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Adenosine A1 receptor-dependent and independent pathways in modulating renal vascular responses to angiotensin II X. Gao,1,* M. Peleli,2,* C. Zollbrecht,2 A. Patzak,3 A. E. G. Persson1 and M. Carlstr€ om2 1 Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden 2 Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden 3 Institute of Vegetative Physiology, Charite-Universit€atsmedizin Berlin, Berlin, Germany
Received 19 June 2014, revision requested 9 August 2014, revision received 19 August 2014, accepted 17 September 2014 Correspondence: M. Carlstr€ om, Department of Physiology and Pharmacology, Nanna Svartz V€ag 2, S-17177 Stockholm, Sweden. E-mail: [email protected]
*These authors equally contributed to this work.
Abstract Aim: Renal afferent arterioles are the effector site for autoregulation of glomerular perfusion and filtration. There is synergistic interaction between angiotensin II (ANG II) and adenosine (Ado) in regulating arteriolar contraction; however, the mechanisms are not clear. In this context, this study investigated the contribution of A1 receptor-dependent and independent signalling mechanisms. Methods: Isolated perfused afferent arterioles from transgenic mice (A1+/+ and A1 / ) were used for vascular reactivity studies. Cultured vascular smooth muscle cells (VSMC) were used for phosphorylation studies of signalling proteins that induce arteriolar contraction. Results: Maximal arteriolar contraction to ANG II was attenuated in A1 / (22%) compared with A1+/+ (40%). Simultaneous incubation with low-dose ado (10 8 mol L 1) enhanced ANG II-induced contraction in A1+/+ (58%), but also in A1 / (42%). An ado transporter inhibitor (NBTI) abolished this synergistic effect in A1 / , but not in wild-type mice. Incubation with Ado + ANG II increased p38 phosphorylation in aortic VSMC from both genotypes, but treatment with NBTI only blocked phosphorylation in A1 / . Combination of ANG II + Ado also increased MLC phosphorylation in A1+/+ but not significantly in A1 / , and NBTI had no effects. In agreement, Ado + ANG II-induced phosphorylation of p38 and MLC in rat pre-glomerular VSMC was not affected by NBTI. However, during pharmacological inhibition of the A1 receptor simultaneous treatment with NBTI reduced phosphorylation of both p38 and MLC to control levels. Conclusion: Interaction between ANG II and Ado in VSMC normally involves A1 receptor signalling, but this can be compensated by receptor independent actions that phosphorylate p38 MAPK and MLC. Keywords afferent arteriole, kidney, microcirculation, myosin light chain, p38 MAP kinase, vascular smooth muscle cell.
The afferent arterioles are the effector site for renal autoregulation where myogenic response and tubuloglomerular feedback (TGF) importantly contribute to 268
the control of glomerular perfusion and filtration (Persson & Wright 1982, Casellas & Moore 1990, Vallon et al. 2006). Various vasoactive substances,
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including angiotensin II (ANG II) and adenosine, modulate the tone and reactivity of the renal resistance vessels (Franco et al. 2009, Persson et al. 2013). ANG II induces constriction of the renal microvasculature via ANG II receptor type 1. Adenosine has a biphasic response with adenosine A1 receptor-mediated contraction in the nanomolar range and A2 receptor-mediated dilatation in the high concentration range (Lai et al. 2006b, 2009, Patzak et al. 2007, Carlstrom et al. 2008). Previous studies have demonstrated a synergistic interaction between the contractile response by ANG II and that of adenosine (Hall et al. 1985, Weihprecht et al. 1994, Hansen et al. 2003b, Franco et al. 2009, Persson et al. 2013). In a physiological concentration, ANG II increased the contractile response to adenosine (Lai et al. 2006b). At the same time, adenosine in low concentrations significantly enhanced the afferent arteriolar response to ANG II (Hansen et al. 2003a, Lai et al. 2006b). Moreover, adenosine A1 gene-deleted mice (A1 / ) displayed attenuated arteriolar responses to ANG II (Hansen et al. 2003b) and had less blood pressure elevation during ANG II infusion (Gao et al. 2011, Lee et al. 2012). The interaction between ANG II and adenosine, in regulating arteriolar contraction, may contribute to the phenomenon of TGF resetting (Persson et al. 2013). The underlying mechanism(s) for this synergism are still under investigation, but increased intracellular Ca2+ sensitivity via modulation of phospholipase C (PLC), p38 mitogen-activated protein kinase (MAPK) and myosin light chain (MLC) phosphorylation have been suggested (Hansen et al. 2003a, Lai et al. 2006a, 2009). Although previous studies have emphasized that A1 receptors are important for the synergistic interaction between adenosine and ANG II (Hansen et al. 2003b, Lai et al. 2006b, 2009, Gao et al. 2011), receptor independent mechanisms may contribute (Lai et al. 2006a). This study aimed to investigate the contribution of adenosine A1 receptor-dependent and independent signalling pathways. It was hypothesized that A1 receptors importantly contribute to the synergism between ANG II and adenosine in regulating renal afferent arteriolar contraction. Moreover, synergistic interaction between adenosine and ANG was also investigated in isolated vascular smooth muscle cells (VSMC) from the mouse aorta and from rat pre-glomerular vessels.
Methods Afferent arteriole measurements Dissection and perfusion of renal cortical afferent arterioles were performed as described previously (Gao et al. 2011). DMEM/F12 with 10 mmol L 1 HEPES
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(Invitrogen AB, Liding€ o, Sweden) was used for dissection, bath and perfusion. The pH was adjusted to 7.4 after addition of bovine serum albumin (BSA: SERVA Electrophoresis Heidelberg, Germany). The concentration of BSA was 0.1% in dissection and bath solutions, and 1% in the perfusion solution. The experiments were digitally recorded and then digitized offline and analysed as described before (Gao et al. 2011). Changes in luminal diameters were measured to estimate the effect of vasoactive substances. In all series, the last 10 s of a control or treatment period were used for statistical analysis of steady state responses. Each experiment used a separate dissected afferent arteriole: only one arteriole was used per animal.
Cell culture of VSMC Aortic VSMC. Isolation and culturing of primary VSMC from A1+/+ and A1 / mice by a modified method of that originally described by Kobayashi and colleagues (Kobayashi et al. 2005). In isoflurane-anesthetized mice, the abdominal aorta was cut at the middle to release blood and then perfused with 1 mL of PBS containing 1000 U mL 1 of heparin (Hospira, Inc. Lake Forest, IL, USA). The aorta was dissected out from the aortic arch to the abdominal aorta and immersed in 20% foetal bovine serum (FBS, from ATCC, Manassas, VA, USA) Dulbecco’s modified eagles medium (DMEM) containing 1000 U mL 1 of heparin. The fat or connecting tissue was rapidly removed with fine forceps under a microscope. The inside of the lumen of aorta was briefly washed with serum-free DMEM. From the other side, it was washed with collagenase type II solution (2 mg mL 1, dissolved in serum-free DMEM) (Sigma–Aldrich Sweden AB, Stockholm, Sweden). After incubation for 45 min at 37 °C, endothelial cells were removed from the aorta by flushing with DMEM containing 10% FBS. The aorta was cut lengthwise, and put onto a 60 mm dish. With a scalpel blade the aorta was cut into almost square pieces and allowed to dry briefly. DMEM with 20% FBS and penicillin/streptomycin was added gently, and the cells were transferred to 48-well culture dishes and placed in an incubator and left undisturbed for a week. All the solutions at every isolation step had antibiotic/antimycotic mix added (Gibco, Carlsbad, CA, USA). After a week, the culture plates were examined under the microscope, to observe the presence of VSMC in the medium. The cells were rinsed two times with 20% FBS-containing DMEM and replaced with fresh medium with antimycotic/antibiotic added. The medium was replaced two times a week. Pre-glomerular VSMC (PG-VSMC). Isolation and culturing of primary PG-VSMC from rats were
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performed as previously described (Luo et al. 2009), and the phenotype was confirmed as described by Dubey and colleagues (Dubey et al. 1992). All experiments were carried out during early passage (between 3 and 9), when it is known that they express smooth muscle cell specific markers. Cells were seeded in 75-cm2 flasks and cultured in DMEM D0572 (Sigma–Aldrich) supplemented with 10% FBS, 2 mM L-Glutamine and 100 U mL 1 Penicillin–Streptomycin (Life Technologies, Grand Island, NY, USA). When used for the experiments, the cells were plated in 60mm dishes until they reached 80% confluence and then serum deprived for 24 h before the treatments.
Cellular protocol The cellular protocol was similar to renal afferent arterioles’. Briefly, the VSMC were divided in three groups: (a) untreated, (b) treated with adenosine (10 8 mol L 1; 15 min) and then ANG II (10 7 mol L 1, 15 min) and (c) pre-treated with NBTI (3 9 10 7 mol L 1; 5 min) and then treated with adenosine (10 8 mol L 1; 15 min) and ANG II (10 7 mol L 1; 15 min). In experiments using the selective adenosine A1 receptors antagonist CPX (5 9 10 8 mol L 1), the cells were pre-incubated with the antagonist for 1 h before the same treatment protocol described above was started. In that case, the control cells were pre-treated with CPX (5 9 10 8 mol L 1, 1 h) and then with NBTI (3 9 10 7 mol L 1; 5 min). After the different treatments, VSMC were placed on ice immediately, washed twice with cold DPBS and lysed in 100ll of lysis buffer supplemented with phosphatase and protease inhibitors (Sigma–Aldrich). Cell lysates were centrifuged (14 000 g for 15 min) and the supernatant was collected and placed in 80 °C until further analysis.
Western blot analysis of p38 MAPK and MLC phosphorylation Protein concentration in the supernatants was determined by Bradford protein assay (Bio-Rad Laboratories AB, Solna, Sweden). Equal protein amounts were separated by 4–20% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Bio-Rad), followed by transfer to a polyvinylidene difluoride membrane (BioRad). After blocking with 5% non-fat dry milk in Tween-containing Tris-buffered saline, membranes were incubated with specific primary antibodies (phospho-p38 MAP Kinase (Thr180/Tyr182) rabbit polyclonal antibody, Cell Signaling #9211, MLC-2B (pSer19) rabbit polyclonal antibody, Acris Antibodies #R1535P or mouse monoclonal antibody, Cell Signaling #3675S, GAPDH mouse monoclonal antibody, 270
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Santa Cruz #sc-47724) and secondary antibodies (horseradish peroxidase-conjugated goat antibodies to rabbit or mouse IgG; DAKO). Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to remove bound antibodies from the membranes, followed by blocking and reprobing the membranes with primary and secondary antibodies. Bands were detected by a SuperSignal West Femto chemiluminescence substrate (Thermo Fisher Scientific), and results were normalized with GAPDH. Images were analysed by a luminescent image analysis system LAS 1000+ (Fujifilm Corp., Tokyo, Japan). The results were quantified by densitometry (Image J, U. S. National Institutes of Health, Bethesda, MD, USA) and reported as relative optical density of the specific proteins.
Drugs and reagents Drugs and chemicals used for this study were obtained from Sigma–Aldrich (St. Louis, MO, USA), unless otherwise stated. In the arteriolar contraction experiments, all compounds were applied to the bath solution. In the cellular experiments, all compounds were applied to the cell culture medium.
Statistical analysis Values are presented as mean SE. Two-way repeated measures analysis of variance (2-way RM ANOVA), followed by Bonferroni’s multiple comparisons test was used to test time- or concentrationdependent changes in the arteriolar diameter and to assess differences between the groups. For multiple comparisons among groups (vascular and cellular studies), one-way ANOVA followed by Tukey’s post hoc test was used to allow for more than one comparison with the same variable. Statistical significance was defined as P < 0.05.
Ethics The experiments were approved by Ethical Committees for Animal Experiments and conducted in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals. The study is conform with Good Publishing Practice in Physiology (Persson & Henriksson 2011).
Results Regulation of afferent arteriolar responses Responses to ANG II. Concentration response curves were obtained by cumulative application of ANG II
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(10–12 to 10–6 mol L 1; each dose applied for 2 min). ANG II constricted afferent arterioles from A1+/+ mice in a concentration-dependent manner, with a maximum response of 40% (Fig. 1). In agreement with that previously described (Gao et al. 2011), afferent arterioles of A1 / mice were less responsive with a maximum contraction of 22% to ANG II (Fig. 1).
reduced arteriolar diameter by 9% in A1+/+ but had no significant effect in A1 / mice (P < 0.05). The subsequent contractile response to ANG II was significantly enhanced in A1+/+ with a maximal contraction of 58% (Fig. 2a). However, the arteriolar responses from A1 / mice were also much potentiated with a maximum contraction of 42% (Fig. 2b).
Responses to adenosine and ANG II. We investigated the synergistic interaction between the contractile response by adenosine and that of ANG II. Incubation with low dose of adenosine (10 8 mol L 1; 15 min)
Responses to adenosine and ANG II during inhibition of adenosine transporters. We investigated the potential role of nucleoside transporters as a receptor independent mechanism. Inhibition of adenosine transporters with NBTI did not significantly alter the contractile responses in A1+/+ (Fig. 2a). However, in A1 gene-deleted mice the sensitizing effect of adenosine on the contractile response to ANG II was abolished (27%), demonstrating an important role of adenosine transporters. Summarized maximal contractile responses in A1+/+ and A1 / mice are demonstrated in Figure 3.
Regulation of p38 MAPK and MLC phosphorylation in aortic VSMC from A1+/+ and A1 / mice To further assess potential differences in cellular signalling, we used aortic primary VSMC from A1+/+ and A1 / to investigate changes in p38 MAPK and MLC phosphorylation (Fig. 4a–d). Figure 1 Contractions to ANG II. Angiotensin II (ANG II)mediated contractions in isolated and perfused afferent arterioles of A1+/+ (n = 6) and A1 / (n = 6) mice. Values are mean SE. * denotes P < 0.05 compared with A1 wild-type mice.
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Adenosine transporter mediates p38 MAPK phosphorylation in A1 / but not in A1+/+. Adenosine signalling is known to induce the phosphorylation of p38 (b)
Figure 2 Contractions to adenosine + ANG II and the effect of adenosine transporter inhibition with NBTI. Effect of low-dose adenosine (10 8 mol L 1) on angiotensin II (ANG II)-mediated contractions in isolated and perfused afferent arterioles of A1+/+ (n = 6) and A1 / (n = 6) mice (a). Effect of low-dose adenosine (Ado; 10 8 mol L 1) on ANG II-mediated contractions, during simultaneous treatment with an adenosine transporter inhibitor (NBTI; 3 9 10 7 mol L 1), in isolated and perfused afferent arterioles of A1+/+ (n = 6) and A1 / (n = 6) mice (b). Values are mean SE. * denotes P < 0.05 compared with A1 knockout mice. © 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12399
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Figure 3 Maximal arteriolar contractions. Summarized maximal afferent arteriolar responses to adenosine (Ado; 10 8 mol L 1) and angiotensin II (ANG II) alone and in combination, and with simultaneous treatment with the adenosine transporter inhibitor (NBTI; 3 9 10 7 mol L 1) in A1+/+ (n = 6) and A1 / (n = 6) mice. Values are mean SE. * denotes P < 0.05 between indicated groups.
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MAPK, which then promotes a phosphorylation cascade leading to VSMC contraction. Pre-treatment of VSMC with adenosine (10 8 mol L 1) and ANG II (10 7 mol L 1) significantly increased p38 MAPK phosphorylation for both A1+/+ (1.92 0.14) and A1 / (1.37 0.07) (Fig. 4c). In VSMC from A1+/+, phosphorylation levels were not significantly changed by simultaneous treatment with the adenosine transporter NBTI compared with that of adenosine + ANG II (1.73 0.23 vs. 1.92 0.14). In contrast, NBTI abolished adenosine + ANG II-induced phosphorylation of p38 MAPK (0.53 0.03 vs. 1.37 0.07) in VSMC from A1 / . Adenosine and ANG II-induced phosphorylation of MLC is lost in A1 / . Phosphorylation of MLC is a key event in inducing the contractile apparatus of VSMC. Pre-treatment of A1+/+ VSMC with adenosine (10 8 mol L 1) and ANG II (10 7 mol L 1) signifi-
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Figure 4 Regulation of p38 MAPK and MLC phosphorylation in aortic vascular smooth muscle cells (VSMC) from A1+/+ and A1 / mice. Effects of adenosine (Ado; 10 8 mol L 1) and angiotensin II (ANG II; 10 7 mol L 1) with or without simultaneous incubation with the adenosine transporter inhibitor NBTI (3 9 10 7 mol L 1) on p38 MAPK (Thr180/Tyr182) and MLC (pSer19/20) phosphorylation in A1+/+ (a & c) and A1 / (b & d) aortic VSMC. Phosphorylation levels were normalized to GAPDH. Panel A and B demonstrate 3–4 representative samples/group from the same gel for p38 MAPK and MLC phosphorylation. Relative changes in p38 MAPK (c) and MLC (d) phosphorylation are demonstrated. Adenosine and angiotensin II (Ado + ANG II) significantly increased the phosphorylation levels of p38 MAPK in both A1+/+ and A1 / . The presence of NBTI reversed this induction in A1 / only but not in A1+/+. Adenosine and angiotensin II (Ado + ANG II) significantly increased the phosphorylation levels of MLC in A1+/+ and NBTI could not significantly reverse this activation. The phosphorylation levels of MLC remained unchanged in VSMC from A1 / . Values are mean SE (n = 5–6/group). * denotes P < 0.05 between indicated groups.
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Figure 5 Regulation of p38 MAPK phosphorylation in pre-glomerular vascular smooth muscle cells (VSMC) during pharmacological inhibition of A1 receptor signalling. Effects of adenosine (Ado; 10 8 mol L 1) and angiotensin II (ANG II; 10 7 mol L 1) with or without simultaneous incubation with the adenosine transporter inhibitor NBTI (3 9 10 7 mol L 1) on p38 MAPK (Thr180/Tyr182) phosphorylation in rat pre-glomerular VSMC in the absence (a & c) or presence (b & d) of an adenosine A1 receptor antagonist (CPX, 0.5 9 10 8 mol L 1). Phosphorylation levels were normalized to GAPDH. Panel A and B demonstrate 3 representative samples/group from the same gel for p38 MAPK phosphorylation. Relative changes in p38 MAPK phosphorylation are demonstrated in the absence (c) or presence (d) of an adenosine A1 receptor antagonist (CPX, 0.5 9 10 8 mol L 1). Panel c: Adenosine and angiotensin II (Ado + ANG II) increased the phosphorylation levels of p38 MAPK, and this was not significantly changed with NBTI. Panel d: During inhibition of A1 receptors adenosine and angiotensin II (Ado + ANG II) increased the phosphorylation levels of p38, and this was significantly reduced by simultaneous treatment with NBTI. Values are mean SE (n = 5–6/group). * denotes P < 0.05 between indicated groups.
cantly increased phosphorylation of MLC (2.48 0.17 vs. control), and this effect was not altered in the presence of NBTI (2.14 0.41 vs. control) (Fig. 4d). The same concentrations of adenosine and ANG II did not significantly increase MLC phosphorylation in the A1 / cells.
Regulation of p38 MAPK and MLC phosphorylation in pre-glomerular VSMC during pharmacological inhibition of A1 receptor signalling Next, we used primary pre-glomerular VSMC isolated from rat to assess effects of adenosine receptordependent and independent signalling in regulation of p38 MAPK (Fig. 5a–d) and MLC (Fig. 6a–d) phosphorylation, during pharmacological inhibition of A1 receptor signalling. Similar to that observed in aortic VSMC, pre-treatment with adenosine and ANG II significantly increased both p38 MAPK (1.89 0.36) (Fig. 5c) and MLC (4.41 0.32) (Fig. 6c) phosphorylation, and this was not significantly reduced by simultaneous treatment with NBTI (3.46 0.17 for
p38 and 3.63 0.27 for MLC respectively). During pharmacological inhibition of A1 receptor signalling, combination with adenosine and ANG II was also associated with increased phosphorylation of both p38 (3.07 0.39) and MLC (2.36 0.45) (Figs 5D and 6D). In both cases, this effect on phosphorylation could be blocked by NBTI (1.65 0.03 for p38 and 0.64 0.08 for MLC), similar to that observed in aortic VSMC from A1 / mice (compare Fig. 4C).
Discussion The present study investigated the roles of receptordependent and independent effects of adenosine on ANG II responses in renal afferent arterioles. We demonstrate that ANG II-mediated arteriolar contraction is attenuated in A1 receptor knockout mice, but simultaneous administration of adenosine sensitizes the contractile response in both wild-type and knockout mice. This finding suggested involvement of a receptor independent mechanism. In A1 knockouts, the adeno-
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(a)
(b)
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Figure 6 Regulation of MLC phosphorylation in pre-glomerular vascular smooth muscle cells (VSMC) during pharmacological inhibition of A1 receptor signalling. Effects of adenosine (Ado; 10 8 mol L 1) and angiotensin II (ANG II; 10 7 mol L 1) with or without simultaneous incubation with the adenosine transporter inhibitor NBTI (3 9 10 7 mol L 1) on MLC (pSer19) phosphorylation in rat pre-glomerular VSMC in the absence (a & c) or presence (b & d) of an adenosine A1 receptor antagonist (CPX, 0.5 9 10 8 mol L 1). Phosphorylation levels were normalized to GAPDH. Panel A and B demonstrate 3–4 representative samples/group from the same gel for MLC phosphorylation. Relative changes in MLC phosphorylation are demonstrated in the absence (c) or presence (d) of an adenosine A1 receptor antagonist (CPX, 0.5 9 10 8 mol L 1). Panel c: Adenosine and angiotensin II (Ado + ANG II) increased the phosphorylation levels of MLC, and this was not significantly changed with NBTI. Panel D: During inhibition of A1 receptors, adenosine and angiotensin II (Ado + ANG II) increased the phosphorylation levels of MLC, and this was significantly reduced by simultaneous treatment with NBTI. Values are mean SE (n = 5–6/group). * denotes P < 0.05 between indicated groups.
sine transporter inhibitor NBTI inhibited the synergistic effect of adenosine on ANG II responses in the renal microvasculature. However, NBTI had no significant effect on arteriolar contraction in wild-type mice. These findings demonstrate the existence of receptor-dependent signalling in the wild type, which is compensated by a receptor independent mechanisms in the A1 knockout mice. The synergistic interaction between adenosine and ANG II-mediated arteriolar contractions is suggested to involve changes in intracellular calcium sensitivity via activation of PLC and/or phosphorylation of p38 MAPK and MLC (Hansen et al. 2003a, Lai et al. 2006a, 2009). Lai and colleagues demonstrated that pre-incubation with adenosine, followed by washout, prevented desensitization of ANG II responses by receptor independent mechanism and enhancement of calcium sensitivity (Lai et al. 2006a). Moreover, pretreatment with adenosine sensitized the contractile response to cumulative ANG II concentrations (Patzak et al. 2007). This synergistic effect was also receptor independent and specific for ANG II, as the arteriolar 274
contractile responses to noradrenalin and endothelin-1 were not influenced by adenosine. In our study, simultaneous administration of low-dose adenosine enhanced maximal contraction to ANG II by approx. 20% in both genotypes and was associated with increased phosphorylation of p38 MAPK and MLC in mouse aortic VSMC. This activation of p38 and MLC is probably associated with reorganization of the contractile apparatus and induction of VSMC constriction (Wynne et al. 2009). Inhibition of NBTI-sensitive adenosine transporters attenuated both arteriolar contraction and p38 MAPK phosphorylation in VSMC from the A1 knockouts, but had no effect in wild-type mice. This suggests that p38 MAPK phosphorylation in A1 / VSMC is dependent on adenosine signalling and regulated by mechanism(s) independent of the adenosine A1 receptor. Surprisingly, although phosphorylation of MLC is a key event in VSMC contractility, the same concentrations of adenosine in combination with ANG II did not significantly increase MLC phosphorylation in the A1 knockout cells. The explanation for this warrants further investi-
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gations, but aortic VSMC are less contractile and may not necessarily reflect the function of the VSMC in afferent arterioles. The rationale for using aortic VSMC is due to the technical issues and the absence of standardized protocol for isolating and culturing VSMC from mouse afferent arterioles. Thus, we next used rat pre-glomerular VSMC which are the true regulators of afferent arteriolar contractility (Hall 1982, Navar et al. 1998). To also distinguish between the roles of chronic and acute inhibition of A1 receptor regulation of p38 and MLC phosphorylation, we used a pharmacological approach. Similar to that observed in aortic VSMC from mice, adenosine + ANG II significantly increased p38 MAPK and MLC phosphorylation, and this activation was not affected by NBTI treatment. During inhibition of the A1 receptor, combination of adenosine and ANG II also induced phosphorylation of p38 MAPK and MLC, and this was markedly reduced during simultaneous inhibition of the adenosine transporters. This suggests that both receptor-dependent and independent mechanisms can contribute to the phosphorylation of p38 MAPK and MLC in VSMC. The intracellular mechanism warrants further investigations, but adenosine transporters would likely increase adenosine levels in VSMC, which may sensitize the contractile machinery similar to that observed during activation of A1 receptors. Adenosine transported into the cell may also stimulate PLC and protein kinase C signalling (Hansen et al. 2003a) or activate RhoA/Rho kinase and p38 MAPK (Lai et al. 2006a, Patzak et al. 2007). All these signalling events by adenosine can enhance calcium sensitivity and increase VSMC contraction (Lai et al. 2006a, 2009, Hansen et al. 2007). From a physiological perspective, adenosine A1 / mice are considered to be normotensive (Lee et al. 2012), although we have previously demonstrated a slightly higher blood pressure level in the knockouts (Brown et al. 2006, Gao et al. 2011). It is unlikely that this would have any major impact on our conclusion, but future studies will determine whether this relative difference in blood pressure could influence vascular reactivity. Finally, this study did not investigate hemodynamic changes in vivo; however, earlier studies demonstrated no major changes in renal blood flow or glomerular filtration rate in the A1 / , despite the absence of TGF (Brown et al. 2001, Sun et al. 2001, Hashimoto et al. 2006, Sallstrom et al. 2010). A possible explanation, and an interpretation of our results, is that the arteriolar contraction in vivo will be similar to that of wild-type mice due to an alternative pathway for activation of key downstream signalling proteins and sensitization of the ANG II response. In conclusion, synergistic interaction between adenosine and ANG II-mediated arteriolar contraction nor-
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mally involves A1 receptor signalling, but this can be compensated by receptor independent mechanism(s) leading to phosphorylation of p38 MAPK and MLC that sensitize the contractile machinery. Future in vivo studies are warranted to investigate the significance of this novel receptor independent pathway for adenosine in modulating renal hemodynamics and filtration properties both during health and disease.
Authors’ contributions M.C. and E.P. designed study and wrote the manuscript. X.G., M.P., C.Z. and M.C. researched and analysed the data. All authors contributed to data research, analysis, discussion and manuscript review. M.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Conflict of interest No potential conflict of interest relevant to this article was reported. This work was supported by grants from the Swedish Research Council (521-2011-2639 and K2009-64X-03522), the Swedish Heart and Lung Foundation (20110589), Jeanssons Foundation (JS2013-00064), the Swedish Society of Medical Research (SSMF), Bodossaki Foundation (Athens, Greece) and by KID-funding from the Karolinska Institutet.
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vascular resistance. Curr Opin Nephrol Hypertens 18, 63–67. Gao, X., Patzak, A., Sendeski, M., Scheffer, P.G., Teerlink, T., Sallstrom, J., Fredholm, B.B., Persson, A.E. & Carlstrom, M. 2011. Adenosine A(1)-receptor deficiency diminishes afferent arteriolar and blood pressure responses during nitric oxide inhibition and angiotensin II treatment. Am J Physiol Regul Integr Comp Physiol 301, R1669– R1681. Hall, J.E. 1982. Regulation of renal hemodynamics. Int Rev Physiol 26, 243–321. Hall, J.E., Granger, J.P. & Hester, R.L. 1985. Interactions between adenosine and angiotensin II in controlling glomerular filtration. Am J Physiol 248, F340–F346. Hansen, P.B., Castrop, H., Briggs, J. & Schnermann, J. 2003a. Adenosine induces vasoconstriction through Gidependent activation of phospholipase C in isolated perfused afferent arterioles of mice. J Am Soc Nephrol 14, 2457–2465. Hansen, P.B., Hashimoto, S., Briggs, J. & Schnermann, J. 2003b. Attenuated renovascular constrictor responses to angiotensin II in adenosine 1 receptor knockout mice. Am J Physiol Regul Integr Comp Physiol 285, R44– R49. Hansen, P.B., Friis, U.G., Uhrenholt, T.R., Briggs, J. & Schnermann, J. 2007. Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine. Acta Physiol (Oxf) 191, 89–97. Hashimoto, S., Huang, Y., Briggs, J. & Schnermann, J. 2006. Reduced autoregulatory effectiveness in adenosine 1 receptor-deficient mice. Am J Physiol Renal Physiol 290, F888– F891. Kobayashi, M., Inoue, K., Warabi, E., Minami, T. & Kodama, T. 2005. A simple method of isolating mouse aortic endothelial cells. J Atheroscler Thromb 12, 138–142. Lai, E.Y., Martinka, P., Fahling, M., Mrowka, R., Steege, A., Gericke, A., Sendeski, M., Persson, P.B., Persson, A.E. & Patzak, A. 2006a. Adenosine restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in renal arterioles. Circ Res 99, 1117–1124. Lai, E.Y., Patzak, A., Steege, A., Mrowka, R., Brown, R., Spielmann, N., Persson, P.B., Fredholm, B.B. & Persson, A.E. 2006b. Contribution of adenosine receptors in the control of arteriolar tone and adenosine-angiotensin II interaction. Kidney Int 70, 690–698. Lai, E.Y., Patzak, A., Persson, A.E. & Carlstrom, M. 2009. Angiotensin II enhances the afferent arteriolar response to
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Adenosine A1 receptor-dependent and independent pathways in modulating renal vascular responses to angiotensin II X. Gao,1,* M. Peleli,2,* C. Zollbrecht,2 A. Patzak,3 A. E. G. Persson1 and M. Carlstr€ om2 1 Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden 2 Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden 3 Institute of Vegetative Physiology, Charite-Universit€atsmedizin Berlin, Berlin, Germany
Received 19 June 2014, revision requested 9 August 2014, revision received 19 August 2014, accepted 17 September 2014 Correspondence: M. Carlstr€ om, Department of Physiology and Pharmacology, Nanna Svartz V€ag 2, S-17177 Stockholm, Sweden. E-mail: [email protected]
*These authors equally contributed to this work.
Abstract Aim: Renal afferent arterioles are the effector site for autoregulation of glomerular perfusion and filtration. There is synergistic interaction between angiotensin II (ANG II) and adenosine (Ado) in regulating arteriolar contraction; however, the mechanisms are not clear. In this context, this study investigated the contribution of A1 receptor-dependent and independent signalling mechanisms. Methods: Isolated perfused afferent arterioles from transgenic mice (A1+/+ and A1 / ) were used for vascular reactivity studies. Cultured vascular smooth muscle cells (VSMC) were used for phosphorylation studies of signalling proteins that induce arteriolar contraction. Results: Maximal arteriolar contraction to ANG II was attenuated in A1 / (22%) compared with A1+/+ (40%). Simultaneous incubation with low-dose ado (10 8 mol L 1) enhanced ANG II-induced contraction in A1+/+ (58%), but also in A1 / (42%). An ado transporter inhibitor (NBTI) abolished this synergistic effect in A1 / , but not in wild-type mice. Incubation with Ado + ANG II increased p38 phosphorylation in aortic VSMC from both genotypes, but treatment with NBTI only blocked phosphorylation in A1 / . Combination of ANG II + Ado also increased MLC phosphorylation in A1+/+ but not significantly in A1 / , and NBTI had no effects. In agreement, Ado + ANG II-induced phosphorylation of p38 and MLC in rat pre-glomerular VSMC was not affected by NBTI. However, during pharmacological inhibition of the A1 receptor simultaneous treatment with NBTI reduced phosphorylation of both p38 and MLC to control levels. Conclusion: Interaction between ANG II and Ado in VSMC normally involves A1 receptor signalling, but this can be compensated by receptor independent actions that phosphorylate p38 MAPK and MLC. Keywords afferent arteriole, kidney, microcirculation, myosin light chain, p38 MAP kinase, vascular smooth muscle cell.
The afferent arterioles are the effector site for renal autoregulation where myogenic response and tubuloglomerular feedback (TGF) importantly contribute to 268
the control of glomerular perfusion and filtration (Persson & Wright 1982, Casellas & Moore 1990, Vallon et al. 2006). Various vasoactive substances,
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including angiotensin II (ANG II) and adenosine, modulate the tone and reactivity of the renal resistance vessels (Franco et al. 2009, Persson et al. 2013). ANG II induces constriction of the renal microvasculature via ANG II receptor type 1. Adenosine has a biphasic response with adenosine A1 receptor-mediated contraction in the nanomolar range and A2 receptor-mediated dilatation in the high concentration range (Lai et al. 2006b, 2009, Patzak et al. 2007, Carlstrom et al. 2008). Previous studies have demonstrated a synergistic interaction between the contractile response by ANG II and that of adenosine (Hall et al. 1985, Weihprecht et al. 1994, Hansen et al. 2003b, Franco et al. 2009, Persson et al. 2013). In a physiological concentration, ANG II increased the contractile response to adenosine (Lai et al. 2006b). At the same time, adenosine in low concentrations significantly enhanced the afferent arteriolar response to ANG II (Hansen et al. 2003a, Lai et al. 2006b). Moreover, adenosine A1 gene-deleted mice (A1 / ) displayed attenuated arteriolar responses to ANG II (Hansen et al. 2003b) and had less blood pressure elevation during ANG II infusion (Gao et al. 2011, Lee et al. 2012). The interaction between ANG II and adenosine, in regulating arteriolar contraction, may contribute to the phenomenon of TGF resetting (Persson et al. 2013). The underlying mechanism(s) for this synergism are still under investigation, but increased intracellular Ca2+ sensitivity via modulation of phospholipase C (PLC), p38 mitogen-activated protein kinase (MAPK) and myosin light chain (MLC) phosphorylation have been suggested (Hansen et al. 2003a, Lai et al. 2006a, 2009). Although previous studies have emphasized that A1 receptors are important for the synergistic interaction between adenosine and ANG II (Hansen et al. 2003b, Lai et al. 2006b, 2009, Gao et al. 2011), receptor independent mechanisms may contribute (Lai et al. 2006a). This study aimed to investigate the contribution of adenosine A1 receptor-dependent and independent signalling pathways. It was hypothesized that A1 receptors importantly contribute to the synergism between ANG II and adenosine in regulating renal afferent arteriolar contraction. Moreover, synergistic interaction between adenosine and ANG was also investigated in isolated vascular smooth muscle cells (VSMC) from the mouse aorta and from rat pre-glomerular vessels.
Methods Afferent arteriole measurements Dissection and perfusion of renal cortical afferent arterioles were performed as described previously (Gao et al. 2011). DMEM/F12 with 10 mmol L 1 HEPES
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(Invitrogen AB, Liding€ o, Sweden) was used for dissection, bath and perfusion. The pH was adjusted to 7.4 after addition of bovine serum albumin (BSA: SERVA Electrophoresis Heidelberg, Germany). The concentration of BSA was 0.1% in dissection and bath solutions, and 1% in the perfusion solution. The experiments were digitally recorded and then digitized offline and analysed as described before (Gao et al. 2011). Changes in luminal diameters were measured to estimate the effect of vasoactive substances. In all series, the last 10 s of a control or treatment period were used for statistical analysis of steady state responses. Each experiment used a separate dissected afferent arteriole: only one arteriole was used per animal.
Cell culture of VSMC Aortic VSMC. Isolation and culturing of primary VSMC from A1+/+ and A1 / mice by a modified method of that originally described by Kobayashi and colleagues (Kobayashi et al. 2005). In isoflurane-anesthetized mice, the abdominal aorta was cut at the middle to release blood and then perfused with 1 mL of PBS containing 1000 U mL 1 of heparin (Hospira, Inc. Lake Forest, IL, USA). The aorta was dissected out from the aortic arch to the abdominal aorta and immersed in 20% foetal bovine serum (FBS, from ATCC, Manassas, VA, USA) Dulbecco’s modified eagles medium (DMEM) containing 1000 U mL 1 of heparin. The fat or connecting tissue was rapidly removed with fine forceps under a microscope. The inside of the lumen of aorta was briefly washed with serum-free DMEM. From the other side, it was washed with collagenase type II solution (2 mg mL 1, dissolved in serum-free DMEM) (Sigma–Aldrich Sweden AB, Stockholm, Sweden). After incubation for 45 min at 37 °C, endothelial cells were removed from the aorta by flushing with DMEM containing 10% FBS. The aorta was cut lengthwise, and put onto a 60 mm dish. With a scalpel blade the aorta was cut into almost square pieces and allowed to dry briefly. DMEM with 20% FBS and penicillin/streptomycin was added gently, and the cells were transferred to 48-well culture dishes and placed in an incubator and left undisturbed for a week. All the solutions at every isolation step had antibiotic/antimycotic mix added (Gibco, Carlsbad, CA, USA). After a week, the culture plates were examined under the microscope, to observe the presence of VSMC in the medium. The cells were rinsed two times with 20% FBS-containing DMEM and replaced with fresh medium with antimycotic/antibiotic added. The medium was replaced two times a week. Pre-glomerular VSMC (PG-VSMC). Isolation and culturing of primary PG-VSMC from rats were
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performed as previously described (Luo et al. 2009), and the phenotype was confirmed as described by Dubey and colleagues (Dubey et al. 1992). All experiments were carried out during early passage (between 3 and 9), when it is known that they express smooth muscle cell specific markers. Cells were seeded in 75-cm2 flasks and cultured in DMEM D0572 (Sigma–Aldrich) supplemented with 10% FBS, 2 mM L-Glutamine and 100 U mL 1 Penicillin–Streptomycin (Life Technologies, Grand Island, NY, USA). When used for the experiments, the cells were plated in 60mm dishes until they reached 80% confluence and then serum deprived for 24 h before the treatments.
Cellular protocol The cellular protocol was similar to renal afferent arterioles’. Briefly, the VSMC were divided in three groups: (a) untreated, (b) treated with adenosine (10 8 mol L 1; 15 min) and then ANG II (10 7 mol L 1, 15 min) and (c) pre-treated with NBTI (3 9 10 7 mol L 1; 5 min) and then treated with adenosine (10 8 mol L 1; 15 min) and ANG II (10 7 mol L 1; 15 min). In experiments using the selective adenosine A1 receptors antagonist CPX (5 9 10 8 mol L 1), the cells were pre-incubated with the antagonist for 1 h before the same treatment protocol described above was started. In that case, the control cells were pre-treated with CPX (5 9 10 8 mol L 1, 1 h) and then with NBTI (3 9 10 7 mol L 1; 5 min). After the different treatments, VSMC were placed on ice immediately, washed twice with cold DPBS and lysed in 100ll of lysis buffer supplemented with phosphatase and protease inhibitors (Sigma–Aldrich). Cell lysates were centrifuged (14 000 g for 15 min) and the supernatant was collected and placed in 80 °C until further analysis.
Western blot analysis of p38 MAPK and MLC phosphorylation Protein concentration in the supernatants was determined by Bradford protein assay (Bio-Rad Laboratories AB, Solna, Sweden). Equal protein amounts were separated by 4–20% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Bio-Rad), followed by transfer to a polyvinylidene difluoride membrane (BioRad). After blocking with 5% non-fat dry milk in Tween-containing Tris-buffered saline, membranes were incubated with specific primary antibodies (phospho-p38 MAP Kinase (Thr180/Tyr182) rabbit polyclonal antibody, Cell Signaling #9211, MLC-2B (pSer19) rabbit polyclonal antibody, Acris Antibodies #R1535P or mouse monoclonal antibody, Cell Signaling #3675S, GAPDH mouse monoclonal antibody, 270
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Santa Cruz #sc-47724) and secondary antibodies (horseradish peroxidase-conjugated goat antibodies to rabbit or mouse IgG; DAKO). Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to remove bound antibodies from the membranes, followed by blocking and reprobing the membranes with primary and secondary antibodies. Bands were detected by a SuperSignal West Femto chemiluminescence substrate (Thermo Fisher Scientific), and results were normalized with GAPDH. Images were analysed by a luminescent image analysis system LAS 1000+ (Fujifilm Corp., Tokyo, Japan). The results were quantified by densitometry (Image J, U. S. National Institutes of Health, Bethesda, MD, USA) and reported as relative optical density of the specific proteins.
Drugs and reagents Drugs and chemicals used for this study were obtained from Sigma–Aldrich (St. Louis, MO, USA), unless otherwise stated. In the arteriolar contraction experiments, all compounds were applied to the bath solution. In the cellular experiments, all compounds were applied to the cell culture medium.
Statistical analysis Values are presented as mean SE. Two-way repeated measures analysis of variance (2-way RM ANOVA), followed by Bonferroni’s multiple comparisons test was used to test time- or concentrationdependent changes in the arteriolar diameter and to assess differences between the groups. For multiple comparisons among groups (vascular and cellular studies), one-way ANOVA followed by Tukey’s post hoc test was used to allow for more than one comparison with the same variable. Statistical significance was defined as P < 0.05.
Ethics The experiments were approved by Ethical Committees for Animal Experiments and conducted in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals. The study is conform with Good Publishing Practice in Physiology (Persson & Henriksson 2011).
Results Regulation of afferent arteriolar responses Responses to ANG II. Concentration response curves were obtained by cumulative application of ANG II
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(10–12 to 10–6 mol L 1; each dose applied for 2 min). ANG II constricted afferent arterioles from A1+/+ mice in a concentration-dependent manner, with a maximum response of 40% (Fig. 1). In agreement with that previously described (Gao et al. 2011), afferent arterioles of A1 / mice were less responsive with a maximum contraction of 22% to ANG II (Fig. 1).
reduced arteriolar diameter by 9% in A1+/+ but had no significant effect in A1 / mice (P < 0.05). The subsequent contractile response to ANG II was significantly enhanced in A1+/+ with a maximal contraction of 58% (Fig. 2a). However, the arteriolar responses from A1 / mice were also much potentiated with a maximum contraction of 42% (Fig. 2b).
Responses to adenosine and ANG II. We investigated the synergistic interaction between the contractile response by adenosine and that of ANG II. Incubation with low dose of adenosine (10 8 mol L 1; 15 min)
Responses to adenosine and ANG II during inhibition of adenosine transporters. We investigated the potential role of nucleoside transporters as a receptor independent mechanism. Inhibition of adenosine transporters with NBTI did not significantly alter the contractile responses in A1+/+ (Fig. 2a). However, in A1 gene-deleted mice the sensitizing effect of adenosine on the contractile response to ANG II was abolished (27%), demonstrating an important role of adenosine transporters. Summarized maximal contractile responses in A1+/+ and A1 / mice are demonstrated in Figure 3.
Regulation of p38 MAPK and MLC phosphorylation in aortic VSMC from A1+/+ and A1 / mice To further assess potential differences in cellular signalling, we used aortic primary VSMC from A1+/+ and A1 / to investigate changes in p38 MAPK and MLC phosphorylation (Fig. 4a–d). Figure 1 Contractions to ANG II. Angiotensin II (ANG II)mediated contractions in isolated and perfused afferent arterioles of A1+/+ (n = 6) and A1 / (n = 6) mice. Values are mean SE. * denotes P < 0.05 compared with A1 wild-type mice.
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Adenosine transporter mediates p38 MAPK phosphorylation in A1 / but not in A1+/+. Adenosine signalling is known to induce the phosphorylation of p38 (b)
Figure 2 Contractions to adenosine + ANG II and the effect of adenosine transporter inhibition with NBTI. Effect of low-dose adenosine (10 8 mol L 1) on angiotensin II (ANG II)-mediated contractions in isolated and perfused afferent arterioles of A1+/+ (n = 6) and A1 / (n = 6) mice (a). Effect of low-dose adenosine (Ado; 10 8 mol L 1) on ANG II-mediated contractions, during simultaneous treatment with an adenosine transporter inhibitor (NBTI; 3 9 10 7 mol L 1), in isolated and perfused afferent arterioles of A1+/+ (n = 6) and A1 / (n = 6) mice (b). Values are mean SE. * denotes P < 0.05 compared with A1 knockout mice. © 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12399
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Figure 3 Maximal arteriolar contractions. Summarized maximal afferent arteriolar responses to adenosine (Ado; 10 8 mol L 1) and angiotensin II (ANG II) alone and in combination, and with simultaneous treatment with the adenosine transporter inhibitor (NBTI; 3 9 10 7 mol L 1) in A1+/+ (n = 6) and A1 / (n = 6) mice. Values are mean SE. * denotes P < 0.05 between indicated groups.
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MAPK, which then promotes a phosphorylation cascade leading to VSMC contraction. Pre-treatment of VSMC with adenosine (10 8 mol L 1) and ANG II (10 7 mol L 1) significantly increased p38 MAPK phosphorylation for both A1+/+ (1.92 0.14) and A1 / (1.37 0.07) (Fig. 4c). In VSMC from A1+/+, phosphorylation levels were not significantly changed by simultaneous treatment with the adenosine transporter NBTI compared with that of adenosine + ANG II (1.73 0.23 vs. 1.92 0.14). In contrast, NBTI abolished adenosine + ANG II-induced phosphorylation of p38 MAPK (0.53 0.03 vs. 1.37 0.07) in VSMC from A1 / . Adenosine and ANG II-induced phosphorylation of MLC is lost in A1 / . Phosphorylation of MLC is a key event in inducing the contractile apparatus of VSMC. Pre-treatment of A1+/+ VSMC with adenosine (10 8 mol L 1) and ANG II (10 7 mol L 1) signifi-
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Figure 4 Regulation of p38 MAPK and MLC phosphorylation in aortic vascular smooth muscle cells (VSMC) from A1+/+ and A1 / mice. Effects of adenosine (Ado; 10 8 mol L 1) and angiotensin II (ANG II; 10 7 mol L 1) with or without simultaneous incubation with the adenosine transporter inhibitor NBTI (3 9 10 7 mol L 1) on p38 MAPK (Thr180/Tyr182) and MLC (pSer19/20) phosphorylation in A1+/+ (a & c) and A1 / (b & d) aortic VSMC. Phosphorylation levels were normalized to GAPDH. Panel A and B demonstrate 3–4 representative samples/group from the same gel for p38 MAPK and MLC phosphorylation. Relative changes in p38 MAPK (c) and MLC (d) phosphorylation are demonstrated. Adenosine and angiotensin II (Ado + ANG II) significantly increased the phosphorylation levels of p38 MAPK in both A1+/+ and A1 / . The presence of NBTI reversed this induction in A1 / only but not in A1+/+. Adenosine and angiotensin II (Ado + ANG II) significantly increased the phosphorylation levels of MLC in A1+/+ and NBTI could not significantly reverse this activation. The phosphorylation levels of MLC remained unchanged in VSMC from A1 / . Values are mean SE (n = 5–6/group). * denotes P < 0.05 between indicated groups.
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Figure 5 Regulation of p38 MAPK phosphorylation in pre-glomerular vascular smooth muscle cells (VSMC) during pharmacological inhibition of A1 receptor signalling. Effects of adenosine (Ado; 10 8 mol L 1) and angiotensin II (ANG II; 10 7 mol L 1) with or without simultaneous incubation with the adenosine transporter inhibitor NBTI (3 9 10 7 mol L 1) on p38 MAPK (Thr180/Tyr182) phosphorylation in rat pre-glomerular VSMC in the absence (a & c) or presence (b & d) of an adenosine A1 receptor antagonist (CPX, 0.5 9 10 8 mol L 1). Phosphorylation levels were normalized to GAPDH. Panel A and B demonstrate 3 representative samples/group from the same gel for p38 MAPK phosphorylation. Relative changes in p38 MAPK phosphorylation are demonstrated in the absence (c) or presence (d) of an adenosine A1 receptor antagonist (CPX, 0.5 9 10 8 mol L 1). Panel c: Adenosine and angiotensin II (Ado + ANG II) increased the phosphorylation levels of p38 MAPK, and this was not significantly changed with NBTI. Panel d: During inhibition of A1 receptors adenosine and angiotensin II (Ado + ANG II) increased the phosphorylation levels of p38, and this was significantly reduced by simultaneous treatment with NBTI. Values are mean SE (n = 5–6/group). * denotes P < 0.05 between indicated groups.
cantly increased phosphorylation of MLC (2.48 0.17 vs. control), and this effect was not altered in the presence of NBTI (2.14 0.41 vs. control) (Fig. 4d). The same concentrations of adenosine and ANG II did not significantly increase MLC phosphorylation in the A1 / cells.
Regulation of p38 MAPK and MLC phosphorylation in pre-glomerular VSMC during pharmacological inhibition of A1 receptor signalling Next, we used primary pre-glomerular VSMC isolated from rat to assess effects of adenosine receptordependent and independent signalling in regulation of p38 MAPK (Fig. 5a–d) and MLC (Fig. 6a–d) phosphorylation, during pharmacological inhibition of A1 receptor signalling. Similar to that observed in aortic VSMC, pre-treatment with adenosine and ANG II significantly increased both p38 MAPK (1.89 0.36) (Fig. 5c) and MLC (4.41 0.32) (Fig. 6c) phosphorylation, and this was not significantly reduced by simultaneous treatment with NBTI (3.46 0.17 for
p38 and 3.63 0.27 for MLC respectively). During pharmacological inhibition of A1 receptor signalling, combination with adenosine and ANG II was also associated with increased phosphorylation of both p38 (3.07 0.39) and MLC (2.36 0.45) (Figs 5D and 6D). In both cases, this effect on phosphorylation could be blocked by NBTI (1.65 0.03 for p38 and 0.64 0.08 for MLC), similar to that observed in aortic VSMC from A1 / mice (compare Fig. 4C).
Discussion The present study investigated the roles of receptordependent and independent effects of adenosine on ANG II responses in renal afferent arterioles. We demonstrate that ANG II-mediated arteriolar contraction is attenuated in A1 receptor knockout mice, but simultaneous administration of adenosine sensitizes the contractile response in both wild-type and knockout mice. This finding suggested involvement of a receptor independent mechanism. In A1 knockouts, the adeno-
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(a)
(b)
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Figure 6 Regulation of MLC phosphorylation in pre-glomerular vascular smooth muscle cells (VSMC) during pharmacological inhibition of A1 receptor signalling. Effects of adenosine (Ado; 10 8 mol L 1) and angiotensin II (ANG II; 10 7 mol L 1) with or without simultaneous incubation with the adenosine transporter inhibitor NBTI (3 9 10 7 mol L 1) on MLC (pSer19) phosphorylation in rat pre-glomerular VSMC in the absence (a & c) or presence (b & d) of an adenosine A1 receptor antagonist (CPX, 0.5 9 10 8 mol L 1). Phosphorylation levels were normalized to GAPDH. Panel A and B demonstrate 3–4 representative samples/group from the same gel for MLC phosphorylation. Relative changes in MLC phosphorylation are demonstrated in the absence (c) or presence (d) of an adenosine A1 receptor antagonist (CPX, 0.5 9 10 8 mol L 1). Panel c: Adenosine and angiotensin II (Ado + ANG II) increased the phosphorylation levels of MLC, and this was not significantly changed with NBTI. Panel D: During inhibition of A1 receptors, adenosine and angiotensin II (Ado + ANG II) increased the phosphorylation levels of MLC, and this was significantly reduced by simultaneous treatment with NBTI. Values are mean SE (n = 5–6/group). * denotes P < 0.05 between indicated groups.
sine transporter inhibitor NBTI inhibited the synergistic effect of adenosine on ANG II responses in the renal microvasculature. However, NBTI had no significant effect on arteriolar contraction in wild-type mice. These findings demonstrate the existence of receptor-dependent signalling in the wild type, which is compensated by a receptor independent mechanisms in the A1 knockout mice. The synergistic interaction between adenosine and ANG II-mediated arteriolar contractions is suggested to involve changes in intracellular calcium sensitivity via activation of PLC and/or phosphorylation of p38 MAPK and MLC (Hansen et al. 2003a, Lai et al. 2006a, 2009). Lai and colleagues demonstrated that pre-incubation with adenosine, followed by washout, prevented desensitization of ANG II responses by receptor independent mechanism and enhancement of calcium sensitivity (Lai et al. 2006a). Moreover, pretreatment with adenosine sensitized the contractile response to cumulative ANG II concentrations (Patzak et al. 2007). This synergistic effect was also receptor independent and specific for ANG II, as the arteriolar 274
contractile responses to noradrenalin and endothelin-1 were not influenced by adenosine. In our study, simultaneous administration of low-dose adenosine enhanced maximal contraction to ANG II by approx. 20% in both genotypes and was associated with increased phosphorylation of p38 MAPK and MLC in mouse aortic VSMC. This activation of p38 and MLC is probably associated with reorganization of the contractile apparatus and induction of VSMC constriction (Wynne et al. 2009). Inhibition of NBTI-sensitive adenosine transporters attenuated both arteriolar contraction and p38 MAPK phosphorylation in VSMC from the A1 knockouts, but had no effect in wild-type mice. This suggests that p38 MAPK phosphorylation in A1 / VSMC is dependent on adenosine signalling and regulated by mechanism(s) independent of the adenosine A1 receptor. Surprisingly, although phosphorylation of MLC is a key event in VSMC contractility, the same concentrations of adenosine in combination with ANG II did not significantly increase MLC phosphorylation in the A1 knockout cells. The explanation for this warrants further investi-
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gations, but aortic VSMC are less contractile and may not necessarily reflect the function of the VSMC in afferent arterioles. The rationale for using aortic VSMC is due to the technical issues and the absence of standardized protocol for isolating and culturing VSMC from mouse afferent arterioles. Thus, we next used rat pre-glomerular VSMC which are the true regulators of afferent arteriolar contractility (Hall 1982, Navar et al. 1998). To also distinguish between the roles of chronic and acute inhibition of A1 receptor regulation of p38 and MLC phosphorylation, we used a pharmacological approach. Similar to that observed in aortic VSMC from mice, adenosine + ANG II significantly increased p38 MAPK and MLC phosphorylation, and this activation was not affected by NBTI treatment. During inhibition of the A1 receptor, combination of adenosine and ANG II also induced phosphorylation of p38 MAPK and MLC, and this was markedly reduced during simultaneous inhibition of the adenosine transporters. This suggests that both receptor-dependent and independent mechanisms can contribute to the phosphorylation of p38 MAPK and MLC in VSMC. The intracellular mechanism warrants further investigations, but adenosine transporters would likely increase adenosine levels in VSMC, which may sensitize the contractile machinery similar to that observed during activation of A1 receptors. Adenosine transported into the cell may also stimulate PLC and protein kinase C signalling (Hansen et al. 2003a) or activate RhoA/Rho kinase and p38 MAPK (Lai et al. 2006a, Patzak et al. 2007). All these signalling events by adenosine can enhance calcium sensitivity and increase VSMC contraction (Lai et al. 2006a, 2009, Hansen et al. 2007). From a physiological perspective, adenosine A1 / mice are considered to be normotensive (Lee et al. 2012), although we have previously demonstrated a slightly higher blood pressure level in the knockouts (Brown et al. 2006, Gao et al. 2011). It is unlikely that this would have any major impact on our conclusion, but future studies will determine whether this relative difference in blood pressure could influence vascular reactivity. Finally, this study did not investigate hemodynamic changes in vivo; however, earlier studies demonstrated no major changes in renal blood flow or glomerular filtration rate in the A1 / , despite the absence of TGF (Brown et al. 2001, Sun et al. 2001, Hashimoto et al. 2006, Sallstrom et al. 2010). A possible explanation, and an interpretation of our results, is that the arteriolar contraction in vivo will be similar to that of wild-type mice due to an alternative pathway for activation of key downstream signalling proteins and sensitization of the ANG II response. In conclusion, synergistic interaction between adenosine and ANG II-mediated arteriolar contraction nor-
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mally involves A1 receptor signalling, but this can be compensated by receptor independent mechanism(s) leading to phosphorylation of p38 MAPK and MLC that sensitize the contractile machinery. Future in vivo studies are warranted to investigate the significance of this novel receptor independent pathway for adenosine in modulating renal hemodynamics and filtration properties both during health and disease.
Authors’ contributions M.C. and E.P. designed study and wrote the manuscript. X.G., M.P., C.Z. and M.C. researched and analysed the data. All authors contributed to data research, analysis, discussion and manuscript review. M.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Conflict of interest No potential conflict of interest relevant to this article was reported. This work was supported by grants from the Swedish Research Council (521-2011-2639 and K2009-64X-03522), the Swedish Heart and Lung Foundation (20110589), Jeanssons Foundation (JS2013-00064), the Swedish Society of Medical Research (SSMF), Bodossaki Foundation (Athens, Greece) and by KID-funding from the Karolinska Institutet.
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