The effect of a neuropeptide Y Y1 receptor antagonist in patients with angina pectoris
L. Gullestada,
T. Bjurøb,
L. Aabergea,
T. Apellanda,
R. Skårdala,
E. Kjekshusa,
M. Nordlanderc,
B. Åbladc and
J. Pernowd,*
a Department of Cardiology Rikshospitalet University Hospital, Oslo, Norway
b Wallenberg Laboratory, Sahlgrens University Hospital, Gothenburg, Sweden
c AstraZeneca R & D, Mölndal, Sweden
d Department of Cardiology, Karolinska Hospital, Stockholm, Sweden
* Correspondence to: John Pernow, MD, PhD, Department of Cardiology, Karolinska Hospital, S-171 76 Stockholm, Sweden. Tel: +46 8 51775876; fax: +46 8 311044
E-mail address: john.pernow{at}ks.se
Received 27 November 2002;
revised 21 February 2003;
accepted 20 March 2003
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Abstract
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Aims Neuropeptide Y (NPY) is a potent vasoconstrictor released during sympathetic activation that may be involved in myocardial ischaemia. We examined the effect of a Y1 receptor antagonist on haemodynamic and ischaemic responses to exercise in patients with coronary artery disease.
Methods and results Eighty-two evaluable male patients were included in a randomized, double blind, two-way crossover study with a low dose (6.7µg/kg/min; n=59)and a high dose (13.3µg/kg/min; n=23) of the Y1 receptor antagonist AR-H040922 given as infusions for 2h or placebo. Myocardial ischaemia during a symptom-limited exercise test was monitored by conventional ST-segment analysis and heart rate (HR)-adjusted ST changes including the ST/HR slope and ST/HR recovery. Administration of the high dose AR-H040922 attenuated systolic blood pressure by 611mmHg (p<0.05) during and after exercise without affecting HR. None of the two doses of AR-H040922 influenced any of the ischaemic parameters or duration of exercise, however. The maximal increase in NPY was higher during AR-H040922 (p<0.05) compared with placebo.
Conclusions Selective NPY Y1 receptor blockade attenuates the increase in blood pressure during exercise indicating a role for endogenous NPY in blood pressure regulation. Despite this effect, the Y1 receptor antagonist did not influence exercise-induced ischaemic parameters in patients with coronary artery disease.
Key Words: Coronary artery disease exercise testing myocardial ischaemia peptides
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1. Introduction
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Myocardial ischaemia during physical exercise in patients with angina pectoris is caused by oxygen demand exceeding the supply, and is mainly due to the activation of the sympathetic nervous system. Release of noradrenaline leads to increased myocardial oxygen demand via a ß-adrenoceptor-mediated increased heart rate and contractility as well as limitation of the increase in oxygen supply due to an
-adrenoceptor-mediated coronary vasoconstriction.1Earlier studies suggest a significant role of
-adrenergic coronary vasoconstriction during exercise-induced myocardial ischaemia in patients with stable angina pectoris.1,2We have also recently demonstrated that the non-adrenergic sympathetic co-transmitter, neuropeptide Y (NPY), increases in plasma duringexercise in patients with angina pectoris and could contribute to myocardial ischaemia, especially in the recovery phase after exercise.3
NPY is particularly abundant in the perivascular sympathetic nerve fibres, where it is co-stored and released with noradrenaline.4Its cardiovascular effects are multiple and not fully explored, but a long-lasting vasoconstriction, which is not mediated via
-adrenergic receptors, is prominent.4In anaesthetized dogs, myocardial release of NPY elicited by cardiac sympathetic nerve stimulation correlated significantly with the coronary vasoconstrictor response which persisted after
- and ß-adrenoceptor blockade.5Intracoronary infusion of NPY in patients with angina pectoris induces myocardial ischaemia with typical chest pain and ECG-changes.6This may indicate that NPY is a mediator of coronary vasoconstriction also in man.
NPY mediates its effects through several receptor subtypes. It is believed that NPY exerts direct vasoconstrictor effects via activation of the Y1 receptor.7In addition to the direct effects, NPY has been demonstrated to potentiate the vasoconstrictor response to noradrenaline.8Accordingly, NPY (Y1) receptor antagonists attenuate both the direct and indirect vasoconstrictor responses evoked by NPY.7
The hypothesis of the present study was that NPY contributes to myocardial ischaemia during exercise in patients with angina. Therefore, we examined the influence of the selective Y1 antagonist AR-H0409229on haemodynamic changes and signs of myocardial ischaemia both during and after exercise in patients with stable angina pectoris.
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2. Methods
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2.1. Patients
Eighty-eight male patients between 3075 years of age, with chronic stable angina pectoris for at least 3 months, with positive (
1mm ST segment depression) symptom limited exercise test, who were referred for coronary angiography for suspected coronary artery disease, were recruited for the study. In six of these patients >1mm ST segment depression was not obtained during the study exercise. These patients were therefore not included. The final study thus included 82 patients. Stable angina was defined as angina that had not changed in characteristics during the last four weeks before inclusion. Significant coronary artery disease was confirmed in all patients, except nine who eventually refused angiography. All patients were in sinus rhythm, with stable clinical condition, without myocardial infarction during the last 3 months, and with unchanged medication (Table 1) during the last month. Excluded from participation werepatients with clinically significant peripheral vascular or cerebral disease, insulin dependent-diabetes mellitus, renal insufficiency (creatinine >180µmol/l), clinically significant liver disease, pulmonary disease or patients using digitalis. Informed consent was obtained from all participants and the Ethics Committee of the hospital (Rikshospitalet, University of Oslo, Norway) approved the protocol.
2.2. Design
The study was performed as a randomized, double blind, two-way crossover, and placebo-controlled study in two separate groups of patients. One group of patients (n=64) received a low dose of the NPY antagonist AR-H040922 at a dose rate of 6.7µg/kg/min or and placebo (NaCl) on two occasions. The other group (n=24) received a high dose of AR-H040922 at a rate of 13.3µg/kg/min and placebo. AR-H040922 and placebo were given as i.v. infusions for 120min. The washout period between the two occasions was at least 1 week. Exercise started 45min after start of the infusion.
2.3. Exercise testing
The subjects were instructed not to take part in physical training the day before the exercise test, and to eat a normal mixed diet. They were not allowed to eat, drink coffee or smoke during the last 4h before the study, and the morning medication was postponed until after the exercise test. The two tests were performed at the same time of the day and under similar conditions.
The bicycle exercise test was carried out on an electrically braked ergometer cycle with a constant pedal rate of 60rpm. Workload was initiated at 40W with subsequent increments of 10W every minute until they stopped because of exhaustion, angina pain, dyspnoea or the occurrence of a systolic blood pressure drop of >10mmHg. Heart rate and blood pressure were recorded before start of the infusion, 45min after start of infusion, every second minute during exercise, at maximal exercise, and at 1, 2, 4, 10 and 30min in the post-exercise period. Blood pressure was determined by the auscultatory method with a blood pressure cuff placed around the upper arm. Heart rate was measured from the ECG recording. After the test the patient remained seated for 1min on the bicycle and thereafter laid down for the rest of the period.
A 12-lead ECG was continuously sampled into a computer before, during and until 10min after exercise. Special care was taken to reduce noise in the analogue ECG by skin preparation and electrode handling. The ST-amplitude was measured 60ms (ST60) after the end of the QRS complex and signal averaging was performed in consecutive 10s intervals so that six ST-values were obtained each minute in 12-leads throughout the entire test. The main variable was time to >1mm ST-depression in lead V5. The 1mm level during exercise was calculated by the computer using linear regression over 2min and related to an average of 12 values at rest before exercise. Secondary variables were absolute ST amplitude changes, the ST/HR slope and the ST-deficit'. Absolute ST amplitude values were measured in all 12 leads as an average of data obtained during the last 30s of exercise. A ST/HR slope10was calculated by linear regression in all leads during the last 4min of exercise and expressed as microvolts per heart beat (µV/bpm). From the ST/HR values we measured the difference between the ST-segment depression during and after exercise at corresponding heart rates.3,11Since the measured value was negative in most cases, it is here referred to as ST deficit'. A time curve for the ST deficit' in each of the first 10min of recovery was constructed.
2.4. Blood sampling
Blood samples were drawn from an arm vein (contra-lateral to the arm in which the infusion was given) at rest before start of exercise, at 2 and 4min after start of exercise, at peak exercise, and after 4, 10 and 30min of recovery. Plasma was separated immediately and frozen at 70°C until analysed. NPY was assessed by radioimmunoassay.12The plasma concentration of AR-H040922 was measured by liquid chromatography with fluorescence detection.9The coefficients of intra-assay variation were 7.0% and 2.9% for NPY and AR-H040922, respectively. In addition routine biochemical analysis for safety reasons were performed at the pre-entry visit and at the follow-up visit 25 days after study day two.
2.5. Statistics
Data from two previous studies (Astra Data on file) indicate that one may expect a within-subject standard deviation of approximately 2.5min for the time to 1mm ST-depression. It is deemed necessary to detect a difference of 0.9min to be able to show that the concept of the drug holds. With 56 patients it will be possible to detect such a difference with an overall power of 0.78 at an overall significance level of 0.085. This calculation assumes an interim analysis after 28 patients and is based on Student's t-test at a significance level of 0.05, both at the interim analysis and the final analysis.
Based on the obtained within subject standard deviation for time to 1mm ST segment depression in the first group of patients receiving the low dose of AR-H040922 of 1.5min, another group of 24 patients were planned to be enrolled into the high dose group. This would allow detection of a difference between active treatment and placebo in mean values of 0.9min with a power of 80% at a significant level of 5% between active treatment and placebo. Clinical measurements were analysed in a mixed ANOVA model with treatment, period, and sequence as fixed effects and patient within sequence as a random effect. Two-sided confidence intervals, with a confidence level of 95%, for the estimated mean and mean difference between values obtained during infusion of AR-H040922 and placebo, respectively, were calculated. Corresponding p-values are given. The calculations are based on Student's t-distribution. Differences were considered significant when p<0.05, two-tailed test. Presented values are mean±SD
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3. Results
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3.1. Patients
A total number of 88 patients were included in the study. Six patients were excluded from the evaluation due to incomplete ECG recordings or due to that significant ST segment depression (>1mm) did not occur during the study exercise. Thus, 59 patients receiving the low dose of AR-H040922 (6.7µg/kg/min) and 23 patients receiving the high dose (13.3µg/kg/min) were included in the final analyses of the study. Some basal characteristics of the patients are presented in.Table 1In the following, data obtained with the two different doses are presented separately.
3.2. Low dose AR-H040922
Administration of AR-H040922 did not affect resting heart rate or systolic blood pressure (Fig. 1). There were no significant differences in duration of exercise or haemodynamic parameters during or after exercise between treatment with AR-H040922 and placebo (Fig. 1, Table 2). Time to >1mm ST segment depression was 469±225s during placebo and 458±192s during administration of AR-H040922 (p=ns; Table 2). Maximal ST depression just before termination of exercise was unaltered by AR-H040922. The ST/HR slope, which has been shown to be a reliable index for myocardial ischaemia at the end of exercise and a reflection of the functional severity of coronary obstruction, was not changed by AR-H040922 (Table 2). Since we previously have suggested a possible involvement of NPY in myocardial ischaemia in the recovery phase after exercise, particular attention was paid tothis phase. However, neither the duration of ST depression or the ST-deficit' was influenced by AR-H040922 (Table 2, Fig. 2).

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Fig. 1 Heart rate and systolic blood before, during and after exercise in patients given the (a) low and (b) high dose of AR-H040922 in comparison with placebo. Data are given as mean±SD. Changes during exercise are calculated from sitting on bicycle and post exercise changes from max workload. Statistically significant differences in changes between AR-H040922 and placebo are indicated. *p<0.005.
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Fig. 2 ST-deficit after the end of exercise in the presence of the low (a) and high dose (b) of placebo (open circles) and AR-H040922 (filled circles). The ST segment amplitude after exercise is compared with that during exercise at corresponding heart rates. A negative value means that the ST segment depression at a given time point after exercise was greater than that observed at a similar heart rate during exercise. Data are given as mean±SD.
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The plasma concentrations of AR-H040922 increased to 2948±661nmol/l at start of exercise and to a maximum of 3569±738nmol/l at the end of exercise. There was a positive interaction between the efficacy variables ST depression and time to >1mm ST segment depression and plasma concentrations of AR-H040922. Based on these findings the study was continued in an additional group receiving a higher infusion rate of AR-H040922, 13.3µg/kg/min.
3.3. High dose AR-H040922
Infusion of the high dose of AR-H040922 resulted in a plasma concentration of 7191±1618nmol/l. The increase in systolic blood pressure during submaximal exercise was significantly lower during administration of AR-H040922 than during placebo (Fig. 1). Likewise, the changes in systolic blood pressure after exercise were greater with AR-H040922 than with placebo (p<0.05). HR was not affected by AR-H040922. Despite this haemodynamic effect of AR-H040922, no significant differences in time to >1mm ST segment depression, exercise time or ECG parameters at the end of exercise were observed between active drug and placebo administration (Table 3, Fig. 2). Furthermore, when the maximal ST segment depression and the ST/HR slopes were compared at highest common workload no differences between drug treatment and placebo were observed.
3.4. NPY levels
Plasma NPY levels increased during exercise both during AR-H040922 and placebo. The increase in plasma concentrations of NPY was significantly greater during exercise with AR-H040922, regardless of dose, than with placebo (Table 4).
3.5. Side effects
Eleven patients reported some kind of side effects during the infusion. Three patients (one in the low dose group and two in the high dose group) developed hypotension during AR-H040922 requiring the infusions to be stopped. In one patient receiving the low dose, the infusion was stopped permanently in the recovery phase after exercise. In the other two patients the infusions were only stopped temporarily for 12 and 13min, respectively. Other side effects were of non-specific character and were equally distributed between active drug and placebo.
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4. Discussion
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Since our previous study3indicated that NPY might be involved as a mediator of myocardial ischaemia during exercise in patients with angina pectoris, administration of an NPY receptor antagonist was expected to be beneficial in patients with exercise-induced angina pectoris. However, the Y1 receptor antagonist AR-H040922 had no effect on exercise-induced ischaemia during or after exercise in patients with coronary artery disease. On the other hand, the Y1 receptor antagonist significantly attenuated the increase in blood pressure during exercise.
Administration of a high dose of the Y1 receptor antagonist resulted in lower systolic blood pressure both during and after exercise. This is to our knowledge the first observation suggesting involvement of endogenous NPY in the regulation of blood pressure during sympathetic activation in humans. The observation that blood pressure was lower also 30min after cessation of the exercise in the presence of AR-H040922 suggests that NPY participates in the regulation of blood pressure for a long period after its release during sympathetic activation. This finding is in accordance with the considerable longer duration of the vasoconstrictor response evoked by NPY than by noradrenaline.4
Several explanations may exist why the selective Y1 receptor antagonist did not reduce the exercise-induced ischaemia. One possibility is that NPY is not involved in the regulation of coronary artery tone and exercise-induced ischaemia. However, considering the vascular effects of NPY it is reasonable to assume that release of NPY during exercise may contribute to myocardial ischaemia by two different mechanisms. NPY may induce coronary constriction and thereby reducing myocardial oxygen supply. In addition, NPY may cause peripheral vasoconstriction and thereby increase afterload14and/or preload,15which will enhance myocardial oxygen demand. Accordingly, the increase in blood pressure during exercise was significantly smaller in the presence of the high dose of AR-H040922, which may support such an effect. Exogenous NPY causes long lasting vasoconstriction in several vascular beds including the coronary circulation of both experimental animals and humans.6,13,16In the coronary circulation this effect is associated with ischaemia and impaired ventricular function.16Furthermore, we have previously demonstrated an association between the increase in plasma NPY and the duration of ST segment depression after exercise and the ST deficit.3These observations suggest an involvement of NPY during exercise-induced myocardial ischaemia. On the other hand, when NPY was infused into the coronary arteries of patients with coronary artery disease only a subgroup of the patients (three of six) responded with coronary constriction whereas NPY was without effect in the rest of the group.6Furthermore, in a recent study on healthy young volunteers i.v. administration of NPY at doses, which elevated plasma NPY levels to 3000pmol/l, did not affect myocardial blood flow.17Thus, it is possible that NPY at concentrations observed in the present study does not produce significant effects on myocardial blood flow in humans.
Another explanation may be that the effect of NPY is mediated by other receptors than Y1. There is evidence for a proportion of post-synaptic Y2 receptors (or other subtypes) since a vasoconstrictor effect has been obtained using NPY fragments specific for the Y2 receptor in certain vascular beds.7The increase in systemic blood pressure and the renal and splanchnic vasoconstrictor response to NPY in humans is blocked by Y1 receptor antagonism18Furthermore, the increase in blood pressure was attenuated by Y1 receptor blockade in the present study. Thus, the Y1 receptor seems to be the dominant receptor subtype in vascular beds of haemodynamic importance.
A third possibility is that the dose of AR-H040922 was inadequate to block NPY-mediated effects. However, available data does not support this. Thus, the observation that systolic blood pressure increased less in the presence of the high dose AR-H040922 clearly suggests that this dose of the antagonist was effective. A previous study in man showed that splanchnic and renal vasoconstriction induced by exogenous NPY was blocked by the low dose of AR-H040922 used in the present study.18Furthermore, it has been clearly demonstrated that the vasoconstrictor response to sympathetic nerve stimulation in the kidney and skeletal muscle of pigs is attenuated by plasma concentrations ofAR-H040922 above 1000nmol/l.9Thus, the presently obtained concentrations of AR-H040922(3000 and 7000nmol/l) are most likely within the therapeutic range.
An observation that may influence the results of the present study is that plasma levels of NPY during exercise increased significantly more in the presence of AR-H040922 than during placebo. Similar findings have been obtained during infusion of NPY in humans.19These observations may indicate that the Y1 receptor participates in the clearance of NPY from the circulation, which is in line with the role of the endothelin B receptor in the clearance of endothelin-1.20Since AR-H040922 is a competitive antagonist at the Y1 receptor, the increased plasma levels of NPY in the presence of the antagonist may reverse the receptor blockade and thereby offset a therapeutic effect.
A limitation of the present protocol is that AR-H040922 was administered as a single i.v. infusion. The rationale for this protocol was the fact that AR-H040922 has poor oral bioavailability and a short plasma half life. Despite this limitation, short term administration of the high dose AR-H040922 did result in a 7 mm Hg reduction of systolic blood pressure for up to 30min after end of exercise. It cannot be excluded, however, that prolonged and repeated administration of an orally active Y1 receptor antagonist may also have additional effects. It is not known whether ongoing medication may have interfered with the NPY receptor antagonist. The patients did not take their morning medication on the day of the exercise test. Thus, the plasma concentrations of concurrent medication are assumed to be low during the exercise tests. It has been described that metoprolol reduces the number and the affinity of NPY binding sites in vascular smooth muscle cells in hypertensive rats.21It is not known, however, whether beta-blockers affect the binding of AR-H040922.
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5. Conclusion
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The present study demonstrates that selective blockade of the Y1 receptor with high dose AR-H040922 significantly inhibits the increase in blood pressure during and after exercise, indicating a role for NPY in blood pressure regulation during and following sympathetic activation in patients with coronary artery disease. However, despite thiseffect AR-H040922 does not influence the degree of myocardial ischaemia during or after exercise.
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Acknowledgments
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We thank Carina Nihlén and Hanne Schulz Jensen for skilful technical assistance. The study was supported by AstraZeneca R&D Mölndal, Sweden, the Swedish Research Council (10857) and the Swedish Heart and Lung Foundation.
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References
|
---|
- Baumgart D, Heusch G. Neuronal control of coronary blood flow. Basic Res Cardiol. 1995;90:142159.[Medline]
- Berkenboom G, Abramowicz M, Vandermoten P et al. Role of alpha-adrenergic coronary tone in exercise-induced angina pectoris. Am J Cardiol. 1986;57:195198.[CrossRef][Medline]
- Gullestad L, Jørgensen B, Bjurø T et al. Post exercise ischemia is associated with increased neuropeptide Y in patients with coronary artery disease. Circulation. 2000;102:987993.[Abstract/Free Full Text]
- Lundberg JM. Pharmacology of cotransmission in the autonomic nervous system: Integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide. Pharmacol Rev. 1996;48:113178.[Medline]
- Tanaka EMH, Chujo M, Yamakawa A et al. Coronary vasoconstrictive effects of neuropeptide T and their modulation by the ATP-sensitive potassium channel in anesthetized dogs. J Am Coll Cardiol. 1997;29:13801389.[CrossRef][Medline]
- Clarke JG, Davies GJ, Kerwin R et al. Coronary artery infusion of neuropeptide Y in patients with angina pectoris. Lancet. 1987;I:10571059.
- Malmstrom RE. Pharmacology of neuropeptide Y receptor antagonists. Focus on cardiovascular functions. Eur J Pharmacol. 2002;447:1130.[CrossRef][Medline]
- Wahlestedt C, Edvinson L, Ekblad E et al. Neuropeptide Y potentiates noradrenaline-evoked vasoconstriction: mode of action. J Pharmacol Exp Ther. 1985;234:735741.[Abstract]
- Malmström RE, Alexandersson A, Balmér K. In vivo Characterization of the novel Neuropeptide Y Y1 receptor antagonist H 409/22. J Cardiovasc Pharmacol. 2000;36:516525.[CrossRef][Medline]
- Okin PM, Kligfield P. Heart rate adjustment of ST segment depression and performance of the exercise electrocardiogram: a critical evaluation. J Am Coll Cardiol. 1995;25:17261735.[CrossRef][Medline]
- Suurkula M, Arvidsson A, Fagerberg B et al. A new method to quantify postexercise ST-deviation the ST deficit. A study in men at high and low risk for coronary heart disease. Clin Physiol. 2001;21:541555.[CrossRef][Medline]
- Thedorsson-Norheim E, Hemsén A, Lundberg JM. Radioimmunoassay for NPY: chromatographic characterization of immunoreactivity in plasma and tissue extracts. Scand J Clin Lab Invest. 1985;45:355365.[Medline]
- Aizawa Y, Satoh M, Aizawa M et al. Potency and receptors involved in coronary vasoconstriction caused by neuropeptide Y (NPY). Jpn Heart J. 1987;28:891898.[Medline]
- Ahlborg G, Weitzberg E, Lundberg JM. Splanchnic and renal vasoconstriction during neuropeptide Y infusion in healthy humans. Clin Physiol. 1992;12:145153.[Medline]
- Linder L, Lautenschlager BM, Haefeli WE. Subconstrictor doses of neuropeptide Y potentiate alpha 1-adrenergic venoconstriction in vivo. Hypertension. 1996;28:483487.[Abstract/Free Full Text]
- Maturi MF, Greene R, Speir E. Neuropeptide Y. A peptide found in human coronary arteries constricts primarily small coronary arteries to produce myocardial ischemia in dogs. J Clin Invest. 1989;83:12171224.[Medline]
- Ullman B, Pernow J, Lundberg JM et al. Cardiovascular effects and cardiopulmonary plasma gradients following intravenous infusion of neuropeptide Y in humans: negative dromotropic effect on atrioventricular node conduction. Clin Sci. 2002;103:535542.[Medline]
- Ahlborg G, Jolin-Mellg
rd
, Wahlquist I et al. NPY-induced splanchnic and renal vasoconstriction in humans is reduced by H 409/22, a novel Y1 receptor antagonist. Abstract book 5th International NPY meeting. Grand Cayman, 1999..
- Nordlander M, Ahlborg G, Ashton M et al. Elevated plasma levels and decreased clearance of NPY during NPY-Y1 receptor blockade by AR-H040922. Abstract book, 6th International NPY Conference Sydney Australia 2001..
- Fukuroda T, Fujikawa T, Ozaki S et al. Clearance of circulating endothelin-1 by ETB receptors in rats. Biochem Biophys Res Comm. 1994;199:14611465.[CrossRef][Medline]
- Zeng C, Wang X, Liu G et al. Effects of ACE inhibitor and beta-adrenergic blocker on plasma NPY and NPY receptors in aortic vascular smooth muscle cells from SHR and WKY rats. Neuropeptides. 2002;36:353361.[CrossRef][Medline]