Intracoronary Ghrelin Infusion Decreases Coronary Blood Flow in Anesthetized Pigs

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Endocrinology 148(2):806 – 812 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1230

Intracoronary Ghrelin Infusion Decreases Coronary Blood Flow in Anesthetized Pigs Elena Grossini, Claudio Molinari, David A. S. G. Mary, Ezio Ghigo, Gianni Bona, and Giovanni Vacca Dipartimento di Medicina Clinica e Sperimentale (E.G., C.M., D.A.S.G.M., G.B., G.V.), Facolta` di Medicina e Chirurgia, Universita` del Piemonte Orientale “A. Avogadro,” 28100 Novara, Italy; Dipartimento di Medicina Interna (E.G.), Universita` degli Studi di Torino, 10124 Torino, Italy iment after coronary flow had returned to the control values observed before infusion. The ghrelin-induced coronary vasoconstriction was not affected by iv atropine (five pigs) or phentolamine (five pigs). This response was abolished by iv butoxamine (five pigs) and intracoronary N␻-nitro-L-arginine methyl ester (five pigs), even after reversing the increase in arterial pressure and coronary vascular resistance caused by the two blocking agents with iv infusion of papaverine. The present study showed that intracoronary infusion of ghrelin primarily caused coronary vasoconstriction. The mechanisms of this response were shown to involve the inhibition of a vasodilatory ␤2-adrenergic receptor-mediated effect related to the release of nitric oxide. (Endocrinology 148: 806 – 812, 2007)

The peptide ghrelin has been linked to the atherosclerotic process and coronary artery disease. We planned to study, for the first time, the primary effects of ghrelin on the intact coronary circulation and determine the mechanisms involved. In 24 sodium pentobarbitone-anesthetized pigs, changes in anterior descending coronary blood flow caused by intracoronary infusion of ghrelin at constant heart rate and arterial pressure were assessed using electromagnetic flowmeters. In 20 pigs, intracoronary infusion of ghrelin decreased coronary blood flow without affecting left ventricular maximum rate of change of left ventricular systolic pressure (dP/dtmax), filling pressures of the heart or plasma levels of GH. In four pigs, this decrease was graded by step increments of infused dose of the hormone. The mechanisms of the above response were studied in the 20 pigs by repeating the exper-

G

HRELIN IS A peptide hormone that has been recently isolated from the stomach and identified as an endogenous ligand for the GH secretagogue receptors (1, 2). In addition to being expressed in the gastrointestinal tract and hypothalamus in which it has been linked to increased appetite and GH secretion (2), such receptors have also been found in many tissues (3) including the coronary arteries (4). Indeed, ghrelin has been associated with atherosclerosis and coronary artery disease (4), although as yet there is little clear-cut information of its effect on the coronary circulation. This is to be expected because the effect of ghrelin may be confounded by factors such as its influence of releasing hormones including GH (5, 6), its ability of reducing arterial pressure (5, 7), and improving cardiac performance (5, 8, 9). However, in the isolated rat heart, ghrelin was found to either improve coronary blood flow (CBF) during reperfusion of ischemic injury (10) or constrict the coronary circulation in isolated rat hearts and arterioles (11). The primary effect of ghrelin on the intact coronary circulation and the mechanisms involved are as yet unknown. This work was therefore planned to study the primary effects of the acute administration of ghrelin on CBF and determine the mechanisms involved. This was achieved by performing experiments of intracoronary administration of small doses of ghrelin and preventing changes in heart rate

and aortic pressure to avoid secondary interference by reflex and local metabolic and physical effects and measuring blood levels of GH to avoid secondary interference elicited by their possible changes. Materials and Methods Experimental animals The experiments were carried out in 24 pigs, weighing 73–77 kg, supplied by an accredited dealer (Azienda Ticozzi, Trecate, Novara, Italy). The animals were fasted overnight and then anesthetized with intramuscular ketamine (20 mg/kg; Parke-Davis, Milan, Italy) followed after about 15 min by iv sodium pentobarbitone (15 mg/kg; Siegfried, Zofingen, Switzerland), after which they were artificially ventilated with oxygen-enriched air using a respiratory pump (Harvard 613; Harvard Apparatus, South Natick, MA). Anesthesia was maintained throughout the experiments by continuous iv infusion of sodium pentobarbitone (7 mg/kg䡠h) and assessed as previously reported (12) from responses of the animals to somatic stimuli. The experiments were carried out in accordance with national guidelines (D.L.G.S. 27/01/1992, license no. 116). The protocol was approved by local ethics committees according to national regulations. This study was monitored in accordance with Good Research Practice Guidelines in the European Community. Pressures in the ascending aorta and right atrium were recorded via catheters connected to pressure transducers (Statham P23 XL; Gould, Valley View, OH) inserted into the right femoral artery and the right external jugular vein, respectively. The chest was opened in the left fourth intercostal space, the pericardium was cut and an electromagnetic flowmeter probe (model BL 613; Biotronex Laboratory Inc., Chester, MD) was positioned around the proximal part of the anterior descending coronary artery to record CBF. Distal to the probe a plastic snare was placed around the artery for zero blood flow assessment. Each probe was calibrated in vitro at the end of each experiment. Ghrelin (NeoMPS, Strasbourg, France) was infused in the coronary artery by using a catheter connected to a butterfly needle inserted into the artery distal to the flowmeter probe. Left ventricular pressure was measured by means of a catheter con-

First Published Online November 16, 2006 Abbreviations: CBF, Coronary blood flow; L-NAME, N␻-nitro-l-arginine methyl ester. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

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nected to a pressure transducer (Gould) inserted through the left atrium. The frequency response of the catheter-manometer system was found to be flat (⫾ 5%) up to 40 Hz. To pace the heart, electrodes were sewn on the left atrial appendage and connected to a stimulator (model S8800; Grass Instruments, Quincy, MA), which delivered pulses of 3–5 V for 2 msec duration at the required frequency. Arterial blood samples were used to measure pH, partial oxygen pressure (PO2) and partial pressure of carbon dioxide (PCO2) (with a gas analyzer, ABL 505; Radiometer, Copenhagen, Denmark) and the hematocrit. The acid-base status of the animals was kept within normal limits as previously reported (12). To prevent changes in aortic pressure during the experiments, a large-bore cannula was introduced into the abdominal aorta through the left femoral artery and connected to a reservoir containing Ringer’s solution and kept at 38 C. The reservoir was pressurized using compressed air, which was controlled with a Starling resistance, and pressure within the reservoir was measured by a mercury manometer. This procedure has been shown to allow the aortic pressure to be maintained at steady levels without significant changes in filling pressures of the heart or the hematocrit (e.g. Refs. 13, 14). Coagulation of the blood was avoided by the iv injection of heparin (Parke Davis; initial doses of 500 IU/kg and subsequent doses of 50 IU/kg every 30 min). The rectal temperature of the pigs was monitored and kept between 38 and 40 C using an electric pad. Mean and phasic aortic blood pressure, mean right atrial pressure, left ventricular pressure, and mean and phasic CBF were monitored and recorded, together with heart rate and left ventricular dP/dtmax by using an electrostatic strip-chart recorder (Gould ES 2000; Gould). The heart rate was obtained from the electrocardiogram. The frequency response of the differentiator used to obtain left ventricular dP/dtmax was flat (⫾ 5%) up to 150 Hz. To calculate coronary vascular resistance, the difference between mean aortic blood pressure and mean left ventricular pressure during diastole was considered as the coronary pressure gradient. Coronary vascular resistance was calculated as the ratio between this pressure gradient and mean diastolic CBF. The diastolic period of measurement was defined as starting when ventricular pressure reached its minimum value after systole and ended when it increased at the end of diastole. The plasma concentration of GH was measured with a RIA kit (PGH46HK; Linco Research, St. Charles, MO). At the end of the experiment, each animal was killed by an iv injection of 90 mg/kg sodium pentobarbitone.

Experimental protocol The experiments were begun after at least 30 min of steady-state conditions with respect to measured hemodynamic variables. In the 24 pigs, the heart was paced to a frequency higher, by 20 bpm, than that observed during the steady-state and the arterial system was connected to the pressurized reservoir. After at least 10 min of steady-state conditions, the experiments were carried out by intracoronary infusing either a solution of ghrelin obtained by dissolving the hormone in 2 ml of distilled water with 4% of mannitol or the vehicle only. The infusions were completed within a period of 2 min by using an infusion pump (model 22; Harvard Apparatus) working at constant rate of 1 ml/min. In each pig, a dose of 0.075 ␮g/min for each milliliter per minute of measured CBF was infused into the coronary artery. This dose was calculated from the reported dose of 10 ␮g/kg body weight given iv in healthy volunteers as bolus administration (5) considering a mean value of cardiac output of 5 liters. After the infusion was stopped, observations were continued for 10 min. Recordings taken for 10 min during the steady-state before infusion of ghrelin were used as control. Measurements of hemodynamic variables were obtained during the last 30 sec of infusion in the steady-state and compared with control values. GH plasma levels were measured during the last 10 sec of ghrelin infusion and compared with the control values obtained before the beginning of infusion. The effect of infusion of ghrelin on CBF and the mechanisms involved were studied in 20 pigs. In the remaining four pigs, the effects of graded administration of the hormone were examined by infusing three subsequent doses of ghrelin of 0.045, 0.060, and 0.075 ␮g/min for each milliliter per minute of measured CBF. Each dose was infused for 2 min. The resulting changes in CBF was compared with control values obtained before starting the infusion. GH plasma levels were measured during the last 10 sec of

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TABLE 1. Changes in hemodynamic variables caused by intracoronary infusion of ghrelin in 20 pigs Data

Control

Test

Change

118.7 ⫾ 12.8 118.7 ⫾ 12.8 0.04 ⫾ 0.1 (96 –140) (96 –140) (⫺0.1 to 0.3) ABP 94.2 ⫾ 10.1 94.4 ⫾ 10.4 0.2 ⫾ 0.6 (79 –115) (79 –116) (⫺1 to 2) dP/dtmax 2498 ⫾ 292 2499 ⫾ 292 1⫾7 (2010 –3012) (2016 –3014) (⫺9 to 18) RAP 3.1 ⫾ 0.5 3.1 ⫾ 0.5 0.02 ⫾ 0.09 (2.2– 4) (2.2– 4) (⫺0.2 to 0.3) LVEDP 5.3 ⫾ 0.8 5.3 ⫾ 0.8 0.03 ⫾ 0.09 (4.1–7.1) (4.4 –7.1) (⫺0.1 to 0.3) CBF (ml/min), mean 62 ⫾ 7.6 55.1 ⫾ 6.8 ⫺7 ⫾ 1.2 (49.2–78.4) (44.2–70.8) (⫺8.7 to ⫺4.7)a HR

Data are means ⫾ SD (range). HR, Heart rate (beats per minute); ABP, mean aortic blood pressure (mm Hg); dP/dtmax, left ventricular dP/dtmax (mm Hg/sec); RAP, mean right atrial pressure (mm Hg); LVEDP, left ventricular end-diastolic pressure (mm Hg). a P ⬍ 0.0005. infusion of the highest dose of ghrelin and compared with the control value obtained before the beginning of the infusion of the smallest dose. The mechanisms of the response of CBF to the infusion of ghrelin were studied in the group of 20 pigs by repeating the experiment after hemodynamic variables had returned to control levels. In five pigs, ghrelin was administered after the iv administration of atropine sulfate (0.5 mg/kg; Sigma Chemical Co., Milan, Italy) to block muscarinic cholinergic receptors, in five pigs after the iv administration of phentolamine (1 mg/kg; Ciba Geigy, Varese, Italy) to block ␣-adrenergic receptors and in five pigs after the iv administration of butoxamine (2.5 mg/kg; Sigma) to block ␤2-adrenergic receptors. In the remaining five pigs, ghrelin was infused after the intracoronary administration of 100 mg of N␻-nitro-l-arginine methyl ester (L-NAME; Sigma) to block the nitric oxide synthase. All the drugs were given in the absence of pacing of the heart and without controlling aortic pressure; their effects on hemodynamic variables were measured in the steady state. In all subsequent experiments, changes in heart rate and aortic pressure were prevented. In two of the L-NAME and two of the butoxamine-treated pigs, the intracoronary infusion of ghrelin was performed when a steady state was attained during a continuous iv infusion of papaverine (Sigma) at a dose of 3.5– 4.5 mg/kg䡠h. This procedure was used to reverse the increase in coronary vascular resistance caused by the two blocking agents.

FIG. 1. The response of CBF to the intracoronary infusion of ghrelin in 20 pigs. The values of CBF obtained during the test period of measurement are plotted on the ordinate against the corresponding values before infusion on the abscissa. The continuous line is the line of equality.

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Statistical analyses Student’s paired t test was used to examine changes in measured variables caused by ghrelin infusion. ANOVA and Student-NewmanKeuls test were used to examine the effect of graded increases in the dose of infused hormone. A value of P ⬍ 0.05 was considered statistically significant. Group data are presented as mean ⫾ sd (range).

Results

In all pigs, recordings commenced approximately 5 h after the induction of anesthesia. The mean pH, PO2, and PCO2 of arterial blood were, respectively, 7.40 ⫾ 0.01 (7.38 –7.43), 116.6 ⫾ 8.8 (104 –137), and 39.7 ⫾ 1 (38 – 41) mm Hg, and the hematocrit was 37.4 ⫾ 1.2 (36 – 40%).

Grossini et al. • Ghrelin and Coronary Blood Flow

1.3 (9.2–13.6%) of the control values and corresponded to an increase in coronary vascular resistance of 11.5 ⫾ 1.8 (7.5– 15%) from a control value of 1.12 ⫾ 0.12 (0.93–1.30) mm Hg/ml䡠min. An example of the above response is shown in Fig. 2. In the 20 pigs examined, the coronary effect of ghrelin began within about 15 sec after starting the infusion and reached a steady state in about 1.2 min. CBF returned to control values within 50 sec after the end of the infusion. Intracoronary infusion of ghrelin did not cause significant changes in the values of plasma concentration of GH. These changes were of 0.1 ⫾ 0.4 (⫺0.6 to 0.9; P ⬎ 0.10) ng/ml from a control value of 4.3 ⫾ 1.2 (2.2– 6.2) ng/ml. Response to graded infusion

Effects of intracoronary infusion of ghrelin

In the group of 20 pigs, intracoronary infusion of the vehicle did not cause changes in the control values of hemodynamic variables. Group values of data and individual changes in mean CBF caused by intracoronary infusion of ghrelin are shown in Table 1 and Fig. 1, respectively. In each pig, intracoronary infusion of ghrelin caused a decrease in mean CBF. Group decrease in this flow amounted to 11.2 ⫾

FIG. 2. Example of experimental recordings showing the hemodynamic effects of the intracoronary infusion of ghrelin in one pig. From top to bottom, heart rate (HR), phasic and mean aortic blood pressure (ABP), left ventricular pressure (LVP), mean right atrial pressure (RAP), left ventricular dP/dtmax (dP/dt), mean and phasic CBF. The arrows indicate the beginning and end of infusion.

In the four pigs, control value of mean CBF was 61.1 ⫾ 11 (50.1–75.8) ml/min. In each of the four pigs, incrementing the dose of infused ghrelin augmented the responses of decreases in CBF (Fig. 3). Group decreases for the three doses were 2.1 ⫾ 0.2 (1.9 –2.3; P ⬍ 0.0005) ml/min, 5.3 ⫾ 0.4 (4.9 – 5.8; P ⬍ 0.0005) ml/min, and 7.2 ⫾ 1.5 (6.2–9.3; P ⬍ 0.0025) ml/min, respectively. ANOVA for repeated measurements showed a significant difference in the responses of CBF to the

Grossini et al. • Ghrelin and Coronary Blood Flow

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tors or after blockade of nitric oxide synthase. The effects of administering the blocking agents on control values of heart rate, mean aortic blood pressure, left ventricular dP/dtmax, and mean CBF are shown in Table 2. In two of the butoxamine-treated pigs and two of the L-NAME-treated pigs, infusion of papaverine decreased mean aortic blood pressure by 11 ⫾ 3.6 (8 –16; P ⬍ 0.005) mm Hg to the same levels observed before giving the blocking agents and increased mean CBF by 4.6 ⫾ 1.4 (3.4 – 6.2; P ⬍ .005) ml/min, with a reduction of coronary vascular resistance of 0.32 ⫾ 0.08 (0.21– 0.38; P ⬍ .0025) mm Hg/ml䡠min. In the same four pigs, the increase in coronary vascular resistance caused by butoxamine and L-NAME alone was 0.24 ⫾ 0.07 (0.16 – 0.32; P ⬍ 0.005) mm Hg/ml䡠min.

FIG. 3. The response of CBF to graded increases of the intracoronary infused dose of ghrelin in four pigs (numbered 1– 4). Ghrelin was infused at doses of 0.045 (solid bars), 0.060 (hatched bars), and 0.075 (open bars) ␮g/min for each milliliter per minute of measured CBF.

three doses (P ⬍ 0.001). The Student-Newman-Keuls test indicated that the response of CBF to the highest dose was significantly greater than that to the middle dose, which in turn was significantly greater than the response caused by the lowest dose. Infusion of ghrelin did not cause significant changes in the values of plasma concentration of GH. These changes were of 0.2 ⫾ 0.4 ng/ml (⫺0.2 to 0.6; P ⬎ 0.15) from a control value of 3.6 ⫾ 0.7 (2.8 – 4.3) ng/ml. Mechanisms of the response

In the 20 pigs, the intracoronary infusion of ghrelin was repeated after blockade of cholinergic and adrenergic recep-

Experiments after blockade of cholinergic receptors. In the five pigs, blockade of cholinergic receptors did not affect the response of CBF to the intracoronary infusion of ghrelin (Fig. 4). Group decrease in this flow was of 7.6 ⫾ 1.1 (6.1 to 9.1; P ⬍ 0.0005) ml/min from a control value of 63.5 ⫾ 11.9 (52.2–76.6) ml/min. In the same pigs, the decrease in mean CBF obtained with intracoronary infusion of ghrelin before giving atropine was 7.3 ⫾ 1.3 (5 to 8.2; P ⬍ 0.0005) ml/min from control values of 63.9 ⫾ 12.4 (49.2–78.4) ml/min. The difference between the two responses before and after blockade of cholinergic receptors was not significant (P ⬍ 0.10). Experiments after blockade of ␣-adrenergic receptors. In the five pigs, blockade of ␣-adrenergic receptors did not affect the response of CBF to the intracoronary infusion of ghrelin (Fig. 4). Group decrease in this flow was of 6.9 ⫾ 1.1 (5.5– 8.1; P ⬍ 0.0005) ml/min from a control value of 59 ⫾ 8.8 (45.8 – 68.5) ml/min. In the same pigs, the decrease in mean CBF obtained with intracoronary infusion of ghrelin before giving phentolamine was 6.8 ⫾ 1.2 (5.7– 8.4; P ⬍ 0.0005) ml/min from control values of 62.3 ⫾ 5.9 (56.1–71.4) ml/min. The differ-

TABLE 2. The effects of blockade of cholinergic receptors, adrenergic receptors, and coronary nitric oxide synthase on hemodynamic variables in 20 pigs

Atropine (n ⫽ 5) Change Phentolamine (n ⫽ 5) Control Change Butoxamine (n ⫽ 5) Control Change (n ⫽ 5) Control

L-NAME

Change

HR

ABP

dP/dtmax

CBF (ml/min), mean

(99 –120) 11.2 ⫾ 1.9 (9 –14)a

(77–103) ⫺0.2 ⫾ 2.4 (⫺3 to 3)

(2243–2430) 50 ⫾ 47 (⫺4 to 113)b

(42.3– 67.4) 1.2 ⫾ 2.1 (⫺1.4 to 3.5)

94 ⫾ 13.4 (80 –111) 13.8 ⫾ 3.4 (10 –19)

96.4 ⫾ 11.4 (82–113) ⫺16.6 ⫾ 3.8 (⫺22 to ⫺12)

2373 ⫾ 318 (1983–2743) ⫺5 ⫾ 66 (⫺91 to 64)

53.8 ⫾ 5.1 (47.7– 61.4) ⫺2.1 ⫾ 4 (⫺7.2 to 3.9)

94 ⫾ 9.5 (79 –103) ⫺9 ⫾ 1.6 (⫺11 to ⫺7)a

91 ⫾ 12.3 (78 –108) 10.6 ⫾ 2.4 (8 –14)a

2380 ⫾ 280 (2083–2683) 1 ⫾ 37 (⫺55 to 45)

53.1 ⫾ 6 (44 –59.8) ⫺5.9 ⫾ 4.2a (⫺13.2 to ⫺3.4)c

95 ⫾ 12.9 (76 –111) ⫺6.8 ⫾ 2.4 (⫺10 to ⫺4)d

89.2 ⫾ 7.2 (79 –97) 14.6 ⫾ 4.8 (9 –21)d

2480 ⫾ 348 (1971–2902) 93 ⫾ 25 (61–131)d

52.3 ⫾ 4.7 (47.2–57.6) ⫺2.3 ⫾ 3 (⫺5.3 to 2.1)

Data are means ⫾ SD (range). HR, Heart rate (beats per minute); ABP, mean aortic blood pressure (mm Hg); dP/dtmax, left ventricular dP/dtmax (mm Hg/sec). a P ⬍ 0.0005. b P ⬍ 0.05. c P ⬍ 0.025. d P ⬍ 0.0025.

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FIG. 4. The response of mean CBF to the intracoronary infusion of ghrelin after blockade of cholinergic receptors, adrenergic receptors, and coronary nitric oxide synthase. The values of CBF obtained in each animal during the test period of measurement are plotted on the ordinate against control values before infusion on the abscissa. The continuous line is the line of equality. E, After blockade of cholinergic receptors; F, after blockade of ␣-adrenergic receptors; 䡺, after blockade of ␤2-adrenergic receptors; f, after blockade of coronary nitric oxide synthase.

ence between the two responses before and after blockade of ␣-adrenergic receptors was not significant (P ⬍ 0.20). Experiments after blockade of ␤2-adrenergic receptors. In the five pigs, blockade of ␤2-adrenergic receptors completely prevented the decrease in CBF caused by intracoronary infusion of ghrelin (Fig. 4). During the test period of measurement, changes in this flow were small and insignificant, amounting to ⫺0.1 ⫾ 0.8 (⫺1.1 to 0.6; P ⬎ 0.35) ml/min from a control value of 55.6 ⫾ 6.8 (49.3– 64.3) ml/min. In the same pigs, the decrease in mean CBF obtained with intracoronary infusion of ghrelin before giving butoxamine was 7 ⫾ 1.6 (4.7– 8.7; P ⬍ 0.0005) ml/min from control values of 61.7 ⫾ 7.2 (51.2–70.3) ml/min. Experiments after blockade of coronary nitric oxide synthase. In the five pigs, blockade of coronary nitric oxide synthase completely prevented the decrease in CBF caused by intracoronary infusion of ghrelin (Fig. 4). During the test period of measurement, changes in this flow were small and insignificant, amounting to ⫺0.1 ⫾ 0.5 (⫺0.8 to 0.5; P ⬎ 0.30) ml/min from a control value of 58.5 ⫾ 6.1 (50.2– 65.3) ml/min. In the same pigs, the decrease in mean CBF obtained with intracoronary infusion of ghrelin before giving L-NAME was 6.8 ⫾ 0.8 (5.9 –7.6; P ⬍ 0.0005) ml/min from control values of 60.3 ⫾ 5.1 (54.3– 65.4) ml/min. Discussion

The present investigation has shown for the first time that intracoronary infusion of ghrelin primarily caused a decrease in CBF and coronary vasoconstriction. The mechanisms of this effect were shown to involve the inhibition of a vasodilatory ␤2-adrenergic receptor-induced release of nitric oxide. The decrease in CBF observed in response to the intracoronary infusion of ghrelin can be attributed to a primary

Grossini et al. • Ghrelin and Coronary Blood Flow

effect of the hormone and not to concomitant changes in hemodynamic variables. The heart rate and aortic pressure were kept constant, and there were not significant changes in the filling pressures of the heart and left ventricular dP/ dtmax. This excluded any interference from reflex, local metabolic and physical effects on the coronary response (15). Moreover, intracoronary infusion of ghrelin did not cause significant changes in the values of plasma levels of GH, thus avoiding the interference of this hormone on the coronary response to ghrelin (16). In addition, intracoronary infusion of the vehicle alone at the same rate as that of ghrelin did not reproduce the effect of infused hormone. A further confirmation of the direct relationship between the hormone and its coronary response is represented by the ability to augment the decrease of CBF by increasing the dose of intracoronary infused ghrelin. Therefore, intracoronary infusion of ghrelin primarily caused coronary vasoconstriction, because this response did not involve changes in other hemodynamic variables or plasma concentration of GH. Previously reported findings obtained in humans and experimental animal models showed positive effects of ghrelin on cardiac performance. For instance, chronic administration of ghrelin has been shown to improve left ventricular dysfunction in rats with ischemic heart failure (8) and to increase cardiac index and stroke volume without altering mean pulmonary arterial pressure or pulmonary capillary wedge pressure in patients with chronic heart failure (9). In these studies, however, ghrelin administration caused an increase in the release of GH, which is known to improve cardiac performance (e.g. 17). In the present study, the acute intracoronary infusion of small doses of ghrelin did not cause changes in the plasma levels of GH or in left ventricular dP/dtmax. This is consistent with previous results in hypophysectomized rats showing that ghrelin exerted a minor role in the control of cardiac function (18) and with previous reported findings showing that administration of a single dose of ghrelin in humans with severe GH deficiency was not accompanied by acute effects on cardiac function (19). Blockade of cholinergic receptors and ␣-adrenergic receptors did not affect the coronary vasoconstriction elicited by intracoronary infusion of ghrelin, a response that was shown to be abolished by blockade of ␤2-adrenergic receptors. The dose of atropine used in this study has been previously used in anesthetized pigs to block cholinergic receptors (20). The dose of 1 mg/kg of phentolamine has been shown in the same experimental model to abolish the reflex coronary vasoconstriction caused by distension of the gallbladder (21) and has been previously used to block coronary ␣-adrenergic receptors (e.g. Refs. 13, 20). The dose of 2.5 mg/kg butoxamine has been shown in anesthetized pigs to abolish the coronary vasoconstriction caused by GH (16). The results obtained with the experiments performed after administering the blocking agents indicate that the mechanisms of the ghrelin-induced coronary vasoconstriction involved ␤2-adrenergic receptor effects. The administration of butoxamine caused an increase in the baseline values of aortic pressure and coronary vascular resistance, but these effects were not involved in the blockade of the coronary response to ghrelin because intracoronary infusion of the hormone did not cause significant changes in CBF even when the increase in aortic

Grossini et al. • Ghrelin and Coronary Blood Flow

pressure and coronary vascular resistance was reversed by papaverine. However, the butoxamine-induced increases in aortic pressure and coronary vascular resistance indicated that ghrelin caused coronary vasoconstriction by blocking a tonic ␤2-adrenergic receptors-mediated vasodilatory effect. This is consistent with previous reported findings showing a tonic ␤2-adrenergic receptors-mediated vasodilatation in the coronary and several other peripheral vascular beds in anesthetized pigs (13, 14, 22). In the present study, the coronary vasoconstriction caused by intracoronary infusion of ghrelin was abolished by blockade of nitric oxide synthase with the intracoronary injection of L-NAME. The dose of 100 mg of the blocking agent has been previously shown in the same experimental model to cause a reduction of the acetylcholine-induced increase in CBF (23, 24), to be a reliable marker of the inhibition of nitric oxide release (25), and to abolish the coronary vasodilation caused by progesterone and testosterone and the coronary vasoconstriction caused dehydroepiandrosterone (13, 20, 26). Also, L-NAME caused an increase in baseline aortic pressure and coronary vascular resistance. This effect, however, did not influence our results because intracoronary infusion of ghrelin did not elicit significant changes in CBF when the increases in aortic pressure and coronary vascular resistance were reversed by papaverine. These results showed that the tonic ␤2-adrenergic receptor-mediated vasodilatory effect blocked by intracoronary infusion of ghrelin involved the endothelial release of nitric oxide. This is consistent with previously reported findings showing that the release of nitric oxide from the endothelium can modulate or mediate ␤-adrenergic effects in the coronary and peripheral vasculature (27–31). Previous reported findings on the role of nitric oxide in the vascular effects of ghrelin are controversial, with either an involvement (32) or not (33, 34). However, these studies have been performed either in isolated vessels or healthy subjects with systemic administration of the hormone, thus without avoiding confounding effects arising from the release of GH (1) and the known centrally induced inhibition of sympathetic neural output (35). The present findings provide important implications regarding the humoral control of the coronary circulation in anesthetized pigs. For instance, previous reports have shown that testosterone, 17␤-estradiol, and progesterone caused coronary vasodilatation through mechanisms which involved the endothelial release of nitric oxide (20, 24, 26). On the other hand, GH and dehydroepiandrosterone have been shown to cause coronary vasoconstriction through blockade of a ␤-adrenergic receptor-mediated tonic vasodilatory effect related to the endothelial release of nitric oxide (13, 16, 36). Finally, insulin has been shown to cause coronary vasoconstriction as the net result of a vasoconstriction mediated by sympathetic ␣-adrenergic effects and a vasodilatation related to the release of nitric oxide (37). It is important to point out that the present results have been obtained with the acute intracoronary administration of ghrelin in experimental conditions that enabled quantifying the primary responses of CBF and avoiding confounding factors. However, these considerations make it possible to suggest a role of ghrelin in the control of the coronary circulation. Thus, the coronary vasoconstriction caused by ghrelin through the blockade of a

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tonic coronary ␤2-adrenergic receptor-mediated vasodilatory effect could be argued to balance the vasodilatory effect caused by the central blockade of the sympathetic vasoconstrictive discharge elicited by the hormone (35). Although the role of vascular GH secretagogue receptors on the coronary response to the acute intracoronary administration has not been examined in the present study. There has been evidence indicating that GH secretagogue receptors are present in cardiovascular tissues, including the coronary arteries of various species and humans (3, 4, 18, 38 – 40). Also, it is worth mentioning that in isolated heart preparation GH secretagogue receptors have been shown to be involved in the coronary vasoconstriction caused by the synthetic GH-releasing hexapeptide hexarelin (41) and that mechanisms of action of ghrelin involving receptors other than GH secretagogue receptors have been described (42). Finally, our findings are consistent with the observation in humans that ghrelin receptor density in the coronary artery is associated with atherosclerosis and coronary artery disease (4). In conclusion, the present study has shown that intracoronary infusion of ghrelin causes coronary vasoconstriction. The mechanisms of this effect were shown to involve the inhibition of a tonic coronary ␤2-adrenergic receptor-mediated vasodilatory effect related to the release of nitric oxide. Acknowledgments We thank the Azienda Ospedaliera Maggiore della Carita` di Novara for its help. Received September 7, 2006. Accepted November 6, 2006. Address all correspondence and requests for reprints to: Dr. E. Grossini, Facolta` di Medicina e Chirurgia, Universita` del Piemonte Orientale “A. Avogadro,” via Solaroli 17, I-28100 Novara, Italy. E-mail: [email protected]. This work was supported by Universita` del Piemonte Orientale “A. Avogadro.” Disclosure Statement: The authors have nothing to disclose.

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