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Phospholipase D1 is involved in ␣1-adrenergic contraction of murine vascular smooth muscle Jörg W. Wegener,*,1 Florian Loga,* David Stegner,† Bernhard Nieswandt,† and Franz Hofmann* *For 923, Institut für Pharmakologie and Toxikologie, Technische Universität München, München, Germany; and †Lehrstuhl für Experimentelle Biomedizin, Universitätsklinikum Würzburg and Rudolf-Virchow-Zentrum, Deutsche Forschungsgemeinschaft (DFG) Forschungszentrum für Experimentelle Biomedizin, Universität Würzburg, Würzburg, Germany ␣1-Adrenergic stimulation increases blood vessel tone in mammals. This process involves a number of intracellular signaling pathways that include signaling via phospholipase C, diacylglycerol (DAG), and protein kinase C. So far, it is not certain whether signaling via phospholipase D (PLD) and PLD-derived DAG is involved in this process. We asked whether PLD participates in the ␣1-adrenergic-mediated signaling in vascular smooth muscle. ␣1-Adrenergic-induced contraction was assessed by myography of isolated aortic rings and by pressure recordings using the hindlimb perfusion model in mice. The effects of the PLD inhibitor 1-butanol (IC50 0.15 vol%) and the inactive congener 2-butanol were comparatively studied. Inhibition of PLD by 1-butanol reduced specifically the ␣1-adrenergic-induced contraction and the ␣1-adrenergic-induced pressure increase by 10 and 40% of the maximum, respectively. 1-Butanol did not influence the aortic contractions induced by high extracellular potassium, by the thromboxane analog U46619, or by a phorbol ester. The effects of 1-butanol were absent in mice that lack PLD1 (Pld1ⴚ/ⴚ mice) or that selectively lack the CaV1.2 channel in smooth muscle (sm-CaV1.2ⴚ/ⴚ mice) but still present in the heterozygous control mice. ␣1-Adrenergic contraction of vascular smooth muscle involves activation of PLD1, which controls a portion of the ␣1-adrenergic-induced CaV1.2 channel activity.—Wegener, J. W., Loga, F., Stegner, D., Nieswandt, B., Hofmann, F. Phospholipase D1 is involved in ␣1-adrenergic contraction of murine vascular smooth muscle. FASEB J. 28, 1044 –1048 (2014). www.fasebj.org ABSTRACT

Key Words: blood pressure 䡠 intracellular signaling 䡠 CaV1.2 Ca2⫹ channel 䡠 U46619

Abbreviations: CPI-17, protein kinase C-potentiated phosphatase inhibitor protein 17; CTR, control; DAG, diacylglycerol; eNOS, endothelial nitric oxide synthase; IBMX, 3-isobutyl-1-methylxanthine; IP3, inositol-1,4,5-trisphosphate; ISR, isradipine; PA, phosphatic acid; PE, phenylephrine; PdBu, phorbol 12,13-dibutyrate; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D 1044

␣1-Adrenergic receptors regulate blood pressure by stimulation of vascular contraction. This process involves several intracellular signaling cascades including Ca2⫹ entry via Ca2⫹ channels, release of Ca2⫹ from intracellular stores, and sensitization of the contractile apparatus to Ca2⫹ (1–3). The prevailing view is that ␣1-adrenergic receptors couple predominantly to G proteins of the Gq/11 family (4). Activated Gq/11 proteins stimulate phospholipase C (PLC) to form the second messengers inositol-1,4,5trisphosphate (IP3), and diacylglycerol (DAG). IP3 acts on the IP3 receptor in the endoplasmic reticulum to release Ca2⫹ into the intracellular compartment. This leads to a Ca2⫹/calmodulin-dependent activation of myosin light chain kinase, phosphorylation of myosin light chain 2, and, finally, to contraction. The other second messenger, DAG, activates conventional members of the protein kinase C (PKC) family by reducing their Ca2⫹ requirement and by enhancing their membrane association (5). In smooth muscle, activated PKC phosphorylates protein kinase C-potentiated phosphatase inhibitor protein 17 (CPI-17), which, in turn, inhibits myosin light chain phosphatase, increases phosphorylation of myosin light chain 2, and enhances contraction. This pathway is part of the so-called sensitization of the contractile filaments (6). ␣1-Adrenergic receptors can also couple to G proteins of the G12/13 family (4). G proteins of the G12/13 family, as well as those of the Gq/11 family (7, 8), activate the small G protein RhoA by the Rho-specific guanine nucleotide exchange factor. RhoA modulates the activities of several enzymes, including the activity of phospholipase D (PLD; refs. 9, 10). Consequently, ␣1-adrenergic stimulation of PLD activity has been shown in fibroblasts and kidney cells (11–13). On stimulation, PLD hydrolyzes phosphatidylcholine to generate phosphatic acid (PA) and free choline. PA is 1 Correspondence: For 923, Institut für Pharmakologie und Toxikologie, TU München, Biedersteiner Str. 29, 80802 München, Germany. E-mail: [email protected] doi: 10.1096/fj.13-237925 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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then directly metabolized to DAG through the actions of PA phosphohydrolase. PLD-derived DAG has been shown to regulate PKC activity in several cell lines in vitro (9). We hypothesized that PLD participates in ␣1-adrenergic-mediated contraction in vascular smooth muscle. Inhibition of PLD by 1-butanol reduced specifically ␣1-adrenergic-mediated contraction in the aorta and hindlimbs. The effects of 1-butanol were not observed in preparations from Pld1⫺/⫺ and from sm-CaV1.2⫺/⫺ mice. These results raise the possibility that PLD1 is involved in ␣1-adrenergic-mediated vascular contraction, probably by partially modulating ␣1-adrenergicinduced CaV1.2 channel activity.

MATERIALS AND METHODS Experiments were performed on mice that globally lack PLD1 (Pld1⫺/⫺ mice) or that specifically lack CaV1.2 in smooth muscle (sm-CaV1.2⫺/⫺ mice) and on the corresponding control (CTR) mice. Handling and generation of the mice were described elsewhere (14, 15). sm-CaV1.2⫺/⫺ mice and the corresponding heterozygous CTR mice were treated with tamoxifen (1 mg/d for 5 consecutive days) to inactivate the CaV1.2 gene and investigated 2 wk later. All experiments complied with the European guidelines for the use of experimental animals and were approved by the local animal ethics committee (Regierung von Oberbayern). Myography was performed isometrically using the Myograph 601 (Danish Myo Technology A/S, Aarhus, Denmark; http://www.dmt.dk). All mice used were euthanized by decapitation. The thoracic aorta was prepared and quickly transferred to buffer solution (137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 12 mM NaHCO3, 0.42 mM NaH2PO4, and 5.6 mM glucose). Buffer solution was bubbled with carbogen (95% O2 and 5% CO2) to give a pH of 7. The thoracic aorta was then cleaned from the connective tissue. Aortic rings of ⬃3 mm in width were mounted in organ baths filled with buffer solution. Rings were prestretched by 3 mN/mm. Experiments were performed at 36 ⫾ 1°C. Substances were added cumulatively to the organ baths. Mean pressure was recorded at a constant perfusion rate using the hindlimb perfusion device UP-100 and the corresponding TAM-D system (Hugo Sachs Elektronik-Harvard Apparatus GmbH, Hugstetten, Germany; http://www.hugosachs.de). After euthanasia, a catheter (0.9-mm diameter) was introduced into the abdominal aorta and advanced to the hindlimb bifurcation. The inferior caval vein was slit open to prevent venous congestion. A roller pump was used to constantly perfuse the hindlimbs with prewarmed buffer solution. The flow rate was adjusted to achieve a mean pressure of ⬃50⫺60 mmHg and was kept constant during the experiment. Substances were added to the perfusion buffer. Latency between application and the vascular effect was ⬃3 min. All experiments were performed at 36 ⫾ 1°C. Western blotting using anti-PLD1 (EP1506Y; Acris Antibodies, Herford, Germany; http://www.acris-antibodies.com), antiCPI-17 (Millipore, Billerica, MA, USA; http://www.millipore. com), and anti-pCPI-17 (Millipore) was performed as described previously (16). In brief, proteins from aorta were isolated by homogenization with SDS lysis buffer (21 mM Tris, pH 8.0; 0.7% SDS; 1.7% ␤-mercaptoethanol; and 0.2 mM phenylmethylsulfonyl fluoride) using a FastPrep device (MP Biomedicals, Irvine, CA, USA; www.mpbio.com) followed by centrifugation (at 13,000 g and 4°C for 10 min). The superPLD1 AND VASCULAR CONTRACTION

natants containing the tissue proteins were stored at ⫺80°C. Western blot analysis of the tissue proteins was performed with selective primary antibodies and with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. All salts and substances used were as pure as commercially available and purchased from Sigma-Aldrich (St. Louis, MO, USA; http://www.sigmaaldrich.com). Intracellular [Ca2⫹] was monitored in freshly isolated aortic smooth muscle using Fura-2/AM at room temperature as described previously (17). [Ca2⫹]i was recorded as fluorescence intensity (at 510 nm) at alternating 350-nm (F350) and 380-nm (F380) excitation wavelengths and their respective ratio (F350/F380) using a TILLvision device (Till Photonics, Gräfelfing, Germany; http://www.till-photonics.de). Stimulation was performed by application of phenylephrine (PE; 30 ␮M) via a syringe device. Results are presented as blots, as original recordings, or as means ⫾ sem. Effects of substances were analyzed under steady-state conditions. Changes in tension or in mean pressure were determined with respect to the maximum of the signals and the baseline. The relaxing effects of substances were determined as the difference between the maximum and the baseline in the presence of 3-isobutyl-1-methylxanthine (IBMX; 100 ␮M) or papaverine (100 ␮M). Statistical comparisons of data sets were performed by a Student’s t test using Prism 5 (GraphPad Software Inc., San Diego, CA, USA; http://www.graphpad.com). Differences were considered significant at a value of P ⬍ 0.05.

RESULTS AND DISCUSSION ␣1-Adrenergic stimulation increases vascular tone by several intracellular signaling cascades (1). We studied the involvement of PLD in these cascades because ␣1-adrenergic stimulation increased PLD activity in heart and aortic smooth muscle (18 –20). PE-contracted aortic rings from mice were treated comparatively with the PLD inhibitor 1-butanol and the inactive congener 2-butanol. The use of these alcohols represents a reliable approach to characterize the participation of PLD in cellular signaling cascades (21); in particular, 1-butanol has been reported to be highly specific for PLD (22, 23). In all experiments, 1- and 2-butanol were used at concentrations of 0.1⫺0.2 vol% to avoid unspecific effects of the alcohol treatment, although the effect of 1-butanol was more obvious at a concentration of 0.5 vol% (Supplemental Fig. S1). Both alcohols relaxed contractions induced by PE in aortic rings (Fig. 1A, B). However, the effect of 1-butanol was significantly larger than that of 2-butanol (Fig. 1A, B and Supplemental Fig. S1), indicating a specific action of this alcohol on ␣1-adrenergic contraction, probably via PLD inhibition. To further characterize the effects of PLD inhibition in murine aorta, the effects of 1-butanol and 2-butanol were comparatively studied on contractions induced by different signaling pathways, i.e., on contractions induced by high extracellular potassium, by the thromboxane analog U46619, and by the phorbol ester, phorbol 12,13-dibutyrate (PdBu). 1-Butanol and 2-butanol showed no different effects in aortic rings that were contracted by high extracellular K⫹ (85 mM) or by PdBu (1␮M; Supplemental Fig. S2). A tiny differ1045

Figure 1. 1-Butanol decreased phenylephrine-induced contraction in aortic rings. Data were normalized to the maximum to allow a better comparison of recordings that were performed in parallel. Absolute values are presented in Fig. 3. A, C, E) Original recordings of tension in aortic rings from wild-type (WT) mice (A), Pld1⫺/⫺ and corresponding CTR mice (C), and sm-CaV1.2⫺/⫺ mice and corresponding CTR mice (E). Bars indicate the presence of PE (3 ␮M), nitro-l-arginine methyl ester (l-NAME; 100 ␮M), 1-butanol or 2-butanol (1-but or 2-but; 0.1 vol%, respectively), ISR (1 ␮M), and IBMX (100 ␮M). Endothelial nitric oxide production was inhibited by l-NAME (100 ␮M). B, D, F) Effect of 1-butanol and/or 2-butanol on PE-induced contraction in rings from wild-type mice (B), from Pld1⫺/⫺ and corresponding CTR mice (D), and from sm-CaV1.2⫺/⫺ mice and corresponding CTR mice (F). Columns represent means ⫾ sem. Numbers represent the number of experiments. n.s., nonsignificant. ***P ⬍ 0.001.

ence was found in rings contracted by U46619 (10 ␮M; Supplemental Fig. S2). These findings show that the effect of 1-butanol is specific for ␣1-adrenergic-mediated contraction and suggest that PLD selectively participates in this signaling pathway. Mammalian PLD exists in 2 isoforms, PLD1 and PLD2 (9). Western blot analysis revealed the presence of PLD1 in murine aorta (Supplemental Fig. S1). To support the notion that PLD1 is involved in ␣1-adrenergic contraction, we analyzed the effects of 1-butanol on PE-induced contractions in aortic rings from Pld1⫺/⫺ mice. 1-Butanol showed no specific effect on PE-induced contraction in rings from Pld1⫺/⫺ mice but reduced contractions in rings from the corresponding CTR mice (Fig. 1C, D). This finding suggests that PLD1 participates in ␣1-adrenergic-mediated contraction. ␣1-Adrenergic contraction in vascular smooth muscle has been shown to activate CaV1.2 channels (2) and, consequently, to depend partially on the presence of functional CaV1.2 channels (14). However, the mechanism by which CaV1.2 channels are activated in smooth muscle is not clear (24). One proposed mechanism is that PLC-derived DAG controls Ca2⫹ channel activity via PKC (24). Recent evidence suggests that PLDderived DAG modulates CaV1.2 channel activity via PKC in urinary bladder smooth muscle (16). Therefore, we analyzed the effects of 1-butanol and 2-butanol in PE-contracted rings from sm-CaV1.2⫺/⫺ mice. In sm-CaV1.2⫺/⫺ mice, 1-butanol showed no specific relaxation when compared with 2-butanol (Fig. 1E, F). In addition, no specific effect of 1-butanol on PE-induced contraction was observed in the presence of the CaV1.2 channel blocker isradipine (ISR) in rings from wildtype mice (Supplemental Fig. S3). These findings imply that the CaV1.2 channel is required for the specific effect of 1-butanol on vascular contraction and PLD1046

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derived DAG may modulate CaV1.2 channel activity in aortic smooth muscle. It has been shown that sustained DAG or PLD activity induced translocation of PKC and had a major affect on PKC activity in in vitro cell systems (25, 26). On receptor activation, a fast peak (⬃15 s) and a sustained peak (⬃5 min) of DAG generation have been reported (9). The initial peak of DAG correlated with the production of IP3 and PLC activity, whereas the second peak was independent of IP3 levels but corresponded with PLD activity. In the present study, we studied tonic contractions of vascular muscle, which developed within 5⫺20 min. In this condition, PLD activity and continuous DAG production may contribute via PKC to contraction of smooth muscle, as proposed previously (27). So far, our results suggest that PLD1 is partially involved in PE-induced contractions in murine aorta. However, the aorta is a conduit vessel and may not be representative for the properties of resistance vessels. To test the involvement of PLD in resistance vessels, we extended the studies described above to the perfused hindlimb model, in which mean pressure was recorded at a constant flow. In wild-type mice, 1-butanol reduced the PE-mediated pressure increase to a larger extent than 2-butanol, demonstrating the specific effect of this alcohol (Fig. 2A, B) even if the substances were applied in the reverse order. In Pld1⫺/⫺ mice, 1- and 2-butanol showed no different effects on the PE-induced pressure increase (Fig. 2C, D). In sm-CaV1.2⫺/⫺ mice, 1-butanol did not influence the PE-induced pressure increase (Fig. 2E, F). Taken together, these results support the concept that PLD1 is involved in PE-induced contraction in vascular smooth muscle and suggest that the effects of 1-butanol require the presence of ␣1-adrenergic-controlled CaV1.2 channel activity. To further test the hypothesis that PLD1/CaV1.2 signal-

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Figure 2. 1-Butanol decreased PE-induced pressure increases during hindlimb perfusion. A, C, E) Original recordings of pressure during hindlimb perfusion in a wild-type (WT) mouse (A), a Pld1⫺/⫺ mouse (C), and a smCaV1.2⫺/⫺ mouse (E). Bars indicate the presence of PE (10 ␮M), 1- or 2-butanol (1-but or 2-but; 0.2 vol%, respectively), ISR (1 ␮M), and IBMX (100 ␮M). Fast downward reflections represent the drop in pressure during the exchange of the perfusion buffer. Flow rate was constant throughout the experiment. B, D, F) Magnitudes of PE-induced pressure increase in wild-type mice (B), in Pld1⫺/⫺ and corresponding CTR mice (D), and in sm-CaV1.2⫺/⫺ and corresponding CTR mice (F). Columns represent means ⫾ sem. Numbers represent the number of experiments. n.s., nonsignificant. **P ⬍ 0.01.

ing is partially involved in ␣1-adrenergic contraction, we analyzed comparatively the magnitudes of agonist-induced contractions and pressure increases in aortas from Pld1⫺/⫺ and sm-CaV1.2⫺/⫺ mice. As expected, the effects of PE and high extracellular potassium were clearly reduced in sm-CaV1.2⫺/⫺ mice (Figs. 2 and 3A), pointing to the major role of the CaV1.2 channel in vascular contraction (14). Similarly, the effects of PdBu were also reduced in aorta from sm-CaV1.2⫺/⫺ mice (Fig. 3C), suggesting that PdBu-activated PKC modulates CaV1.2 channel activity as proposed (24, 28). The effects of U46619 were only marginally attenuated in aorta from sm-CaV1.2⫺/⫺ mice (Fig. 3D). We further expected that the absence of PLD1 should also reduce the PE-mediated effects because the signaling of PLD1 to CaV1.2 should be diminished. Unexpectedly, we did not find differences in the magnitudes of PE-induced contractions and pressure increases between prepara-

tions from wild-type and Pld1⫺/⫺ mice (Figs. 2 and 3A). Further analysis revealed that the magnitudes of contractions in aorta induced by high potassium or by U46619 were also not different between CTR and Pld1⫺/⫺ mice (Fig. 3B, D). In contrast, the magnitudes of contraction induced by PdBu were increased in aorta from Pld1⫺/⫺ mice (Fig. 3C). These results raise the possibility that PE-activated contractions in aorta from Pld1⫺/⫺ mice are mainly mediated by an up-regulated Ca2⫹-independent contraction pathway to compensate for the putative missing PLD1/CaV1.2 signaling. This view is supported by the findings that blockade of CaV1.2 channels by ISR had a reduced effect on PEinduced contractions but was similarly effective on PdBu- and K85-induced contractions in aorta from Pld1⫺/⫺ mice (Fig. 1C and Supplemental Fig. S4), PE-induced Ca2⫹ signals were attenuated in isolated aortic smooth muscle cells from Pld1⫺/⫺ mice (Supple-

Figure 3. Magnitudes of agonist-induced contraction in murine aorta. All magnitudes were assessed in the presence of nitro-l-arginine methyl ester (100 ␮M) to exclude the vasodilator tone by endothelial nitric oxide production. Aortas from Pld1⫺/⫺, sm-CaV1.2⫺/⫺, and their respective CTR mice were stimulated with PE (3 ␮M, A), with high extracellular potassium [85 mM (K85); B], with PdBu (1 ␮M; C), and with U46619 (10 ␮M; D). Columns represent means ⫾ sem. Numbers represent the number of experiments. n.s., nonsignificant. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001. PLD1 AND VASCULAR CONTRACTION

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mental Fig. S4), and enhanced phosphorylated CPI-17 was observed after the aorta from Pld1⫺/⫺ mice was stimulated with PdBu (Supplemental Fig. S4). Phosphorylated CPI-17 is a main mediator of DAG/PKC-induced, Ca2⫹-independent contraction in smooth muscle (6). A similar up-regulation of the adrenergic activation of PKC/ CPI-17 signaling has been reported in arteries from diabetic rats (29). Other evidence for expression of compensatory mechanisms in knockout mouse models comes from endothelial nitric oxide synthase (eNOS)deficient mice, in which the neuronal isoform of nitric oxide synthase substitutes for eNOS in the responses of cerebral arterioles to acetylcholine (30). In summary, these data support our hypothesis that sustained ␣1-adrengic-mediated contraction of vascular smooth muscle encloses a signaling pathway involving PLD1 that partially controls CaV1.2 channel activity, probably via DAG/PKC. It remains to be elucidated which subtype of aortic ␣1-adrenergic receptors may couple to PLD1 (31). Vascular PLD1 may represent a new target for the therapy of vascular dysfunction. The authors thank Theodora Kernel for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (For 923, WE2650/4-1 to J.W.W. and Ni556/8-1 to B.N.).

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Received for publication July 9, 2013. Accepted for publication November 11, 2013.

WEGENER ET AL.