© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Sphingosine 1-phosphate induces sinus tachycardia and coronary vasoconstriction in the canine heart
aDepartment of Pharmacology, Yamanashi Medical University, Tamaho-cho, Nakakoma-gun, Yamanashi 409-3898, Japan
bDepartment of Laboratory Medicine, Yamanashi Medical University, Yamanashi, Japan
* Corresponding author. Tel.: +81-55-273-9504; fax: +81-55-273-6739 atsushis{at}res.yamanashi-med.ac.jp
Received 19 November 1999; accepted 4 January 2000
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Abstract |
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 Top Abstract 1 Introduction3 Results4 DiscussionReferences |
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Objective: Sphingosine 1-phosphate is a naturally occurring biologically active lysophospholipid. Recent studies suggested that sphingosine 1-phosphate is released into the blood flow from activated platelets upon stimulation to exert multiple biological phenomenon. The purpose of this study was to assess the effects of sphingosine 1-phosphate on sinus automaticity, ventricular contraction and coronary blood flow. Methods: The canine isolated, blood-perfused sinoatrial node and papillary muscle preparations were used. Results: Sphingosine 1-phosphate increased the sinoatrial rate, while it decreased the coronary blood flow, which was followed by a weak negative inotropic effect. These positive chronotropic and coronary vasoconstrictor effects were not attenuated by the β- and
-adrenoceptor antagonists atenolol and prazosin, respectively. Furthermore, sphingosine 1-phosphate did not affect the adenylate cyclase activity of the membrane preparations made from the canine right atrium and right ventricle, indicating the involvement of a novel signaling pathway in sphingosine 1-phosphate-induced cardiac effects. Conclusions: These results may provide a clue to better understanding the physiological as well as the pathophysiological regulation of sphingosine 1-phosphate in the heart.
KEYWORDS Lipid metabolism; Platelets; Second messengers; Sinus node; Vasoconstriction/dilation; Signal transduction
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1 Introduction |
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TopAbstract1 Introduction3 Results4 DiscussionReferences |
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Sphingolipid breakdown products play an important role as intercellular as well as intracellular signaling molecules [1]. Sphingosine 1-phosphate (Sph-1-P) is the initial product of the catabolism of sphingosine (Sph) by Sph kinase, and is then generally cleaved by Sph-1-P lyase to yield ethanolamine phosphate and a fatty aldehyde in most of the cells [1]. While Sph-1-P had been regarded as an intermediary metabolite in sphingolipid metabolism in the cells, recent studies have shown that Sph1-P exhibits several important and specific biological functions besides the role as an intermediary lipid metabolite [1–4].
Unlike other cells, platelets lack the degrading enzyme of Sph-1-P, namely Sph-1-P lyase, but possess strong Sph kinase activity [2,3]. This enzymatic imbalance causes the accumulation of Sph-1-P in platelets, which can be released extracellularly upon stimulation by agonists including thrombin, the final product of the clotting cascade [2,3]. Recent studies demonstrated that Sph-1-P released from activated platelets may be involved in a variety of physiological and pathophysiological processes including hemostasis, thrombosis and atherosclerosis [1,5,6]; however, a precise analysis of acute cardiovascular effects of Sph-1-P is still lacking.
The purpose of the present study was to assess the direct and acute effects of Sph-1-P on the sinus automaticity, ventricular contraction and coronary blood flow using the canine isolated, blood-perfused sinoatrial node and papillary muscle preparations [7–9]. After the cardiovascular effects of Sph-1-P were assessed, the preparation was treated with the β-adrenoceptor antagonist atenolol or -adrenoceptor antagonist prazosin to pharmacologically assess the possibility that Sph-1-P may release norepinephrine from the viable sympathetic nerve endings in the isolated preparations. Furthermore, the effects of Sph-1-P on the adenylate cyclase activity were examined in the cardiac tissue for the first time to explore the signal transduction systems of Sph-1-P in the heart.
2 Methods
Animals were obtained through the Animal Laboratory for Research of Yamanashi Medical University. All experiments were performed in accordance with Guidelines for Animal Experiments of Yamanashi Medical University.
2.1 Canine isolated, blood-perfused heart preparations
Experiments were carried out using the canine isolated sinoatrial node and papillary muscle preparations cross-circulated with heparinized arterial blood of the blood-donor dog [7–9].
2.1.1 Isolated preparations
The preparations were obtained from a beagle dog of either sex, weighing approximately 10 kg. The dog was anesthetized with pentobarbital sodium (30 mgkg–1, i.v.), given heparin calcium (500 Ukg–1, i.v.), and exsanguinated. The heart was excised and plunged into cold Tyrode's solution kept at about 4°C. The sinoatrial node preparation consisted of the entire right atrium. The sinus node artery was cannulated through the right coronary artery. Bipolar silver–silver chloride electrodes were attached onto the sinus nodal region. The papillary muscle preparation consisted of the anterior papillary muscle of the right ventricle and the interventricular septum. The anterior septal artery, the sole nutrient artery of this preparation, was directly cannulated. Bipolar silver–silver chloride pacing electrodes were attached onto the His-bundle region. Then the papillary muscle preparation was sutured onto an acrylic plate in a size of 30x75 mm to the reduce motion artifact derived from the ventricular contraction.
2.1.2 Blood-donor dog
Adult beagle dogs of either sex, weighing 12–15 kg, were used as the blood-donor dog. The dog was anesthetized with pentobarbital sodium (30 mgkg–1, i.v.) and mechanically ventilated (Shinano, SN-480-3, Tokyo, Japan). Anesthesia was maintained with supplemental doses of pentobarbital as required. At the start of cross-circulation, heparin calcium (500 Ukg–1, i.v.) was given, and an additional dose of 200 Ukg–1 was intravenously supplemented every hour. The ECG lead II and systemic blood pressure were continuously monitored using a polygraph system (Nihon-Kohden, RM-6000, Tokyo, Japan). Arterial blood gases were kept within the physiological range by adjusting the respiratory rate and oxygen supplementation.
2.1.3 Cross-circulation
The preparations were placed in a double-wall glass jacket maintained at 38°C by circulating warm water, and perfused with arterial blood from the carotid artery of the donor dog. The perfusion pressure was kept at 120 mmHg using a peristaltic pump (Cole-Parmer, 7553-20, Chicago, IL) and a Starling's pneumatic resistance placed parallel to the perfusion circuit. The coronary blood flow through each nutrient artery was continuously monitored using an electromagnetic flowmeter (Nihon-Kohden, MVF3200). Venous blood from the preparations and excess blood passing through the pneumatic resistance were collected in a blood reservoir and returned to the jugular vein of the donor dog.
2.1.4 Parameters measured
The spontaneously beating rate of the sinoatrial node preparation, i.e. the sinoatrial rate, was measured using a cardiotachograph (Nihon-Kohden, AT-601G) triggered by the bipolar electrograms obtained from the sinus nodal region. The papillary muscle preparation was electrically driven at a cycle length of 500 ms using a stimulator (Nihon-Kohden, SEN-7203) with an isolation unit (Nihon-Kohden, SS-201J). The stimulation pulses were rectangular in shape, 1–3 V amplitude (about 20% above the threshold voltage) and 5 ms duration. The developed tension of the papillary muscle preloaded with 2 g weight was measured isometrically using a force displacement transducer (Dia Medical, DRM-T200, Tokyo, Japan) and an amplifier (Dia Medical, DRM-T20). The sinoatrial rate and developed tension of the papillary muscle together with the coronary blood flow through each nutrient artery were continuously recorded on a rectilinear recorder (Nihon-Kohden, RJG-4124) at a paper speed of 25 mmmin–1.
2.2 Experimental protocol
Once the preparations were stabilized, Sph-1-P in doses of 0.01–10 µg (26.4 pmol–26.4 nmol) or its vehicle solution was injected into each nutrient artery using a small microsyringe (Ito, Tokyo, Japan) in a volume of 10–100 µl over 4 s. Physiological recordings were performed for 10 min after each dose. Because a relatively small amount of the substance was administered to the preparations compared to that needed in a whole animal model and the preparations were continuously perfused with Sph-1-P free arterial blood from the donor dog, multiple doses of the substance were studied in the same preparation [7–9]. The effluent blood through each preparation immediately after the injection of the substance was discarded to eliminate the effects of the substance on the donor dog. Having assessed the effects of Sph-1-P and vehicle solution on each parameter, β-adrenoceptor antagonist atenolol in a dose of 10 µg or -adrenoceptor antagonist prazosin in a dose of 10 µg was administered to each preparation. Then, the same range of doses of Sph-1-P was administered to compare the effects on the sinoatrial rate and coronary blood flow with those recorded before the treatment of each adrenoceptor antagonist.
2.3 Measurement of adenylate cyclase activity
2.3.1 Production of the plasma membrane preparation
After the experiment, the heart of the blood-donor dog was excised. The sinus nodal area of the right atrium and the papillary muscle of the right ventricle, both weighing 50–100 mg, were trimmed, placed in the microcentrifugation tube and homogenized with 1 ml of ice-cold SET buffer (0.25 moll–1 sucrose; 0.1 mmoll–1 EDTA; 5.0 mmoll–1 Tris–HCl) using a pellet mixer. The homogenate was filtered through a Nitex filter (Tetko, CA, USA) and centrifuged at 10 000 g for 5 min at 4°C. The pellet was resuspended in 1 ml of SET buffer and the mixture was centrifuged three more times. The final pellet was resuspended in 500 µl of SET buffer. Protein analysis was performed using a commercially available protein assay reagent (Pierce, Rockford, IL). The membrane suspension was diluted with SET buffer to a concentration of 3–5 mg proteinml–1, and it was stored at –80°C until its adenylate cyclase activity was measured.
2.3.2 Enzymatic fluorometric assay of adenylate cyclase activity
The adenylate cyclase activity of the membrane preparation was measured with an enzymatic fluorometric assay technique which was essentially the same as that previously described [10–12]. Fifty µl of adenylate cyclase mix (100 mmoll–1 Trisacetate, pH 7.4; 20 mmoll–1 KCl; 10 mmoll–1 MgCl2; 20 mmoll–1 phosphoenolpyruvate; 2 mmoll–1 ATP; 20 µmoll–1 GTP; 2 mmoll–1 dithiothreitol; 0.4 mgl–1 bovine serum albumin; 0.1 mmoll–1 3-isobutyl-1-methylxanthine (IBMX); 100 µgml–1 pyruvate kinase) was added to each microcentrifugation tube in duplicate with or without either of Sph-1-P (2x10–8 to 2x10–4 moll–1) or forskolin (2x104 moll–1). Next, the membrane suspension in a volume of 50 µl was added to each tube. The reaction mixture and membrane suspension, both before and after being mixed, were maintained at 4°C to ensure the same starting time for all assay tubes. The reaction was initiated by placing the tubes in a water bath maintained at 37°C. After 30 min, the reaction was terminated by heating at 90°C for 3 min. The mixture was vortexed three times and centrifuged at 10 000 g for 5 min. A volume of 5 µl of the supernatant was transferred to a 10x75 mm disposable assay tube (Iwaki Lab Ware, Tokyo, Japan) in triplicate. For the cyclic AMP standard, 5 µl of a known amount of cyclic AMP was added to the tubes. The cyclic AMP concentration was assayed using the enzymatic fluorometric method as previously described [10–12].
2.4 Drugs and biochemicals
The following drugs were purchased: pentobarbital sodium (Tokyo-Kasei, Tokyo, Japan), heparin calcium (Mitsui, Tokyo, Japan), acetylcholine chloride (Daiichi, Tokyo, Japan), atropine sulfate (Tanabe, Osaka, Japan) and Sph-1-P (Biomol, Plymouth Meeting, PA). Atenolol, isoproterenol, prazosin, phenylephrine, apyrase, phosphoenolpyruvate, phosphodiesterase, IBMX, sucrose, fructose, dithiothreitol and forskolin were obtained from Sigma Chemical Company (St. Louis, MO, USA). Sph1-P (MW: 379.5) was dissolved in phosphate-buffered saline with 4 mgml–1 of bovine serum albumin (Sigma) in a concentration of 100 µgml–1, and diluted with the same solvent to prepare 10 µgml–1 and 1 µgml–1 solutions. We examined various types of solvent to dissolve Sph-1-P in higher concentration, since a larger volume of injection to the nutrient coronary artery may affect the cardiovascular variables via the dilution of the arterial blood. We found the currently used solvent to be the most effective for the present study, which can provide 100 µgml–1 of Sph-1-P solution. All other enzymes and substrates were obtained from Boehringer-Mannheim (Indianapolis, IN, USA).
2.5 Data analysis and statistics
The data are presented as the mean±S.E. The statistical comparisons of mean values were carried out using one-way repeated-measures ANOVA followed by Contrast, or a t-test for paired data. A P value less than 0.05 was considered significant.
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3 Results |
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TopAbstract1 Introduction3 Results4 DiscussionReferences |
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3.1 Effects of Sph-1-P on the sinoatrial rate, developed tension of the papillary muscle and coronary blood flow
The sinoatrial node preparation showed spontaneous regular automaticity of 80±2 beatsmin–1 (n=6). Typical tracings showing the effects of Sph-1-P on the sinoatrial rate are shown in Fig. 1(A) (left), and its dose–response curve is shown in Fig. 1 (right). Administration of a vehicle solution in a volume of 100 µl did not affect the sinoatrial rate, while Sph-1-P increased it in a dose-dependent manner. Significant differences between Sph-1-P and the vehicle were observed at 3–10 µg for the sinoatrial rate.
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The basal developed tension of the papillary muscle was 6.1±1.3 g (n=6) and the basal coronary blood flow through the anterior septal artery was 9.1±0.7 mlmin–1 (n=6). Typical tracings showing the effects of Sph-1-P on the developed tension and the coronary blood flow are shown in Fig. 1(B) (left), and their dose–response curves are shown in Fig. 1(B) (right). Administration of the vehicle solution in a volume of 100 µl affected neither the developed tension nor the coronary blood flow, while Sph-1-P decreased both of them in a dose-dependent manner. Significant differences between Sph-1-P and the vehicle were observed at 3–10 µg for the developed tension, and at 0.3–10 µg for the coronary blood flow.
As another series of experiments, we performed the cumulative study for the vehicle solution. The repeated administration of the currently used vehicle solution up to 10 times hardly affected the cardiovascular variables measured in this study (n=4).
3.2 Pharmacological analysis of Sph-1-P induced positive chronotropic and coronary vasoconstrictor effects
A typical experiment showing the effects of atenolol in a dose of 10 µg, which effectively blocked the β-adrenoceptor for at least 30 min in the current model, on the Sph-1-P induced positive chronotropic effect is shown in Fig. 2(A) (left), and a summary of the results is shown in Fig. 2(A) (right, n=6). The administration of atenolol significantly attenuated the effects of β-adrenoceptor agonist isoproterenol, but it did not affect the positive chronotropic effects of Sph-1-P in doses of 0.01–10 µg (the results for 0.01–3 µg of Sph-1-P were not shown in the figure).
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A typical experiment showing the effects of prazosin in a dose of 10 µg, which effectively blocks the
-adrenoceptor at least for 20 min in the current model, on the Sph-1-P induced coronary vasoconstrictor effect is shown in Fig. 2(B) (left), and a summary of the results is shown in Fig. 2(B) (right, n=4). The administration of prazosin significantly attenuated the effects of the -adrenoceptor agonist phenylephrine but it did not affect the coronary vasoconstrictor action of Sph-1-P in doses of 0.01–10 µg (the results for 0.01–3 µg of Sph-1-P were not shown in the figure).
3.3 Effects of Sph-1-P on the adenylate cyclase activity
The effects of Sph-1-P and forskolin on the adenylate cyclase activity in membrane preparations obtained from the right atrium and right ventricle are summarized in Table 1 (n=5). Forskolin (10–4 moll–1) significantly increased the adenylate cyclase activity of the membrane preparations, while Sph-1-P in concentrations of 10–8 to 10–4 moll–1 did not affect the adenylate cyclase activity of these membrane preparations.
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4 Discussion |
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TopAbstract1 Introduction3 Results4 DiscussionReferences |
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Since information regarding the cardiovascular effects of Sph-1-P is still lacking [1], we assessed the effects of Sph-1-P on sinoatrial automaticity and ventricular contraction in addition to the coronary blood flow using the well-established canine isolated, blood-perfused sinoatrial node and papillary muscle preparations [7–9]. As clearly demonstrated in the present study, Sph-1-P increased the sinoatrial rate, while it decreased the coronary blood flow and ventricular contraction. This is the first report describing the positive chronotropic and coronary vasoconstrictor effects of Sph1-P, while it has been reported that Sph-1-P together with sphingosine play mediatory roles in the immediate negative inotropic effects of TNF
on cardiac myocytes [13]. Since the negative inotropic effect was less potent than either of the other effects and appeared after coronary vasoconstriction as shown in Fig. 1(B) (left), the currently observed negative inotropic effect of Sph-1-P may be at least in part induced by a transient myocardial ischemia besides its direct action. On the other hand, Sph-1-P in broad concentrations did not affect the adenylate cyclase activity of the membrane preparations, suggesting that Sph-1-P does not stimulate the adenylate cyclase activity in particulate. However, this result will not necessarily exclude the hypothesis that Sph-1-P could increase cyclic AMP production through an indirect activation of adenylate cyclase undetectable on particulate fraction. An important finding of this study is a positive chronotropic effect of Sph-1-P. Previous studies have shown that Sph-1-P activates muscarinic potassium current IK(ACh) in guinea-pig atrial myocytes [14] and rabbit sinoatrial node cells [15], and suggested that Sph-1-P may exert the bradycardic effect in vivo [16,17]. Contrary to this previous knowledge, the present study showed that Sph-l-P has the β-adrenoceptor as well as adenylate cyclase on particulate fraction independent positive chronotropic effect, indicating the presence of a novel, unidentified signaling pathway in Sph-1-P induced tachycardia. One of the possible reasons for these contradictory observations may be the difference in the route of Sph-1-P administration. In our study we administered Sph-1-P through the nutrient coronary artery with the arterial blood from the donor dog, while in other studies [14–17] Sph-1-P was directly superfused on the surface of the preparation. One can speculate that unidentified factors might be synthesized by Sph-1-P during its coronary circulation to modulate the sinus nodal function. The other possibility could be the species difference in the tissue distribution of the Sph-1-P receptor-coupled signal transduction system. Although subfamilies of G protein-coupled receptors encoded by the endothelial differentiation genes (edgs) have been found to be functional Sph-1-P receptors [18], one(s) transducing Sph-1-P effects in the canine heart need to be clarified. Moreover, it remains to be determined whether other mechanisms, such as intracellular actions, also contribute to the observed effects of Sph-1-P.
The observed -adrenoceptor independent coronary vasoconstrictor action of Sph-1-P also deserves a comment. While there is no report describing the action of Sph-1-P on the coronary blood flow, it has been shown that Sph-1-P potently increases [Ca2+]i in human arterial smooth muscle cells [4]. Thus, the present results together with previous knowledge suggest that Sph-1-P may regulate a coronary vascular tone. More importantly, since the source for Sph-1-P in the coronary vascular system may be activated platelets [2,3,5], Sph-1-P may play an important role in the pathophysiology of the acute coronary syndrome [19]. In addition, the current results may also explain several cardiac phenomenon in patients with thrombotic disorders, including blood transfusion toxicity.
In summary, Sph-1-P induced a sinus tachycardia and coronary vasoconstriction followed by a weak negative inotropic effect in the canine isolated heart models under physiologically maintained electrical and mechanical conditions. These observations will provide a clue to better understanding the physiological as well as the pathophysiological regulation of Sph-1-P in the heart.
Time for primary review 19 days.
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Acknowledgements |
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This study was supported in part by Grant-in-Aid for Scientific Research from the TV Yamanashi Science Development Fund.
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