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Keywords:

  • purinoceptors;
  • uridine adenosine tetraphosphate;
  • P2 receptors;
  • rat isolated perfused kidney;
  • vasoconstriction;
  • vasodilation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

BACKGROUND AND PURPOSE Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine tetraphosphate (Up4A) as a novel and highly potent endothelial-derived contracting factor. Up4A induces strong vasoconstrictive effects in the renal vascular system mainly by P2X1 receptor activation. However, other purinoceptors are also involved and were analysed here.

EXPERIMENTAL APPROACH The rat isolated perfused kidney was used to characterize vasoactive actions of Up4A.

KEY RESULTS After desensitization of the P2X1 receptor by α,β-methylene ATP (α,β-meATP), Up4A showed dose-dependent P2Y2-mediated vasoconstriction. Continuous perfusion with Up4A evoked a biphasic vasoconstrictor effect: there was a strong and rapidly desensitizing vasoconstriction, inhibited by P2X1 receptor desensitization. In addition, there is a long-lasting P2Y2-mediated vasoconstriction. This vasoconstriction could be blocked by suramin, but not by PPADS or reactive blue 2. In preparations of the rat isolated perfused kidney model with an elevated vascular tone, bolus application of Up4A showed a dose-dependent vasoconstriction that was followed by a dose-dependent vasodilation. The vasoconstriction was in part sensitive to P2X1 receptor desensitization by α,β-meATP, and the remaining P2Y2-mediated vasoconstriction was only inhibited by suramin. The Up4A-induced vasodilation depended on activation of nitric oxide synthases, and was mediated by P2Y1 and P2Y2 receptor activation.

CONCLUSIONS AND IMPLICATIONS Up4A activated P2X1 and P2Y2 receptors to act as a vasoconstrictor, whereas endothelium-dependent vasodilation was induced by P2Y1/2 receptor activation. Up4A might be of relevance in the physiology and pathophysiology of vascular tone regulation.


Abbreviations
α,β-meATP

α,β-methylene ATP

AngII

angiotensin II

ApnA

diadenosine-n-phophate (n: number of phosphates)

ApnG

adenosine-guanosine-n-phosphate (n: number of phosphates)

ApoE

apolipoprotein E

CI

confidence interval

DMSO

dimethyl sulphoxide

eNOS

endothelial NOS

GpnG

diguanosine-n-phosphate (n: number of phosphates)

l-NAME

NG-nitro-l-arginine methyl ester

MAP

mean arterial blood pressure

MCP-1

monocyte chemoattractant protein-1

MRS2179

2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate

PPADS

pyridoxal-phosphate-6-azophenyl-2;4-disulphonic acid

RB2

reactive blue 2

Up4A

uridine adenosine tetraphosphate

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Over the past two decades, there has been an increase in evidence that the purinoceptor system is involved in vascular tone control (van der Giet et al., 2002a; Buvinic et al., 2006), and also potentially involved in the pathogenesis of hypertension (Jankowski et al., 2005; Tolle et al., 2008). In the mid-1990s, a new group of purinergic compounds, the so-called diadenosine polyphosphates, were identified as highly potent vasoactive substances (Schluter et al., 1994; Gabriels et al., 2002). In the following years, it became evident that these dinucleoside polyphosphates induce vasoconstriction in various vascular systems, mainly via P2X1 receptor activation. However, some of the vasoactive effects observed with diadenosine pentaphosphate (Ap5A) and the corresponding hexaphosphate (Ap6A) were also mediated via activation of G-protein-coupled P2Y receptors (Gabriels et al., 2002). In the following years, more dinucleoside polyphosphates containing either two adenosines, one adenosine and one guanosine, or two guanosines were identified. A complex family of purinergic dinucleoside polyphosphates with a variable phosphate chain of 2–7 phosphates (Jankowski et al., 2009) has now been described. Adenosine-containing dinucleoside polyphosphates act more as vasoconstrictive agents, and guanosine-containing dinucleoside polyphosphates act as cell-proliferating agents. One of the guanosine-containing dinucleoside polyphosphates is of special interest. Diguanosine pentaphosphate (Gp5G) is a potent activator of Rho-kinase and modulates the vasoactive responses of other known vasoactive substances such as angiotensin II (AngII) (Tolle et al., 2006). Gp5G activates P2Y2 receptors (receptor nomenclature follows Alexander et al., 2009) to induce calcium sensitization, and such sensitization is believed to be an important mechanism in the control of vascular tone and blood pressure.

Recently, our group identified the first pyrimidine-containing dinucleoside polyphosphate as a highly potent, new endothelial-derived vasoconstrictive factor. The substance was characterized as uridine adenosine tetraphosphate (Up4A) (Jankowski et al., 2005). Up4A shares properties of both P2X receptor and P2Y receptor agonists. In the first study, we demonstrated that Up4A acts as a vasoconstrictive agent by P2X1 receptor activation (Jankowski et al., 2005). The response was only partially inhibited by the P2X1 and P2X3 receptor desensitizer α,β-methylene ATP (α,β-meATP), indicating the activation of other purinoceptors. It was proposed that P2Y receptors are responsible for the remaining vasoactive properties of Up4A. These Up4A-activated P2Y receptors have not yet been characterized in depth.

There is some evidence that Up4A might have implications in the pathogenesis of human hypertension. Up4A plasma levels in young hypertensives are significantly increased compared to age-matched controls. The Up4A concentration is significantly correlated with the left ventricular mass and intima media wall thickness in these young hypertensives (Jankowski et al., 2007).

The purpose of the current work was to identify all purinoceptors other than P2X1 which are activated by Up4A. It is necessary to know more about the activation of the P2 receptors other than P2X1 receptors activated by Up4A. We sought to understand the complex vasoregulatory properties of Up4A as a novel endothelium-derived vascoconstrictive factor. In this study, we focused on the P2Y receptor-mediated physiological vasoactive actions of Up4A. To study the vasoactive effects of Up4A, we used the model of the rat isolated perfused kidney. We demonstrated that Up4A exerted vasoconstriction not only via P2X1 receptors, but also by activation of P2Y2 receptors. The Up4A-induced vasodilation was mediated via endothelial activation of P2Y1 and P2Y2 receptors.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Animal experiments

All animal care and experimental procedures were approved by the State Ethics committee Landesamt für Gesundheit, Ernährung und Technische Sicherheit Berlin.

Preparation of the rat isolated perfused kidney

Adult male Wistar–Kyoto rats (4–6 months old) were anaesthetized with ketamine (50 mg·kg–1, intraperitoneally)/xylazine (10 mg·kg–1, intraperitoneally). The abdominal cavity was opened by a midventral incision. The aorta and the left kidney were carefully isolated from adhesive tissue by blunt dissection. Ligatures were placed around the left renal artery and the infrarenal aorta. A polyethylene catheter (20-gauge) was placed in the distal aorta. Immediately after the insertion of the catheter, 500 U of heparin sodium was injected. Perfusion was then started. The catheter was gently advanced into the left renal artery without interruption of flow. The kidney was excised and immediately mounted in the perfusion system (Hugo Sachs Electronic, Freiburg, Germany).

Perfusion system

The perfusion procedure generally followed the description given by Hofbauer et al. (1973). The kidney was perfused at a constant flow rate using a peristaltic pump equilibrated to a perfusion pressure of about 70 mmHg Tyrode's solution of the following composition (in mM): NaCl, 137; KCl, 2.7; CaCl2, 1.8; MgCl2, 1.1; NaHCO3, 12; NaH2PO2, 0.42; and glucose, 5.6 gassed with 95% O2–5% CO2 and maintained at 37°C was used as perfusate. The pH was measured continuously by a pH sensor included in the perfusion system and was held between 7.35 and 7.45. Responses were measured as changes in perfusion pressure (mmHg) with a pressure transducer (Statham Transducer P23Gb, Siemens, Erlangen, Germany) on a side arm of the perfusion catheter, connected to a bridge amplifier (Hugo Sachs), and recorded digitally. Preparations were allowed to equilibrate for 30 min prior to experimentation.

Basal tone preparations

Vasoconstrictor responses of preparations to doses of Up4A, α,β-me-ATP, UTP, UDP or AngII were assessed at basal tone. For each substance, dose–response curves were constructed, with a minimum of 20 min being allowed to elapse between consecutive doses to avoid desensitization. This procedure allowed dose–response curves for several agonists to be constructed for the same preparation. A significant degree of cross-desensitization or auto-desensitization was not detected. The procedure has been described previously (van der Giet et al., 1999). The non-selective P2 receptor antagonists suramin (100 µM), pyridoxal phosphate 6-azophenyl-2;4-disulphonic acid (PPADS; 10 µM), reactive blue 2 (RB2, 100 µM), selective P2Y1 receptor antagonist MRS2179 (10 µM) or the P2X1/3 receptor desensitizing agent α,β-meATP (10 µM) were added to the perfusate 30 min before challenge with Up4A. In some experiments, NOS was inhibited by continuous perfusion with NG-nitro-l-arginine methyl ester (l-NAME) (100 µM). In some experiments, we performed endothelial cell removal prior to the experiments. Endothelium removal was performed with Triton X-100. The endothelium was removed by perfusion of the isolated kidney for 5 s with 0.1% Triton X-100. The lack of response to acetylcholine was used to check endothelium removal. Unaffected contraction to K+ (130 mM bolus) indicated an intact vascular smooth muscle cell function, which was tested before and after endothelium removal.

Continuous perfusion with Up4A

Vasoconstrictor responses to continuous perfusion with Up4A were assessed at basal tone. Dose–response curves were constructed for each substance, with 20 min being allowed to elapse between consecutive continuous perfusions. A significant cross-desensitization or auto-desensitization was not detected when substances were being given in intervals of at least 20 min. Desensitization was tested prior to experiments with all substances used (data not shown), and the results were compatible with previous observations with purinergic substances (van der Giet et al., 1999).

Continuous perfusion with P2X receptor antagonists

The non-selective P2 receptor antagonist suramin (100 µM) and the P2X receptor antagonist PPADS (10 µM) were added to the perfusate 30 min before challenge with Up4A. In an additional experiment, the P2X receptor agonist α,β-meATP (10 µM) was also perfused before challenge with Up4A.

Raised tone preparations

Vasodilator responses to doses of Up4A and ACh were assessed in raised-tone preparations. Perfusion pressure was increased by continuous perfusion with AngII (200 nM). The resistance of vasodilator responses to desensitization and the reproducibility of responses with time-allowed dose–response curves for several agonists to be constructed for the same preparation. The P2 receptor antagonist suramin (100 µM), PPADS (10 µM, 100 µM), RB2 (100 µM), MRS 2179 (10 µM) and the antagonist of NOS l-NAME (100 µM) were added to the perfusate 30 min before challenge with Up4A.

Assessment of oedema during perfusion experiments

To assess the development of oedema during the perfusion experiments, rat kidneys were weighed before and after the experiments. After perfusion, the weight was 132 ± 16% of the initial weight, indicating that a slight oedema of the kidneys developed. The response to 10 nM AngII at the end of the experiments was 106 ± 9% of the initial response.

Data analysis

Responses were measured as changes in perfusion pressure (mmHg), and results presented as the means ± SEM and if necessary their 95% confidence interval (95% CI). Statistical analysis was performed using Friedman's test. To compare columns for statistical variance, we applied Dunn's correction where applicable. P < 0.05 were considered significant.

Materials

All vasoactive substances were applied as 100 µL bolus into a valve proximal to the perfused kidney preparation. Drug dilutions were performed daily from stock solutions of 10 mM (concentrates stored frozen) in HPLC grade water or HPLC grade dimethyl sulphoxide (DMSO) unless otherwise indicated. Heparin (sodium salt), suramin (hexasodium salt), α,β-meATP and ketamine/xylazine were purchased from Sigma Aldrich (Schnelldorf, Germany). Up4A was purchased from Jena Bioscience (Jena, Germany). Prior to use, Up4A was purified according to a procedure described by Heidenreich et al. (1995).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Vasoconstrictor responses in basal tone preparations

The baseline perfusion pressure of the rat isolated perfused kidneys decreased by 3.0 ± 0.5 mmHg during the first, and by 2.0 ± 1.5 mmHg during the second hour of perfusion. Vascular reactivity to vasoactive agents did not diminish during this time. After the equilibration period, the baseline pressure was 68 ± 2 mmHg (n= 73). The addition of suramin (100 µM) to the perfusate caused an increase of perfusion pressure of 7 ± 5 mmHg. After addition of l-NAME (100 µM), non-significant increases of perfusion pressure of 10 ± 4 mmHg and for RB2 (100 µM) of 6 ± 2 mmHg were observed. The addition of MRS2179 (10 µM), PPADS (10 µM) to the perfusate did not induce any change in baseline perfusion pressure (data not shown).

At basal tone, Up4A caused a dose-dependent vasoconstriction (EC50[log mol]=–8.3 ± 0.1 and maximal change in perfusion pressure (Vmax) of 107 ± 8 mmHg, n= 7, Figure 1A,B). In the presence of the P2X1 receptor desensitizer α,β-meATP (10 µM) in the perfusate, responses to bolus application of Up4A were significantly decreased, but not completely abolished (EC50[log mol]=–8.6 ± 0.2, Vmax= 32 ± 3 mmHg, n= 7, Figure 1A,B). This remaining vasoconstriction could be almost totally blocked by the non-selective P2 receptor antagonist suramin (100 µM), whereas the selective P2Y1 receptor antagonist MRS2179 (10 µM), the non-selective P2 receptor antagonist PPADS (10 µM) or RB 2 (100 µM) had no effect (Figure 1C). UTP induced a dose-dependent vasoconstriction [EC50[log mol]= 7.9 ± 0.2 and maximum change in perfusion pressure (Vmax) of 39.0 ± 1.9 mmHg, n= 7, Figure 1D]. UDP only showed a very small change of perfusion pressure at high dosages. In the presence of suramin, there was a profound and significant (P < 0.05) inhibition of UTP-induced vasoconstriction. PPADS also showed a significant (P < 0.05) inhibitory effect. MRS2179 showed no significant effect. (Figure 1D).

image

Figure 1. (A) Original tracing of a representative experiment of the rat isolated perfused kidney showing the dose-dependent increase of the perfusion pressure (MAP) induced by Up4A in the absence (1–4) and the presence of α,β-meATP (1 µM; 5–8) (1:100 pmol; 2:1 nmol; 3:10 nmol; 4:100 nmol; 5:100 pmol; 6:1 nmol; 7:10 nmol; 8:100 nmol). (B) Dose–response curve of changes in perfusion pressure in the rat isolated perfused kidney induced by Up4A in the absence and presence of α,β-meATP. Each point is the mean of seven determinations, and vertical lines show SEM. For abbreviations, see text. Where error bars do not appear in figures, errors are within the symbol size. *<0.05 significant difference from baseline perfusion pressure. (C) Dose–response curve of changes in perfusion pressure in the rat isolated perfused kidney induced by Up4A in the presence of α,β-meATP (10 µM) and in the presence of the P2Y receptor antagonists suramin (50 µM), PPADS (10 µM), the specific P2Y1 receptor antagonist MRS2179 (10 µM), and RB2 (100 µM). Each point is the mean of seven determinations and vertical lines show SEM. Where error bars do not appear in figures, errors are within the symbol size. *<0.05 significant difference from baseline perfusion pressure. (D) Dose–response curve of changes in perfusion pressure in the rat isolated perfused kidney induced by UDP and UTP in the presence of the P2 receptor antagonists suramin (100 µM), PPADS (10 µM) and the specific P2Y1 receptor antagonist MRS2179 (10 µM). Each point is the mean of seven determinations, and vertical lines show SEM. Where error bars do not appear in figures, errors are within the symbol size. *<0.05 significant difference from baseline perfusion pressure.

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Continuous perfusion with Up4A

Continuous perfusion with Up4A led to a concentration-dependent increase of the perfusion pressure (Figure 2A). The perfusion pressure increase was divided into two phases. The first phase consists of a fast vasoconstrictive response with a rapid desensitization (EC50[log mol·L–1]=–6.6 ± 0.1, Vmax= 96.2 ± 9.2 mmHg, n= 8, Figure 2B). The second phase was characterized by a long-lasting, stable vasoconstriction (EC50[log mol·L–1]=–6.6 ± 0.1, Vmax= 42 ± 2 mmHg, n= 8, Figure 2C). Both phases showed a concentration-dependent vasoconstriction. The first, fast desensitization effect of Up4A could be completely inhibited by parallel continuous perfusion with α,β-meATP, suramin and PPADS. MRS2179 and RB2 had no significant (P < 0.05) inhibitory effect (Figure 2D).

image

Figure 2. (A) Original tracing of a representative experiment of the rat isolated perfused kidney showing the concentration-dependent increase of the perfusion pressure induced by the continuous perfusion with various concentrations of Up4A (1 = 1 nM; 2 = 10 nM; 3 = 50 nM; 4 = 100 nM, 5 = 500 nM; 6 = 1 µM; 7 = 5 µM; 8 = 10 µM). A biphasic vasoconstrictor response was observed; an initial transient effect and a subsequent sustained phase of vasoconstriction. (B) Concentration–response curve of the first short-acting part of the perfusion pressure change induced by continuous perfusion with Up4A. Each point is the mean of eight experiments, and vertical lines show SEM. Significant difference (*P < 0.05) from baseline perfusion pressure of the Up4A concentration (bolus application). Where error bars do not appear in figures, errors are within the symbol size. (C) Concentration–response curve of the sustained part of the perfusion pressure change induced by continuous perfusion with Up4A. Each point is the mean of eight experiments, and vertical lines show SEM. Significant difference (*P < 0.05) from baseline perfusion pressure of the Up4A concentration (bolus application). Where error bars do not appear in figures, errors are within the symbol size. (D) Influence of different P2 antagonists on the first short-acting part of the perfusion pressure increase induced by Up4A (5 µM). The P2X1 and P2X3 desensitizer α,β-meATP (10 µM), and the non-selective P2 receptor antagonist suramin (100 µM) and PPADS (10 µM) significantly inhibited (*P < 0.05) the Up4A-induced perfusion pressure increase, whereas the selective P2Y1 receptor antagonist MRS2179 (10 µM) and the non-selective P2 receptor antagonist RB2 (100 µM) had no significant influence. (E) Influence of different P2 antagonists on the second sustained part of the perfusion pressure increase induced by Up4A (5 µM). In the presence of the P2X1 and P2X3 desensitizer α,β-meATP (10 µM), there was no significant effect on the Up4A-induced sustained perfusion pressure. In the presence of the non-selective P2 receptor antagonist suramin (100 µM), the sustained perfusion pressure increase was significantly decreased (*P < 0.05), whereas the selective inhibition of the P2Y1 receptor by MRS2179 (10 µM) and the non-selective P2 antagonists PPADS (10 µM) and RB2 (100 µM) had no significant effect.

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The second, long-lasting vasoconstrictive effect of Up4A was significantly (P < 0.05) blocked by suramin, whereas PPADS, MRS2179 and RB2 showed no significant inhibition (Figure 2E).

Vasoactive responses in raised tone preparations

The basal pressure of the rat isolated perfused kidney was raised by continuous perfusion with AngII (200 nM) by 76.2 ± 4.2 mmHg. Under conditions of raised perfusion pressure, Up4A induced a dose-dependent increase of the perfusion pressure (EC50[log M]=–8.7 ± 0.1, Vmax= 87.5 ± 3.7 mmHg, n= 7, Figure 3A,B). This vasoconstriction was followed by a dose-dependent decline of perfusion pressure (EC50[log M]=–8.0 ± 0.1, Vmax=–65.7 ± 9.3 mmHg, n= 7, Figure 3A,C).

image

Figure 3. (A) Original tracing of a representative experiment performed on the rat isolated perfused kidney showing the effect of bolus application of Up4A under raised tone with AngII (200 nM). The bolus application of Up4A induced a dose-dependent, rapid increase of the perfusion pressure with a subsequent dose-dependent vasodilatation. 1: Up4A 100 pmol; 2: Up4A 500 pmol; 3: Up4A 1 nmol; 4: Up4A 5 nmol; 5: Up4A 10 nmol; 6: Up4A 50 nmol; 7: Up4A 100 nmol; 8: ACh 1 nmol; 9: wash-out. (B) Dose–response curve of the first vasoconstrictor effect of Up4A under raised basal tone. Each point is the mean of seven determinations, and vertical lines show SEM. Significant difference (*P < 0.05) from increased perfusion pressure. For abbreviations, see text. Where error bars do not appear in figures, errors are within the symbol size. (C) Dose–response curve of the second vasodilator effect of Up4A under raised basal tone. Each point is the mean of six determinations, and vertical lines show SEM. Significant difference (*P < 0.05) from baseline perfusion. For abbreviations, see text. Where error bars do not appear in figures, errors are within the symbol size.

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As was the case under basal conditions, the initial increase of the perfusion pressure was significantly attenuated by α,β-meATP, suramin and PPADS, whereas MRS2179 and RB2 had no significant effect (Figure 4A). Under conditions of continuous perfusion with AngII (200 nM) and α,β-meATP (10 µM), a residual perfusion pressure increase by Up4A could be detected. This remaining vasoconstriction was significantly blocked by suramin. PPADS, RB2 and MRS2179 had no significant effect on this remaining perfusion pressure increase (Figure 4B).

image

Figure 4. (A) Influence of different P2 antagonists on the first short-acting part of the perfusion pressure increase induced by Up4A (5 µM). The P2X1 and P2X3 receptor desensitizer α,β-meATP (10 µM), and the non-selective P2 receptor antagonist suramin (100 µM) and PPADS (10 µM) significantly inhibited (*P < 0.05) the Up4A-induced perfusion pressure increase, whereas the selective P2Y1 receptor antagonist MRS2179 (10 µM) and the non-selective P2 receptor antagonist RB2 (100 µM) had no significant influence. (B) Influence of different P2 antagonists on the second sustained part of the perfusion pressure increase induced by Up4A (5 µM). In the presence of the P2X1 and P2X3 receptor desensitizer α,β-meATP (10 µM), the Up4A-induced sustained perfusion pressure increase was significantly enhanced. In the presence of the non-selective P2 receptor antagonist suramin (100 µM), the sustained perfusion pressure increase was significantly decreased (*P < 0.05), whereas the selective inhibition of the P2Y1 receptor by MRS2179 (10 µM) and the non-selective P2 antagonists PPADS (10 µM) and RB2 (100 µM) had no significant effect. (C) In the presence of an intact endothelium, the eNOS antagonist l-NAME (300 µmol·L–1) significantly reduced (*P < 0.05) the Up4A- (10 nmol) induced decrease of the perfusion pressure. After removal of the endothelium, Up4A (10 nmol) could not induce any significant decrease of perfusion pressure. (D) The Up4A-induced perfusion pressure decrease could be significantly inhibited (*P < 0.05) by the non-selective P2 receptor antagonist suramin (100 µM), the selective P2Y1 receptor antagonist MRS2179 (10 µM), the non-selective P2 receptor antagonist PPADS (10 µM) and RB2 (10 µM).

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The Up4A (10 pmol)-induced decrease of the perfusion pressure could be significantly diminished by continuous perfusion with the non-selective NOS antagonist l-NAME and after chemical removal of the endothelium with Triton X-100 (Figure 4C). The vasodilatation could also be significantly attenuated by the non-selective P2 receptor antagonists suramin, PPADS and RB2, and by the selective P2Y1 receptor antagonist MRS2179 (Figure 4D).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Our results clearly demonstrated that Up4A activated at least three different purinoceptor subtypes in the kidney to induce a complex vasoactive response. Besides the P2X1 receptor, which induces vasoconstriction (Jankowski et al., 2005), we infer from our data that there was also a P2Y2 receptor activation. The P2Y2 receptor is responsible for additional, long-lasting vasoconstriction. Activation of the P2Y1/P2Y2 receptors results in an endothelium-dependent, NOS-mediated vasodilation.

Studying the purinoceptor subtypes involved in the vasoconstriction and vasodilation observed in the current experiments is a complex undertaking. There are no specific pharmacological active agonists and antagonists available for purinoceptors. The purinoreceptor expression profile in kidney tissue has been extensively studied, but there are many open questions. Turner and coworkers demonstrated the expression of P2X1, P2X2 and P2Y1 receptors in rat renal vascular smooth muscle cells (Turner et al., 2003). Using a pharmacological approach, Inscho and colleagues identified a receptor-mediating vasoactive response in renal tissue that was activated by UTP (Inscho et al., 1998). As UTP can activate P2Y2/4 receptors, it is tempting to speculate that these receptors can activate response in renal tissue. Potentially, these receptor subtypes are expressed at a very low level and therefore they cannot be detected by immunohistochemistry (Turner et al., 2003). In the present work, we demonstrated that UTP showed potent vasoactive actions, which were inhibited by suramin, but not PPADS, indicating that the P2Y2 receptor might be a receptor with vasoactive actions in the kidney. UDP, which is an agonist at P2Y6 receptors, induced only a very mild vasoconstriction at high doses. In 2005, we identified Up4A as a potent, endothelium-derived vasoactive substance, and we identified the P2X1 receptor as the main receptor mediating the observed vasoconstrictor effects. There were hints that further P2 receptors are involved in the Up4A-induced vasoconstriction (Jankowski et al., 2005). Here, we could show that the Up4A-induced vasoconstriction depends not only on the activation of the P2X1 receptor (Jankowski et al., 2005), but also on the activation of the P2Y2 receptor. Under basal conditions, the bolus application of Up4A induced a dose-dependent vasoconstriction in the rat isolated perfused kidney. The Up4A-induced vasoconstriction was blocked by the P2X1 receptor desensitizer α,β-meATP, the non-selective P2 receptor antagonist suramin and PPADS. After α,β-meATP-induced desensitization of the P2X1 receptor, the bolus application of Up4A induced a dose-dependent vasoconstriction. This vasoconstriction was completely blocked by the non-selective P2 receptor antagonist suramin. Suramin mainly inhibits activation of P2Y1 and P2Y2 receptors, and shows only weak low affinity at the P2Y6 receptor (von Kugelgen, 2006). There was no significant inhibition of Up4A-induced vasoconstriction by the non-selective P2 receptor antagonist RB2. RB2 inhibits activation of the P2Y1, P2Y4 and P2Y6 receptors. There is also a very low inhibitory affinity at the P2Y2 receptor (von Kugelgen, 2006). PPADS is a potent antagonist of the P2Y1 and P2Y4 receptors (von Kugelgen, 2006), whereas MRS2179 is the only tested selective antagonist at the P2Y1 receptor (von Kugelgen, 2006). PPADS and MRS2179 showed no significant antagonistic effects on Up4A-induced vasoconstriction. Taken together, these observations suggest that the P2Y2 receptor is responsible for the residual vasoconstriction induced by Up4A. The observations are in line with recent findings showing that the P2Y2 receptor mediates the UTP-induced vasoconstriction in porcine isolated arteries. The P2Y4 and P2Y6 receptors were not involved (Rayment et al., 2007). In control experiments, we can show that the UTP-induced vasoconstriction in the rat isolated perfused kidney was inhibited by suramin and not by PPADS.

Continuous perfusion of the rat isolated perfused kidney with Up4A induced a fast, concentration-dependent perfusion pressure increase with a subsequent desensitization. In addition, continuous perfusion with Up4A induced a sustained increase in perfusion pressure without signs of desensitization. The first part of the Up4A-induced vasoconstriction was attenuated by α,β-meATP, suramin and PPADS, whereas RB2 and MRS2179 again had no significant effect. Thus, this part of the vasoconstriction is due to an activation of the P2X1 receptor. The second part of the Up4A-induced vasoconstriction was significantly attenuated by suramin, whereas α,β-meATP, PPADS, RB2 and MRS2179 had no effect. The continuous activation of the P2Y2 receptor was responsible for this sustained Up4A-induced vasoconstriction. Recently, it was shown in young hypertensive patients that the Up4A concentration correlates with blood pressure, left ventricular mass and intima media thickness (Jankowski et al., 2007). It is possible that P2Y2 receptor activation is involved in the physiology and pathophysiology of blood pressure regulation. After inter-arterial application of Up4A in rats, there is a potent transient increase in the mean arterial blood pressure (MAP) with signs of desensitization (Jankowski et al., 2005). After the initial increase in blood pressure, there is a sustained increase in MAP. This is further evidence that Up4A is a compound that regulates and increases blood pressure (Jankowski et al., 2005).

In the experiments with AngII-induced raised perfusion pressure in the rat isolated perfused kidney, we observed a biphasic, vasoactive response to bolus application of Up4A. The first response was a dose-dependent increase in vascular tone that was followed by a sustained decrease of perfusion pressure. As was the case under basal conditions, the perfusion pressure increase could be significantly attenuated by suramin, α,β-meATP and PPADS, whereas MRS2179 and RB2 showed no effect. In the presence of α,β-meATP, a perfusion pressure increase could be seen, which was blocked by suramin, whereas PPADS, RB2 and MRS2179 had no effect. Therefore, this remaining perfusion pressure increase was due to the activation of the P2Y2 receptor. The Up4A-induced perfusion pressure decrease was not present after de-endothelialization or after inhibition of NOS by l-NAME. Up4A activates NOS by stimulating P2Y1 and P2Y2 receptors on endothelial cells. The Up4A-induced perfusion pressure decrease was partially inhibited by the P2Y1 receptor antagonist MRS2179. In addition, the non-selective P2Y receptor antagonist RB2, which is a potent antagonist of the P2Y1 and P2Y6 receptors, and a weak antagonist of the P2Y2 and P2Y4 receptors, and the non-selective P2Y receptor antagonist PPADS, a potent antagonist of the P2Y1 receptor, and a weak antagonist of the P2Y4 and P2Y6 receptors inhibited the Up4A-induced vasodilation. A nearly complete inhibition of the Up4A-induced reduction of perfusion pressure could be observed using suramin, a potent antagonist of the P2Y1 and P2Y2 receptors, and a weak antagonist of the P2Y6 receptor. Thus, Up4A appears to activate NOS via stimulation of the P2Y1 and P2Y2 receptors. Up4A-induced vasodilation is inhibited by the P2Y1 receptor antagonist MRS2179 and even more pronounced inhibition by suramin. Suramin is not an antagonist of the P2Y4, but a potent antagonist of the P2Y2 receptor. These findings are in accordance with recent findings showing the activation of eNOS by stimulation of the P2Y1, P2Y2 and possibly the P2Y4 receptors (da Silva et al., 2009). Currently, it is not possible to study single P2Y receptor subtypes due to the lack of selective antagonists. From our experiments, we cannot exclude the possibility that the P2Y4 receptor might play a role in NOS activation, but the expression of this receptor is low in the rat kidney.

We and others were able to demonstrate previously that diadenosine polyphosphates such as Ap4A activate various purinoceptor subtypes to induce vasoconstriction and vasodilation in isolated arterial vessels or in the rat isolated perfused kidney. Ap4A mainly induces vasoconstriction in the rat isolated perfused kidney and in the mesenteric bed via activation of P2X1 receptors (van der Giet et al., 1998; Gabriels et al., 2002). In addition, Ap4A could also induce an endothelium-dependent vasodilation in isolated mesenteric arteries which was mainly attributable to the activation of endothelial expressed P2Y receptors (Busse et al., 1988). However, those diadenosine polyphosphates with more than four phosphate groups mainly activate P2X receptors to induce vasoconstriction in most vascular models tested (Gabriels et al., 2002). Using the rat isolated perfused kidney model, no vasodilation was observed in response to Ap5A or Ap6A (van der Giet et al., 1997). However Gabriels and coworkers reported a possible vasodilation induced by Ap5A or Ap6A in the renal microcirculation (Gabriels et al., 2000), which might potentially be attributable to the degradation products of Ap5A and Ap6A. Ap4G, Ap5G and Ap6G are also potent vasoconstrictors activating P2X1/3 receptors in the rat isolated perfused kidney (Cinkilic et al., 2001; van der Giet et al., 2001). There was no Ap5G- or Ap6G-induced vasodilation observed in the rat isolated perfused kidney, whereas in coronary arteries we were able to show that Ap5A, Ap5G, Ap6A and Ap6G can activate P2Y1 receptors with consequent activation of eNOS (van der Giet et al., 2002b). The adenosine- and guanosine-containing dinucleoside polyphosphates mainly activate P2X1 receptors to induce vasoconstriction in vascular models, and P2Y receptor-mediated vasoactive effects are rarely reported for these substances (Gabriels et al., 2002). In the present work, we showed that the uridine-containing dinucleoside Up4A robustly activated P2Y1/2 receptors, as well as P2X1 receptors. This is of interest as there are some reports that activation of the P2Y2 receptor is involved in the regulation of blood pressure. It was shown that mice lacking P2Y2 receptor have salt-resistant hypertension and facilitated renal Na+ and water reabsorption (Rieg et al., 2007). In addition, there is paracrine regulation of the epithelial Na+ channel in the mammalian collecting duct by purinergic P2Y2 receptors (Pochynyuk et al., 2008). All these mechanisms are believed to be relevant in blood pressure regulation. In addition, there are reports that activation of P2Y2 receptor signalling might be potentially relevant in pro-inflammatory vascular disease conditions like atherosclerosis. Activation of P2Y2 receptors in peritoneal macrophages can lead to the production of the chemokine CCL2 (monocyte chemoattractant protein 1; MCP-1) (Stokes and Surprenant, 2007), and this is a key chemokine in the initiation and progression of atherosclerotic disease (Viedt et al., 2002). Activation of the P2Y1 receptor also contributes to the pathogenesis of atherosclerosis, as elegantly demonstrated with P2Y1/ApoE double knock-out mice (Hechler et al., 2008). Unfortunately, the precise mechanism is not fully understood to date.

In summary, we were able to demonstrate that Up4A induced renal vasoconstriction in perfused kidneys via activation of the P2X1 receptor, which shows fast desensitization, and via activation of the P2Y2 receptor, which induces sustained vasoconstriction. Up4A activated NOS via stimulation of the endothelial P2Y1 and P2Y2 receptors. Elevated serum concentrations of Up4A can potentially induce a significant elevation of the blood pressure. P2Y receptor signalling initiated by Up4A might be relevant in blood pressure control, in the pathogenesis of human hypertension and in the progression of vascular inflammatory disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

The study was supported by a grant from the Deutsche Nierenstiftung (M.T.), Deutsche Forschungsgemeinschaft (JA972/11-1, M.vdG. and J.J.), Sonnenfeld-Stiftung (M.vdG. and M.T.), BMBF (0313920D), Deutsche Hochdruckliga (M.T.), Dr Robert-Pfleger-Stiftung (M.T. and M.vdG.), Manchot-Stiftung (M.T.) and the Dr Werner Jackstädt-Stiftung (M.vdG. and M.T.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Supporting Information: Teaching Materials; Figs 1–4 as PowerPoint slide.

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BPH_914_sm_Figs1-4.pptx384KSupporting info item

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