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Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction
VPH-06018; No of Pages 7 Vascular Pharmacology xxx (2013) xxx–xxx
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Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction I.V. Strielkov ⁎, I.V. Kizub, A.S. Khromov, A.I. Soloviev
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Department of Experimental Therapeutics, Institute of Pharmacology and Toxicology of NAMS of Ukraine, 14 E. Potier Str., 03680, Kyiv, Ukraine
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Article history: Received 29 October 2012 Received in revised form 31 January 2013 Accepted 4 February 2013 Available online xxxx
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The aim of the study was to investigate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in hypoxic pulmonary vasoconstriction (HPV) and elucidate its possible interactions within HPV mechanism. Inhibition of PC-PLC with D609 (30 μM) resulted in partial reduction of the transient phase and almost complete abolition of the sustained phase of HPV in isolated rat intrapulmonary arteries (IPAs). Intravenous injection of D609 (5 mg/kg) 30 min before the onset of hypoxia prevented the development of acute hypoxic pulmonary hypertension (AHPH) in rats. D609 also inhibited pulmonary vasoconstriction induced with a generator of superoxide anions LY83583, but not the one induced with hydrogen peroxide. Protein kinase C (PKC) inhibition with Ro-31-8220 partially diminished the transient phase of hypoxic contraction in IPA while the sustained phase remained unchanged. Phosphocholine (70 mg/kg), known to be released due to phosphatidylcholine breakdown by PC-PLC, induced sustained contraction in isolated IPA and also transient pulmonary and systemic hypertension if administered intravenously. We conclude that PC-PLC plays an important role in sustained HPV possibly through the activation of PKC-independent mechanism, which may be coupled with phosphocholine release. © 2013 Published by Elsevier Inc.
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Keywords: Hypoxia Hypoxic pulmonary vasoconstriction Pulmonary hypertension Phospholipase C
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1. Introduction
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Pulmonary arteries possess the ability to contract in hypoxia. Currently hypoxic pulmonary vasoconstriction (HPV) is generally recognized as a two-phased reaction (Bennie et al., 1991; Leach et al., 1994; Robertson et al., 1995) initiated by the changes in reactive oxygen species (ROS) generation rate in the mitochondrial electron transport chain (discussed by Sylvester et al., 2012). According to numerous reports, the first phase is characterized by the transient rise in intracellular Ca +2 levels in pulmonary artery smooth muscle cells (PASMCs), while the second phase is associated with Ca 2+-sensitization of their contractile apparatus (Robertson et al., 1995, 2003, 2001) and is dependent on endothelium (Dipp et al., 2001; Leach et al., 1994). Despite the long research history, intrinsic mechanisms underlying the phenomenon of HPV remain unclear.
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Abbreviations: AHPH, acute hypoxic pulmonary hypertension; DAG, diacylglycerol; HPV, hypoxic pulmonary vasoconstriction; IPA, intrapulmonary arteries; MAP, mean arterial pressure; PASMC, pulmonary artery smooth muscle cells; PC-PLC, phosphatidylcholine-specific phospholipase C; PI-PLC, phosphatidylinositolspecific phospholipase C; PKC, protein kinase C; pO 2m , oxygen tension in skeletal muscles; PSS, physiological saline solution; ROS, reactive oxygen species; RVP, right ventricular pressure; TK , tension elicited by 80 mM K +. ⁎ Corresponding author. Tel.: +380 68 804 39 23; fax: +380 44 536 13 41. E-mail addresses: [email protected] (I.V. Strielkov), [email protected] (I.V. Kizub), [email protected] (A.S. Khromov), [email protected] (A.I. Soloviev).
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It has been demonstrated earlier that diacylglycerol (DAG) levels increase in PASMCs during hypoxia (Weissmann et al., 2006). This fact points towards possible involvement of at least one of phospholipase C isoforms in HPV. Therefore, we turned our attention to phosphatidylcholine-specific phospholipase C (PC-PLC), which is known to catalyze the breakdown of phosphatidylcholine to DAG and phosphocholine. DAG produced by PC-PLC has distinctive combinations of fatty acid residues including more palmitic, oleic, and linoleic acids compared to DAG released through the phosphotidylinositol breakdown, which has more residues of stearic and arachidonic acids. Phosphatidylcholine-derived DAG is released slower evoking prolonged protein kinase C (PKC) activation (Ha and Exton, 1993). Evidence of the involvement of PC-PLC in classical (α), novel (ε) and atypical (ζ) PKC isozyme activation has been obtained (Dijk et al., 1997; Goldberg et al., 1997; Ha and Exton, 1993), although it has been suggested earlier that only novel isoforms may be susceptible (Ha and Exton, 1993). Phosphocholine released through phosphatidylcholine breakdown is in most cases not considered to be an active intracellular messenger capable of influencing vascular tone. It has been previously shown that PC-PLC activation may lead to the development of pulmonary hypertension (Witzenrath et al., 2006). There is evidence that PC-PLC is activated in hypoxia at least in some cases. For instance, the involvement of this enzyme in hypoxic translocation of PKC-α in endothelium and PKC-α, PKC-ε in cardiomyocytes has been discovered (Goldberg et al., 1997; Lo et al., 2001). PC-PLC inhibitor D609 has been demonstrated to abolish hypoxia-induced DAG increase in the different types of cells (Temes
1537-1891/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.vph.2013.02.002
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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All experimental procedures conformed to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and were approved by the Ethics committee of the Institute of Pharmacology and Toxicology of NAMS of Ukraine.
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2.2. In vitro tension measurements
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Adult male Wistar rats (250–275 g) were killed by cervical dislocation. The heart and lungs were rapidly removed and placed into cold Krebs physiological saline solution (PSS; in mM: 118 NaCl, 24 NaHCO3, 1 MgSO4, 0.44 NaH2PO4, 4 KCl, 5.5 glucose, and 1.8 CaCl2). Small intrapulmonary arteries (IPA; 150–400 μm in diameter) were dissected free and cleaned of connective and adipose tissue. Arteries were treated with special care in order to keep the endothelium intact. They were then mounted on a Mulvany–Halpern wire myograph (Danish Myo Technology A/S, Aarhus, Denmark), bathed in PSS gassed with 5% CO2 and balance air (pH 7.4), and warmed to 37 °C. Vessels were stretched to a tension equivalent to 30 mm Hg, which is close to the physiological pulmonary artery blood transmural pressure. Then IPAs were stimulated by repeated 2-min exposures to 80 mM K + containing PSS (in mM: 38 NaCl, 24 NaHCO3, 1 MgSO4, 0.44 NaH2PO4, 80 KCl, 5.5 glucose, and 1.8 CaCl2) until the resulting contraction reached a stable level. The intact status of the endothelium was verified by a relaxation to acetylcholine (100 nM) under the preconstriction induced by 20 nM synthetic thromboxane agonist U46619. In order to elicit a full contractile response to hypoxia, arteries were preconstricted with 20 nM U46619 or 24 mM K + to achieve the level that was 10–15% of the maximal contraction produced by 80 mM K +. Arteries were then exposed to a hypoxic gas (5% CO2, balance nitrogen) for 40 min. Vessels were allowed to recover in PSS under normoxic conditions for at least 1 h between successive exposures to hypoxia. Three HPV responses were elicited in each artery; the third one was preceded by preincubation with drugs. The latter were added to the solution at least 15 min before the onset of hypoxia. Oxygen tension in the myograph chamber was continuously monitored via the dissolved oxygen meter (Diamond General oxygen electrode, Ann Arbor, MI; Strathkelvin oxygen meter, Glasgow, UK). During the hypoxic challenge the chamber pO2 was typically 2–3 mm Hg, as compared with the control pO2 of 135–145 mm Hg.
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Experiments were conducted on adult white male-rats (240–280 g), which were kept on the vivarium standard diet. Animals were anesthetized with a single injection of chloralose–urethane (1:10, 40 mg of urethane per 100 g of body weight, i.p.). Tracheostomy was applied. The left jugular vein and the left common carotid artery were catheterized with teflon catheters, then heparin was injected (50 U per 100 g of body weight). The catheterization of the right ventricle was performed through the right jugular vein. Polarographic electrodes were placed under the stomach skin (a silver-chloride electrode) and in inner thigh muscles (a platinum electrode). Initial values of the parameters were registered in 20–30 min necessary for the blood flow stabilization.
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NaCl, KCl, glucose CaCl2 were obtained from BDH (UK); MgSO4, CaCl2, heparin sodium salt, U46619, U73343, U73122, D609, LY83583, Ro-31-8220, phosphocholine, hexamethonium chloride, and imipramine hydrochloride were from Sigma (USA); NaHCO3 was from Fisher Scientific (UK); and gases were from BOC (UK). In our in vivo experiments we used a minimal dose of phosphocholine of those shown to have an effect (Cansev et al., 2007). This dose (70 mg/kg) was required in order to induce reaction of significant duration considering the high rate of phosphocholine degradation in the bloodstream. Phosphocholine concentration for in vitro studies was calculated based upon the in vivo injection dose and circulating blood volume.
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Registration of vascular rings contractile activity and analysis were made using “Acquisition Engine 1.1.7” software (Cairn Research Ltd., UK). Tension is presented as a percentage of the maximum constriction obtained in response to the final exposure to 80 mM K+ during the equilibration procedure (TK). Recordings and data analysis for in vivo experiments were made using “Chart 5” software (ADInstruments, Australia). Data were statistically analyzed with “Statistica 8” (StatSoft Inc., USA). Concentration–response curve was fitted using “Origin 7.5” software (OriginLab Corp., USA). All values are expressed as means ± SEM (standard error of the mean). Comparisons of the effects of treatments against control and of inhibitors against treatments were performed by one-way ANOVA with appropriate post hoc tests. Pair-wise comparisons were made by Student t test. Difference was considered significant if the confidence range was no less than 95% or p b 0.05.
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3. Results
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3.1. Effect of PC-PLC inhibition on HPV
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We conducted a preliminary set of experiments in order to assess the concentration of D609, which can effectively inhibit vasoconstriction in rat IPA (Fig. 1). Basing on the obtained results we chose the concentration of 30 μM (minimal concentration of D609 that almost completely abolishes vasoconstriction) to use in experiments with HPV. Hypoxia induced a biphasic contractile response in IPA preconstricted with 20 nM U46619. The first (transient) phase peaked within 2–4 min after the onset of hypoxia with maximal tension value of 44.2±6.0% TK (pb 0.001, n=7). The transient phase was followed by a slowly developing sustained phase that persisted during the rest of the 40-min time course of hypoxic challenge and finally reached 16.4±5.6% of TK (pb 0.001). Reoxygenation resulted in the return of tension to the initial U46619-induced values in 4–8 min and, on subsequent washing with PSS, to the original baseline values preceding the application of the preconstricting agonist.
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Hypoxic hypoxia was achieved by mechanical lung ventilation with the gas mixture containing 10% of O2, balance nitrogen (app Ugo Basile 7025; minute volume was about 140 ml/min). The duration of hypoxia was 40 min. The systolic pressure in the right ventricle and in the carotid artery was measured using ISOTEC pressure transducers (HSE, Germany). The mean arterial pressure (MAP) was calculated. Electrochemical (polarographic) measurements of oxygen tension in skeletal muscles (pO2m) were taken using PA 3 polarograph (Laboratorní přístroje, Czech Republic). The animals were euthanized at the end of the experiment by means of intravenous urethane injection (400 mg/100 g of body weight).
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et al., 2004). Furthermore, it appears that PC-PLC may be activated by ROS (Liu et al., 2007). Having analyzed the data above, we suggested that PC-PLC activation may be involved in HPV. In the present study, we tested this hypothesis and also investigated some of the possible up- and downstream interactions of PC-PLC in HPV mechanism.
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Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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Since the application of the PC-PLC inhibitor D609 (30 μM) attenuated contractile responses to U46619, the concentration of the latter was therefore titrated upwards (to approximately 220 nM) until an equivalent degree of pretone to that prior to the control hypoxic challenge was elicited. Inhibition of PC-PLC with D609 diminished the transient phase by 67% (to 14.6 ± 1.8% TK, p b 0.01, n = 7) and abolished the sustained phase (− 6.6 ± 0.6% TK, p b 0.01) (Fig. 2A). Considering that the inhibitory effect D609 on U46619-induced pretone may affect the comparability of the obtained results with control, we conducted further in vitro experiments on IPA with depolarization-induced tone. The latter was elicited with 24 mM K +. D609 had no effect on pretone in this case and hypoxia induced much lower peak tension values during both HPV phases: 6.8 ± 1.7% TK (p b 0.05, n = 6) in the first phase and 12.9 ± 4.3 TK (p b 0.05) in the second phase, respectively. D609 reduced the transient phase of HPV by 59% (to 2.8 ± 1.0% TK, p b 0.05, n = 6) and completely suppressed the sustained phase (− 1.7 ± 3.64, p b 0.001). It should also be noted that additional transient contraction has been observed on reoxygenation (Fig. 2B).
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Fig. 1. Effect of D609 on U46619 (50 nM) induced vasoconstriction in isolated rat IPA.
Fig. 2. Effect of PC-PLC inhibition on response to hypoxia (0% O2) in rat IPA. (A) Effect of D609 on tension in IPA preconstricted with U46619 (20–220 nM) under hypoxia: example traces (left) and mean values ± SE (right, n = 7 for each group). * p b 0.05, ** p b 0.01 compared to control. (B) Effect of D609 on tension in IPA preconstricted with physiological salt solution containing 30 mM of K+ (KPSS) under hypoxia: example traces (left) and mean values ± SE (right, n= 6 for each group). * pb 0.05, ** p b 0.01 compared to control. (C) Effect of D609 on tension in IPA in the absence of preconstriction under hypoxia: example traces (left) and mean values±SE (right, n=7 for each group). * pb 0.05, ** pb 0.01 compared to control.
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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3.4. Effect of PC-PLC inhibition on ROS-induced constriction in IPA Considering that the increase in ROS generation is proposed as one of the most probable HPV triggering mechanisms (Leach et al., 2001; Waypa et al., 2001, 2002; Weissmann et al., 2003), we suggested that PC-PLC, being involved in HPV, may be activated by ROS and thus contributes to ROS-induced contraction in IPA. Hydrogen peroxide (100 μM) induced a sustained slowly developing reversible rise in IPA tension that reached 5.5 ± 0.6% TK in 40 min (n = 8, p b 0.05). Application of D609 15 min before the hydrogen peroxide exposure had no effect on the reaction (4.9 ± 0.5% TK, p > 0.05, n = 6). On the contrary, the superoxide generator LY83583 (10 μM), applied for 10 min to non-preconstricted IPA, evoked a contraction (28.7 ± 1.4% TK, p b 0.001, n = 6), which was reduced by a preliminary application of the PC-PLC inhibitor by 60% (to 11.5 ± 1.1, p b 0.001, n = 6).
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3.5. Effect of protein kinase C inhibition on HPV and AHPH development 288 Speaking of possible PC-PLC downstream interactions in HPV, we suggest that PKC activation through DAG generation may be regarded as the most predictable event. Generally PKC is considered to be involved in HPV (Barman, 2001; Littler et al., 2003; Rathore et al., 2006; Weissmann et al., 1999), but there is no consensus regarding the phase to which its activity contributes (Aaronson et al., 2012; Robertson et al., 1995; Zhao et al., 1996). Thus, we investigated the role of a PKC in sustained HPV in vitro. Application of the specific PKC inhibitor Ro-31-8220 (3 μM) 15 min before the onset of hypoxia reduced the first phase of HPV
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The initial right ventricle systolic pressure (RVP) in rats with catheterized right heart amounted to 29.7 ± 1.1 mm Hg (n= 8). Mechanical lung ventilation with the hypoxic gas mixture provoked the development of pulmonary hypertension and systemic hypotension. The RVP increase was biphasic apparently reflecting the hypoxic tone increase pattern in IPA. The first phase has been observed for 4–6 min from the beginning of hypoxia. The peak value of RVP during this period was registered at 2.4± 0.2 min of hypoxia and it was 42% higher than the initial level (41.8± 1.2 mm Hg, p b 0.001). The second phase represented a sustained pulmonary hypertension that persisted during the rest of the 40-minute hypoxic challenge. RVP at that period amounted to 40.4 ± 1.3 mm Hg (pb 0.001), which was 36% higher than the initial level (Fig. 3). Mean arterial pressure (MAP) decreased under hypoxia by 38% (from 109.4 ± 4.1 to 67.6± 5.4 mm Hg, p b 0.01), and pO2m decreased more than twofold (from 34.5 ± 1.2 to 14.9 ± 1.1 mm Hg, p b 0.01). An intravenous administration of D609 (5 mg/kg) per se had no effect on pulmonary and systemic hemodynamic as well as on pO2m in normoxia (p>0.05, n = 6). An injection of this PC-PLC inhibitor 30 min before hypoxia almost completely prevented the development of pulmonary hypertension. RVP amounted to 30.1± 1.0 mm Hg (p> 0.05, n = 8) at 3 min of hypoxia and 33.1±1.8 mm Hg (p>0.05, n = 8) at 40 min (Fig. 3). PC-PLC inhibitor did not affect the decrease in MAP (to 76.2± 6.3 mm Hg, p >0.05) and pO2m (to 16.5 ± 1.2 mm Hg, p > 0.05) in hypoxia.
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In view of the fact that D609 is the only commercially available PC-PLC inhibitor and is used by some researchers as phosphatidylinositolspecific phospholipase C (PI-PLC) inhibitor (Goldberg et al., 1997), we have compared the effect of D609 with the effect of specific PI-PLC inhibitor U73122 (Fig. 4). The latter (3 μM) suppressed the transient phase of HPV in isolated IPA preconstricted by 24 mM K+ (to 1.3±1.1% TK, pb 0.05, n=6), but had no effect on the sustained phase (12.1±3.7% TK, p>0.05, n=6). U73122 did not influence HPV in isolated non-preconstricted IPA. The tension value achieved after 40 min of hypoxia amounted to 4.4±0.8% TK (p>0.05, n=6). U73343, the inactive form of U73122, had no effect on pulmonary vascular tone in normoxia and hypoxia.
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3.3. Effect of phosphatidylinositol-specific phospholipase C inhibition on 259 HPV 260
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It is known that IPAs are able to exhibit HPV even in the absence of preconstriction (Dipp et al., 2001; Leach et al., 1994), thus allowing to study this phenomenon irrespectively of the nature of the preconstrictor effect. In our experiments, hypoxia induced a relatively small sustained contraction in non-preconstricted IPA during 40 min of hypoxic challenge (5.0 ± 1.1% TK, p b 0.01, n = 7). Phases of the reaction were not clearly separable. As it can be seen in Fig. 2C, D609 reduced HPV by 60% (2.0 ± 0.7% TK, p b 0.01). It is known that D609 is also able to inhibit sphingomyelin synthase (Adibhatla et al., 2011).We conducted additional experiments in which we excluded the influence of sphingomyelin synthesis/breakdown pathway on HPV using the sphingomyelinase inactivator imipramine as it was previously implemented by Witzenrath et al. (2006). Imipramine (10 μM) had no significant effect on HPV in non-preconstricted IPA as well as in IPA preconstricted with 24 mM K+ (data not shown) suggesting that the effect of D609 in hypoxia relies on PC-PLC inhibition rather than on the changes in synthesis or breakdown of sphingomyelin.
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Fig. 3. Effect of D609 (5 mg/kg) on the right ventricular pressure in normoxia (n = 6) and hypoxic hypoxia induced by mechanical ventilation with 10% O2. Each point is the mean of 6–8 experiments. * Significant difference (p b 0.05) between hypoxia and hypoxia + D609, ** significant difference (p b 0.01) between hypoxia and hypoxia + D609.
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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Having established that PKC inhibitor is ineffective towards sustained HPV in preconstricted IPA (which is also supported by the data obtained by Robertson et al. (1995)), while PC-PLC inhibitor completely abolishes it, we hypothesized that DAG-independent vasoconstricting mechanisms may exist. Phosphocholine, being along with DAG a product of PC-PLC-induced hydrolysis of phosphotidylcholine, is generally accepted to be inactive towards vascular tone. Cansev et al. (2007) have shown that phosphocholine can induce an increase in arterial blood pressure possibly acting without interfering with neurogenic mechanisms. Thus, we investigated the effect of phosphocholine on tone of rat IPA. Phosphocholine at the concentration of 2.5 mM elicited significant sustained constriction in IPA in the presence of depolarizationinduced pretone (to 9.7 ± 2.1% TK, p b 0.05, n = 6). This reaction was not significantly affected by atropine (9.6 ± 2.3% TK, p > 0.05, n = 5). An in vivo injection of phosphocholine (70 mg/kg, i.v.) elicited significant transient synchronous rise in RVP (from 32.8 ± 1.3 to 39.3 ± 1.1 mm Hg, p b 0.01, n = 6) and MAP (from 125.5 ± 5.3 to 143.2 ± 8.6 mm Hg, p b 0.01), which had a duration of approximately 3– 4 min. A repetition of such injection in 20 min induced a diminished systemic blood pressure response (132.2 ± 8.0 mm Hg, p b 0.01, n = 6), but the transient rise of RVP remained unchanged (40.4 ± 2.6 mm Hg, p b 0.05). The third phosphocholine administration in another 20 min interval had no effect on MAP (118.8 ± 8.5 mm Hg, p > 0.05, n = 6), while RVP increase was even slightly greater (to 43.7 ± 3.6 mm Hg, p b 0.05, Fig. 5). Considering that phosphocholine degrades to choline in blood flow (Cansev et al., 2007), there is a possibility that the RVP and MAP increase occurred due to the activation of nicotinic acetylcholine receptors in postganglionic neurons of sympathetic nervous system. Therefore, we investigated the effect of nicotinic acetylcholine receptor inhibition with hexamethonium on phosphocholine-induced blood pressure rise. An intravenous administration of hexamethonium (15 mg/kg) resulted in the development of profound sustained pulmonary and systemic hypotension. RVP and MAP reached nadir in 4 min decreasing by approximately 36%: from 31.9 ±1.7 to 20.4 ± 2.2 mm Hg (pb 0.05, n = 5) and from 110.9± 11.0 to 69.9± 6.3 mm Hg (pb 0.01, n = 5), respectively. The injection of phosphocholine, following the stabilization of hemodynamics, elicited transient pulmonary and systemic blood pressure increase, which was approximately equal in absolute magnitude to the one observed previously (to 26.0± 1.4 and 88.9 ±10.3 mm Hg, respectively; p b 0.05, n =5).
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Our results indicate a difference in underlying mechanisms between HPV elicited in preconstricted and non-preconstricted arteries. Hypoxia in the presence of the pretone induces two-phase vasoconstriction in which the transient phase is highly susceptible to PI-PLC inhibitor and partially susceptible to PC-PLC and PKC inhibitors. The sustained phase is resistant to PI-PLC and PKC inhibitors, while completely abolished by PC-PLC inhibitor. On the other hand, reaction in nonpreconstricted IPA (exhibiting no clearly separable phases) is partially diminished by PC-PLC and PKC inhibitors, while unaffected by PI-PLC inhibitor. Such inconsistency between the two models of experimental HPV has been also recently demonstrated by Aaronson et al. (2012). Results obtained by these authors in the experiments on nonpreconstricted IPA significantly differ from the results obtained by some of them earlier with the use of preconstriction (Robertson et al., 1995, 2000b). Taking together the available data, we presume that the mechanism of HPV observed in non-preconstricted IPA may be a sort of a combination of the mechanisms underlying transient and sustained phases of HPV in IPA with pretone. However, we are strongly convinced that pulmonary arteries in vivo are in somewhat constricted state due to various factors, such as endothelin release (Sato et al., 2000), circulating vasoconstrictor substances in plasma (McMurtry, 1984) and others, so the properties of HPV observed in the presence of preconstriction have more physiological relevance. Earlier an important role of PKC and TRPC6 channels in transient phase of HPV has been shown (Robertson et al., 1995; Weissmann et al., 2006). Both these intracellular effectors are known to be activated by DAG, while a significant increase in DAG levels during hypoxia was observed in PASMCs as well (Weissmann et al., 2006). Having established that the transient phase appears, to a considerable degree, to be dependent on PC-PLC activation, we suggest that this enzyme may provide DAG for PKC and/or TRPC6 activation during this period. Inhibition of PC-PLC reduces transient phase almost two times as effectively as PKC inhibition, which may possibly be attributed to the diminished TRPC6 activation (Weissmann et al., 2006). It should be noted that the transient phase reduction due to the D609 influence may also be (at least in part) a result of the absence of the sustained HPV, since phases superimpose. Inhibition of PC-PLC and PKC reduces HPV in non-preconstricted IPA approximately by the same amount, which may be a sign of PC-PLC-dependent PKC activation under these conditions. Judging by the obtained results, PC-PLC may play one of the key roles in the mechanism of sustained HPV. Activation of this enzyme also appears to mediate superoxide-induced IPA constriction, so we hypothesize that PC-PLC may be activated by ROS suggested to be excessively generated under hypoxia (discussed by Sylvester et al., 2012). Previously, basing on the fact that intracellular Ca2+ levels in PASMCs remain unchanged, it has been proposed that Ca2+-sensitization of
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in IPA preconstricted with 24 mM K + by approximately 38% (to 4.2 ± 1.9 TK, p b 0.05, n = 6) and had no effect on the sustained phase (12.5 ± 4.1 TK, p > 0.05, n = 6). Ro-31-8220 reduced HPV in non-preconstricted IPA by 55% (from 4.6 ± 0.2 to 2.0 ± 0.4% TK, p b 0.001, n = 7).
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Fig. 4. Effect of PI-PLC inhibition on response to hypoxia (0% O2) in rat IPA preconstricted with 20 nM U46619 (left, n = 7 for control, n= 6 for U73122) and in the absence of preconstriction (right, n = 7 for control, n= 6 for U73122). * p b 0.05 compared to control.
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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contractile apparatus occurs during the sustained phase (Robertson et al., 2003, 2001). In addition, it has been shown that this process may be predominantly dependent on Rho-kinase (Robertson et al., 2000a). Both PC-PLC and Rho-kinase appear to mediate constriction in IPA induced by superoxide anion (Knock et al., 2009). Taken together, these data allow us to suggest that PC-PLC activity during the sustained HPV may be involved in PKC-independent Ca2+-sensitization processes in PASMCs. In addition, Rho-kinase activity in hypoxia may be associated with PC-PLC activity through a pathway yet unknown. On the other hand, it should be noted that Weissmann et al. (2006) reported the significant rise in intracellular Ca2+ levels in PASMCs during the sustained phase of HPV. Our data suggest that the transient phase of HPV strongly depends on PI-PLC activation. This suggestion conforms to the results obtained earlier on isolated mice lungs by Fuchs et al. (2011). Taken together, it appears that both phospholipases (PC-PLC and PI-PLC) are involved in HPV development but act separately each in its respective phase. Surprisingly, earlier biphasic DAG increase has been observed in hamster fibroblasts activated by α-thrombin (Ha and Exton, 1993). The first phase was rapid (with the peak at 15 s) and associated with the hydrolysis of phosphatidylinositol 4,5-bisphosphate, while the second was slow (peak at 5–15 min) and resulted from the phosphatidylcholine hydrolysis. It appears that the same events may occur in pulmonary vessels in hypoxia. The phases of RVP increase during hypoxic challenge observed in vivo, judging by their durations and proportions, correspond to the phases of HPV observed in experiments on isolated IPA. Therefore, we conclude that the first and the second phase of the RVP increase are manifestations of the respective phases of HPV on the systemic level. PC-PLC inhibition with D609 abolished the development of AHPH, which is consistent with the results obtained in isolated IPA.
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Fig. 5. Effect of phosphocholine (120 mg/kg) on the right ventricular (RVP) and mean arterial (MAP) pressure during 3 consecutive bolus injections made with 20-minute interval. (А) Example traces. (B) Mean relative maximum RVP and MAP increase observed after phosphocholine injections (n = 6). * Significant difference (pb 0.05) between the initial values and maximum values after phosphocholine injection.
As we previously noted, D609 had no effect on systemic hemodynamics and pO2m under normoxic and hypoxic conditions. While D609 has been shown to possess a very low toxicity (Bai et al., 2004), we suggest that this compound has a significant potential to be used for the creation of a new drug against hypoxic pulmonary hypertension. As we mentioned before, while it appears that PKC and TRPC6 solely contribute to the transient phase of HPV, there is a possibility that other PC-PLC-activated but DAG-independent mechanisms may exist. Searching for the alternative mechanisms of implementation of the vasoconstricting effect of PC-PLC activation we turned our attention to another product of phosphotidylcholine hydrolysis, phosphocholine. This metabolite induces sustained contraction in IPA. Furthermore, phosphocholine injected intravenously evokes significant transient rise in RVP and MAP and, what is even more intriguing, this effect on pulmonary circulation is stable throughout the repeated administrations, while gradual desensitization to phosphocholine has been observed in systemic circulation. The observed transient hypertension apparently has a non-neurogenic origin as the reaction is not substantially affected by hexamethonium. Taken together, these data suggest that phosphocholine is able to induce vasoconstriction (primarily pulmonary) and thus we hypothesize that it may act as a messenger along with DAG released during PC-PLC activation. The idea of differential sensitivity of arteries to phosphocholine is supported by the fact that this compound has no effect on the tone of isolated rat aorta (Cansev et al., 2007).
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5. Conclusions
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In summary, the obtained results demonstrate the important role of 452 PC-PLC in the mechanism of HPV. We conclude that this phospholipase 453 is partially involved in the transient phase presumably initiating PKC 454
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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This study was supported by the International Junior Research Grant of The Physiological Society. The authors would like to thank Prof. J.P.T. Ward for his kind support of the present study and Dr. V.A. Snetkov for his assistance in the experimental work.
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activation. PC-PLC contribution to the sustained HPV appears to be critical and may involve the activation of other vasoconstricting mechanisms along with PKC. They may rely on phosphocholine released due to PC-PLC-catalyzed phosphotidylcholine breakdown. According to our findings, phosphocholine is able to induce IPA constriction in vitro as well as AHPH in vivo. The underlying mechanisms of this reaction are yet to be elucidated. Contrary to PC-PLC, PI-PLC is not involved in sustained HPV. Considering that PC-PLC contributes to the constriction of IPA induced by superoxide anion, we assume that PC-PLC may be activated in hypoxia as a result of increased ROS generation, which has been previously proposed as HPV triggering event. We suggest that the demonstrated prevention of AHPH with D609 may become a basis for the development of a new anti-hypertensive therapy.
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Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction I.V. Strielkov ⁎, I.V. Kizub, A.S. Khromov, A.I. Soloviev
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Department of Experimental Therapeutics, Institute of Pharmacology and Toxicology of NAMS of Ukraine, 14 E. Potier Str., 03680, Kyiv, Ukraine
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Article history: Received 29 October 2012 Received in revised form 31 January 2013 Accepted 4 February 2013 Available online xxxx
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The aim of the study was to investigate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in hypoxic pulmonary vasoconstriction (HPV) and elucidate its possible interactions within HPV mechanism. Inhibition of PC-PLC with D609 (30 μM) resulted in partial reduction of the transient phase and almost complete abolition of the sustained phase of HPV in isolated rat intrapulmonary arteries (IPAs). Intravenous injection of D609 (5 mg/kg) 30 min before the onset of hypoxia prevented the development of acute hypoxic pulmonary hypertension (AHPH) in rats. D609 also inhibited pulmonary vasoconstriction induced with a generator of superoxide anions LY83583, but not the one induced with hydrogen peroxide. Protein kinase C (PKC) inhibition with Ro-31-8220 partially diminished the transient phase of hypoxic contraction in IPA while the sustained phase remained unchanged. Phosphocholine (70 mg/kg), known to be released due to phosphatidylcholine breakdown by PC-PLC, induced sustained contraction in isolated IPA and also transient pulmonary and systemic hypertension if administered intravenously. We conclude that PC-PLC plays an important role in sustained HPV possibly through the activation of PKC-independent mechanism, which may be coupled with phosphocholine release. © 2013 Published by Elsevier Inc.
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Keywords: Hypoxia Hypoxic pulmonary vasoconstriction Pulmonary hypertension Phospholipase C
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Pulmonary arteries possess the ability to contract in hypoxia. Currently hypoxic pulmonary vasoconstriction (HPV) is generally recognized as a two-phased reaction (Bennie et al., 1991; Leach et al., 1994; Robertson et al., 1995) initiated by the changes in reactive oxygen species (ROS) generation rate in the mitochondrial electron transport chain (discussed by Sylvester et al., 2012). According to numerous reports, the first phase is characterized by the transient rise in intracellular Ca +2 levels in pulmonary artery smooth muscle cells (PASMCs), while the second phase is associated with Ca 2+-sensitization of their contractile apparatus (Robertson et al., 1995, 2003, 2001) and is dependent on endothelium (Dipp et al., 2001; Leach et al., 1994). Despite the long research history, intrinsic mechanisms underlying the phenomenon of HPV remain unclear.
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Abbreviations: AHPH, acute hypoxic pulmonary hypertension; DAG, diacylglycerol; HPV, hypoxic pulmonary vasoconstriction; IPA, intrapulmonary arteries; MAP, mean arterial pressure; PASMC, pulmonary artery smooth muscle cells; PC-PLC, phosphatidylcholine-specific phospholipase C; PI-PLC, phosphatidylinositolspecific phospholipase C; PKC, protein kinase C; pO 2m , oxygen tension in skeletal muscles; PSS, physiological saline solution; ROS, reactive oxygen species; RVP, right ventricular pressure; TK , tension elicited by 80 mM K +. ⁎ Corresponding author. Tel.: +380 68 804 39 23; fax: +380 44 536 13 41. E-mail addresses: [email protected] (I.V. Strielkov), [email protected] (I.V. Kizub), [email protected] (A.S. Khromov), [email protected] (A.I. Soloviev).
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It has been demonstrated earlier that diacylglycerol (DAG) levels increase in PASMCs during hypoxia (Weissmann et al., 2006). This fact points towards possible involvement of at least one of phospholipase C isoforms in HPV. Therefore, we turned our attention to phosphatidylcholine-specific phospholipase C (PC-PLC), which is known to catalyze the breakdown of phosphatidylcholine to DAG and phosphocholine. DAG produced by PC-PLC has distinctive combinations of fatty acid residues including more palmitic, oleic, and linoleic acids compared to DAG released through the phosphotidylinositol breakdown, which has more residues of stearic and arachidonic acids. Phosphatidylcholine-derived DAG is released slower evoking prolonged protein kinase C (PKC) activation (Ha and Exton, 1993). Evidence of the involvement of PC-PLC in classical (α), novel (ε) and atypical (ζ) PKC isozyme activation has been obtained (Dijk et al., 1997; Goldberg et al., 1997; Ha and Exton, 1993), although it has been suggested earlier that only novel isoforms may be susceptible (Ha and Exton, 1993). Phosphocholine released through phosphatidylcholine breakdown is in most cases not considered to be an active intracellular messenger capable of influencing vascular tone. It has been previously shown that PC-PLC activation may lead to the development of pulmonary hypertension (Witzenrath et al., 2006). There is evidence that PC-PLC is activated in hypoxia at least in some cases. For instance, the involvement of this enzyme in hypoxic translocation of PKC-α in endothelium and PKC-α, PKC-ε in cardiomyocytes has been discovered (Goldberg et al., 1997; Lo et al., 2001). PC-PLC inhibitor D609 has been demonstrated to abolish hypoxia-induced DAG increase in the different types of cells (Temes
1537-1891/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.vph.2013.02.002
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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All experimental procedures conformed to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and were approved by the Ethics committee of the Institute of Pharmacology and Toxicology of NAMS of Ukraine.
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2.2. In vitro tension measurements
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Adult male Wistar rats (250–275 g) were killed by cervical dislocation. The heart and lungs were rapidly removed and placed into cold Krebs physiological saline solution (PSS; in mM: 118 NaCl, 24 NaHCO3, 1 MgSO4, 0.44 NaH2PO4, 4 KCl, 5.5 glucose, and 1.8 CaCl2). Small intrapulmonary arteries (IPA; 150–400 μm in diameter) were dissected free and cleaned of connective and adipose tissue. Arteries were treated with special care in order to keep the endothelium intact. They were then mounted on a Mulvany–Halpern wire myograph (Danish Myo Technology A/S, Aarhus, Denmark), bathed in PSS gassed with 5% CO2 and balance air (pH 7.4), and warmed to 37 °C. Vessels were stretched to a tension equivalent to 30 mm Hg, which is close to the physiological pulmonary artery blood transmural pressure. Then IPAs were stimulated by repeated 2-min exposures to 80 mM K + containing PSS (in mM: 38 NaCl, 24 NaHCO3, 1 MgSO4, 0.44 NaH2PO4, 80 KCl, 5.5 glucose, and 1.8 CaCl2) until the resulting contraction reached a stable level. The intact status of the endothelium was verified by a relaxation to acetylcholine (100 nM) under the preconstriction induced by 20 nM synthetic thromboxane agonist U46619. In order to elicit a full contractile response to hypoxia, arteries were preconstricted with 20 nM U46619 or 24 mM K + to achieve the level that was 10–15% of the maximal contraction produced by 80 mM K +. Arteries were then exposed to a hypoxic gas (5% CO2, balance nitrogen) for 40 min. Vessels were allowed to recover in PSS under normoxic conditions for at least 1 h between successive exposures to hypoxia. Three HPV responses were elicited in each artery; the third one was preceded by preincubation with drugs. The latter were added to the solution at least 15 min before the onset of hypoxia. Oxygen tension in the myograph chamber was continuously monitored via the dissolved oxygen meter (Diamond General oxygen electrode, Ann Arbor, MI; Strathkelvin oxygen meter, Glasgow, UK). During the hypoxic challenge the chamber pO2 was typically 2–3 mm Hg, as compared with the control pO2 of 135–145 mm Hg.
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Experiments were conducted on adult white male-rats (240–280 g), which were kept on the vivarium standard diet. Animals were anesthetized with a single injection of chloralose–urethane (1:10, 40 mg of urethane per 100 g of body weight, i.p.). Tracheostomy was applied. The left jugular vein and the left common carotid artery were catheterized with teflon catheters, then heparin was injected (50 U per 100 g of body weight). The catheterization of the right ventricle was performed through the right jugular vein. Polarographic electrodes were placed under the stomach skin (a silver-chloride electrode) and in inner thigh muscles (a platinum electrode). Initial values of the parameters were registered in 20–30 min necessary for the blood flow stabilization.
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NaCl, KCl, glucose CaCl2 were obtained from BDH (UK); MgSO4, CaCl2, heparin sodium salt, U46619, U73343, U73122, D609, LY83583, Ro-31-8220, phosphocholine, hexamethonium chloride, and imipramine hydrochloride were from Sigma (USA); NaHCO3 was from Fisher Scientific (UK); and gases were from BOC (UK). In our in vivo experiments we used a minimal dose of phosphocholine of those shown to have an effect (Cansev et al., 2007). This dose (70 mg/kg) was required in order to induce reaction of significant duration considering the high rate of phosphocholine degradation in the bloodstream. Phosphocholine concentration for in vitro studies was calculated based upon the in vivo injection dose and circulating blood volume.
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Registration of vascular rings contractile activity and analysis were made using “Acquisition Engine 1.1.7” software (Cairn Research Ltd., UK). Tension is presented as a percentage of the maximum constriction obtained in response to the final exposure to 80 mM K+ during the equilibration procedure (TK). Recordings and data analysis for in vivo experiments were made using “Chart 5” software (ADInstruments, Australia). Data were statistically analyzed with “Statistica 8” (StatSoft Inc., USA). Concentration–response curve was fitted using “Origin 7.5” software (OriginLab Corp., USA). All values are expressed as means ± SEM (standard error of the mean). Comparisons of the effects of treatments against control and of inhibitors against treatments were performed by one-way ANOVA with appropriate post hoc tests. Pair-wise comparisons were made by Student t test. Difference was considered significant if the confidence range was no less than 95% or p b 0.05.
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3.1. Effect of PC-PLC inhibition on HPV
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We conducted a preliminary set of experiments in order to assess the concentration of D609, which can effectively inhibit vasoconstriction in rat IPA (Fig. 1). Basing on the obtained results we chose the concentration of 30 μM (minimal concentration of D609 that almost completely abolishes vasoconstriction) to use in experiments with HPV. Hypoxia induced a biphasic contractile response in IPA preconstricted with 20 nM U46619. The first (transient) phase peaked within 2–4 min after the onset of hypoxia with maximal tension value of 44.2±6.0% TK (pb 0.001, n=7). The transient phase was followed by a slowly developing sustained phase that persisted during the rest of the 40-min time course of hypoxic challenge and finally reached 16.4±5.6% of TK (pb 0.001). Reoxygenation resulted in the return of tension to the initial U46619-induced values in 4–8 min and, on subsequent washing with PSS, to the original baseline values preceding the application of the preconstricting agonist.
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Hypoxic hypoxia was achieved by mechanical lung ventilation with the gas mixture containing 10% of O2, balance nitrogen (app Ugo Basile 7025; minute volume was about 140 ml/min). The duration of hypoxia was 40 min. The systolic pressure in the right ventricle and in the carotid artery was measured using ISOTEC pressure transducers (HSE, Germany). The mean arterial pressure (MAP) was calculated. Electrochemical (polarographic) measurements of oxygen tension in skeletal muscles (pO2m) were taken using PA 3 polarograph (Laboratorní přístroje, Czech Republic). The animals were euthanized at the end of the experiment by means of intravenous urethane injection (400 mg/100 g of body weight).
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et al., 2004). Furthermore, it appears that PC-PLC may be activated by ROS (Liu et al., 2007). Having analyzed the data above, we suggested that PC-PLC activation may be involved in HPV. In the present study, we tested this hypothesis and also investigated some of the possible up- and downstream interactions of PC-PLC in HPV mechanism.
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Since the application of the PC-PLC inhibitor D609 (30 μM) attenuated contractile responses to U46619, the concentration of the latter was therefore titrated upwards (to approximately 220 nM) until an equivalent degree of pretone to that prior to the control hypoxic challenge was elicited. Inhibition of PC-PLC with D609 diminished the transient phase by 67% (to 14.6 ± 1.8% TK, p b 0.01, n = 7) and abolished the sustained phase (− 6.6 ± 0.6% TK, p b 0.01) (Fig. 2A). Considering that the inhibitory effect D609 on U46619-induced pretone may affect the comparability of the obtained results with control, we conducted further in vitro experiments on IPA with depolarization-induced tone. The latter was elicited with 24 mM K +. D609 had no effect on pretone in this case and hypoxia induced much lower peak tension values during both HPV phases: 6.8 ± 1.7% TK (p b 0.05, n = 6) in the first phase and 12.9 ± 4.3 TK (p b 0.05) in the second phase, respectively. D609 reduced the transient phase of HPV by 59% (to 2.8 ± 1.0% TK, p b 0.05, n = 6) and completely suppressed the sustained phase (− 1.7 ± 3.64, p b 0.001). It should also be noted that additional transient contraction has been observed on reoxygenation (Fig. 2B).
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Fig. 1. Effect of D609 on U46619 (50 nM) induced vasoconstriction in isolated rat IPA.
Fig. 2. Effect of PC-PLC inhibition on response to hypoxia (0% O2) in rat IPA. (A) Effect of D609 on tension in IPA preconstricted with U46619 (20–220 nM) under hypoxia: example traces (left) and mean values ± SE (right, n = 7 for each group). * p b 0.05, ** p b 0.01 compared to control. (B) Effect of D609 on tension in IPA preconstricted with physiological salt solution containing 30 mM of K+ (KPSS) under hypoxia: example traces (left) and mean values ± SE (right, n= 6 for each group). * pb 0.05, ** p b 0.01 compared to control. (C) Effect of D609 on tension in IPA in the absence of preconstriction under hypoxia: example traces (left) and mean values±SE (right, n=7 for each group). * pb 0.05, ** pb 0.01 compared to control.
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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3.4. Effect of PC-PLC inhibition on ROS-induced constriction in IPA Considering that the increase in ROS generation is proposed as one of the most probable HPV triggering mechanisms (Leach et al., 2001; Waypa et al., 2001, 2002; Weissmann et al., 2003), we suggested that PC-PLC, being involved in HPV, may be activated by ROS and thus contributes to ROS-induced contraction in IPA. Hydrogen peroxide (100 μM) induced a sustained slowly developing reversible rise in IPA tension that reached 5.5 ± 0.6% TK in 40 min (n = 8, p b 0.05). Application of D609 15 min before the hydrogen peroxide exposure had no effect on the reaction (4.9 ± 0.5% TK, p > 0.05, n = 6). On the contrary, the superoxide generator LY83583 (10 μM), applied for 10 min to non-preconstricted IPA, evoked a contraction (28.7 ± 1.4% TK, p b 0.001, n = 6), which was reduced by a preliminary application of the PC-PLC inhibitor by 60% (to 11.5 ± 1.1, p b 0.001, n = 6).
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3.5. Effect of protein kinase C inhibition on HPV and AHPH development 288 Speaking of possible PC-PLC downstream interactions in HPV, we suggest that PKC activation through DAG generation may be regarded as the most predictable event. Generally PKC is considered to be involved in HPV (Barman, 2001; Littler et al., 2003; Rathore et al., 2006; Weissmann et al., 1999), but there is no consensus regarding the phase to which its activity contributes (Aaronson et al., 2012; Robertson et al., 1995; Zhao et al., 1996). Thus, we investigated the role of a PKC in sustained HPV in vitro. Application of the specific PKC inhibitor Ro-31-8220 (3 μM) 15 min before the onset of hypoxia reduced the first phase of HPV
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The initial right ventricle systolic pressure (RVP) in rats with catheterized right heart amounted to 29.7 ± 1.1 mm Hg (n= 8). Mechanical lung ventilation with the hypoxic gas mixture provoked the development of pulmonary hypertension and systemic hypotension. The RVP increase was biphasic apparently reflecting the hypoxic tone increase pattern in IPA. The first phase has been observed for 4–6 min from the beginning of hypoxia. The peak value of RVP during this period was registered at 2.4± 0.2 min of hypoxia and it was 42% higher than the initial level (41.8± 1.2 mm Hg, p b 0.001). The second phase represented a sustained pulmonary hypertension that persisted during the rest of the 40-minute hypoxic challenge. RVP at that period amounted to 40.4 ± 1.3 mm Hg (pb 0.001), which was 36% higher than the initial level (Fig. 3). Mean arterial pressure (MAP) decreased under hypoxia by 38% (from 109.4 ± 4.1 to 67.6± 5.4 mm Hg, p b 0.01), and pO2m decreased more than twofold (from 34.5 ± 1.2 to 14.9 ± 1.1 mm Hg, p b 0.01). An intravenous administration of D609 (5 mg/kg) per se had no effect on pulmonary and systemic hemodynamic as well as on pO2m in normoxia (p>0.05, n = 6). An injection of this PC-PLC inhibitor 30 min before hypoxia almost completely prevented the development of pulmonary hypertension. RVP amounted to 30.1± 1.0 mm Hg (p> 0.05, n = 8) at 3 min of hypoxia and 33.1±1.8 mm Hg (p>0.05, n = 8) at 40 min (Fig. 3). PC-PLC inhibitor did not affect the decrease in MAP (to 76.2± 6.3 mm Hg, p >0.05) and pO2m (to 16.5 ± 1.2 mm Hg, p > 0.05) in hypoxia.
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In view of the fact that D609 is the only commercially available PC-PLC inhibitor and is used by some researchers as phosphatidylinositolspecific phospholipase C (PI-PLC) inhibitor (Goldberg et al., 1997), we have compared the effect of D609 with the effect of specific PI-PLC inhibitor U73122 (Fig. 4). The latter (3 μM) suppressed the transient phase of HPV in isolated IPA preconstricted by 24 mM K+ (to 1.3±1.1% TK, pb 0.05, n=6), but had no effect on the sustained phase (12.1±3.7% TK, p>0.05, n=6). U73122 did not influence HPV in isolated non-preconstricted IPA. The tension value achieved after 40 min of hypoxia amounted to 4.4±0.8% TK (p>0.05, n=6). U73343, the inactive form of U73122, had no effect on pulmonary vascular tone in normoxia and hypoxia.
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It is known that IPAs are able to exhibit HPV even in the absence of preconstriction (Dipp et al., 2001; Leach et al., 1994), thus allowing to study this phenomenon irrespectively of the nature of the preconstrictor effect. In our experiments, hypoxia induced a relatively small sustained contraction in non-preconstricted IPA during 40 min of hypoxic challenge (5.0 ± 1.1% TK, p b 0.01, n = 7). Phases of the reaction were not clearly separable. As it can be seen in Fig. 2C, D609 reduced HPV by 60% (2.0 ± 0.7% TK, p b 0.01). It is known that D609 is also able to inhibit sphingomyelin synthase (Adibhatla et al., 2011).We conducted additional experiments in which we excluded the influence of sphingomyelin synthesis/breakdown pathway on HPV using the sphingomyelinase inactivator imipramine as it was previously implemented by Witzenrath et al. (2006). Imipramine (10 μM) had no significant effect on HPV in non-preconstricted IPA as well as in IPA preconstricted with 24 mM K+ (data not shown) suggesting that the effect of D609 in hypoxia relies on PC-PLC inhibition rather than on the changes in synthesis or breakdown of sphingomyelin.
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Fig. 3. Effect of D609 (5 mg/kg) on the right ventricular pressure in normoxia (n = 6) and hypoxic hypoxia induced by mechanical ventilation with 10% O2. Each point is the mean of 6–8 experiments. * Significant difference (p b 0.05) between hypoxia and hypoxia + D609, ** significant difference (p b 0.01) between hypoxia and hypoxia + D609.
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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Having established that PKC inhibitor is ineffective towards sustained HPV in preconstricted IPA (which is also supported by the data obtained by Robertson et al. (1995)), while PC-PLC inhibitor completely abolishes it, we hypothesized that DAG-independent vasoconstricting mechanisms may exist. Phosphocholine, being along with DAG a product of PC-PLC-induced hydrolysis of phosphotidylcholine, is generally accepted to be inactive towards vascular tone. Cansev et al. (2007) have shown that phosphocholine can induce an increase in arterial blood pressure possibly acting without interfering with neurogenic mechanisms. Thus, we investigated the effect of phosphocholine on tone of rat IPA. Phosphocholine at the concentration of 2.5 mM elicited significant sustained constriction in IPA in the presence of depolarizationinduced pretone (to 9.7 ± 2.1% TK, p b 0.05, n = 6). This reaction was not significantly affected by atropine (9.6 ± 2.3% TK, p > 0.05, n = 5). An in vivo injection of phosphocholine (70 mg/kg, i.v.) elicited significant transient synchronous rise in RVP (from 32.8 ± 1.3 to 39.3 ± 1.1 mm Hg, p b 0.01, n = 6) and MAP (from 125.5 ± 5.3 to 143.2 ± 8.6 mm Hg, p b 0.01), which had a duration of approximately 3– 4 min. A repetition of such injection in 20 min induced a diminished systemic blood pressure response (132.2 ± 8.0 mm Hg, p b 0.01, n = 6), but the transient rise of RVP remained unchanged (40.4 ± 2.6 mm Hg, p b 0.05). The third phosphocholine administration in another 20 min interval had no effect on MAP (118.8 ± 8.5 mm Hg, p > 0.05, n = 6), while RVP increase was even slightly greater (to 43.7 ± 3.6 mm Hg, p b 0.05, Fig. 5). Considering that phosphocholine degrades to choline in blood flow (Cansev et al., 2007), there is a possibility that the RVP and MAP increase occurred due to the activation of nicotinic acetylcholine receptors in postganglionic neurons of sympathetic nervous system. Therefore, we investigated the effect of nicotinic acetylcholine receptor inhibition with hexamethonium on phosphocholine-induced blood pressure rise. An intravenous administration of hexamethonium (15 mg/kg) resulted in the development of profound sustained pulmonary and systemic hypotension. RVP and MAP reached nadir in 4 min decreasing by approximately 36%: from 31.9 ±1.7 to 20.4 ± 2.2 mm Hg (pb 0.05, n = 5) and from 110.9± 11.0 to 69.9± 6.3 mm Hg (pb 0.01, n = 5), respectively. The injection of phosphocholine, following the stabilization of hemodynamics, elicited transient pulmonary and systemic blood pressure increase, which was approximately equal in absolute magnitude to the one observed previously (to 26.0± 1.4 and 88.9 ±10.3 mm Hg, respectively; p b 0.05, n =5).
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Our results indicate a difference in underlying mechanisms between HPV elicited in preconstricted and non-preconstricted arteries. Hypoxia in the presence of the pretone induces two-phase vasoconstriction in which the transient phase is highly susceptible to PI-PLC inhibitor and partially susceptible to PC-PLC and PKC inhibitors. The sustained phase is resistant to PI-PLC and PKC inhibitors, while completely abolished by PC-PLC inhibitor. On the other hand, reaction in nonpreconstricted IPA (exhibiting no clearly separable phases) is partially diminished by PC-PLC and PKC inhibitors, while unaffected by PI-PLC inhibitor. Such inconsistency between the two models of experimental HPV has been also recently demonstrated by Aaronson et al. (2012). Results obtained by these authors in the experiments on nonpreconstricted IPA significantly differ from the results obtained by some of them earlier with the use of preconstriction (Robertson et al., 1995, 2000b). Taking together the available data, we presume that the mechanism of HPV observed in non-preconstricted IPA may be a sort of a combination of the mechanisms underlying transient and sustained phases of HPV in IPA with pretone. However, we are strongly convinced that pulmonary arteries in vivo are in somewhat constricted state due to various factors, such as endothelin release (Sato et al., 2000), circulating vasoconstrictor substances in plasma (McMurtry, 1984) and others, so the properties of HPV observed in the presence of preconstriction have more physiological relevance. Earlier an important role of PKC and TRPC6 channels in transient phase of HPV has been shown (Robertson et al., 1995; Weissmann et al., 2006). Both these intracellular effectors are known to be activated by DAG, while a significant increase in DAG levels during hypoxia was observed in PASMCs as well (Weissmann et al., 2006). Having established that the transient phase appears, to a considerable degree, to be dependent on PC-PLC activation, we suggest that this enzyme may provide DAG for PKC and/or TRPC6 activation during this period. Inhibition of PC-PLC reduces transient phase almost two times as effectively as PKC inhibition, which may possibly be attributed to the diminished TRPC6 activation (Weissmann et al., 2006). It should be noted that the transient phase reduction due to the D609 influence may also be (at least in part) a result of the absence of the sustained HPV, since phases superimpose. Inhibition of PC-PLC and PKC reduces HPV in non-preconstricted IPA approximately by the same amount, which may be a sign of PC-PLC-dependent PKC activation under these conditions. Judging by the obtained results, PC-PLC may play one of the key roles in the mechanism of sustained HPV. Activation of this enzyme also appears to mediate superoxide-induced IPA constriction, so we hypothesize that PC-PLC may be activated by ROS suggested to be excessively generated under hypoxia (discussed by Sylvester et al., 2012). Previously, basing on the fact that intracellular Ca2+ levels in PASMCs remain unchanged, it has been proposed that Ca2+-sensitization of
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in IPA preconstricted with 24 mM K + by approximately 38% (to 4.2 ± 1.9 TK, p b 0.05, n = 6) and had no effect on the sustained phase (12.5 ± 4.1 TK, p > 0.05, n = 6). Ro-31-8220 reduced HPV in non-preconstricted IPA by 55% (from 4.6 ± 0.2 to 2.0 ± 0.4% TK, p b 0.001, n = 7).
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Fig. 4. Effect of PI-PLC inhibition on response to hypoxia (0% O2) in rat IPA preconstricted with 20 nM U46619 (left, n = 7 for control, n= 6 for U73122) and in the absence of preconstriction (right, n = 7 for control, n= 6 for U73122). * p b 0.05 compared to control.
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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contractile apparatus occurs during the sustained phase (Robertson et al., 2003, 2001). In addition, it has been shown that this process may be predominantly dependent on Rho-kinase (Robertson et al., 2000a). Both PC-PLC and Rho-kinase appear to mediate constriction in IPA induced by superoxide anion (Knock et al., 2009). Taken together, these data allow us to suggest that PC-PLC activity during the sustained HPV may be involved in PKC-independent Ca2+-sensitization processes in PASMCs. In addition, Rho-kinase activity in hypoxia may be associated with PC-PLC activity through a pathway yet unknown. On the other hand, it should be noted that Weissmann et al. (2006) reported the significant rise in intracellular Ca2+ levels in PASMCs during the sustained phase of HPV. Our data suggest that the transient phase of HPV strongly depends on PI-PLC activation. This suggestion conforms to the results obtained earlier on isolated mice lungs by Fuchs et al. (2011). Taken together, it appears that both phospholipases (PC-PLC and PI-PLC) are involved in HPV development but act separately each in its respective phase. Surprisingly, earlier biphasic DAG increase has been observed in hamster fibroblasts activated by α-thrombin (Ha and Exton, 1993). The first phase was rapid (with the peak at 15 s) and associated with the hydrolysis of phosphatidylinositol 4,5-bisphosphate, while the second was slow (peak at 5–15 min) and resulted from the phosphatidylcholine hydrolysis. It appears that the same events may occur in pulmonary vessels in hypoxia. The phases of RVP increase during hypoxic challenge observed in vivo, judging by their durations and proportions, correspond to the phases of HPV observed in experiments on isolated IPA. Therefore, we conclude that the first and the second phase of the RVP increase are manifestations of the respective phases of HPV on the systemic level. PC-PLC inhibition with D609 abolished the development of AHPH, which is consistent with the results obtained in isolated IPA.
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Fig. 5. Effect of phosphocholine (120 mg/kg) on the right ventricular (RVP) and mean arterial (MAP) pressure during 3 consecutive bolus injections made with 20-minute interval. (А) Example traces. (B) Mean relative maximum RVP and MAP increase observed after phosphocholine injections (n = 6). * Significant difference (pb 0.05) between the initial values and maximum values after phosphocholine injection.
As we previously noted, D609 had no effect on systemic hemodynamics and pO2m under normoxic and hypoxic conditions. While D609 has been shown to possess a very low toxicity (Bai et al., 2004), we suggest that this compound has a significant potential to be used for the creation of a new drug against hypoxic pulmonary hypertension. As we mentioned before, while it appears that PKC and TRPC6 solely contribute to the transient phase of HPV, there is a possibility that other PC-PLC-activated but DAG-independent mechanisms may exist. Searching for the alternative mechanisms of implementation of the vasoconstricting effect of PC-PLC activation we turned our attention to another product of phosphotidylcholine hydrolysis, phosphocholine. This metabolite induces sustained contraction in IPA. Furthermore, phosphocholine injected intravenously evokes significant transient rise in RVP and MAP and, what is even more intriguing, this effect on pulmonary circulation is stable throughout the repeated administrations, while gradual desensitization to phosphocholine has been observed in systemic circulation. The observed transient hypertension apparently has a non-neurogenic origin as the reaction is not substantially affected by hexamethonium. Taken together, these data suggest that phosphocholine is able to induce vasoconstriction (primarily pulmonary) and thus we hypothesize that it may act as a messenger along with DAG released during PC-PLC activation. The idea of differential sensitivity of arteries to phosphocholine is supported by the fact that this compound has no effect on the tone of isolated rat aorta (Cansev et al., 2007).
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In summary, the obtained results demonstrate the important role of 452 PC-PLC in the mechanism of HPV. We conclude that this phospholipase 453 is partially involved in the transient phase presumably initiating PKC 454
Please cite this article as: Strielkov, I.V., et al., Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction, Vascul. Pharmacol. (2013), http://dx.doi.org/10.1016/j.vph.2013.02.002
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Acknowledgments
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This study was supported by the International Junior Research Grant of The Physiological Society. The authors would like to thank Prof. J.P.T. Ward for his kind support of the present study and Dr. V.A. Snetkov for his assistance in the experimental work.
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References
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activation. PC-PLC contribution to the sustained HPV appears to be critical and may involve the activation of other vasoconstricting mechanisms along with PKC. They may rely on phosphocholine released due to PC-PLC-catalyzed phosphotidylcholine breakdown. According to our findings, phosphocholine is able to induce IPA constriction in vitro as well as AHPH in vivo. The underlying mechanisms of this reaction are yet to be elucidated. Contrary to PC-PLC, PI-PLC is not involved in sustained HPV. Considering that PC-PLC contributes to the constriction of IPA induced by superoxide anion, we assume that PC-PLC may be activated in hypoxia as a result of increased ROS generation, which has been previously proposed as HPV triggering event. We suggest that the demonstrated prevention of AHPH with D609 may become a basis for the development of a new anti-hypertensive therapy.
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