sucrose gradient centrifugation. [3H]PIP2 was concentrated in caveolae, along with G{alpha}q and PLCbeta1b. Caveolae contained only 27.3 ± 6.9% (means ± SE, n = 6) of the total {alpha}1-adrenergic receptor complement of the heart. These did not migrate to PIP2-containing caveolar fractions with norepinephrine stimulation under normoxic conditions, even though caveolar PIP2 was depleted. In contrast, [3H]PIP2 in caveolae increased during 2 min of reperfusion, independently of norepinephrine release and thus of {alpha}1-adrenergic receptor activation. The increased PIP2 in the caveolar fractions where signaling proteins are concentrated may be critical for the heightened generation of Ins(1,4,5)P3 in early reperfusion.

lipid signaling; light lipid rafts; phospholipase C; Gq; caveolin

BRIEF PERIODS OF ISCHEMIA-REPERFUSION in the rat and mouse heart cause norepinephrine release, activation of {alpha}1-adrenergic receptors ({alpha}1-AR), and substantial generation of inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] associated with the onset of arrhythmogenesis (1, 2, 13, 24, 41). Ins(1,4,5)P3 is generated from sarcolemmal phosphatidylinositol(4,5)bisphosphate (PIP2) following activation of phospholipase C (PLC). We addressed the possibility that increased availability of PIP2 or factors required for PLC activation contributed to the heightened generation of Ins(1,4,5)P3 during postischemic reperfusion.

PIP2 is generated from phosphatidylinositol most commonly by phosphorylation to PI(4)P, followed by further phosphorylation to PI(4,5)P2 (9) and to a lesser extent by sequential 5' and 4' phosphorylation (23). PIP2 is critically involved in myocardial responses. In addition to Ins(1,4,5)P3, PLC cleavage of PIP2 generates sn-1,2-diacylglycerol, an activator of PKC isoforms (31). PIP2 is also the precursor of PIP3, an important factor in cardiomyocyte development and cytoprotection (43). Further to these roles as precursor to other active molecules, PIP2 itself acts as a second messenger by activating phospholipase D activity in the heart (25). PIP2 is critically important in stabilizing various ion channels and transporters (22, 32) and also regulates the cytoskeleton by virtue of its ability to interact specifically with actin-binding proteins via their plextrin homology (PH) domains (32). With this complexity in mind, we sought to define the localization of PIP2 in the heart in relation to signaling proteins involved in Ins(1,4,5)P3 generation, specifically {alpha}1-AR, Gq, and PLCbeta, and to establish how the localization of these various factors was influenced by ischemia and reperfusion.

The sarcolemma is highly organized, and it is now recognized that many signaling responses are localized to cholesterol-rich, sphingolipid-rich regions called light lipid rafts to reflect their low buoyant densities (20, 37). Important among this heterogeneous fraction are the caveolae, which in the heart are characterized by the presence of caveolin-3 (47). The concentration of receptors and downstream factors within caveolae facilitates regulation and potentially enhances specificity by physically limiting available signaling partners (20, 28, 37). In the heart, the caveolar fraction has been reported to contain {alpha}1-AR, Gq, and the subtypes of PLCbeta (15), but the localization of PIP2 and its precursors have not been defined.

In the current study, we show that PIP2, as well as its precursors PIP and PI, is highly enriched in the caveolar fraction along with the lower molecular weight "b" splice variant of PLCbeta1 (4). Other proteins potentially involved in {alpha}1-AR signaling were not exclusively localized in caveolae, suggesting a previously unrecognized level of specificity for PLCbeta1b. We show that caveolar-localized PIP2 is rapidly depleted by {alpha}1-AR activation, but surprisingly that brief ischemia followed by reperfusion increases the content of PIP2 in caveolae independently of norepinephrine release and {alpha}1-AR activation. This rise in PIP2 in the vicinity of PLCbeta1b may contribute to the enhanced Ins(1,4,5)P3 generation observed under these conditions.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of perfused rat hearts, ischemia-reperfusion, and norepinephrine depletion. All procedures were approved by the Alfred Medical and Education Precinct animal ethics committee and all followed the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Adult male Sprague-Dawley rats were killed by decapitation. Hearts were isolated and perfused by the Langendorf method with HEPES-buffered Krebs medium, pH 7.4, at 7 ml/min, as previously described in detail (1). The medium contained the following (in mM): 126 NaCl, 25 NaHCO3, 1.85 CaCl2, 1.05 MgCl2, 0.5 NaH2PO4, 4 KCl, 11 glucose, and 20 HEPES buffer, pH 7.4. Inositol phospholipids were labeled by perfusing the hearts with medium containing 2 µCi/ml [3H]inositol for 2 h, at 7 ml/min, followed by washing with nonradioactive medium. LiCl (10 mM) and propranolol (1 µM) were then added to the perfusate to inhibit inositol phosphate metabolism and beta-adrenergic receptor activation, respectively (1).

For studies of ischemia and reperfusion, [3H]inositol-labeled hearts were subjected to 20 min global zero-flow ischemia by turning off the perfusion pump for 20 min. Reperfusion was initiated by restarting flow at 7 ml/min. For studies of norepinephrine, stimulation under normoxic conditions norepinephrine (100 µM) was added to [3H]inositol-labeled hearts for 2 min. After treatment, ventricles were excised at the atrioventricular junction and were rapidly frozen in liquid N2 and stored at –80°C. Protocols are outlined in Fig. 1.


Figure 1
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  		Fig. 1. Protocols used for perfused rat heart experiments. [3H]Inositol-labeled hearts are treated with 10 mM LiCl and 1 µM propranolol (pro) at the times indicated by shaded arrows. Addition of 100 µM norepinephrine (Nor) is indicated by hatched arrows. Collection of the ventricles is indicated by open arrows. Isch, ischemia; reper, reperfusion.

 
Depletion of norepinephrine was achieved by treating the animals with reserpine (5 mg/kg body wt ip) for 18 h. This reduced norepinephrine levels in the hearts from 24.4 ± 10.4 pmol/mg protein (means ± SE, n = 4) to undetectable levels. Norepinephrine was measured as described previously (14).

Separation of different membrane fractions from isolated perfused rat hearts. All solutions for preparation of membrane fractions contained the following proteinase and phosphatase inhibitors: 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, 20 mM NaF, 1 mM Na3VO4, and 1 mM sodium pyrophosphate. Frozen hearts were homogenized in 2.5 ml of 0.5 M NaCO3, pH 11, by using a Polytron homogenizer at maximum speed. Homogenates were sonicated three times for 20 s each and then mixed with an equal volume of 80% sucrose in MES-buffered saline (25 mM MES, pH 6.5, 0.15 M NaCl), as described elsewhere (39). Tubes were overlayed with 4 ml of 35% sucrose and 3 ml of 5% sucrose and centrifuged at 38,000 rpm for 24 h at 4°C using a SW-41 rotor in a Beckman L-90K ultracentrifuge. Gradients were fractionated from the bottom using a peristaltic pump. Fractions (1 or 2 ml) were collected. The same fractions were used for lipid analysis and for estimation of protein content. The fractions were divided into two equal aliquots, one being used for lipid extractions, as described below. The other aliquot was used for measurement of membrane protein content. These aliquots were diluted to 10 ml with MES-buffered saline, and membranes were separated from soluble material by centrifugation in a 70.1 Ti rotor at 50,000 rpm for 30 min. Membrane pellets were used for protein analysis and {alpha}1-AR quantification, as outlined below.

Measurement of [3H]inositol phospholipids. Phospholipids were extracted from the fractions using an equal volume of CHCl3/CH3OH/HCl (200/100/1 vol/vol/vol). Phases were separated by adding 2 mM EDTA, and the lipid phase was evaporated under vacuum. Dried lipids were deacylated using methylamine/CH3OH/butanol (42/47/9 vol/vol/vol) for 45 min at 50°C, followed by evaporation. Dried deacylated lipids were extracted with petroleum spirit-butanol-ethyl formate, as described previously (49). The deacylated lipids were separated into PI, PIP, and PIP2 fractions using 1-ml columns of Dowex-1 (formate form), as described previously (49). Samples were counted in a beta counter.

Measurement of [3H]PIP2 and [3H]Ins(1,4,5)P3 in intact rat ventricle. Isolated perfused rat hearts were labeled with [3H]inositol (2 µCi/ml) for 2 h and subjected to ischemia-reperfusion, as described. [3H]Ins(1,4,5)P3 and [3H]PIP2 were extracted as described in detail previously (1, 50).

Measurement of {alpha}1A- and {alpha}1B-AR. The two subtypes of {alpha}1-AR were identified and quantified using 125I-labeled 2-[beta-(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone ([125I]HEAT) (ProSearch, Melbourne, Australia) in competition binding studies with the {alpha}1A-selective antagonist KMD-3213 (53). Membranes, prepared as described above (100 µl), were mixed with 50 µl of [125I]HEAT (50–100 pM, 50,000–100,000 counts/min) and 50 µl of KMD-3213 (10–11 to 10–5 M) in 20 mM Tris·HCl, 10 mM MgCl2, pH 7.4, for 60 min at 25°C. Bound ligand was separated from unbound by rapid filtration through Whatman GF/C glass fiber filters under vacuum, followed by rapid washing with 20 ml of the above buffer. Filters were counted in a gamma counter. Binding data were analyzed using Prism 4.0 (GraphPad Software, San Diego, CA) to provide best-fit estimates for receptor concentration and affinity for both receptor subtypes (26).

Measurement of membrane proteins. Pelleted membranes, prepared as described above, were solubilized in SDS-PAGE sample buffer (8). Protein concentration was measured by a modified Lowry estimation (33). Proteins (100 µg) were separated on gradient SDS-PAGE (7.5–15% acrylamide) and electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Membranes were stained with Ponceau S (Sigma) before blocking and subsequent development with antibodies. The antibodies used were the following: G{alpha}q/11 (1/250, COOH-terminal directed, Santa Cruz), PLCbeta1 (1 µg/ml, Santa Cruz), and PLCbeta3 (1 µg/ml, Santa Cruz). Secondary antibodies were from Amersham Life Sciences, and bound antibody was detected using chemiluminescence-employing ECL reagents from Amersham Life Sciences (Bucks, UK). Band density was quantified from scanned images using Optimas 6.5 software (Media Cybernetics, Silver Spring, MD).

Statistical analysis. Comparisons between two groups were made using Student’s unpaired t-test. Where comparisons involved more than two groups, data analysis was performed using a two-way ANOVA followed by Tukey’s test. Analyses were performed using Sigma Stat 2.03 for Windows (Systat Software, Point Richmond, CA).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Reperfusion of ischemic rat hearts increases PIP2 independently of norepinephrine release. Isolated perfused [3H]inositol-labeled rat hearts were subjected to 20 min global zero-flow ischemia followed by reperfusion for 0.5–3 min and then snap frozen (1). [3H]Ins(1,4,5)P3 and [3H]PIP2 were extracted and quantified. Reperfusion caused an increase in [3H]Ins(1,4,5)P3 that was maximal 1–2 min after reinitiation of flow (Fig. 2A). Despite the clear activation of PLC and Ins(1,4,5)P3 generation, there was no decrease in [3H]PIP2 over this time period. Loss of PIP2 associated with generation of Ins(1,4,5)P3 might have been masked by increased generation of PIP2 caused by the ischemia-reperfusion procedure itself. To investigate this possibility, hearts were depleted of norepinephrine to prevent {alpha}1-adrenergic receptor-mediated PLC activation. Depletion of norepinephrine prevented the [3H]Ins(1,4,5)P3 response over 3 min of postischemic reperfusion, as reported previously (1), and caused a marked increase in [3H]PIP2 after reinitiation of flow (Fig. 2).


Figure 2
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  		Fig. 2. Postischemic reperfusion causes generation of inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] (B) as well as increases in phosphatidylinositol(4,5)bisphosphate (PIP2) (A). Isolated perfused rat hearts, both intact and norepinephrine depleted, were labeled with [3H]inositol and subsequently subjected to 20 min of zero-flow ischemia followed by reperfusion at 7 ml/min for the indicated time. [3H]Ins(1,4,5)P3 and [3H]PIP2 were extracted and quantified. Values shown are expressed as [3H]counts/minute (CPM)/g wet wt; means ± SE, n = 6. Open symbols, norepinephrine-depleted hearts; filled symbols, intact hearts. *P < 0.05 and **P < 0.01 relative to 20 min ischemia.

 
To ensure that the rise in PIP2 in norepinephrine-depleted hearts reflected a loss of PLC activity, a number of experiments were performed. First, 100 µM norepinephrine was added to the perfusate of the norepinephrine-depleted hearts during 2 min of reperfusion, resulting in the generation of Ins(1,4,5)P3 with no increase in PIP2 (Fig. 3). Second, 1.5 mM gentamicin was added during reperfusion of intact hearts (not reserpine treated) to inhibit PLC (24). Treatment with gentamicin prevented Ins(1,4,5)P3 generation, as reported previously (13), and also resulted in increased PIP2 (Fig. 3). Thus the ischemia-reperfusion procedure generates PIP2 independently of norepinephrine release. When PLC is activated by {alpha}1-adrenergic receptor activation, this PIP2 increase is not observed, implying that the increased PIP2 provides a substrate for the activated PLC.


Figure 3
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  		Fig. 3. A: PIP2 increases when phospholipase C (PLC) is inhibited during postischemic reperfusion. B: isolated perfused rat hearts, both intact and norepinephrine depleted, were labeled with [3H]inositol and subsequently subjected to 20 min of zero-flow ischemia followed by reperfusion at 7 ml/min for 2 min. [3H]Ins(1,4,5)P3 and [3H]PIP2 were extracted and quantified. Open bars, values after 20 min ischemia (isch); solid bars, catecholamine-depleted hearts (reserpine); gray bars, catecholamine-depleted hearts with added 100 µM norepinephrine (res + nor); hatched bars, intact hearts, no reserpine (intact); crosshatched bars, intact hearts treated with 1.5 mM gentamicin. Value are expressed as [3H]CPM/g wet wt; means ± SE, n = 6. *P < 0.05 and **P < 0.01 relative to 20 min ischemia.

 
PIP2 is concentrated in caveolar fractions of rat heart membranes, along with G{alpha}q and PLCbeta1. The localization of the PIP2 in cardiac sarcolemma was investigated to establish whether the PIP2 that increased during reperfusion was likely to be available for receptor-activated PLC hydrolysis to Ins(1,4,5)P3. Previous studies have reported that proteins required for {alpha}1-AR signaling are localized to the caveolar fractions isolated from rat hearts (12, 15). However, these studies did not define the localization of PI, PIP, and PIP2. To examine the localization of these lipids in rat heart membranes, perfused [3H]inositol-labeled rat hearts were homogenized, and membrane fractions were separated on the basis of their buoyant densities, as described in METHODS. [3H]PI, [3H]PIP, and [3H]PIP2 were concentrated in the light lipid raft fractions along with caveolin-3 (Fig. 4). Membrane-bound PLCbeta1 was almost exclusively found in the caveolar fractions (Fig. 4, Table 1). In the adult rat heart, we observed only the lower molecular mass splice variant of PLCbeta1, PLCbeta1b (140 kDa mol mass). The two splice variants of PLCbeta1, PLCbeta1a, and PLCbeta1b (150 and 140 kDa mol mass, respectively) described in other cells types (4) are detectable in neonatal rat cardiomyocytes but not in caveolae from adult rat heart (Fig. 4). G{alpha}q was found in the caveolar fraction as well as in higher density membranes, although relative to protein content G{alpha}q was highly enriched in the caveolae fraction (Fig. 4, Table 1). In marked contrast to PLCbeta1b, membrane fractions contained relatively little PLCbeta3 (only 1–5% of the total cellular content of PLCbeta3), and this was not highly enriched in caveolae (Fig. 4). Other proteins found in the caveolar fractions included PLC{delta}1, epidermal growth factor receptors, and PI(4)P 5-kinase1{alpha}. PI(4)P 5-kinase1beta was not detected in rat hearts (data not shown).


Figure 4
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  		Fig. 4. Inositol phospholipids localize to caveolae along with immediate signaling proteins in rat heart membranes. Isolated perfused rat hearts were labeled with [3H]inositol and subsequently harvested, and membranes of different buoyant densities were separated on sucrose gradients. Membranes were harvested by centrifugation, and the lipid and protein contents were analyzed in the same fractions. A: values are expressed as [3H]PI, [3H]PIP, or [3H]PIP2 total CPM per fraction. B: total protein content of fractions. C: Western blots showing signaling proteins in membrane fractions, 100 µg protein per lane. Figures shown are representative experiments. Quantified data for PLCbeta1b and G{alpha}q are shown in Table 1. PLCbeta1 from neonatal rat cardiomyocytes (NRVM) is shown for comparison.

 

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  		Table 1. Percentage of G{alpha}q, PLCbeta1, and {alpha}1-AR in caveolae relative to total membrane content

 
{alpha}1-AR are not concentrated in caveolae in rat hearts. The caveolar localization of {alpha}1A- and {alpha}1B-AR was then assessed. Overall rat heart membranes contained 745.1 ± 161.8 fmol/heart of {alpha}1-AR, and of these, 79.3 ± 7.3% were the {alpha}1B-subtype (means ± SE, n = 6). However, only 27.3 ± 6.9% of the total {alpha}1-AR complement was found in the caveolar fractions (Fig. 5, Table 1) where [3H]PIP, [3H]PIP2, and PLCbeta1b are concentrated (Fig. 4, Table 1). There was no difference between the {alpha}1A- and {alpha}1B-subtypes in terms of localization (Fig. 5). The small percentage of {alpha}1-AR located in the caveolar fraction along with its substrate PIP2 and its immediate signaling proteins G{alpha}q and PLCbeta1 suggested that localization may limit the activity of {alpha}1-AR signaling.


Figure 5
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  		Fig. 5. {alpha}1-Adrenergic receptors ({alpha}1-ARs) are primarily located outside the light lipid raft fractions in rat heart membranes. Membrane fractions of different buoyant densities were prepared from rat hearts and analyzed for {alpha}1A- and {alpha}1B-AR content using 125I-labeled 2-[beta-(4-hydroxyphenol)-ethyl-aminomethyl]tetralone ([125I]HEAT) in competition binding studies with the {alpha}1A-selective antagonist KMD-3213. Receptor concentration and affinity were calculated using nonlinear regression analysis to obtain best-fit values. A: representative competition binding curve; B and C: {alpha}1A- and {alpha}1B-AR concentrations in the different membrane fractions. Light lipid raft fractions are 9–12 ml from bottom and are indicated by darker gray bars. Data shown are {alpha}1-AR concentrations, means ± SE; n = 6.

 
We next examined whether norepinephrine stimulation under normoxic conditions or postischemic reperfusion caused movement of {alpha}1-AR into caveolae or alternatively moved G{alpha}q or PLCbeta1 to membranes of higher density where most of the {alpha}1-AR are located. [3H]inositol-labeled rat hearts were subjected to ischemia-reperfusion, and membrane fractions were separated on the basis of their buoyant densities. {alpha}1-AR (ligand binding), PLCbeta1, and G{alpha}q (Western blots) were measured in the same membrane fractions as used for inositol phospholipid quantifications (shown below). Western blots were quantified, and the percentage of the particular protein in the caveolar fractions was estimated relative to the content of all membrane fractions added together. As shown in Table 1, stimulation with 100 µM norepinephrine for 2 min in normoxia did not cause any detectable change in the distribution of any of these proteins. Reperfusion for 2 min after 20 min ischemia, however, caused a selective loss of {alpha}1-AR from the caveolar fraction. Loss of {alpha}1-AR from caveolae, rather than providing an explanation for the heightened response in reperfusion, most likely reflects receptor desensitization (45).

Ischemia-reperfusion increases PIP2 in caveolar fractions. The localization of the [3H]PIP2 that increases during postischemic reperfusion was then assessed. [3H]inositol-labeled hearts were subjected to 20 min ischemia, followed by 2 min reperfusion, and membrane fractions were prepared as described above. Neither 20 min ischemia nor 2 min postischemic reperfusion caused any change in the caveolar content of PIP2 in norepinephrine replete hearts (Fig. 6B). However, when catecholamine-depleted hearts were used, [3H]PIP2 increased in the caveolar fractions over the 2-min period of reperfusion (Fig. 6A), but there was no increase in [3H]PIP2 in noncaveolar fractions. This implies that activation of PLC under reperfusion conditions reduces the [3H]PIP2 in the caveolar fractions by hydrolyzing it to [3H]Ins(1,4,5)P3. To confirm that norepinephrine stimulation for 2 min reduces [3H]PIP2 in caveolae, similar experiments were performed using [3H]inositol-labeled hearts under normoxic conditions. Stimulation with 100 µM norepinephrine for 2 min caused a reduction in [3H]PIP2 in the caveolar fractions (Fig. 6C). Thus our data suggest that postischemic reperfusion increases the generation of PIP2 in caveolae independently of norepinephrine release and {alpha}1-AR activation. This increase in PIP2 in caveolae, where G{alpha}q and PLCbeta1 are concentrated, potentially provides substrate for activated PLC, thereby reducing the caveolar PIP2 content by generating Ins(1,4,5)P3.


Figure 6
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  		Fig. 6. Norepinephrine depletion uncovers an increase in caveolar [3H]PIP2 during 2 min postischemic reperfusion. A and B: [3H]inositol-labeled rat hearts were subjected to 20 min global zero-flow ischemia (isch) or 20 min ischemia followed by 2 min reperfusion (reper) in the presence of 1 µM propranolol and 10 mM LiCl. Membranes of different buoyant densities were separated on sucrose gradients. [3H]PIP2 was extracted from fractions and quantified. A: norepinephrine-depleted hearts. B: intact rat hearts. C: normoxic conditions; [3H]inositol-labeled rat hearts were stimulated with 100 µM norepinephrine (Nor) or no additions (NA) in the presence of 1 µM propranolol and 10 mM LiCl. Values are [3H]PIP2/g wet wt; means ± SE, n = 5. *P < 0.05 relative to 20 min ischemia. **P < 0.05 relative to NA.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Reperfusion of ischemic rat or mouse hearts causes norepinephrine release from the cardiac sympathetic nerves (40), activation of {alpha}1-AR (42), and consequent generation of Ins(1,4,5)P3 by PLC hydrolysis of PIP2 (1). The rise in Ins(1,4,5)P3 is associated with the onset of arrhythmias that can be prevented by inhibiting PLC activity (13, 24) or by depleting the hearts of norepinephrine. {alpha}1-AR-mediated Ins(1,4,5)P3 generation is markedly enhanced over the first 3 min after initiation of reperfusion compared with responses in normoxic myocardium, and we sought to define the mechanisms responsible. Ins(1,4,5)P3 is generated from the sarcolemmal phospholipid PIP2 following activation of {alpha}1-AR, Gq, and PLCbeta. In the current study we addressed the possibility that ischemia and reperfusion alter the availability of PIP2 for PLCbeta-mediated hydrolysis to Ins(1,4,5)P3.

In heart membranes, we showed that PIP2 and its precursors PIP and PI are localized in the caveolar fractions along with caveolin-3 (Fig. 4). Membrane-bound PLCbeta1b was also localized exclusively in these fractions (Fig. 4, Table 1) where it is well placed to hydrolyze PIP2 to Ins(1,4,5)P3. Rat heart caveolae exhibited only the lower molecular weight "b" splice variant of PLCbeta1 (4), in contrast to neonatal rat cardiomyocytes that express both PLCbeta1a and PLCbeta1b (3) (Fig. 4). PLCbeta1b is important in nuclear responses in some cell types (11), and the significance of its caveolar localization in the heart is not known. In marked contrast to PLCbeta1, the PLCbeta3 subtype was largely in the soluble fraction, and the membrane-bound component was mostly found in noncaveolar membranes essentially devoid of PIP and PIP2 (Fig. 4). We have previously reported that {alpha}1-AR couples exclusively to PLCbeta1 in neonatal rat cardiomyocytes, even though these cells express both PLCbeta1 and PLCbeta3 (3). At least in some cell types, PLCbeta3 can be targeted to particular membrane domains via its COOH-terminal, PDZ-binding domain (7), and it is likely that the different PDZ-binding domains of the PLCbeta subtypes are responsible for their subtype-specific localizations in cardiac membrane fractions (44). Data shown in Fig. 4 suggest the possibility that {alpha}1-AR preferentially hydrolyze PIP and PIP2 via PLCbeta1 because of its localization in caveolae along with these lipids. In contrast to the inositol phospholipids and PLCbeta1, G{alpha}q was found in both caveolar and nonraft membrane fractions, although relative to protein content, it was highly concentrated in the caveolar fractions (Fig. 4, Table 1). Thus in the rat heart, most of the factors required for generation of Ins(1,4,5)P3 are concentrated in caveolae. For this reason it was surprising that {alpha}1-AR were largely located outside of these fractions (Fig. 5, Table 1). This is especially puzzling in light of a recent study showing close association between PIP2 and PLC-coupled receptors in atrial myocytes (10). Whereas heavier membrane fractions containing {alpha}1-AR also contained G{alpha}q, they were essentially devoid of PLCbeta1 as well as the lipid substrates PIP and PIP2. One possible consequence of this is that the majority of the {alpha}1-AR population in the heart is unable to activate PLC. If this is so, it may explain the comparatively weak PLC response to {alpha}1-AR activation in heart preparations compared with other cell types (6, 34, 49). Another possibility is that either the {alpha}1-AR or the lipids translocate with stimulation. Our studies, however, provided no evidence to suggest movement of {alpha}1-AR into caveolae or movement of PLCbeta1 or PIP2 to membranes of higher density when {alpha}1-AR were stimulated.

The current studies involve the use of a perfused rat ventricle that contains a number of other cell types in addition to cardiomyocytes. We have previously shown that PLC responses in this model reflect primarily responses in cardiomyocytes (52). Specifically, we detected no measurable response to angiotensin II or compound 48/80, strong stimuli of vascular smooth muscle and mast cell, respectively. {alpha}1-AR are not detected on mast cells or cardiac fibroblasts and thus contribution to norepinephrine-dependent responses from these cell types can be discounted. Furthermore, PLC responses in cardiomyocytes differ in terms of rate and in inositol phosphate isomers generated from responses in other cell types (51).

Having identified caveolae as the site where PIP2 is hydrolyzed by PLCbeta1 to generate inositol phosphates, we next examined how signaling factors in caveolae were influenced by ischemia and reperfusion. Importantly, we found that postischemic reperfusion, but not ischemia itself, caused a selective loss of both {alpha}1-AR from caveolae (Table 1), even though Ins(1,4,5)P3 generation is maximal under these conditions (Fig. 2B). Rather than contributing to the heightened activity, this loss of {alpha}1-AR most likely reflects desensitization, although treatment with maximal concentrations of norepinephrine did not cause such changes during normoxia (Table 1). Such a conclusion is consistent with reports showing movement of beta2-AR out of caveolae following agonist stimulation of isolated cardiomyocytes (38).

The current studies show that postischemic reperfusion causes heightened generation of PIP2 in caveolae independently of norepinephrine release and {alpha}1-AR activation (Fig. 6). This PIP2 is available for {alpha}1-AR initiated hydrolysis when stimulated by norepinephrine released under these pathological conditions. Thus the heightened availability of caveolar PIP2 may contribute to the enhanced generation of Ins(1,4,5)P3 and thereby to the initiation of arrhythmias under conditions of ischemia and reperfusion. Ins(1,4,5)P3 causes Ca2+ release from receptors localized on the sarcoplasmic reticulum and the perinuclear membrane (5, 29). Subsarcolemmal IP3 receptors on the sarcoplasmic reticulum are clearly present in atrial myocytes (29), but their expression is undetectably low in ventricle. The possibility remains that ischemia causes an increase in subsarcolemmal IP3 receptor. The conducting myocytes express a higher concentration of IP3 receptor (type 1) than working myocytes (46, 19), and some of these are subsarcolemmal and thus may contribute to arrhythmogenesis. Even though the expression of IP3 receptor is low in heart, there is evidence that IP3 receptor activation can perturb the Ca2+-induced Ca2+ release program orchestrated by the more prevalent ryanodine receptors and lead to increased Na+/Ca2+ exchange (30), a possible mediator of the observed arrhythmias. Furthermore, removal of the type 2 IP3 receptor, the subtype expressed in working cardiomyocytes (17), prevented arrhythmogenic responses to PLC activation in mouse atrial myocytes (27). Importantly, increased IP3 receptor expression is seen in failed human ventricular tissue (18) and in atrial samples from patients predisposed to atrial fibrillation (54), suggesting a contribution of Ins(1,4,5)P3 to human pathology. Whereas our studies and those of others have pointed to Ins(1,4,5)P3 as an initiator of arrhythmias, some recent studies have suggested the possibility of a protective role against postischemic infarction (35, 36, 48). This may explain our previous finding that the absence of Ins(1,4,5)P3 generation in mouse hearts expressing constitutively active {alpha}1B-AR reduced arrhythmias but did not limit infarction (16, 21).

In conclusion, the current studies show that brief periods of ischemia and reperfusion in rat hearts cause a number of changes in the caveolar fraction of rat heart sarcolemma that may be related to enhanced Ins(1,4,5)P3 generation. Of these, the loss of caveolar {alpha}1-AR likely reflects heightened receptor activation under these conditions. However, the increased synthesis of PIP2 in caveolae in early reperfusion may be important as a source of substrate for the generation of the Ins(1,4,5)P3 when {alpha}1-AR are activated following release of norepinephrine from the sympathetic nerves.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Australian National Health and Medical Research Council Grants 317802 and 826921 and a Principal Research Fellowship (E. A. Woodcock) Grant 317803.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Arthur Christopoulos, Dept. of Pharmacology University of Melbourne, for help with receptor binding analysis.

Present address of L. Turnbull: Dept. of Microbiology, Monash University, Wellington Road, Clayton, 3800 Victoria, Australia. Present address of J. F. Arthur: Dept. of Biochemistry, Monash University, Wellington Road, Clayton, 3800 Victoria, Australia.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. A. Woodcock, Cellular Biochemistry Laboratory, Baker Heart Research Institute, PO Box 6492, St. Kilda Road Central, Melbourne, Victoria 8008, Australia (e-mail: liz.woodcock{at}baker.edu.au)

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
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 DISCUSSION
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