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Am J Physiol Heart Circ Physiol 288: H2611-H2619, 2005. First published January 28, 2005; doi:10.1152/ajpheart.00549.2004
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Arachidonic acid incorporation and turnover is decreased in sympathetically denervated rat heart

Casey B. Patrick,1 Jane McHowat,2 Thad A. Rosenberger,1 Stanley I. Rapoport,1 and Eric J. Murphy1,3

1Section on Brain Physiology and Metabolism, National Institute on Aging, National Institutes of Health, Bethesda, Maryland; 2Department of Pathology, Saint Louis University, St. Louis, Missouri; and 3Department of Pharmacology, Physiology, and Therapeutics, and Department of Chemistry, University of North Dakota, Grand Forks, North Dakota

Submitted 16 June 2004 ; accepted in final form 18 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Heart sympathetic denervation can accompany Parkinson's disease, but the effect of this denervation on cardiac lipid-mediated signaling is unknown. To address this issue, rats were sympathetically denervated with 6-hydroxydopamine (6-OHDA, 50 mg/kg ip) and infused with 170 µCi/kg of either [1-14C]palmitic acid ([1-14C]16:0) or [1-14C]arachidonic acid ([1-14C]20:4 n-6), and kinetic parameters were assessed using a steady-state radiotracer model. Heart norepinephrine and epinephrine levels were decreased 82 and 85%, respectively, in denervated rats, and this correlated with a 34% reduction in weight gain in treated rats. Fatty acid tracer uptake was not significantly different between groups for either tracer, although the dilution coefficient {lambda} was increased in [1-14C]20:4 n-6-infused rats, which indicates that less 20:4 n-6 was recycled in denervated rats. In [1-14C]16:0-infused rats, incorporation rate and turnover values of 16:0 in stable lipid compartments were unchanged, which is indicative of preservation of {beta}-oxidation. In [1-14C]20:4 n-6-infused rats, there were dramatic reductions in incorporation rate (60–84%) and turnover value (56–85%) in denervated rats that were dependent upon the lipid compartment. In addition, phospholipase A2 activity was reduced 40% in treated rats, which is consistent with the reduction observed in 20:4 n-6 turnover. These results demonstrate marked reductions in 20:4 n-6 incorporation rate and turnover in sympathetic denervated rats and thereby suggest an effect on lipid-mediated signal transduction mediated by a reduction in phospholipase A2 activity.

palmitic acid; signal transduction; phospholipase A2; catecholamines; phospholipids

UNDER NORMAL PHYSIOLOGICAL conditions, the mammalian heart uses palmitic acid (16:0) as a primary source of metabolic energy via {beta}-oxidation (11), but the heart also takes up polyunsaturated fatty acids such as arachidonic acid (20:4 n-6; Refs. 22, 36). The uptake rate and ultimate deposition of fatty acids depend in part upon chain length (11). Under physiological plasma glucose levels, saturated and monounsaturated fatty acids are esterified into heart triacylglycerol (TAG) pools and are used almost exclusively for {beta}-oxidation (10, 25, 46). Palmitic acid, a saturated fatty acid, is targeted for esterification into heart TAG pools and is used to meet energy demands (11, 25, 36), whereas 20:4 n-6 is primarily esterified into heart phospholipid pools (36).

The roles of phospholipids and their constitutive fatty acids in lipid-mediated signal transduction in heart are becoming more appreciated. It is accepted that a number of signal transduction pathways in heart are mediated through increased phospholipase A2 (PLA2) activity and include release of 20:4 n-6 (33). In cultured rat ventricular myocytes, IL-1{beta} activates a membrane-associated, plasmalogen-selective, calcium-independent PLA2 (iPLA2) through a receptor-linked mechanism to release 20:4 n-6 (34). TNF-{alpha} mediates 20:4 n-6 release by increasing the activity of a cytosolic PLA2 in rat ventricular myocytes (28). This release of 20:4 n-6 is associated with both the positive and negative effects of TNF-{alpha} on heart function (3). Angiotensin II stimulates the release of 20:4 n-6 and inositol phosphates through activation of multiple receptor subtypes coupled to PLA2 and phospholipase C activation (29). Thrombin stimulates choline plasmalogen (PlsCho) turnover via the plasmalogen-selective iPLA2 to release 20:4 n-6 and lysoPlsCho, both of which are potent second messengers (32). {beta}2-Adrenergic receptor stimulation also leads to 20:4 n-6 release through activation of a PLA2 in the cytosol (37), which suggests a role for 20:4 n-6 in regulating heart ionotropic and chronotropic events (45).

Recent evidence indicates that many patients with Parkinson's disease have complete sympathetic cardiac denervation, thereby removing sympathetic nervous system input into the heart (17, 18). Treating rats with 6-hydroxydopamine (6-OHDA) effectively models cardiac sympathetic denervation and decreases heart tyrosine hydroxylase activity, which causes a concomitant decrease in heart norepinephrine and dopamine levels (24). Sympathetic denervation of rat heart with 6-OHDA (100 mg/kg ip) may also increase the number of {beta}-adrenergic receptors in heart (50), although others have demonstrated no effect of denervation on {beta}-adrenergic receptor number (51). A similar level of denervation is accomplished by injecting rats with 6-OHDA (50 mg/kg ip) on two consecutive days, 10 days before studies (20). Similar to rats, dogs injected with 6-OHDA have increased heart {beta}-adrenergic receptor density, whereas heart muscarinic receptor density remains unchanged (49). In rabbits, sympathetic denervation by 6-OHDA does not alter {beta}-adrenergic receptor number but does reduce isoproterenol-induced cAMP production and cardiac output (48). Similarly, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in mice decreases {beta}-adrenergic receptor number and reduces myocyte functional properties (42). Although sympathetic denervation may or may not alter the number of {beta}-adrenergic receptors, denervation does reduce the effectiveness of receptor coupling to adenyl cyclase (48) to result in decreased heart function (42, 48). Because {beta}2-receptors are also linked to a PLA2 in cytosol, sympathetic denervation may result in a decrease in receptor-stimulated PLA2 activity and thereby cause a reduction in 20:4 n-6 incorporation and turnover.

To address this issue, we injected rats with 6-OHDA (50 mg/kg for 2 consecutive days) 10 days before infusing the rats with either [1-14C]16:0 or [1-14C]20:4 n-6 (170 µCi/kg). Sympathetic denervation was confirmed by measurement of heart catecholamine levels in 6-OHDA-injected rats. Using a steady-state radiotracer kinetic model, we calculated incorporation rate, fractional turnover, and half-life values for 16:0 and 20:4 n-6 in stable heart lipid compartments. We found a dramatic reduction (45–70%) in [1-14C]20:4 n-6 turnover with a concomitant increase in half-life values in heart lipid compartments of sympathetically denervated rats, whereas [1-14C]16:0 incorporation rate and turnover were unaffected.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats (body wt, 150–200 g) were obtained from Harlan Laboratories (St. Louis, MO) and maintained on standard laboratory rat diet and water ad libitum. The animal protocol used in this study was approved by the National Institute of Child Health and Human Development IACUC and was conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication 80-23).

Treatment with 6-OHDA. Before treatment with 6-OHDA, rats were handled daily for five consecutive days to acclimate them to the investigators. The rats were divided into two groups. Each group was injected with either vehicle or 6-OHDA (50 mg/kg ip per injection) on two consecutive days and used on day 10 after the initial injection. After each injection, the rats were monitored for responses to catecholamine release including rapid respiration, slow locomotion, and piloerection. The rats were weighed on days 0, 3, 7, and 10.

Animal surgery. After rats were anesthetized with halothane (1–3%), the femoral artery and vein were catheterized with polyethylene-50 tubing. The wound area was anesthetized with lidocaine (1%) and was closed using standard surgical staples. The hindquarters of the rat was placed in a plaster body cast, taped loosely to a wooden block, and maintained postoperatively in a temperature-controlled environment for 3–4 h. The rat's body temperature was maintained at 37°C by a feedback heating device and a rectal thermocouple.

Intravenous infusion. Awake adult male rats were infused with 170 µCi/kg of [1-14C]16:0 or [1-14C]20:4 n-6 into the femoral vein over 10 min using a variable-rate infusion paradigm to achieve steady-state plasma radioactivity. Before and during the experimental period, arterial blood samples (200 µl) were obtained to determine plasma radioactivity. After infusion, the rat was killed by infusion of pentobarbital sodium (100 mg/kg iv). The heart was rapidly removed, bisected, and rinsed twice with ice-cold phosphate-buffered saline to remove residual blood. After it was blotted dry, the heart was frozen in liquid N2 and stored at –80°C until it was used. The time from pentobarbital administration to freezing was reproducibly 1.3 ± 0.2 min.

Radiotracer preparation. Radiotracer (Moravek Biochemical; Brea, CA) was prepared by taking an aliquot of tracer in ethanol and evaporating the ethanol under a constant stream of N2 at 50°C. Before use, radiotracer purity was assessed by TLC and was found to be >97% pure. The fatty acid tracer was solubilized in 5 mM HEPES (pH 7.4) buffer that contained "essentially fatty acid-free" bovine serum albumin (50 mg/ml; Sigma Chemical; St. Louis, MO). Solubilization was facilitated by sonication in a bath sonicator for 40 min at 45°C. Radioactivity was determined using liquid scintillation counting and was adjusted to 100 µCi/ml. The appropriate amount of radiotracer was prepared for each rat using the animal's weight based upon the infusion parameters of 170 µCi/kg (16).

Catecholamine analysis. Control and denervated rats were subjected to the surgical procedures and postsurgical conditions and were infused with vehicle. The hearts of these rats were removed as previously described (see Intravenous infusion) and frozen in liquid N2. After pulverization, catecholamines were extracted using 0.4 M perchloric acid. Catecholamines were separated by high-performance liquid chromatography, and mass values were determined using electrochemical detection.

Plasma extraction. Arterial blood samples, obtained at fixed times during the infusion period, were used to determine plasma radioactivity in the organic fraction via a two-phase extraction system (14). Phases were thoroughly mixed and separated overnight in a –20°C freezer. The upper phase was removed, and the lipid-containing lower phase was rinsed with 0.63 ml of theoretical upper phase to remove any aqueous soluble contaminants (14). The upper phase was discarded, a portion of the lower phase was dried, and its radioactivity was quantified with a 2200 CA Tri-Carb liquid scintillation counter (Packard Instruments; Downers Grove, IL). Another portion was used for lipid separation by TLC, and the fractions were analyzed as described (see Heart acyl-CoA extraction and analysis).

Blood extraction. Whole blood was extracted to correct for residual blood radioactivity after the procedure described above for plasma. The lipid fractions were separated as described below. Total radioactivity and radioactivity values of each individual lipid fraction were used to correct for residual radioactivity contributed by blood left in the heart.

Heart lipid extraction. Frozen heart tissue was pulverized under liquid-N2 temperatures, and the lipids in the resulting powder were extracted with a Tenbroeck tissue homogenizer using a two-phase system (14). The upper phase and proteinaceous interface were removed and saved in a 20-ml glass scintillation vial. The lower organic phase was washed twice with 2 ml of theoretical upper phase, and phase separation was facilitated by centrifugation between washes. The washes were removed and combined with the previously removed upper phase. The washed lower phase was dried under a stream of N2, and the lipids were redissolved in 3 ml of a 3:2 (vol/vol) n-hexane-2-propanol solution that contained 5.5% H2O.

Heart acyl-CoA extraction and analysis. For heart acyl-CoA extraction, pulverized heart tissue was homogenized at 4°C by sonication in 2 ml of 100 mM KH2PO4 buffer (pH 4.9) after addition of 17:0 CoA and 14:0 CoA as internal standards (12, 13). After sonication, 2-propanol (2 ml) was added, and the homogenate was mixed and sonicated again for 20 s. Proteins were precipitated by addition of 0.25 ml of saturated ammonium sulfate (4 M), and acetonitrile (4 ml) was added followed by centrifugation at 1,600 g for 5 min. The supernatant was removed and diluted with 10 ml of 100 mM KH2PO4 buffer (pH 4.9). A low-pH buffer was used to minimize losses of acyl-CoA by heart acyl-CoA hydrolase activity, which is inactive at pH < 6 (26).

The acyl-CoA fraction was then concentrated using solid-phase extraction with an oligonucleotide purification cartridge (Applied Biosystems; Foster City, CA; Ref. 12). The sample was loaded onto the washed cartridge and the polar components were eluted with 5 ml of 25 mM KH2PO4 buffer (pH 4.9). The acyl-CoA fraction was eluted using a 75:25 (vol/vol) solution of 0.15 ml of 2-propanol and 1 mM acetic acid (39). A portion of this eluent was directly used for separation of the acyl-CoA species on a Phenomenex Luna C-18(2) column (4.6 x 250 mm), and the individual acyl-CoA values were quantified by UV absorbance measurements.

Acyl-CoA was separated using high-performance liquid chromatography and was detected by UV absorbance at 260 nm. The acyl-CoA species were eluted from the Phenomenex Luna C-18(2) column with a binary gradient using 75 mM KH2PO4 buffer (pH 4.9) as solvent A and acetonitrile as solvent B. The flow rate was 1 ml/min, and the initial A-to-B solvent proportions were 56:44%. An acetonitrile gradient was used to elute the acyl-CoA as the acetonitrile was increased from 44 to 49% over 25 min. Hydrophobic molecules were eluted by increasing the percentage of acetonitrile to 70% over 5 min. Acyl-CoA concentrations were calculated based on the peak area of a standard acyl-CoA mixture. Two internal standards that were eluted at different times were used to calculate the effects of peak broadening and the sample recovery.

Aqueous fraction. The aqueous fraction was initially processed for liquid scintillation counting by drying it at 80°C for 18 h to remove 14CO2, thus providing a measure of nonvolatile byproducts of {beta}-oxidation. The dried material was then solubilized in 2 ml of Soluene (Packard Instruments) in tightly capped scintillation vials heated at 80°C for 2 h. Radioactivity was determined after addition of 10 ml of Ready-Solv liquid scintillation cocktail (Beckman; Fullerton, CA) using a Packard 2200 CA Tri-Carb liquid scintillation counter.

Thin-layer chromatography. Phospholipids and neutral lipids were separated by TLC. Phospholipids were separated on heat-activated Whatman silica gel-60 plates (20 x 20 cm, 250 µm) and developed in a 60:30:3:1 (vol/vol/vol/vol) chloroform-methanol-acetic acid-water solution. This solvent system resolves cardiolipin, phosphatidic acid, and ethanolamine glycerophospholipids (EtnGpl) but not phosphatidylinositol and phosphatidylserine. Phosphatidylserine and phosphatidylinositol were resolved using heat-activated Whatman silica gel-60 plates and were developed in a 50:37.5:3.5:2 (vol/vol/vol/vol) chloroform-methanol-acetic acid-water solution (23). Neutral lipids were separated using heat-activated silica G plates (Analtech; Newark, DE) developed in a 70:30:1.3 (vol/vol/vol) petroleum ether-diethyl ether-acetic acid solution (30). This solvent system resolves cholesteryl esters, diacylglycerols (DAGs), nonesterified fatty acids, and TAGs. Lipid fractions were identified using authentic standards (Doosan-Serday; Englewood Cliffs, NJ, and NuChek Prep; Elysian, MN).

Nonesterified fatty acid quantitation. To determine the mass and specific activity values of the fatty acid of interest in the plasma, fatty acids in plasma were separated (by TLC) from other lipid components and esterified using an acid-catalyzed esterification (1). After esterification, individual fatty acids were separated by gas-liquid chromatography (SP-2330 column, 0.32 mm id, 30 ml) using a Hewlett-Packard model 5890 series II (King of Prussia, PA) gas-liquid chromatograph and were quantified by flame ionization detection. Fatty acids were quantified using an external standard method (17:0), and a set of standard curves was derived using a set of commercially prepared standards (NuChek Prep). Correlation coefficients for each standard curve were ≥0.96.

Esterified fatty acid quantitation. To determine the mass and specific activity value of the fatty acid of interest esterified into individual lipids, individual phospholipid or neutral lipids were separated (by TLC) and the acyl chains were transesterified in methanol to form the methyl esters. Briefly, silica containing the lipid class of interest was incubated with 0.5 M KOH in anhydrous methanol for 20 min at 35°C (5). The fatty acid methyl esters were extracted using petroleum ether, and the mass of each fatty acid was determined as described above for plasma free fatty acids.

Liquid scintillation counting. Bands corresponding to the appropriate lipid fractions were scraped into 20-ml liquid scintillation vials, and 0.5 ml of H2O was added followed by 10 ml of Beckman Ready-Solv liquid scintillation cocktail. Samples were mixed and quantified by liquid scintillation counting at least 1 h after addition of the cocktail.

PLA2 activity. Powdered heart tissue was homogenized in ice-cold PLA2 assay buffer that contained 250 mM sucrose, 10 mM KCl, 10 mM imidazole, 5 mM EDTA, 2 mM dithiothreitol and 10% glycerol, pH 7.8 with 10 N KOH (Sigma Chemical). The homogenate was centrifuged at 14,000 g for 10 min, and the supernatant was then centrifuged at 100,000 g for 60 min to separate the membrane fraction (pellet) from the cytosolic fraction (supernatant). The membranes were washed twice and centrifuged after each wash at 100,000 g for 60 min. The washed membranes were then resuspended in PLA2 assay buffer, and PLA2 activity was assayed as previously described (33). The substrates were 100 µM of either 1-O-alk-1'-enyl-2-acyl glycerophosphocholine (PlsCho) or phosphatidylcholine (PtdCho) that contained 16:0 in the sn-1 position and [3H]18:1 in the sn-2 position. Released radiolabeled fatty acid was isolated by application of 25 µl of the butanol phase to channeled silica gel G plates, development in a 70:30:1 (vol/vol/vol) petroleum ether-diethyl ether-acetic acid solution, and subsequent quantification by liquid scintillation counting. The reaction conditions selected resulted in linear reaction velocities with respect to both time and total protein concentration for each substrate examined. Protein content of each sample was determined by the Lowry method using freeze-dried bovine serum albumin as the protein standard as described previously (31).

Fatty acid model and calculations. A method has been developed to quantify the rate of entry and incorporation of fatty acids into rodent brain in vivo (40, 41, 44) using standard steady-state radiotracer kinetic analysis. This method can be universally applied to any tissue and takes into account the recycling of endogenously released fatty acids back to stable lipid compartments, thereby making its use ideal for measuring the kinetics of heart fatty incorporation and turnover. To calculate fatty acid incorporation rates from plasma into stable lipid compartments, the specific activities of the fatty acid in the plasma and tissue compartments must be measured. This is done using liquid scintillation counting to measure the radioactivity and gas-liquid chromatography to measure fatty acid mass in any given compartment. Similarly, the specific activity of the acyl-CoA fraction is measured using a combination of liquid scintillation counting and mass measurement as determined by UV detection after separation by HPLC. The equations used in this model have been described in detail elsewhere (40, 41, 44) and have been applied to heart fatty acid uptake and incorporation into stable lipid compartments as previously described.

Recently this method was used to measure the kinetics of fatty acid uptake and disposition into rat heart using fatty acid tracer bound to bovine serum albumin (36). Under physiological conditions, fatty acids are delivered to the heart in either an albumin-bound form or through triglycerides that are rapidly hydrolyzed by lipoprotein lipase to release the fatty acids (4). Heart, unlike other organs, is uniquely able to rapidly hydrolyze triglycerides to provide sufficient free fatty acids for its use (4). Nonetheless, the infusion of either triglyceride or free fatty acid bound to albumin does not appear to affect the kinetics of tracer movement from the plasma compartment into heart (4, 47, 52). The rapid interchange of fatty acids hydrolyzed from triglycerides with the free fatty acid pool negates any difference between these two different routes of delivery (47). Furthermore, infusion of [1-3H]hexadecanol has been used to quantify the kinetics of heart ether-lipid biosynthesis (15) and indicate that the use of high-specific-activity radiotracers was successful in estimating heart lipid biosynthesis or fatty acid uptake (4, 35, 36, 47, 52). Thus the method used in this study is applicable to studying the kinetics of heart fatty acid uptake, incorporation into lipid pools, and subsequent turnover.

Statistical analysis. Differences between group means were determined using a one-way or two-way Student's t-test where appropriate. Statistical significance was defined as P ≤ 0.05. All values are expressed as means ± SD.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Physiological parameters. Physiological parameters were measured to assess postsurgical recovery and to demonstrate effects of sympathetic denervation. In both groups, there was a postsurgical elevation in PCO2, decrease in PO2, and reduction in temperature, all of which returned to normal values within the 3-h postsurgical recovery time (Table 1). Blood pressure and heart rate were measured before infusion (Table 2). No significant differences were observed in either of these two parameters between groups.


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  		Table 1. Physiological parameters for treated and control rats

 

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  		Table 2. Preinfusion physiological parameters for treated and control rats

 
Catecholamine levels. Catecholamine levels were measured in denervated and control rats to demonstrate a marked reduction in catecholamine levels consistent with sympathetic denervation (38). Norepinephrine levels were significantly reduced 82% by 6-OHDA treatment; control values were 26,733 ± 6,180 and values for 6-OHDA-treated rats were 4,761 ± 841 ng/mg of tissue mass. As reported elsewhere, heart epinephrine levels were decreased 85% in the denervated compared with the control rats (Fig. 1). Heart dopamine levels were reduced 60% in the 6-OHDA-treated compared with the control rats. Neither the levels of 3,4-dihydroxyphenyl acetic acid, which is a product of dopamine catabolism produced by the sequential action of monoamine oxidase and aldehyde dehydrogenase on dopamine, or the levels of L-dihydroxyphenyl alanine, the precursor for dopamine, were different between groups (Fig. 1). Thus the 6-OHDA-treated rats demonstrated a robust reduction in heart catecholamine levels consistent with sympathetic denervation (38).



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  		Fig. 1. Heart epinephrine, dopamine, 3,4-dihydroxyphenyl acetic acid (DOPAC), and L-dihydroxyphenyl alanine (L-DOPA) levels in treated and control rats. Values are means ± SD, n = 7–8 rats. *P < 0.0001, significance from control as determined using one-way, paired Student's t-test.

 
Weight gain. Body weights of control and denervated rats were measured on days 0, 7, and 10. Control rats gained 72.8 ± 10.6 g (n = 8), whereas denervated rats gained 48.6 ± 7.6 g (n = 7; P = 0.0002). Thus there was a significant decrease in weight gain in treated rats, and this 34% decrease positively correlated with the reduction in heart catecholamine levels (data not shown). A reduction in weight gain was used to ensure that 6-OHDA-treated rats used for fatty acid infusion were sympathetically denervated.

Plasma curves. The average area under the curve for the [1-14C]16:0-infused rats was 2,091 ± 507 nCi·min–1·ml–1 (n = 13), whereas the average area for [1-14C]20:4 n-6 rats was 1,768 ± 207 nCi·min–1·ml–1 (n = 14). These areas are not significantly different.

Fatty acid uptake and distribution. Total fatty acid tracer uptake and distribution into aqueous and organic compartments was assessed in control and denervated rats (Table 3). There were no significant differences in [1-14C]16:0 total uptake or distribution between groups. Similarly, there were no significant differences in [1-14C]20:4 n-6 total uptake or distribution between groups. However, more [1-14C]20:4 n-6 (79%) was targeted to the total organic fraction compared with the aqueous fraction relative to [1-14C]16:0 (69%) infused rats. Similar results were seen when these values were expressed as the unilateral incorporation coefficient k*.


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  		Table 3. Total tracer uptake and distribution into aqueous and organic compartments

 
Distribution of each tracer in individual lipid compartments in the organic fraction was expressed as mean k*. For [1-14C]16:0, there was no significant difference between the two groups (Table 4); most of the tracer was esterified in TAG with a lesser amount in the phospholipids. For [1-14C]20:4 n-6, there was significantly more tracer in the choline glycerophospholipids (ChoGpl) in the denervated compared with the control rats. No other significant change was observed, although most of the tracer was targeted to the ChoGpl and to a lesser extent to the TAG. Thus sympathetic denervation did not have any dramatic effect on the distribution and targeting of either tracer.


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  		Table 4. Distribution of [1-14C]16:0 and [1-14C]20:4 n-6 into stable heart lipid compartments

 
Fatty acid incorporation rate, turnover, and half-life. Distribution alone is not indicative of tracer movement through a given lipid compartment. To address the kinetics of fatty acid incorporation and turnover, a steady-state radiotracer model was used. For [1-14C]20:4 n-6, there were many significant alterations in incorporation rate, turnover, and half-life of the tracer in stable heart lipid compartments (Table 5). The dilution coefficient {lambda} was significantly elevated 5.25-fold in denervated rats compared with control rats. The increase in {lambda} indicates a lesser degree of fatty acid recycling of 20:4 n-6 from heart phospholipid pools in treated rats, with a greater contribution of labeled 20:4 n-6 from the plasma compared with the use of endogenous recycled, unlabeled 20:4 n-6.


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  		Table 5. Effects of 6-OHDA treatment on 20:4 n-6 incorporation rate, fractional turnover, and half-life in stable heart lipid pools

 
For every compartment listed, there was a large decrease in the 20:4 n-6-incorporation rate in treated compared with control rats. The reduction in the incorporation was dependent on the lipid compartment with reductions of 59% for ChoGpl, 60% for EtnGpl, 63% for the combined phosphatidylinositol and phosphatidylserine (PtdIns-Ser), 75% for TAG, and 84% for DAG. The rank order for incorporation through the lipid compartments was ChoGpl > TAG >> EtnGpl > PtdIns-Ser = DAG.

The decrease in incorporation rate was accompanied by a decrease in fractional turnover and elevation of half-life. The reduction in turnover was dependent on the lipid compartment with reductions of 61% for ChoGpl, 56% for EtnGpl, 82% for PtdIns-Ser, 68% for TAG, and 83% for DAG. The reductions in fractional turnover were the same as for reductions in the incorporation rate, as 6-OHDA treatment did not significantly alter cold fatty acid concentrations. The rank order for fractional turnover was TAG = DAG > PtdIns-Ser > ChoGpl > EtnGpl. The half-lives have the same rank order, which for control rats ranged from 0.05 h for TAG to 3.9 h for EtnGpl. Thus in denervated rats, there was a significant reduction in fatty acid incorporation rate and turnover in heart lipid compartments with a concomitant increase in half-life.

For [1-14C]16:0, there was no significant change in incorporation rate, turnover, or half-life between 6-OHDA-treated and control groups. However, the incorporation rate through the TAG pool was nearly 20,000 nmol·h–1·g–1, which is consistent with the use of 16:0 in heart {beta}-oxidation. The rank order for the incorporation rate was TAG > DAG = ChoGpl > EtnGp = PtdIns-Ser. The rank order for turnover was DAG > TAG = PtdIns-Ser > EtnGpl = ChoGpl. The half-life values of the TAG, DAG, and PtdIns-Ser pools were the shortest, on the order of 0.010 h, whereas the longest half-life was for the ChoGpl compartment at 0.52 h. Thus sympathetic denervation did not alter the kinetics of 16:0 incorporation, turnover, or half-life in heart stable lipid compartments, which is consistent with a preservation of the hearts' ability to maintain {beta}-oxidation capacity in the treated rats.

PLA2 activity. Because of the decreases observed in 20:4 n-6 incorporation rate, turnover, and half-life in the stable lipids of the denervated rat hearts, PLA2 activities were also measured. Two different substrates, PtdCho and PlsCho, were used to measure activity in both cytosolic and membrane fractions in the presence of Ca2+. Using the PlsCho substrate, there was a significant 40% reduction in activity of membrane-bound PLA2 (Table 6). However, when PtdCho was used as a substrate, there was a 40% reduction in the activity of PLA2 localized in the cytosol. Hence these data demonstrate decreased PLA2 activity in treated rats, which is consistent with the observed reduction in 20:4 n-6 incorporation rate and turnover.


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  		Table 6. Effects of 6-hydroxydopamine treatment on heart phospholipase A2 activity

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cardiac sympathetic denervation is associated with Parkinson's disease (2, 17–19, 21, 27). The loss of sympathetic innervation is positively correlated with disease severity (21) and demonstrates a progressive decrease with disease progression (21, 27). Although a majority of patients with Parkinson's disease have some degree of sympathetic denervation, not every patient has orthostatic hypotension; however, patients with orthostatic hypotension have a significantly greater sympathetic degree of denervation (19). Evidence suggests that the sympathetic denervation is the result of nerve-terminal "die back" as opposed to loss of the cell body (19, 27). Despite the evidence supporting an association of sympathetic denervation with Parkinson's disease, there is limited information regarding the effects of denervation on heart function and signal transduction.

A recent study using mice treated with MPTP (30 mg/kg ip) to produce heart sympathetic denervation demonstrated a functional alteration of myocytes (42). These myocytes had reduced mechanical properties and also had altered Ca2+-handling properties. Furthermore, there was a 67% decrease in the number of {beta}-receptors and reduced response to norepinephrine (1 µM) challenge in myocytes from the MPTP-treated mice, which suggests an alteration in receptor coupling to intracellular signaling mechanisms. In 6-OHDA-treated rabbits, the number of {beta}-receptors was unchanged, although there was a marked reduction (78%) in isoproterenol-induced cAMP production (48), which suggests aberrant signal transduction coupling. Thus both models of denervation demonstrated an altered response to {beta}-receptor stimulation. Because {beta}2-receptor stimulation can also increase 20:4 n-6 release mediated through PLA2 activation (37), the lack of responsiveness in either model may also be attributed to an attenuation in the coupling of PLA2 to the {beta}2-receptor. This hypothesis is consistent with the results presented herein that demonstrate a marked reduction in [1-14C]20:4 n-6 turnover (see Table 5) and PLA2 activity (see Table 6) in denervated hearts.

In 6-OHDA-treated rats, we demonstrated a marked reduction in the 20:4 n-6-incorporation rate into stable lipid compartments and a marked reduction in turnover. These reductions were accompanied by an increase in the dilution coefficient {lambda}, which indicates a lesser contribution of 20:4 n-6 coming from endogenous 20:4 n-6 released from heart lipid pools and an increase in 20:4 n-6 coming from plasma. This reduction is consistent with a reduction in 20:4 n-6 release via PLA2 activity. A similar situation was seen in Li+- and valproic acid-treated rats (6–8), where there was a selective 40–80% reduction in 20:4 n-6 incorporation rate and turnover in the brain as well as an increase in {lambda} for 20:4 n-6. This increase in {lambda} was of a similar magnitude to that observed in 6-OHDA-treated rat hearts. In Li+-treated rats, there was a 50% decrease in cytosolic PLA2 (type IV, cPLA2) activity (9) accompanied by a 40% reduction in cPLA2 enzyme levels (43). A similar reduction in PLA2 enyzmic activity occurred in 6-OHDA-treated rats (see Table 6) because of either a reduction in enzyme expression or an alteration in posttranslational modification of PLA2 downstream from receptor stimulation.

As identified in brain after Li+ treatment in rats, there were no marked changes in [1-14C]16:0 kinetic parameters in the 6-OHDA-treated rats (Table 7) despite a reduction in [1-14C]20:4 n-6 (see Table 5). We showed that the TAG pool had a very rapid incorporation rate and turnover of 16:0, which suggests that the passage of this fatty acid through this pool is obligatory in the trafficking of 16:0 from the membrane to the mitochondria for {beta}-oxidation. The rapid turnover of 16:0 in the DAG pool suggests that DAG is a metabolic intermediate in the biosynthesis of TAG or a pool representing the catabolism of TAG associated with release of a single fatty acid for {beta}-oxidation, rather than a pool resulting from phosphatidylinositol hydrolysis by phospholipase C activation. The rates of 16:0 incorporation and turnover were much greater than those observed for 20:4 n-6 except for the ChoGpl fraction, where the rates for both tracers were of a similar magnitude. Thus these results are suggestive of a preservation of heart {beta}-oxidation in 6-OHDA-treated rats, which is consistent with the physiological data that demonstrates no overt alterations in heart rate or pressure in the treated rats.


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  		Table 7. Effects of 6-OHDA treatment on 16:0 incorporation rate, fractional turnover, and half-life in stable heart lipid pools

 
However, it is important to note that we did not measure heart mechanical function in this study. Although no changes were observed in heart rate or blood pressure in the treated rats, we did not use any sophisticated measures of heart function. Furthermore, we did not challenge the treated rats with a {beta}-receptor agonist to determine responsiveness to {beta}-adrenergic stimulation. As noted above, others have demonstrated in models of sympathetic denervation a reduction in mechanical function (42) and aberrant receptor coupling to adenyl cyclase (48). Our results of a reduction in 20:4 n-6 incorporation and a reduction in PLA2 activity are consistent with the reduction in adenyl cyclase activity (48).

Although only a moderate amount of 20:4 n-6 was targeted to TAG in rat hearts (36), a significant proportion was used for {beta}-oxidation, albeit much less than for 16:0. The incorporation rate and turnover of 20:4 n-6 in TAG were reduced 75 and 70%, respectively, in denervated rats (see Table 5). The reduction was greater than that observed for ChoGpl, which is a substrate for iPLA2 and cPLA2. There was no measurable decrease in the amount of [1-14C]20:4 n-6 used in {beta}-oxidation between groups (see Table 3), but there was a 10% increase in the targeting of [1-14C]20:4 n-6 into the ChoGpl pool (see Table 4). Collectively, these results suggest that under conditions of sympathetic denervation, there is an alteration in 20:4 n-6 trafficking so that more 20:4 n-6 is targeted to the ChoGpl pool for lipid-mediated signal transduction rather than to TAG, where it would be used for {beta}-oxidation. An alternative hypothesis is that TAG represents a quickly releasable pool of 20:4 n-6 that is then targeted for esterification into ChoGpl. The first hypothesis suggests an attempt to preserve heart function by limiting the reduction in ChoGpl 20:4 n-6 pool size. It is consistent with the more limited reductions in turnover and incorporation rate of tracer in the ChoGpl pool relative to TAG pools and with the increased targeting of [1-14C]20:4 n-6 to the ChoGpl.

In summary, there was a marked reduction in the incorporation rate and turnover of 20:4 n-6 in stable lipid compartments in rats treated with 6-OHDA. These rats had a substantial reduction in heart catecholamine levels, which is consistent with sympathetic denervation. The lack of changes in 16:0 incorporation rate or turnover indicates a selective process for 20:4 n-6, although changes in the kinetics of other polyunsaturated fatty acids cannot be ruled out at this time. Nonetheless, there appears to be a preservation of the heart's capacity for {beta}-oxidation and a reduction in the capacity for lipid-mediated signaling via 20:4 n-6 as evidenced by reductions in 20:4 n-6 incorporation and turnover. This supposition is further supported by the measured 40% reduction in PLA2 activity in treated rats. At this time, the isoform of PLA2 that accounts for this reduction in 20:4 n-6 turnover has not been identified, but this is the subject of future studies. Nonetheless, in sympathetic denervated heart, the selective, marked reduction in 20:4 n-6 incorporation rate and turnover is consistent with an uncoupling of heart receptors from intracellular signaling processes mediated via increased PLA2 activity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The completion of this work was partially supported by American Heart Association Grant 0151121Z (to E. J. Murphy).


   ACKNOWLEDGMENTS
 
The authors thank Courtney Holmes for HPLC analysis of heart catecholamines, Dr. David S. Goldstein for helpful and substantive discussions, Drs. Paula Castagnet and Carole Haselton for manuscript review, and Cindy Murphy for typed manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. J. Murphy, Dept. of Pharmacology, Physiology, and Therapeutics, School of Medicine and Health Sciences, Univ. of North Dakota, 501 N. Columbia Rd., Rm. 3700, Grand Forks, ND 58202-9037 (E-mail: emurphy{at}medicine.nodak.edu)

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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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