Skip Navigation

Cardiovascular Research 1999 42(1):48-56; doi:10.1016/S0008-6363(98)00298-3
© 1999 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kurz, T.
Right arrow Articles by Richardt, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kurz, T.
Right arrow Articles by Richardt, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1999, European Society of Cardiology

Alpha1-adrenergic receptor-mediated increase in the mass of phosphatidic acid and 1,2-diacylglycerol in ischemic rat heart

Thomas Kurz*, Ines Schneider, Ralph Tölg and Gert Richardt

Medizinische Klinik II, Medizinische Universität zu Lübeck, Ratzeburger Alle 160, 23538 Lübeck, Germany

* Corresponding author. Tel.: +49-451-500-2500; fax: +49-451-500-6437.

Received 8 June 1998; accepted 21 September 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: 1,2-Diacylglycerol (1,2-DAG) and phosphatidic acid (PA) are produced by phospholipase C and D activity and play a key role as second messengers in receptor-mediated signal transduction. So far, little is known about alterations of endogenous 1,2-DAG and PA production during myocardial ischemia. Methods: Rat isolated perfused hearts were subjected to global ischemia, total lipids were extracted, and separated by thin-layer chromatography. The mass of PA and 1,2-DAG were quantified using laserdensitometric analysis of visualized lipids. Results: Compared to normoxic control values (1,2- DAG 713±45 ng/mg protein, PA 171±11 ng/mg protein), the myocardial content of 1,2-DAG and PA was unaltered after 10 min of ischemia. Prolonged myocardial ischemia (20 min), however, which was accompanied by marked overflow of endogenous norepinephrine, significantly increased the mass of both second messengers (1,2-DAG 1062±100 ng/mg protein, PA 340±29 ng/mg protein). The increase in PA and 1,2-DAG in response to ischemia was abolished by inhibition of ischemia-induced norepinephrine release as well as by alpha1-adrenergic blockade but unaffected by β-adrenergic blockade. While inhibition of diacylglycerol kinase did not affect ischemia-induced increase in PA and 1,2-DAG, inhibition of phosphatidylinositol-specific phospholipase C activity significantly suppressed ischemia-induced increase in 1,2-DAG but did not affect endogenous production of PA indicating phospholipase C-independent formation of PA and activation of both, phospholipase C and D, in the ischemic heart. Conclusions: Ischemia elicits an alpha1-adrenergic receptor-mediated increase in the mass of myocardial PA and 1,2-DAG. The increase in endogenous PA is suggested to be due to the activation of myocardial phospholipase D, whereas 1,2-DAG is formed predominantly by activation of phospholipase C in the ischemic heart.

KEYWORDS Phosphatidic acid; Diacylglycerol; Myocardial ischemia; Adrenergic receptor; Norepinephrine


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Receptor-mediated activation of phospholipases is a central biochemical event in cellular signaling to form second messengers and biological mediators. Among the phospholipases, the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C is an established pathway in hormone-regulated signal transduction in a variety of tissues. The initial products of phospholipase C mediated hydrolysis of PIP2, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, play pivotal roles as intracellular second messengers through the mobilization of calcium from intracellular stores and activation of protein kinase C [1, 2]. The role of phospholipids in receptor-mediated signal transduction, however, is not restricted to phosphoinositides, but also applies to phosphatidylcholine. Phospholipase D (EC 3.1.4.4) appears to play a key role in the receptor-mediated breakdown of choline phospholipids [3]. The primary lipid product of phospholipase D is phosphatidic acid (PA), and in several types of cells and tissues a major metabolic fate of PA is dephosphorylation by phosphatidate phosphohydrolase to 1,2-diacylglycerol (1,2-DAG), representing an alternative pathway for 1,2-DAG formation to the hydrolysis of PIP2 by phospholipase C. The molecular species of 1,2-DAG derived from phosphatidylcholine may have a different pattern of activating some isoforms of protein kinase C and, thus, may serve another function than the PIP2-derived one [2, 4]. Furthermore, formation of 1,2-DAG from phosphatidylcholine has been proposed to cause a delayed, prolonged increase in 1,2-DAG as compared with a rapid, but transient increase in 1,2-DAG produced by hydrolysis of PIP2 by phospholipase C, and thus, the agonist-induced stimulation of phospholipase D may be responsible for sustained activation of specific isoforms of protein kinase C [5]. This pathway could be of great physiological importance because sarcolemma contains much more phosphatidylcholine than phosphoinositides. Moreover, recent studies have demonstrated the potential role for PA as a second messenger per se. These studies have implicated PA in the regulation of DNA synthesis [6, 7], mobilization of intracellular calcium [6, 8, 9], stimulation of Na+–Ca2+ exchange [10], induction of proto-oncogene expression [6], stimulation of protein synthesis, [11]and direct activation of diacylglycerol-independent isoforms of protein kinase C [4]. Furthermore, we have shown previously that exogenous PA can directly increase PIP2-specific phospholipase C activity in cardiac myocytes, leading to an increase in 1,4,5-inositol trisphosphate and 1,3,4,5-tetrakisphosphate [12]due to binding of PA to myocardial phospholipase C-{delta}1 [13].

Although cellular phospholipase D is emerging as a central compound in signal transduction, the information on myocardium is very limited. Myocardial phospholipase D is primarily localized at the sarcolemmal level [14]and its activity has been demonstrated to be increased during myocardial ischemia [15], however, the mechanism of this activation has not been studied. Previously, we have shown that norepinephrine increases phospholipase D activity in adult ventricular myocytes via activation of alpha1-adrenergic receptors [16]. As myocardial ischemia is associated with the release of massive amounts of norepinephrine within the ischemic myocardium [17], the present study was designed to characterize the effect of myocardial ischemia on accumulation of endogenous PA and 1,2-DAG in the rat heart. The problem of dissecting and understanding the complicated intracellular signaling mechanisms after receptor activation is compounded by difficulties in analyzing minute levels of these lipid mediators. In contrast to most studies to date, which have relied on the use of radiolabels to tag, follow and quantify the lipid mediators, we determined the mass of endogenous PA and 1,2-DAG by a novel photodensitometric method. Therefore, we were able to avoid problems associated with the use of radiolabelling procedures such as lack of equilibrium labeling and uncertainty in the involvement of unlabelled pools in the signaling pathway.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Heart perfusion
Hearts were obtained from male Wistar rats (231±21 g; Charles River, Germany) anaesthetized with thiopental (100 mg/kg) injected intraperitoneally. Following anesthesia the peritoneal cavity was opened and 500 I.U. heparin injected into the inferior vena cava. Then the thorax was opened, the heart was rapidly excised, weighed (0.91±0.08 g) and rinsed in ice-cold saline. The ascending aorta was cannulated for isolated perfusion (Langendorff technique) as previously described [18]. The hearts were perfused at a constant flow-rate of 8 ml/min with a modified Krebs–Henseleit buffer composed of (mmol/l): NaCl 125, NaHCO3 16.9, Na2HPO4 0.2, KCl 4.0, CaCl2 1.85, MgCl2 1.0, glucose 11, Na2EDTA 0.027. The temperature of the perfusate was adjusted to 37.5°C at the point of entry into the ascending aorta and the pH of the solution was adjusted to 7.4 by gassing with O2–CO2 (95:5).

For ischemia experiments, the hearts were subjected to global ischemia by stopping perfusion flow, and were subsequently reperfused for 5 min at the initial flow-rate. If drugs were used, they were added to the perfusion buffer starting 10 min prior to ischemia and were resumed during reperfusion. Samples for determination of norepinephrine were taken throughout the last minute before ischemia and during the first 5 min of the reperfusion period. Cumulative norepinephrine overflow was determined from the reperfusion sampling period. At the end of the perfusion, the hearts were frozen immediately in liquid nitrogen.

Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Medizinische Universität zu Lübeck and the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1985).

2.2 Extraction, separation, and quantification of lipids
Frozen hearts were minced and homogenized in ice-cold buffer (0.1 mmol/l HCl, 0.1 mmol/l EDTA) by using an Ultra-Turrax blender (Polytron, two 15-s bursts at setting 5), followed by homogenization in a PTFE–glass Potter-Elvehjem homogenizer (ten up-and-down strokes of a tightly fitting pestle, slowly rotating). Total lipids were extracted from the homogenate using the Bligh and Dyer procedure [19]. The aqueous phase of the extraction mixture was supplemented with NaCl (final concentration in aqueous phase 150 mmol/l NaCl) to facilitate partitioning of PA into the chloroform enriched organic phase. The chloroform phase (containing 0.01% butylated hydroxytoluene as an antioxidant) was separated and dried under a stream of N2. The dried extract was solubilized in 100 µl of chloroform and aliquots were used for separation of individual phospholipid fractions by thin-layer chromatography. With modifications of methods reported previously, [20]PA and 1,2-DAG were resolved from other lipids by TLC using a short bed/continuous development (SB/CD) chamber (Regis). The bottom of the chamber has five stop positions. The TLC plate is placed in the chamber against one of the stops and the back of the plate is then leaned diagonally against the wall so that part of the plate extends out of the chamber. A glass cover plate is placed against the TLC plate, holding it in place and partially sealing the tank. PTFE wings are then slid up to the sides of the TLC plate so that the tank becomes completely sealed and the plate is held firmly in place. When the solvent advances up the TLC plate and passes through the lip of the chamber it evaporates resulting in a steady flow of solvent into and up the plate. The distance on the TLC plate from the solvent level to the point of solvent evaporation is the bed length. Thus, selection of the stop position against which the TLC plate is placed in the development chamber determines the actual bed length and thereby the velocity of the solvent.

For analysis of 1,2-DAG, aliquots (10 µl) of total myocardial lipids were applied to silica gel G plates (Macherey-Nagel, SIL-G25) impregnated with boric acid in order to inhibit acyl migration. Plates were placed against stop position 1 and developed in 50 ml diethylether for 2 min. The plates were air-dried and developed a second time (in the same direction) at stop position 3 for 60 min, using a solvent system of benzene–hexane–diethylether–formic acid (65:50:2:0.2, v/v/v/v). Using this solvent system, 1,2-DAG was well resolved from other lipids, particularly from 1,3-DAG.

For analysis of PA, aliquots (25 µl) of total myocardial lipids were applied to unmodified silica gel G TLC plates (Merck, Kieselgel 60). Plates were developed in a solvent system of benzene–chloroform–pyridine–formic acid (45:35:4:3, v/v/v/v) at stop position 5 for 120 min.

TLC plates were removed from the SB/CD chamber and heated at 50°C for 90 min to evaporate the residual solvents. The plates were stained with Coomassie Brilliant Blue R250 (0.03% in 30% methanol 100 mmol/l NaCl) for 30 min and destained for 5 min in 30% methanol 100 mmol/l NaCl[21]. Stained lipid spots were quantitated using a Hoefer GS 300 scanning densitometer in the reflectance mode at 580 nm (LED). The mass of cellular lipid was interpolated from standard curves established from known quantities of dioleoylglycerol (seven standards ranging from 100 to 1600 ng) or PA (seven standards ranging from 100 to 1000 ng) which were spotted on corresponding plates. Photodensitometric peak areas were integrated using the Hoefer GS 370 densitometry software. The integrated peak areas were best fitted to a second-order polynomial curve using a least squares method (Fig. 1). Correlation coefficients of the standard curves were between 1.00 and 0.99. Recoveries of 1,2-DAG and PA were examined by adding selected amounts of exogenous PA and dioleoylglycerol standard, respectively, to aliquots of myocardial homogenate and the samples were then treated as described above. After quantification of each sample, the mass of PA and 1,2-DAG, respectively, in each sample was compared to the control level without addition of exogenous standard. The recoveries of PA and 1,2-DAG were 85.6 (S.E.M. 2.3)% and 94.7 (S.E.M. 2.0)%, respectively.


Figure 1
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Resolution of 1,2-DAG and PA from other lipids by short bed/continuous development thin-layer chromatography (TLC) and quantification by photodensitometry. Total myocardial lipids were separated by TLC as described in Section 2. (A) Representative TLC plate containing resolved total lipids (sample) and diacylglycerol standards. (C) Representative TLC plate containing resolved total lipids (sample) and PA standards. Integrated peak areas of diacylglycerol (B) and PA (D) standards were best fitted to a second order polynomial curve.

 
The mass of PA and 1,2-DAG were expressed as ng/mg protein. Protein measurements were made by a modification of the method of Lowry et al. [22]using bovine serum albumin as the standard.

2.3 Determination of norepinephrine
Samples for determination of endogenous norepinephrine were cooled on ice, and stabilized by the addition of Na2EDTA to a final concentration of 10 mmol/l. Samples were stored at –60°C until assayed. Norepinephrine was quantified by high-performance liquid chromatography and electrochemical detection as described previously [18]. The drugs used here did not interfere with the extraction, separation or detection of norepinephrine.

2.4 Materials
1,2-Diacyl-sn-glycero-3-phosphate (Sigma, from egg yolk lecithin) and 1,2-dioleoyl-sn-glycerol (Avanti Polar-Lipids) were used as the phosphatidic acid and 1,2-diacylglycerol standards, respectively. Desipramine HCl, S-(–)-propranolol HCl, prazosin HCl were from Sigma. S-(–)-norepinephrine bitartrate, R59949 [GenBank] (3-{2-(4-(bis-(4-fluorophenyl)methylene)-1-piperidinyl)-ethyl}-2,3-dihydro-2-thioxo- 4(1H)quinazolinone}), U 73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), and U 73344 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5- pyrrolidinedione) were from Calbiochem.

2.5 Statistical analysis
In text and figures results are expressed as means±S.E.M. The significance of differences was assessed by analysis of variance followed by Scheffe’s F test. A P value of less than 0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of ischemia and reperfusion on myocardial content of PA and 1,2-DAG
Hearts were subjected to selected periods of myocardial ischemia followed by 5 min of reperfusion (Fig. 2). After 10 min of ischemia the masses of PA and 1,2-DAG were not significantly different from control values of either continuously perfused hearts or freshly excised hearts, which were not mounted on the Langendorff apparatus (n=4; PA: 186.0±11.6 ng/mg protein; 1,2-DAG: 807.8±52.4 ng/mg protein). After 20 min of ischemia the myocardial content of PA and 1,2-DAG increased significantly and remained elevated after 30 min of ischemia.


Figure 2
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of ischemia/reperfusion on myocardial content of phosphatidic acid (PA; left graph) and 1,2-diacylglycerol (1,2-DAG; right graph). Rat hearts were either subjected to global ischemia followed by 5 min reperfusion (ischemia) or continuously perfused for the indicated time periods (control). Data are arithmetic mean±S.E.M., n=6–8 each group.

 
When hearts were subjected to 20 min of ischemia followed by 20 min of reperfusion, the myocardial content of PA and 1,2-DAG (n=4; PA: 211.9±13.6 ng/mg protein; 1,2-DAG: 702.7±14.8 ng/mg protein) was not significantly different from control values of hearts, which were continuously perfused for the same time period (n=4; PA: 172.9±15.3 ng/mg protein; 1,2-DAG: 798.1±68.6 ng/mg protein).

The increase in the myocardial content of PA and 1,2-DAG was accompanied by a progressive overflow of norepinephrine after 20 and 30 min of ischemia (20 min: 195.0±45.6 pmol/g; 30 min: 421.9±29.9 pmol/g) comparable to the time course of norepinephrine release previously demonstrated [17].

In another series of experiments, rat hearts were subjected to selected periods of ischemia without reperfusion in order to evaluate whether the observed alterations in the masses of PA and 1,2-DAG were due to ischemia per se or to the subsequent reperfusion period. After 10 min of ischemia without reperfusion the myocardial content of PA and 1,2-DAG was not significantly different from control values of continuously perfused hearts (PA: 214.9±18.9 vs. 173.6±6.3 ng/mg protein; 1,2-DAG: 877.1±80.9 vs. 757.8±49.7 ng/mg protein). Ischemic periods of 20 and 30 min, respectively, increased the masses of PA and 1,2-DAG comparable to the rise of PA and 1,2-DAG in ischemia/reperfusion (20 min ischemia; PA: 264.5±19.7 vs. 168.9±10.8 ng/mg protein; 1,2-DAG: 1305.9±153.4 vs. 738.7±81.5 ng/mg protein; 30 min ischemia; PA: 312.3±14.4 vs. 165.0±10.2 ng/mg protein; 1,2-DAG: 1118.5±78.5 vs. 753.8±64.4 ng/mg protein). Thus, myocardial ischemia per se induces an increase in the mass of PA and 1,2-DAG, which is maintained during subsequent reperfusion.

3.2 Norepinephrine-mediated increase in myocardial PA and 1,2-DAG during ischemia
Inhibition of ischemia-induced norepinephrine release by desipramine almost completely suppressed the increase in the masses of PA and 1,2-DAG induced by 20 min ischemia followed by 5 min reperfusion (Fig. 3). The effect of desipramine seems to be specifically related to inhibition of ischemia-induced norepinephrine release (without desipramine: 247.3±31.3 pmol/g; with desipramine: 49.8±7.3 pmol/g; P<0.01), since desipramine had no effect on myocardial content of PA and 1,2-DAG in continuously perfused hearts. Furthermore, perfusion of normoxic rat hearts with exogenous norepinephrine (1 µmol/l) for 20 min resulted in a significant increase in the mass of PA and 1,2-DAG (PA: 317.3±37.6 vs. 159.7±9.7 ng/mg protein; 1,2-DAG: 1249.7±81.5 vs. 787.3±75.8 ng/mg protein).


Figure 3
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effect of inhibition of ischemia-induced norepinephrine release on the accumulation of phosphatidic acid (PA; left graph) and 1,2-diacylglycerol (1,2-DAG; middle graph) in ischemic myocardium. Rat hearts were subjected to 20 min global ischemia followed by 5 min reperfusion either in the absence (open bars) or presence (filled bars) of desipramine (100 nmol/l). The addition of desipramine was started 10 min prior to ischemia. The effect of desipramine on the accumulation of PA and 1,2-DAG was also studied in normoxically perfused hearts. Rat hearts were perfused for 20 min either in the absence (open bars) or presence (filled bars) of desipramine (100 nmol/l). Data are arithmetic mean±S.E.M., n=6–9 each group.

 
The increase in the masses of PA and 1,2-DAG after 20 min ischemia followed by 5 min reperfusion was unaltered in the presence of the β-adrenergic blocking agent L-propranolol, but almost completely abolished in the presence of the alpha1-adrenergic receptor antagonist prazosin (Fig. 4). Neither L-propranolol nor prazosin had any significant effect on myocardial content of PA and 1,2-DAG in hearts which were normoxically perfused for 20 min (L-propranolol: PA: 169.8±14.3 ng/mg protein; DAG: 760.2±101.2 ng/mg protein; prazosin: PA: 186.5±24.0 ng/mg protein; DAG: 713.0±102.8 ng/mg protein).


Figure 4
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effect of β-adrenoceptor blockade or alpha1-adrenoceptor blockade on the accumulation of phosphatidic acid (PA; left graph) and 1,2-diacylglycerol (1,2-DAG; right graph) in ischemic myocardium. Rat hearts were subjected to 20 min global ischemia followed by 5 min reperfusion either in the absence of drugs (open bars) or the presence of propranolol (1 µmol/l; hatched bars) or prazosin (1 µmol/l; filled bars). Drugs were added 10 min prior to ischemia and continued until the end of the experiment. Data are arithmetic mean±S.E.M. n=6 each group.

 
3.3 Involvement of phospholipase C and phospholipase D
The ischemia/reperfusion-induced increase in PA and 1,2-DAG was not affected by inhibition of diacylglycerol kinase with R59949 [GenBank] even at a concentration about two orders of magnitude higher than its known IC50-value [23](Table 1). Likewise, in normoxically perfused hearts R59949 [GenBank] did not affect the myocardial content of PA and 1,2-DAG. Inhibition of phosphatidylinositol-specific phospholipase C with U-73122 had no effect on the increase in the mass of PA elicited by 20 min ischemia/reperfusion, but significantly suppressed the ischemia-induced increase in 1,2-DAG (Table 1). In contrast, the inactive analog U-73343 had no effect on both the ischemia-induced increase in PA and 1,2-DAG. In normoxically perfused hearts, neither U-73122 nor U-73343 affected the myocardial content of PA and 1,2-DAG.


View this table:
[in this window]
[in a new window]

  		Table 1 Effect of inhibition of diacylglycerol kinase or phospholipase C on the accumulation of phosphatidic acid and 1,2-diacylglycerol in normoxic and ischemic myocardium

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This paper is the first to describe the effects of myocardial ischemia and reperfusion on the myocardial content of endogenous PA and 1,2-DAG in the heart. The present study demonstrates that myocardial ischemia is associated with an increase in the second messengers PA and 1,2-DAG which is due to ischemia-induced release of norepinephrine and subsequent stimulation of myocardial alpha1-adrenergic receptors. The ischemia-induced rise in myocardial PA and 1,2-DAG is transient returning to basal levels upon prolonged reperfusion. Our results indicate functional coupling of the alpha1-adrenergic receptor in the ischemic myocardium not only to PIP2-specific phospholipase C activity, resulting in the formation of 1,2-DAG, but also to phosphatidylcholine-specific phospholipase D activity, resulting in PA generation.

The present findings were obtained using the measurement of the mass of PA and 1,2-DAG rather than radiolabelling procedures. Hence, it was possible to define accurately the precise ratio of both second messengers during the time-course of myocardial ischemia. Basal levels of 1,2-DAG were about 4-fold higher than myocardial levels of PA. Since myocardial ischemia induced a maximum 2-fold increase of both second messengers, the increase in absolute amounts was much more pronounced for 1,2-DAG as compared to PA. This may reflect either an attenuated formation or an enhanced metabolism of PA as compared to 1,2-DAG. It should be noted that besides direct formation of 1,2-DAG through phospholipase C, dephosphorylation of PA by phosphatidic acid phosphohydrolase may contribute to the production of 1,2-DAG. The significance of this pathway for the generation of DAG during myocardial ischemia is supported by findings of a previous study [15]. Inhibition of phosphatidic acid phosphohydrolase by high-dose propranolol (100 µmol/l) [24]led to an increase in PA which was accompanied by a decrease in DAG suggesting that PA formed by phospholipase D activity indirectly contributes to DAG generation. Although phosphatidic acid phosphohydrolase can be effectively inhibited by high-dose propranolol in vitro, the β-adrenergic receptor antagonist at this concentration causes complete arrest of the heart [25]. Thus, in the present study we could not investigate this pathway due to the lack of other more specific inhibitors of this enzyme [26]. Diacylglycerol can also intracellularly be converted to PA. This conversion, which is catalyzed by diacylglycerol kinase, has been suggested to be the means whereby the cell controls the levels of diacylglycerol. In the present study, inhibition of diacylglycerol kinase [27, 23]did not affect the ischemia-induced formation of endogenous PA. This suggests that phospholipase D activity accounts exclusively for PA generation in the ischemic heart. Furthermore, while inhibition of PIP2-specific phospholipase C activity [28, 29]almost completely suppressed ischemia-induced increases in 1,2-DAG, inhibition of this enzyme did not significantly affect formation of PA. Taken together, the findings of the present study are consistent with phospholipase C-independent formation of PA and suggest that both, phospholipase C and phospholipase D, are activated in the ischemic heart.

The increase in PA and 1,2-DAG in ischemic myocardium was closely related to ischemia-induced release of norepinephrine providing first evidence for an adrenergic mechanism underlying the activation of phospholipase C and phospholipase D during myocardial ischemia. While ischemic periods up to 10 min which did not induce cardiac release of norepinephrine were not accompanied by significant increases in both second messengers, prolonged myocardial ischemia caused extensive release of norepinephrine as well as a pronounced rise in myocardial PA and 1,2-DAG. Previously, ischemia-induced norepinephrine release could be assigned to a carrier-mediated norepinephrine transport of cardiac sympathetic neurons in the rat [18]and human heart [30]. This local release of norepinephrine can be specifically inhibited by blockers of the neuronal catecholamine carrier such as desipramine [18]. Consistent with an adrenergic mechanism, inhibition of ischemia-induced norepinephrine release prevented the increase in PA and 1,2-DAG. In addition, adrenergic stimulation of normoxic hearts by exogenous norepinephrine caused increases in both second messengers comparable to those during ischemia. Thus, these findings provide strong evidence that adrenergic mechanisms are instrumental in inducing the increase in phospholipase C and phospholipase D activity in the ischemic heart. We recently showed that stimulation of alpha1-adrenergic receptors leads to an increase in endogenous PA in ventricular myocytes and that activation of phospholipase D contributes to this increase in mass of myocytic PA [16]. Here we demonstrate that the ischemia-induced increase in the mass of PA and 1,2-DAG is mediated by the alpha1-adrenergic receptor. Likewise, the release of 1,4,5-inositoltrisphosphate in postischemic reperfusion has been demonstrated to be mediated by alpha1-adrenergic receptors [31]. Thus, myocardial ischemia induces the stimulation of the phospholipase C and phospholipase D signal transduction pathway, both of which are activated by the alpha1- adrenergic receptor. However, bloodborne factors such as thrombin, endothelin, and bradykinin can also stimulate the phospholipase C and phospholipase D signaling pathway in cardiac myocytes or other cells [16, 32–37], and many of these factors are found to be present in myocardial ischemia. Thus, ischemia-induced activation of these signal transduction pathways in vivo may have more complex origins than those in our in vitro model. Furthermore, it should be noted that our finding of an alpha1-adrenergic receptor-mediated increase in both second messengers in ischemic rat heart cannot be extrapolated directly to the human heart due to potential species differences which cannot be excluded in the present study.

While activation of phospholipase C has been demonstrated to contribute importantly to the development of ventricular arrhythmias under ischemic conditions [38, 39], activation of phospholipase D has been suggested to improve functional recovery of ischemic hearts [15]and to be involved in the protection of ischemic preconditioning in the heart [25]. Hence, alpha1-adrenergic receptor blockade would be expected to cause beneficial and detrimental effects in myocardial ischemia at the same time. Further studies are required to clarify the specific role of the signal transduction pathways downstream the alpha1-adrenergic receptor as well as the contribution of alpha1-adrenergic receptor subtypes in ischemic myocardium. However, particularly the exploration of the phospholipase D pathway will have to await the development of agents or experimental systems that will permit the selective and specific activation of the enzyme.

Time for primary review 30 days.


   Acknowledgements
 
We would like to thank Maike Kurz for her excellent technical assistance. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ku 774/2-1).


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

  1. Berridge MJ. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Ann Rev Biochem (1987) 56:159–193.[Web of Science][Medline]
  2. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science (1992) 258:607–614.[Abstract/Free Full Text]
  3. Exton JH. Phospholipase D: Enzymology, mechanisms of regulation, and function. Physiolog Rev (1997) 77:303–320.[Abstract/Free Full Text]
  4. Limatola C, Schaap D, Moolenaar WH, van Blitterswijk WJ. Phosphatidic acid activation of protein kinase C-{partial} overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids. Biochem J (1994) 304:1001–1008.[Web of Science][Medline]
  5. Exton JH. Phosphatidylcholine breakdown and signal transduction. Biochim Biophys Acta (1994) 1212:26–42.[Medline]
  6. Moolenaar WH, Kruijer W, Tilly BC, Verlaan I, Bierman AJ, de Laat SW. Growth factor-like action of phosphatidic acid. Nature (1986) 323:171–173.[CrossRef][Medline]
  7. Knauss TC, Jaffer FE, Abboud HE. Phosphatidic acid modulates DNA synthesis, phospholipase C, and platelet-derived growth factor mRNAs in cultured mesangial cells. J Biol Chem (1990) 265:14457–14463.[Abstract/Free Full Text]
  8. Kroll MH, Zavoico GB, Schafer AI. Second messenger function of phosphatidic acid in platelet activation. J Cell Physiol (1989) 139:558–564.[CrossRef][Web of Science][Medline]
  9. Xu YJ, Panagia V, Shao Q, Wang X, Dhalla NS. Phosphatidic acid increases intracellular free Ca2+ and cardiac contractile force. Am J Physiol (1996) 271:H651–H659.[Medline]
  10. Philipson KD, Nishimoto AY. Stimulation of Na+–Ca2+ exchange in cardiac sarcolemmal vesicles by phospholipase D. J Biol Chem (1984) 259:16–19.[Abstract/Free Full Text]
  11. Xu YJ, Yau L, Yu LP, Elimban V, Zahradka P, Dhalla NS. Stimulation of protein synthesis by phosphatidic acid in rat cardiomyocytes. Biochem Pharmacol (1996) 52:1735–1740.[CrossRef][Web of Science][Medline]
  12. Kurz T, Wolf RA, Corr PB. Phosphatidic acid stimulates inositol 1,4,5-trisphosphate production in adult cardiac myocytes. Circ Res (1993) 72:701–706.[Abstract/Free Full Text]
  13. Henry RA, Boyce SY, Kurz T, Wolf RA. Stimulation and binding of myocardial phospholipase C by phosphatidic acid. Am J Physiol (1995) 269:C349–C358.[Web of Science][Medline]
  14. Panagia V, Ou C, Taira Y, Dai J, Dhalla NS. Phospholipase D activity in subcellular membranes of rat ventricular myocardium. Biochim Biophys Acta (1991) 1064:242–250.[Medline]
  15. Moraru II, Popescu LM, Maulik N, Liu X, Das DK. Phospholipase D signaling in ischemic heart. Biochim Biophys Acta (1992) 1139:148–154.[Medline]
  16. Ye H, Wolf RA, Kurz T, Corr PB. Phosphatidic acid increases in response to noradrenaline and endothelin-1 in adult rabbit ventricular myocytes. Cardiovasc Res (1994) 28:1828–1834.[Abstract/Free Full Text]
  17. Schömig A. Catecholamines in myocardial ischemia. Systemic and cardiac release. Circulation (1990) 82(II):13–22.
  18. Schömig A, Fischer S, Kurz T, Richardt G, Schömig E. Nonexocytotic release of endogenous noradrenaline in the ischemic and anoxic rat heart: Mechanism and metabolic requirements. Circ Res (1987) 60:194–205.[Abstract/Free Full Text]
  19. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol (1959) 37:911–917.[Medline]
  20. Welsh CJ, Schmeichel K. Assays for investigations of signal transduction mechanisms involving phospholipase D: Mass measurements of phosphatidate, phosphatidylethanol, and diacylglycerol in cultured cells. Anal Biochem (1991) 192:281–292.[CrossRef][Web of Science][Medline]
  21. Nakamura K, Handa S. Coomassie Brilliant Blue staining of lipids on thin-layer plates. Anal Biochem (1984) 142:406–410.[CrossRef][Web of Science][Medline]
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem (1951) 193:265–275.[Free Full Text]
  23. Rodriguez-Linares B, Walker T, Watson S. The diacylglycerol kinase inhibitor, R59949, potentiates secretion but not increased phosphorylation of 47 kDalton protein in human platelets. Biochem Pharmacol (1991) 41:835–838.[CrossRef][Web of Science][Medline]
  24. Billah M.M, Anthes J.C. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J (1990) 269:281–291.[Web of Science][Medline]
  25. Cohen M.V, Liu Y, Liu G.S, et al. Phospholipase D plays a role in ischemic preconditioning in rabbit heart. Circulation (1996) 94:1713–1718.[Abstract/Free Full Text]
  26. Sozzani S, Agwu D.E, McCall C.E, et al. Propranolol, a phosphatidate phosphohydrolase inhibitor, also inhibits protein kinase C. J Biol Chem (1992) 267:20481–20488.[Abstract/Free Full Text]
  27. de Chaffoy de Courcelles D, Roevens P, Van Belle H, Kennis L, Somers Y, De Clerck F. The role of endogenously formed diacylglycerol in the propagation and termination of platelet activation. J Biol Chem (1989) 264:3274–3285.[Abstract/Free Full Text]
  28. Smith R.J, Sam L.M, Justen J.M, Bundy G.L, Bala G.A, Bleasdale J.E. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther (1990) 253:688–697.[Abstract/Free Full Text]
  29. Thompson A.K, Mostafapour S.P, Denlinger L.C, Bleasdale J.E, Fisher S.K. The aminosteroid U- 73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation. J Biol Chem (1991) 266:23856–23862.[Abstract/Free Full Text]
  30. Kurz T, Richardt G, Hagl S, Seyfarth M, Schömig A. Two different mechanisms of noradrenaline release during normoxia and simulated ischemia in human cardiac tissue. J Mol Cell Cardiol (1995) 27:1161–1172.[CrossRef][Web of Science][Medline]
  31. Anderson K.E, Dart A.M, Woodcock E.A. Inositol phosphate release and metabolism during myocardial ischemia and reperfusion in rat heart. Circ Res (1995) 76:261–268.[Abstract/Free Full Text]
  32. Kuraja I.J, Tanner J.K, Woodcock E.A. Endothelin stimulates phosphatidylinositol turnover in rat left and right atria. Eur J Pharmacol (1990) 189:299–306.[CrossRef][Web of Science][Medline]
  33. Steinberg S.F, Robinson R.B, Lieberman H.B, Stern D.M, Rosen M.R. Thrombin modulates phosphoinositide metabolism, cytosolic calcium, and impulse initiation in the heart. Circ Res (1991) 68:1216–1229.[Abstract/Free Full Text]
  34. Minshall R.D, Nakamura F, Becker R.P, Rabito S.F. Characterization of bradykinin B2 receptors in adult myocardium and neonatal rat cardiomyocytes. Circ Res (1995) 76:773–780.[Abstract/Free Full Text]
  35. Rubin R. Phosphatidylethanol formation in human platelets: Evidence for thrombin-induced activation of phospholipase D. Biochem Biophys Res Commun (1988) 156:1090–1096.[CrossRef][Web of Science][Medline]
  36. Clark K.J, Murray A.W. Evidence that the bradykinin-induced activation of phospholipase D and of the mitogen-activated protein cascade involve different protein kinase C isoforms. J Biol Chem (1995) 270:7097–7103.[Abstract/Free Full Text]
  37. Clerk A, Sugden P.H. Regulation of phospholipases C and D in rat ventricular myocytes: Stimulation by endothelin-1, bradykinin and phenylephrine. J Mol Cell Cardiol (1997) 29:1593–1604.[CrossRef][Web of Science][Medline]
  38. Woodcock E.A, Lambert K.A, Du X-J. Ins(1,4,5)P3 during myocardial ischemia and its relationship to the development of arrhythmias. J Mol Cell Cardiol (1996) 28:2129–2138.[CrossRef][Web of Science][Medline]
  39. Du X, Anderson K.E, Jacobsen A, Woodcock E.A, Dart A.M. Suppression of ventricular arrhythmias during ischemia-reperfusion by agents inhibiting Ins(1,4,5)P3 release. Circulation (1995) 91:2712–2716.[Abstract/Free Full Text]

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


This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. Luodonpaa, H. Leskinen, M. Ilves, O. Vuolteenaho, and H. Ruskoaho
Adrenomedullin modulates hemodynamic and cardiac effects of angiotensin II in conscious rats
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2004; 286(6): R1085 - R1092.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. Mier, D. Kemken, H. A. Katus, G. Richardt, and T. Kurz
Adrenergic activation of cardiac phospholipase D: role of {alpha}1-adrenoceptor subtypes
Cardiovasc Res, April 1, 2002; 54(1): 133 - 139.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
I. Lemire, A. Ducharme, J.-C. Tardif, F. Poulin, L. R. Jones, B. G. Allen, T. E. Hebert, and H. Rindt
Cardiac-directed overexpression of wild-type {alpha}1B-adrenergic receptor induces dilated cardiomyopathy
Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H931 - H938.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Takeda, Y. Kagaya, J. Takahashi, T. Sugie, J. Ohta, J. Watanabe, K. Shirato, H. Kondo, and K. Goto
Gene Expression and In Situ Localization of Diacylglycerol Kinase Isozymes in Normal and Infarcted Rat Hearts: Effects of Captopril Treatment
Circ. Res., August 3, 2001; 89(3): 265 - 272.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kurz, T.
Right arrow Articles by Richardt, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kurz, T.
Right arrow Articles by Richardt, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?