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Cardiovascular Research 1999 43(1):200-209; doi:10.1016/S0008-6363(99)00062-0
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Enhanced acetylcholine and P2Y-receptor stimulated vascular EDHF-dilatation in congestive heart failure

Malin Malmsjöa, Anders Bergdahla, Xiao-He Zhaob, Xiang-Ying Sunb, Thomas Hednerb, Lars Edvinssona and David Erlingea,*

aDivision of Experimental Vascular Research, Department of Internal Medicine, Lund University Hospital, Lund, Sweden
bDepartment of Clinical Pharmacology, Gothenburg University, Gothenburg, Sweden

* Corresponding author. Tel.: +46-46-17-35-45; fax: +46-46-32-82-42 david.erlinge{at}med.lu.se

Received 5 October 1998; accepted 12 January 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Objective: Congestive heart failure (CHF) is accompanied by impaired peripheral blood flow and endothelial dysfunction with decreased release of nitric oxide (NO). Strong evidence supports the existence of another vasodilatory substance, endothelium derived hyperpolarising factor (EDHF), which has not previously been studied in CHF. Method: CHF was induced by left coronary artery ligation resulting in a reproducible myocardial infarction in Sprague Dawley rats. Vasodilatory responses to acetylcholine and extracellular nucleotides (ATP, ADPβS, ADP and UTP) were examined in cylindrical segments of the mesenteric artery, precontracted with noradrenaline. The combined NO- and EDHF-dilatation (after inhibition of cyclo-oxygenase pathways) was called "total dilatation", as indomethacin had only minor effects in this system. NO-dilatation was studied in segments pretreated with indomethacin and the potassium channel inhibitors charybdotoxin (10–7.5 M) and apamin (10–6 M), while EDHF-dilatations were studied in the presence of indomethacin (10–5 M) and L-NOARG (10–3.5 M). Results: EDHF-dilatations in CHF were strongly up-regulated for ACh (36% vs. 73%; sham vs. CHF operated rats), ADPβS (10% vs. 42%), ADP (0% vs. 21%) and UTP (3% vs. 35%). These dilatations were abolished by a combination of charybdotoxin and apamin, confirming that they were mediated by EDHF. The NO-dilatations on the other hand were down-regulated in CHF as compared to sham operated rats for ACh (93% vs. 76%; sham vs. CHF operated rats), ADPβS (61% vs. 37%). ADP (60% vs. 30%), ATP (49% vs. 34%) and UTP (65% vs. 47%), while a minor decrease was seen in the total dilatation for ACh (87% vs. 75%; sham vs. CHF operated rats), ADPβS (47% vs. 42%), ADP (59% vs. 39%), ATP (52% vs. 39%) and UTP (59% vs. 44%). Conclusion: In this model of non-atherosclerotic CHF there was a minor decrease in the total dilatation and a marked down-regulation of the NO-mediated dilatation, while the EDHF-dilatation was up-regulated. Increased EDHF-activity in CHF may represent a compensatory response to decreased NO-activity to preserve endothelial function and tissue perfusion.

KEYWORDS Adenosine; Endothelial function; Heart failure; Nitric oxide; Regional bloodflow


   1 Introduction
Top
 Abstract
 1 Introduction
2 Material and methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Congestive heart failure (CHF) is characterised by reduced cardiac function resulting in inadequate tissue perfusion. CHF also induces changes in several cardiovascular regulatory systems, such as vascular smooth muscle cell (VSMC) hypercontractility, endothelial cell dysfunction, activation of the renin-angiotensin and the sympathetic nervous system [1–3].

Endothelial cells induce vasodilatation when stimulated by acetylcholine (ACh) and extracellular nucleotides such as ATP, UTP and ADP. The dilatory mediators, released by ACh, have so far mainly been characterised as nitric oxide (NO), prostaglandins and endothelium-derived hyperpolarising factor (EDHF). We have recently shown that extracellular nucleotides not only stimulate release of NO and prostaglandins [4], but also EDHF by activation of endothelial P2Y-receptors [5,6]. Altered endothelial cell function during CHF may result from an increased activity from circulating vasoconstrictors such as catecholamines [7,8], endothelin [9,10], arginine vasopressin [11,12], cytokines such as TNF{alpha} [7,13,14], or an altered endothelial shear stress [15,16]. Investigation of endothelial function and stimulated release of NO in CHF has classically been performed by the use of ACh [17]. It has been shown that ACh-induced NO-release is decreased in CHF, and that endothelium-dependent dilatation of resistance vessels is blunted in patients with severe CHF [18–20]. In conductance vessels, flow-dependent dilatation is reduced in CHF, reflecting endothelial dysfunction. Previous reports suggests that decreased levels of NO may be associated with increased activity of EDHF, that in a compensatory manner, maintains endothelial vasodilator function during vascular diseases such as hypertension [21], diabetes [22] and hypercholesterolemia [23].

ATP is stored in and may be released from most cells, e.g. endothelial cells exposed to hypoxia or shear stress as well as trombocytes during aggregation. ATP is also a co-transmitter with noradrenaline (NA) in perivascular sympathetic nervous system [24], which is known to possess increased activity in CHF [25,26]. Recent studies have shown that plasma adenosine levels are increased in patients with ischemic and non-ischemic chronic CHF, and that adenosine-levels are correlated to the severity of the disease [27]. Since circulating (adenosine triphosphate) ATP is rapidly degraded to adenosine by ectonucleotidases, present preferentially on endothelial cells ([28]), the elevation of plasma adenosine may reflect an increased release of ATP in CHF. Thus, it is of considerable interest to study the cardiovascular effects of extracellular ATP in CHF, in order to elucidate possible adaptations to its responses. The present study was designed to examine endothelial function in CHF by investigating the potential alterations in ACh and P2Y-receptor stimulated EDHF- and NO-dilatation. To our knowledge there are no previous studies on EDHF-dilatation in CHF.


   2 Material and methods
Top
 Abstract
 1 Introduction
 2 Material and methods
3 Results
 4 Discussion
 5 Conclusions
 References
 
2.1 Experimental animals
Experiments were conducted on male Sprague-Dawley rats (ALAB, Sollentuna, Sweden) weighing approximately 500 g (2–3 months old). All animals were maintained on standard rat pellets and tap water ad libitum and housed in cages in groups of five animals, at +26°C, with 60% humidity and a 05.00 a.m. to 07.00 p.m. light/dark regimen. Animals were caged individually after surgical operation. The protocol was approved by the Ethics Committees for Animal Experiments at the University of Gothenburg and at the Lund University, both in Sweden.

2.2 Induction of myocardial infarction
During a shortlasting methohexital sodium anaesthesia, 60 mg/kg i.p., the rats were intubated and artificially ventilated with a respirator (Carlsson ventilator, No 8908, Mölndal, Sweden). A left thoracotomy was performed, exposing the left ventricular wall. The left coronary artery was ligated by positioning a suture between the pulmonary artery outflow tract and the left atrium. The lungs were thereafter hyperinflated using positive end-expiratory pressure, the thorax was immediately closed and the rats were allowed to recover for 4–8 weeks before the experiments were started. Rats which underwent the same surgical procedure, but without coronary artery ligation, served as controls (sham operated rats). Early mortality (1 day) after surgery was 10% and late mortality (1 month) 2%.

2.3 Evaluation of infarction size
To confirm the induced myocardial infarction in the operated animals a histological follow-up was performed. During the tissue preparation (see below) the hearts from the control and CHF rats were removed and immersed into a 6% formaline solution. The ventricular region of the heart was then cut from the apex to the base in four slices and from each slice 10 µm thin sections were prepared and stained for microscopical evaluation. Photographs were made at x10 magnification and the endocardial circumference of the left ventricle and the extent of the fibrotic area from each slice was measured. Infarct size was evaluated according to [29], i.e. the fibrotic fraction in per cent of the total cross-sectional endocardial circumference of the left ventricle. Rats with a myocardial infarction comprising more than 30% of the left ventricular wall were included in the study.

2.4 In vitro experiments
The rats were anaesthetised by inhalation of CO2 (carbon dioxide), and killed by a cardiac cut. The mesenteric artery was gently removed and immersed in a cold oxygenated bicarbonate based buffer solution (for composition, se below) where after the superior mesenteric artery before branching was dissected free of adhering tissue under a microscope. The vessels were cut into cylindrical segments (2–3 mm long) which were immediately used in the experiments. Each cylindrical segment was mounted on two L-shaped metal prongs, one of which was connected to a force displacement transducer (FT03C) for continuous recording of the isometric tension, and the other to a displacement device [30]. The position of the holder could be changed by means of a movable unit allowing fine adjustments of the vascular resting tension by varying the distance between the metal prongs. The mounted artery segments were immersed in temperature controlled (37°C) tissue baths containing bicarbonate based buffer solution of the following composition (mM): NaCl 119, NaHCO3 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5 and glucose 5.5. The solution was continuously gassed with 5% CO2 in O2 resulting in a pH of 7.4. Special care was taken to ensure that the endothelium was not damaged. The arterial segments were allowed to stabilise at a resting tension of 2 mN for 1 h, after which the contractile capacity of each vessel segment was examined by exposure to a potassium-rich (60 mM) buffer solution in which NaCl was exchanged for an equimolar concentration of KCl (for composition see above). When two reproducible contractions had been achieved the vessels were used for further studies.

2.5 Measurement of mechanical responses
Relaxation was studied in vascular segments precontracted by 10–6 M NA. Agonists were added cumulatively to determine concentration–response relationships. Twelve ring segments were studied at the same time in separate tissue baths. To avoid desensitisation of receptors, agonists were only added once to each segment. Thus, comparisons were made between the responses in different vessel segments. Antagonists were administered 30 min before application of agonists. All experiments were performed with indomethacin present to abolish prostacyclin-induced dilatory effects. Endothelium-denudation was performed on the dissected intact vessel before mounting by perfusion for 5 s with 0.1% Triton X followed by another 5 s of perfusion with a buffer solution using a fine needle. Endothelium denudation was checked by monitoring responses to ACh. Abolished dilatation indicated a properly removed endothelium. To eliminate the possibility of SMC damage by Triton X, vasodilatory responses to the endothelium-independent vasodilator SIN-1 was assessed (see results).

2.6 Antagonists of the different dilatory mediators
The EDHF-mediated dilatory response was studied after eliminating the prostacyclin- and NO-mediated dilatations by administration of indomethacin and L-NOARG, while the NO-dilatation was studied in artery segments pretreated with indomethacin, charybdotoxin and apamin to abolish all EDHF-effects. Dilatations recorded with only indomethacin present were called "total dilatation" as indomethacin only had minor effects in this system. To block all dilatory mediators, a combination of indomethacin, L-NOARG, charybdotoxin and apamin was used.

2.7 Drugs
Methohexital sodium (Brietal®, Lilly Inc., Indianapolis, IN, USA), adenosine-triphosphate (ATP, 10–9–10–3.5 M), adenosine-diphosphate (ADP, 10–9–10–3.5 M), adenosine 5’O-thiodiphosphate (ADPβS, 10–9–10–4 M), uridine-triphosphate (UTP, 10–9–10–3.5 M), acetylcholine (ACh, 10–9–10–5 M), indomethacin (10–5 M), noradrenaline (NA, 10–6–10–5 M), N{omega}-nitro-L-arginine (L-NOARG, 10–3.5 M), 3-morpholino-synonimine (SIN-1, 10–7–10–3 M), charybdotoxin (10–7.5 M) and apamin (10–6 M) (Sigma Co., USA). All drugs were dissolved in 0.9% saline.

2.8 Calculations and statistics
The amplitude of contraction before application of relaxatory agonists was set to 100%. The negative logarithm of the drug concentration eliciting 50% relaxation (EC50) was determined by linear regression analysis using the values immediately above and below half-maximum response. Rmax refers to maximum relaxation. Values are presented as mean±SEM. All experiments were performed on 5–18 segments (animals). Statistical significance was defined as P<0.05, ANOVA by repeated measures for the curves (see figures) and by Students t-test for the difference between Rmax values (see Tables).


   3 Results
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
4 Discussion
 5 Conclusions
 References
 
3.1 CHF status
3.1.1 Myocardial infarction size
Hearts, in which the left coronary artery had been ligated, demonstrated pathological hallmarks of myocardial infarction such as myocyte degeneration, fibrosis in the left ventricular region and cardiac dilatation. The infarction size of the ligated hearts were 38.1±3.5% (mean±SD) of the left ventricular circumference. No myocardial degeneration was seen in the sham operated rats.

3.1.2 Other signs of congestive heart failure
Congestion of the lungs and abdominal organs and of the portal and the lienal veins were seen in the CHF operated rats, supporting the establishment of congestive heart failure. These observations correspond with our findings of increased heart- (0.49±0.03% vs. 0.34±0.02%, CHF vs. sham operated rats) and lung-weights (0.69±0.05% vs. 0.41±0.04%) in CHF rats, when calculated as percentage of total body weight.

3.2 Dilatory responses
Dilatory responses were examined in the rat mesenteric artery. The contractile response to 60 mM K+ did not differ between sham (5.70±0.15 mN), and CHF operated rats (5.85±0.13 mN). Neither did the NA-induced precontraction differ (6.24±0.19 mN vs. 6.32±0.25 mN, CHF vs. sham operated rats).

3.2.1 Endothelium denudation and SIN-1 dilatations
Relaxation to each agonist was totally abolished by endothelium denudation in both CHF and sham operated rats. SIN-1 induced dilatations did not differ between control vessel segments (Rmax 103±5% and pEC50 5.12±0.03) and denuded vessel segments (Rmax 97±4% and pEC50 5.05±0.04), indicating intact function of the VSMC’s after denudation. The SIN-1-induced dilatations did neither differ between CHF (Rmax 96±3% and pEC50 5.01±0.04) and sham operated rats (Rmax 101±2% and pEC50 4.99±0.05), indicating that CHF does not affect the dilatory capacity to NO of the smooth muscle cells.

3.2.2 Indomethacin
Indomethacin did not affect the dilatory responses to any of the agonists significantly (reduction of dilatation <5%), indicating a very minor contribution of cyclo-oxygenase products.

3.2.3 Inhibition of NO, EDHF and cyclo-oxygenase pathways
When the artery segments were pretreated with a combination of indomethacin, L-NOARG, charybdotoxin and apamin, all dilatory responses were abolished in both CHF and sham operated rats, indicating that the L-NOARG and indomethacin resistant dilatation was indeed mediated by EDHF.

3.2.4 Total dilatation
Dilatations recorded with only indomethacin present were called "total dilatation" as indomethacin only had minor effects in this system. CHF induced a minor decrease in the total dilatation for ACh, ADPβS, ADP, ATP and UTP (Table 1, Figs. 1a, 2a, 3a, 4a and 5aGoGoGoGo).


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  		Table 1 Relaxation maximum (Rmax) and pEC50 values for the total dilatation for different agonists in sham operated and CHF rats. There were no statistical difference between Rmax (sham vs. CHF, Student’s t-test). N=number of different animals

 

Figure 1
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  		Fig. 1 ACh. Concentration dependent relaxations to ACh in segments of mesenteric artery of CHF and sham operated rats. Concentration–response curves were constructed in the presence of indomethacin (10–5 M) to examine the total dilatation of the vessel (a), indomethacin (10–5 M), charybdotoxin (10–7.5 M) and apamin (10–6 M) to study the NO-dilatation (b), indomethacin (10–5 M) and L-NOARG (10–3.5 M) to study the EDHF-dilatation (c). Dilatory responses are expressed as percentage of an initial contraction induced by NA. Data are shown as means±SEM of 9–18 experiments.

 

Figure 2
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  		Fig. 2 ADPβS. Concentration dependent relaxations to ADPβS in segments of mesenteric artery of CHF and sham operated rats. Concentration–response curves were constructed in the presence of indomethacin (10–5 M) to examine the total dilatation of the vessel (a), indomethacin (10–5 M), charybdotoxin (10–7.5 M) and apamin (10–6 M) to study the NO-dilatation (b), indomethacin (10–5 M) and L-NOARG (10–3.5 M) to study the EDHF-dilatation (c). Dilatory responses are expressed as percentage of an initial contraction induced by NA. Data are shown as means±SEM of 7–16 experiments.

 

Figure 3
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  		Fig. 3 ADP. Concentration dependent relaxations to ADP in segments of mesenteric artery of CHF and sham operated rats. Concentration–response curves were constructed in the presence of indomethacin (10–5 M) to examine the total dilatation of the vessel (a), indomethacin (10–5 M), charybdotoxin (10–7.5 M) and apamin (10–6 M) to study the NO-dilatation (b), indomethacin (10–5 M) and L-NOARG (10–3.5 M) to study the EDHF-dilatation (c). Dilatory responses are expressed as percentage of an initial contraction induced by NA. Data are shown as means±SEM of 5–9 experiments.

 

Figure 4
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  		Fig. 4 ATP. Concentration dependent relaxations to ATP in segments of mesenteric artery of CHF and sham operated rats. Concentration–response curves were constructed in the presence of indomethacin (10–5 M) to examine the total dilatation of the vessel (a), indomethacin (10–5 M), charybdotoxin (10–7.5 M) and apamin (10–6 M) to study the NO-dilatation, and indomethacin (10–5 M) and L-NOARG (10–3.5 M) to study the EDHF-dilatation (b). Dilatory responses are expressed as percentage of an initial contraction induced by NA. Data are shown as means±SEM of 5–8 experiments.

 

Figure 5
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  		Fig. 5 UTP. Concentration dependent relaxations to UTP in segments of mesenteric artery of CHF and sham operated rats. Concentration–response curves were constructed in the presence of indomethacin (10–5 M) to examine the total dilatation of the vessel (a), indomethacin (10–5 M), charybdotoxin (10–7.5 M) and apamin (10–6 M) to study the NO-dilatation (b), indomethacin (10–5 M) and L-NOARG (10–3.5 M) to study the EDHF-dilatation (c). Dilatory responses are expressed as percentage of an initial contraction induced by NA. Data are shown as means±SEM of 5–9 experiments.

 
3.2.5 NO-dilatation
The NO-dilatation was studied in artery segments pretreated with indomethacin, charybdotoxin and apamin. Concentration-dependent dilatations, that were significantly lower in CHF as compared to sham operated rats, could be observed for ACh, ADPβS, ADP, ATP and UTP (Table 2, Figs. 1c, 2c, 3c, 4b and 5cGoGoGoGo).


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  		Table 2 Relaxation maximum (Rmax) and pEC50 values for the NO dilatation for different agonists in sham operated and CHF rats. Statistical differences between Rmax (sham vs. CHF, Student’s t-test) are indicated (*, P<0.05). N=number of different animals

 
3.2.6 EDHF-dilatation
EDHF-dilatation was studied in artery segments pretreated with indomethacin and L-NOARG. ACh-, ADP-, ADPβS- and UTP-induced concentration-dependent dilatations that were markedly up-regulated in the CHF rats for ACH, ADPβS, ADP and UTP (Table 3, Figs. 1c, 2c, 3c, and 5cGoGoGo). ATP did not induce EDHF-dilatation either in CHF or in sham operated rats (Table 3 and Fig. 4b).


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  		Table 3 Relaxation maximum (Rmax) and pEC50 values for the EDHF dilatation for different agonists in sham operated and CHF rats. Statistical differences between Rmax (sham vs. CHF, Student’s t-test) are indicated (**, P<0.01; ***, P <0.001). N=number of different animals

 
3.2.7 Relative involvement of EDHF and NO in ach stimulated vasodilatation in sham and CHF operated rats
Based on the calculated area under the curves in Fig. 1a, b and c, the total dilatation for CHF as compared to sham was 78%. The relative shares of the dilatory mediators was in CHF, 57% for NO and 43% for EDHF, and in sham operated rats 77% for NO and 23% for EDHF (Fig. 6).


Figure 6
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  		Fig. 6 Schematic figure of the relative involvement of EDHF and NO in ACh stimulated vasodilatation in sham and CHF operated rats, based on the calculated area under the curves in Fig. 1a, b and c. The total dilatation for CHF as compared to sham amounts to 78%. The relative shares of the dilatory mediators is in CHF, 57% for NO and 43% for EDHF, and in sham operated rats 77% for NO and 23% for EDHF.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
5 Conclusions
 References
 
The most interesting finding was that this model of non-atherosclerotic CHF induced an up-regulation of the ACh and P2Y-receptor stimulated EDHF-dilatation, while the NO-dilatation was down-regulated.

4.1 The CHF model
A heart failure model was used where the left anterior descending coronary artery was ligated inducing an acute infarction, where after the rats were left to recover for 4–8 weeks. During this period a heart failure state was developed. A myocardial infarction comprising more than 30% of the left ventricular wall has in this heart failure model been shown to decrease cardiac output and increase left ventricular end-diastolic pressure [29]. Furthermore, the infarct size is positively correlated with left ventricular volume and negatively correlated with left ventricular ejection fraction [31]. Other studies of this heart failure model have revealed increased plasma levels of catecholamines and ANP, as well as pulmonary oedema, hepatic congestion and pleural effusion [26,32,33]. The present study shows increased heart- (0.34% of total body weight vs. 0.49%, sham operated vs. CHF) and lung-weights (0.41% vs. 0.69%) in CHF rats. Earlier studies of this heart failure model have shown that the impairment of the left ventricular function is directly related to the loss of myocardium [29], with the infarction-sizes being divided into small (5–19%), moderate (20–39%) and large (>40%) [34]. Loss of more then 30% of the myocardium of the left ventricle (referring to the method described above) results in a pump-dysfunction with reduced values of peak flow, systolic and mean arterial pressure. The damage of the left ventricular wall was in the present study histologically proven, and showed a myocardial infarction comprising 38±4% of the endocardial circumference. Also, we noted signs of organic congestion as described above. Our model can therefore be considered successful according to CHF criteria denoted by the literature. We used this model to examine the endothelial function in CHF by investigating potential alterations in ACh- and P2Y-receptor-stimulated EDHF- and NO-dilatation.

4.2 Total dilatation and NO-mediated dilatation
Investigation of endothelial function and stimulated release of NO in CHF have classically been performed by the use of ACh [17]. Endothelium-dependent dilatation of resistance arteries is blunted in patients with severe CHF [18]. In conductance vessels, flow-dependent dilatation is reduced. These findings reflect endothelial dysfunction in CHF. The ACh-stimulated release of NO is impaired while the bloodflow response to nitroglycerin or sodium nitroprusside, both endothelium-independent vasodilators, is preserved indicating a normal response of the VSMC’s to exogenous NO (Drexler and Hornig, 1996). In accordance to this, our experiments demonstrate a decreased NO-dilatation for ACh, and for the P2Y-receptor agonists ADPβS, ADP, ATP and UTP, indicating a CHF induced endothelial dysfunction (Table 2, Figs. 1b, 2b, 3b, 4b and 5bGoGoGoGoGo). The NO-donor SIN-1 induced relaxations that were unaffected by CHF, indicating preserved VSMC response to exogenous NO.

The total dilatation of the vessel, induced by ACh and P2Y-receptor stimulation, was slightly decreased, indicating general endothelial dysfunction (Table 1, Figs. 1a, 2a, 3a, 4a and 5aGoGoGoGoGo). The decrease in total dilatation was not as pronounced as for the NO-dilatation, indicating a mechanism that compensates for the decreased NO-effect.

4.3 EDHF-dilatation
The vasodilatory effects of ACh have so far mainly been ascribed to NO and prostaglandins. Since Taylor and Weston [35] demonstrated endothelium-dependent relaxation by a factor that could cause relaxation by increasing the membrane potential of the VSMC’s, it has been generally accepted that ACh also induce release of EDHF. Although the identity of EDHF remains unknown it has been proposed that EDHF represents epoxyeicosanoic acids [36]. According to the criteria denoted by the literature identification of EDHF requires evidence that it is released by the endothelium, not inhibited by antagonists of NO-synthetase or cyclo-oxygenase pathways and that it induces hyperpolarisation and relaxation of VSMC’s that can be blocked by the potassium channel inhibitors charybdotoxin and apamin [37–40]. In the mesenteric artery activation of P2Y-receptors induce endothelium-dependent vasodilatation mediated by a factor distinct from NO and prostacyclin that is blocked by charybdotoxin and apamin indicating that it is EDHF [5]. This was confirmed by electrophysiological experiments in which both ACh and P2Y-receptor agonists induce endothelium dependent hyperpolarisation of the VSMC’s [6]. The hyperpolarisations were antagonised by charybdotoxin and apamin, confirming the use of this combination to inhibit EDHF.

The present study shows endothelium-dependent dilatations, distinct from NO and prostacyclin, to ACh, ADPβS, ADP and UTP since they were not blocked by L-NOARG and indomethacin. A combination of charybdotoxin and apamin totally abolished these dilatations indicating that they were mediated by EDHF. The high concentration of L-NOARG (10–3.5 M) used in the present experiments has in previous studies been shown to abolish the release of NO [41]. Furthermore, addition of the NO-scavenger PTIO to L-NOARG (10–3.5) did not reduce the dilatation further, indicating that all the NO-production was blocked (data not shown). We have previously examined the specificity for charybdotoxin and apamin as EDHF-inhibitors on other vasodilators acting independently of the endothelium. They did not modify the relaxation to the NO-donor SIN-1 and thus we conclude that they do not affect the direct NO-mediated activation of the VSMC’s [5].

ACh, ADPβS-, ADP- and UTP-induced EDHF-dilatations were not as effective as previously shown in young healthy rats [5]. These differences might be age-related as the rats used in this previous study were approximately 300 g lighter. Previous reports have shown that the vasodilator response to ACh decreases with age, which is due to inactivation of K+ channels, while the NO-mediated dilatation is preserved [42]. The lack of ATP-mediated EDHF-dilatation in both CHF and sham operated rats depends on simultaneous ATP-activation of contractile P2X-receptors on the VSMC’s [6]. ATP in the circulation mediates vasodilatation through its degradation product ADP, acting on endothelial ADP-preferring P2Y1-receptors. UTP on the other hand mediates dilatation via pyrimidine-sensitive P2Y-receptors on the endothelium (P2Y2, P2Y4 or P2Y6).

Previous reports suggest that decreased levels of NO may be associated with increased activity of EDHF [21]. During physiological conditions, the production of EDHF is damped by NO. Impaired NO-synthesis, alleviates this intrinsic inhibition of EDHF and endothelial vasodilator function is maintained [43]. Up-regulated EDHF-activity may compensate for an increased vascular tonus elicited by decreased NO-synthesis in hypercholesterolemia [23] and hypertension [21], or by increased vasoconstriction in diabetes [22]. Our experiments showed EDHF-mediated dilatations that were several-fold stronger in the CHF rats for ACh, ADPβS, ADP and UTP (Table 3, Figs. 1c, 2c, 3c and 5cGoGoGoGo). We therefore speculate that a compensatory up-regulation of EDHF-mediated dilatation may represent a balancing adaptation to endothelial dysfunction and decreased NO-release and tissue perfusion in CHF (Fig. 6). The mechanism underlying the increased EDHF-dilatation is not known. It could depend on an enhanced synthesis of EDHF triggered by reduced levels of NO or by some of the many humoral factors that are increased in CHF. On the other hand the up-regulated EDHF-dilatation might depend on a greater responsiveness of the VSMC’s to EDHF.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 5 Conclusions
References
 
The present study demonstrates that CHF induces a slight decrease in the total dilatation of the blood vessel while the relationship between the dilatory mediators is altered. EDHF-dilatations stimulated by ACh and P2Y-receptor agonists are up-regulated, while the NO-dilatations are down-regulated (Fig. 6). The increased EDHF-dilatation may be due to a general adaptation process, which compensates for a decreased NO-activity and peripheral tissue perfusion in CHF.

Time for primary review 21 days.


   Acknowledgements
 
The study was supported by the Swedish Hypertension Society, the Royal Physiographic Society, Lund, the Thelma Zoegas Foundation, the Crafoord Foundation, the Jeanson foundation, the Tore Nilsson Foundation, the Svensson Foundation, the Medical Faculty, Lund and Gothenburg University and the Swedish Medical Research Council (grant no 5958 and 8642).


   References
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 

  1. Holton F.A, Holton P. The possibility that ATP is a transmitter at sensory nerve endings. J Physiol (London) (1953) 119:50P–51P.[Medline]
  2. Dzau V.J, Colucci W.S, Hollenberger N.K, Williams G.H. Relation of the renin-angiotensin-aldosterone system to clinical state in congestive heart failure. Circulation (1981) 63:645–651.[Free Full Text]
  3. Chen G, Suzuki H, Weston A.H. Acetylcholine releases endothelial-derived hyperpolarising factor and EDRF from rat blood vessels. Br J Pharmacol (1988) 95:1165–1174.[Web of Science][Medline]
  4. Burnstock G. Vascular control by purines with emphasis on the coronary system. Eur Heart J (1989) 10(suppl_F):15–21.[Abstract/Free Full Text]
  5. Malmsjö M, Edvinsson L, Erlinge D. P2U-receptor mediated endothelium-dependent but nitric oxide independent vascular relaxation. Br J Pharmacol (1998) 123(4):719–729.[CrossRef][Web of Science][Medline]
  6. Malmsjö M, Erlinge D, Högestätt E, Zygmunt P. Endothelial P2Y receptors induce hyperpolarisation of vascular smooth muscle by release of endothelium derived hyperpolarising factor. Eur J Pharmacol (1999) 364:169–173.[CrossRef][Web of Science][Medline]
  7. Chidsey C.A, Braunwald E, Morrow A.G. Catecholamine excretion and cardiac stores of norephinephrine in congestive heart failure. Am J Med (1965) 39:442–451.[CrossRef][Web of Science][Medline]
  8. Edvinsson L, Ekman R, Hedner P, Valdemarsson S. Congestive heart failure: involvement of perivascular peptides reflecting activity in sympathetic, parasympathetic and afferent fibres. Eur J Clin Invest (1990) 20:85–89.[Web of Science][Medline]
  9. McMurray J.J, Ray S.G, Abdullah I, Dargie H.J, Morton J.J. Plasma endothelin in chronic heart failure. Circulation (1992) 85:1374–1379.[Abstract/Free Full Text]
  10. Rodeheffer R.J, Lerman A, Heublein D.M, Burnett J.C. Increased levels of endothelin in congestive heart failure. Mayo Clin Proc (1992) 67:719–724.[Web of Science][Medline]
  11. Gunther A, Riegger J, Liebau G, Kochsiek K. Antidiuretic hormone in congestive heart failure. Am J Med (1982) 72:49–52.[CrossRef][Web of Science][Medline]
  12. Goldsmith S.R, Francis G.S Jr., Cowley A.W, Levine T.B, Cohn J.N. Increased plasma arginine vasopressin levels in patients with congestive heart failure. J Am Coll Cardiol (1983) 1:1385–1390.[Web of Science][Medline]
  13. Holtz J, Förstermann U, Pohl U, Giesler M, Bassenge E. Flow-dependent, endothelium-mediated dilation of epicardial coronary arteries in conscious dogs: effects of cyclooxygenase inhibition. J Cardiovasc Pharmacol (1984) 6:1161.[Web of Science][Medline]
  14. Morris S.M, Billiar T.R. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol (1994) 266:E829–E839.[Web of Science][Medline]
  15. Pohl U, Holz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension (1986) 8:37.[Abstract/Free Full Text]
  16. Rubanyi G.M, Romero J.C, Vanhoutte P.M. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol (1986) 231:H405.
  17. Vallance P Collier J Moncada S. Lancet. (1989) 997.
  18. Drexler H.D, Hornig B. Importance of endothelial function in chronic heart failure. J Card Pharmacol (1996) 27:S9–S12.[CrossRef][Web of Science][Medline]
  19. Katz S.D, Schwartz M, Yuen J, LeJemtel T.H. Impaired acethylcholine-mediated vasodilation in patients with congestive heart failure. Role of endothelium-derived vasodilating and vasoconstricting factors. Circulation (1993) 88:55–61.[Abstract/Free Full Text]
  20. Lindsay D.C, Jiang C, Brunotte F, Adamopoulos S, Coats A.J.S, Rajagopalan B, Poole-Wilson P.A, Collins P. Impairment of endothelium dependent responses in a rat model of chronic heart failure: effects of an exercise training protocol. Cardiovasc Res (1992) 26:694–697.[Abstract/Free Full Text]
  21. Vargas F, Osuna A, Fernandez-Rivas A. Vascular reactivity and flow-pressure curve in isolated kidneys from rats with N-nitro-L-arginine methyl ester-induced hypertension. J Hypertens (1996) 14:373–379.[CrossRef][Web of Science][Medline]
  22. Makino A, Kamata K. Eleveted plasma endothelin-1 in streptozotocin-induced diabetic rats and responsiveness of the mesenteric arterial bed to endothelin-1. Br J Pharmacol (1998) 123:1065–1072.[CrossRef][Web of Science][Medline]
  23. Brandes R.P, Behra A, Lebherz C, Boger R.H, Bode-Boger S.M, Phivthong-Ngam L, Mugge A. N(G)-nitro-L-arginine- and indomethacin-resistant endothelium-dependent relaxation in the rabbit renal artery: effect of hypercholesterolemia. Atherosclerosis (1997) 135:49–55.[CrossRef][Web of Science][Medline]
  24. Burnstock G. Dual control of local blood flow by purines. Biological actions of extracellular ATP. Ann NY Acad Sci (1990) 603:31–45.
  25. Valdemarsson S, Bergdahl A, Edvinsson L. Relationships between plasma levels of catecholamines and neuropeptides and the survival times in patients with congestive heart failure. J Int Med (1994) 235:595–601.[Web of Science][Medline]
  26. Bergdahl A, Valdemarsson S, Pantev E, Ottosson A, Feng Q.-P, Sun X.-Y, Hedner T, Edvinsson L. Modulation of vascular contractile response to {alpha}1- and {alpha}2-adrenergic and neuropeptide Y receptor stimulation in rats with ischaemic heart failure. Acta Physiol Scand (1995) 154:429–437.[Web of Science][Medline]
  27. Funaya H, Kitakaze M, Node K, Minamino T, Komamura K, Hori M. Plasma adenosine levels increase in patients with chronic heart failure. Circulation (1997) 95:1363–1365.[Abstract/Free Full Text]
  28. Gordon J.L. Extracellular ATP: Effects, sources and fate. Biochem J (1986) 233:309–319.[Web of Science][Medline]
  29. Pfeffer M.A, Pfeffer J.M, Fishbein M.C, Fletcher P.J, Spadaro J, Kloner R.A, Braunwald E. Myocardial infarction size and ventricular function in rats. Circ Res (1979) 44:503–512.[Abstract/Free Full Text]
  30. Högestätt E, Andersson K.-E, Edvinsson L. Mechanical properties of rat cerebral arteries as studied by a sensitive device for recording of mechanical activity in isolated small blood vessels. Acta Physiol Scand (1983) 117:49–61.[Web of Science][Medline]
  31. Pfeffer M.A, Braunwald E. Ventricular remodeling after myocardial infarction: experimental observations and clinical implications. Circulation (1990) 81:1161–1172.[Abstract/Free Full Text]
  32. Tikkanen T, Tikkanen I, Fyhrquist F. Elevated plasma atrial natriuretic peptid in rats with myocardial infarcts. Life Sci (1987) 40:659–663.[CrossRef][Web of Science][Medline]
  33. Valdemarsson S, Bergdahl A, Edvinsson L. Relationships between plasma levels of catecholamines and neuropeptides and the survival time in patients with congestive heart failure. J Intern Med (1994) 235:595–601.[Web of Science][Medline]
  34. Pfeffer M.A, Pfeffer J.M, Steinberg C, Finn P. Survival after an experimental myocardial infarction: beneficial effects of long-term therapy with catopril. Circulation (1985) 72:406–412.[Abstract/Free Full Text]
  35. Taylor S.G, Weston A.H. Endothelium-derived hyperpolarising factor: a new endogenous inhibitor from the vascular endothelium. Trends Pharmacol Sci (1988) 9:272–274.[CrossRef][Medline]
  36. Campbell W.B, Gebremedhin D, Pratt F.P, Harder D.R. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factor. Circ Res (1996) 78:415–423.[Abstract/Free Full Text]
  37. Corriu C, Feletou M, Canet E, Vanhoutte P.M. Endothelium-derived factors and hyperpolarization of the carotid artery of the guinea-pig. Br J Pharmacol (1996) 119:959–964.[Web of Science][Medline]
  38. Zygmunt P.M, Högestätt E.D. Role of potassium channels in endothelium-dependent relaxation resistant to nitroarginine in the rat hepatic artery. Br J Pharmacol (1996) 117:1600–1606.[Web of Science][Medline]
  39. Chataigneau T, Feletou M, Thollon C, Villeneuve N, Vilaine J.P, Duhault J, Vanhoutte P.M. Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries. Br J Pharmacol (1998) 123:968–974.[CrossRef][Web of Science][Medline]
  40. Zygmunt P.M, Plane F, Paulsson M, Garland C.J, Högestätt E.D. Interactions between endothelium-derived relaxing factors in the rat hepatic artery: focus on regulation of EDHF. Br J Pharmacol (1998) 124:992–1000.[CrossRef][Web of Science][Medline]
  41. Zygmunt P.M, Grundemar L, Högstätt E.D. Endothelium-dependent relaxation resistant to N{omega}-nitro-L-arginine in the rat hepatic artery and aorta. Acta Physiol Scand (1994) 152:107–114.[Web of Science][Medline]
  42. Mantell L, Amerini S, Ledda F. Roles of nitric oxide and endothelium-derived hyperpolarizing factor in vasorelaxant effect of acetylcholine as influenced by aging and hypertension. J Cardiovasc Pharmacol (1995) 25:595–602.[Web of Science][Medline]
  43. Bauersachs J, Popp R, Hecker M, Sauer E, Flemming I, Busse R. Nitric oxide attenuates the release of endothelium-derived hyperpolarizing factor. Circulation (1996) 94:3341–3347.[Abstract/Free Full Text]

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