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Gen. Pharmac. Vol. 26, No. I, 143-148, 1995 pp. Copyright 0 1995 Elsevier Science Ltd

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Effect of N”-nitro-L-arginine Methyl Ester on Cardiac Haemodynamic Responses to

Adenosine Infusion in Conscious Rats ISABEL HERNANDEZ, TOM& QUESADA and LUIS F. CARBONELL

Departamento de Fisiologia, Facultad de Medicina, 300100: Murcia, Spain /Tel: 34 68 3639521

(Received 26 April 1994)

Abstract-l. The aim of the present study was to evaluate the role of NO in the cardiovascular effects of adenosine in conscious rats.

2. Cardiac index was determinated by thermodilution. In a group of rats, three doses of adenosine were infused (iv.) at a rate of 150, 300 and 450pg/kg/min in the absence and in the presence of L-NAME (10 mg/kg). In a second group of rats, the experimental protocol was the same as that of the first group, except an infusion of methoxamine (50 pg/kg/min) was given during the second adenosine administration, instead of L-NAME.

3. In the absence of L-NAME or methoxamine, adenosine induced a dose-dependent decrease in mean arterial pressure and an increase in vascular conductance although adenosine did not affect cardiac index.

4. L-NAME administration attenuated the decreasing effect on the mean arterial pressure in response to the two lower doses of adenosine. In the presence of L-NAME, adenosine induced a significant increase in cardiac index from 18.7 f 1.5 to 29.1 + 1.9 and 26.2 k 1.4 ml/min/lOO g. Administration of L-NAME significantly attenuated the adenosine-induced increase in vascular conductance.

5. Methoxamine infusion induced an enhanced response to adenosine infusion. In the presence of methoxamine, adenosine induced a significant greater decrease in mean arterial pressure, and increase in cardiac index and vascular conductance.

6. These results indicate that part of the cardiovascular effects of adenosine can be mediated by NO, since L-NAME administration partially blocked the adenosine-induced vasodilatation.

Key Words: N’“-nitro-L-arginine methyl ester (L-NAME), adenosine, haemodynamic, cardiac index, vascular conductance

INTRODUCTION

The relaxing effect of endothehum by vasoactive agents has been extensively studied as a modulating factor in vascular responses (Furchgott, 1984; Tesfamarian and Halpern, 1988). Adenosine, an en- dogenous nucleoside that participates in a multitude of biochemical and physiological process, relaxes blood vessels by acting on A, receptors, and are often associated with activation of adenyl cyclase (Londons and Wolff, 1977). Vascular endothelial cells have been shown to release several vasoactive agents either spontaneously or in response to chemical or mechanical stimuli, and an important endothelium- derived relaxing factor derived from L-arginine is nitric oxide (NO) (Ignarro et al.. 1981; Moncada et al., 1991).

Synthesis of NO can be blocked by analogues of

L-arginine, such as N”‘-nitro-L-arginine methyl ester (L-NAME) (Moore et al., 1990). Recently several studies have been published about the haemodynamic effects of L-arginine analogues. L-NAME, adminis- tered in vivo, increases peripheral resistance and aortic pressure and reduces cardiac output, evidently by preventing a normal continuous release of NO from endothelial cells (Gardiner et al., 1990; Van Gelderen et al., 1991; Bower and Law, 1993). On the other hand, adenosine receptor agonist reduced mean arterial pressure and increased the systemic vascular conductance index and cardiac index in anaesthetized dogs (Gerencer et al., 1992).

Inasmuch as the adenosine-induced vasorelaxation was not abolished by removing the endothehum (Furchgott, 1983) adenosine relaxes vascular smooth muscle in an endothelium-independent manner. However, it was reported in several studies that

143

144 Isabel Hernindez et al.

adenosine also causes endothelium-dependent vaso- relaxation in canine coronary arteries (Rubanyi and Vanhoute, 1985) guinea pig aorta (Headrick and Berne, 1990) and rat aorta (Moritoki et al., 1990). All these studies were performed in vitro on isolated blood vessels and no in vivo studies have been reported.

Therefore, the purpose of the present study was to investigate the role of NO in the cardiovascular action of adenosine in conscious rats. First, we compared the haemodynamic effects of adenosine before and after the inhibition of NO synthesis by the administration of L-NAME. Second, because the administration of L-NAME induced a rise in blood pressure, we compared the adenosine effects in the presence of an c( -antagonist vasoconstrictor (methoxamine) at a dose that increases blood pressure at the same level as that observed with NO inhibition.

MATERIALS AND METHODS

Experiments were performed on male Sprague- Dawley rats (350-450 g). All the experimental proto- cols were carried out in previously instrumentized conscious rats.

Surgical Procedures

Catheters were placed into the left femoral artery for measurement of arterial pressure and heart rate (Tygon microbore tubing, S-S4HL), and into the left femoral vein for infusions (Clear vinyl tube, double lumen i.d. 0.41 mm, o.d. l.Omm). A right atria1 catheter (Silastic, Dow Corning, 1 mm o.d., 0.5 mm id.) and a thoracic aortic thermocouple (Columbus Instruments) were implanted via the right external jugular vein and right carotid artery, respectively. The catheters were brought out through the skin on the dorsal side of the neck. Finally, the distal ends of these lines were thread through a lightweight flexible spring connected to a swivel. All surgical procedures were performed under aseptic techniques. Rats were placed in plastic cages with the swivels mounted above, allowing complete freedom of movement and free access to chow and tap water. Two full days were permitted for recovery from surgery and acclimatiz- ation to the environment, and haemodynamic studies were conducted while the rats rested quietly in their home cages.

Cardiac output was measured by thermodilution as previously described in our laboratory (Hernandez et al., 1991). The thermodilution curve and the pressure signal were processed with a microcomputer system (Cardiomax IIR, Columbus Instruments). Haemodynamic values were the mean of two determi-

nations. Cardiac output was measured by rapid injec- tion of 200~1 0.9% saline at room temperature (20°C) through the jugular catheter, using a spring- loaded constant-rate, constant-volume syringe (Hamilton, model CR 700-200). Cardiac output and mean arterial pressure were digitally obtained by the microcomputer. Cardiac index was calculated by dividing cardiac output by animal weight (100 g) and total peripheral conductance by dividing cardiac ina- dex by mean arterial pressure.

Experimental Protocols

Effect of L-NAME on adenosine-induced hemo- dynamic changes

Group 1 (n = 7). On the day of experiment, after an equilibration period, baseline measurements of heart rate (HR), mean arterial pressure (MAP), cardiac index (CI) and total peripheral conductance (TPC) were made. An i.v. infusion of adenosine (Sigma) was then started at a rate of 150 pg/kg/min. Ten minutes later, when the haemodynamics had stabilized, HR, MAP, CI and TPC were measured again. The adenosine infusion rate was then increased, and after at least 10 min of infusion of the new dose of adenosine and no less than 15 min after the last determination, haemodynamics, including MAP, CI and TPC were determinated. In this control period, a total of three doses of adenosine were adminis- trated, 150, 300 and 450 pg/kg/min. 20-30 min were allowed for restoration of basal hemodynamic parameters, then an i.v. bolus of N”‘-nitro-L-arginine methyl ester hydrochloride (Sigma), (L-NAME, 30mg/kg b.w.) was administered to the animals. MAP were continuously recorded until blood press- ure stabilization, and haemodynamic variables were determined again and a second infusion of adenosine was given at the same three doses as before.

Effect of methoxamine on adenosine -induced hemo - dynamic changes

Group 2 (n = 5). After the first infusion of the three doses of adenosine with a recovery period of 30 min, we infused methoxamine (wellcome) at a rate of 50 pg/kg/min in order to achieve the same level of MAP as in group 1 with L-NAME. The infusion was maintained during the remainder of the protocol and a second infusion of adenosine was given. The rest of the experimental protocol was the same as for group 1.

Statistical Analysis

Values are mean f SEM. A two-way repeated measure analysis of variance (ANOVA) was used to

L-NAME and adenosine-induced vasodilatation

Table I. Haemodynamic variables before and I5 min after administration of L-NAME (IO mg/kg) or methoxamine (50 &kg/min)

Baseline L-NAME Baseline Methoxamine

Heart rate (beats/min) 386 f 16 276 + 21. 345 f 8 308 f IS* Mean arterial pressure (mmHg) ll4&3 I51 + 5’ lO7&6 154*7* Cardiac index (ml/min/lOO g) 41&3 I9 + 28 42 4 4 40 + 4t Stroke volume (PI/beat) IlO? IO 70 * 10’ 120 4 IO 130 f lot Total peripheral conductance (~l/min/mmHg/lOO g) 370 f 30 120 * 200 390 f 30 260 f 20*t

Values are mean f SE, *P < 0.05 vs baseline; tP < 0.05 vs L-NAME.

145

analyze the data. Significant differences between indi- vidual means were determined using a Duncan mul- tiple range test. Values of P < 0.05 were considered significant.

RESULTS

Basal values and the effect of L-NAME and methoxamine on heart rate, mean arterial pressure, cardiac index and total peripheral conductance in groups 1 and 2 are shown in Table 1. A similar significance increase (P < 0.05) in mean arterial pressure was achieved with both drugs. Mean arterial pressure increases from 114 + 3 to 151 + 5 mmHg with L-NAME, and from 106 + 6 to 154 f 7 mmHg with methoxamine. The increase in mean arterial pressure with L-NAME was accompanied by a signifi- cant decrease in cardiac index, around 50% (P < 0.05). In contrast, no change in cardiac index was detected with the same increase in blood pressure induced by methoxamine. Total peripheral conduc- tance decreased with both drugs. Whilst with L- NAME total peripheral conductance fell 70% from the basal value, with methoxamine the decrease in this parameter was significantly lower, around 35% (P < 0.05).

EfSect of L-NAME on adenosine-induced haemo- dynamic changes

The effects of L-NAME on adenosine-induced changes in mean arterial pressure are shown in Fig. 1 (panel A). In the absence of L-NAME, adenosine induced a dose-dependent decrease of mean arterial pressure (P < 0.05). In the presence of L-NAME there were significant attenuations of the responses to the two lower doses, but not to the higher dose of adenosine. Adenosine-induced changes in cardiac index are summarized in Fig. 1 (panel B). In the absence of L-NAME, adenosine did not affect cardiac index but a slight, although significant, increase was seen after administration of L-NAME, with the higher doses of adenosine, although there were no differences before or after L-NAME administration. Figure 1 (panel C) shows changes in total peripheral

conductance in response to adenosine. There was a dose-dependent increase in total peripheral conduc- tance in response to adenosine in the absence of L-NAME. In the presence of L-NAME, the rise in total peripheral conductance in response to the three doses of adenosine was clearly attenuated.

25 A r

-15 : C 150 300 450

Adenosine (pglkglmin)

25r B

-10 3 C 150 300 450

Adenosine (pglkglmin)

o.6 r c

-0.2 1 I I I I C 150 300 450

Adenosine (pglkglmin) Fig. I. Changes in cardiovascular variables during infusions of three doses of adenosine in the absence (0) and in the presence (0) of NC-nitro+arginine methyl ester (L-NAME) at a dose of IO mg/kg. Values are mean and bars show SEM *P <0.05 vs baseline; tP < 0.05 between

groups. (Mean arterial pressure, MAP.)

146 Isabel Hernindez et al.

-15 - C 150 300 450

Adenosine (pg/kg/min) 30 25

20 I5 IO 5 0

-5 -10 t

B * T .at

s::, , C 150 300 450

Adenosine (kg/kg/min) 0.6 C

0.5

0.4

0.3

0.2

0.1

0.0 !

C I50 300 450

Adenosine (pg/kg/min)

Fig. 2. Changes in cardiovascular variables during infusions of three doses of adenosine in the absence (0) and in the presence (0) of methoxamine infusion at a rate of 50pg/kg/min. Values are means and bars show SEM, *P < 0.05 vs baseline; tP < 0.05 between groups. (Mean

arterial pressure, MAP.)

EfSect qf methoxamine on adenosine-induced haemo- dynamic changes

In group 2, treated with methoxamine, adenosine had a dose-dependent effect, decreasing mean arte- rial pressure in the absence of methoxamine infusion (Fig. 2, panel A). In the presence of methoxamine, there was a significant enhancement of hypotensive response to the three doses of adenosine. In the absence of methoxamine, cardiac index was un- affected by either dose of adenosine (Fig. 2, panel B), whereas in the presence of methoxamine there was a significant augmentation in cardiac index in response to the higher dose of adenosine, although the lower dose of adenosine did not change this parameter. As shown in Fig. 2 (panel C) in the absence of methoxamine, adenosine caused a dose-

dependent increase in total peripheral conductance, and in the presence of methoxamine, this effect on vascular conductance was significantly augmented in response to the higher doses, but not to the lower dose of adenosine.

DISCUSSION

In the present work, we compared the haemo- dynamic effects of adenosine in the absence and in the presence of L-NAME, a potent inhibitor of nitric oxide synthesis (Moore et al., 1990; Ress et al., 1990), in order to investigate the role of NO in the cardio- vascular action of adenosine. Administration of L-NAME caused hypertension associated with re- ductions in cardiac index and total vascular conduc- tance (Table 1). These findings are consistent with L-arginine-nitric oxide pathways acting tonically in vivo to control vascular conductance, and are in concordance with those reported by Gardiner et al.

(1990) in conscious Long-Evans rats. On the other hand, a similar increase in blood pressure induced by methoxamine was accompanied by no change in cardiac index and less reduction in total vascular conductance. Since, the blood pressure levels are the same with methoxamine as with L-NAME, the differ- ence in response of cardiac index and vascular con- ductance to these two drugs cannot be attributed to a major sympathetic baroreflex-mediated inhibition in the presence of L-NAME.

As shown in Fig. 1, the administration of adenosine alone caused a dose-related decrease in blood pressure, whereas in the presence of L-NAME there was a significant attenuation of the response to the lower doses, but not to the higher dose of adenosine. From the results of blood pressure the involvement of NO in the response of adenosine is not clear, since L-NAME did not attenuate the hypotensive effect of the higher dose of adenosine. In order to a properly assess the involvement of NO on the vasoactive effect of adenosine, other haemo- dynamic parameters such as cardiac index and vascular conductance were determined.

As with previous results, reported by Gerencer et al. (1992) using two different adenosine analogues, in the present study, adenosine caused a slight but not significant increase in CI with a significant increase in vascular conductance when administrated alone. This result and the concomitant decrease in blood pressure are in concordance with arteriolar dilatation. On the other hand, the combination of adenosine with L-NAME resulted in slight increases in CI and in a marked attenuation of vascular conductance adenosine-induced response, suggesting that part

L-NAME and adenosine-induced vasodilatation 147

of the adenosine-induced increase in vascular conductance could be mediated by NO.

In general, the vasodilator effect of adenosine has been found not to depend on endothelium (White and Angus, 1987; Mathie et al., 1991), but other in vitro studies by Rubanyi and Vanhoute (1985) show evi- dence that part of the vasorelaxation induced by adenosine is endothelium-dependent in addition to the endothelium-independent relaxation of vascular smooth muscle strips. More recent investigations by Headrick and Berne (1990) and Moritoki et al. (1990) have revealed that the activation of the A2 adenosine receptor is responsible for the endothelium-depen- dent vasorelaxation induced by adenosine. We found that in conscious rats, inhibition of NO synthesis by L-NAME infusion considerably attenuated the in- creased adenosine-induced vascular conductance. These results are in concordance with an other in vivo study by Gardiner et al. (1991) where they showed a differential involvement of L-NAME-susceptible vasodilator responses to A, receptor agonist (NECA) in different vascular beds. From that study it appears that the hindquarters and internal carotid vascular beds showed vasodilator responses to NECA that are attenuated by L-NAME.

It is known that when vasculature is vasocon- stricted, the administration of a vasodilator agent results in an enhanced response. This is the case of adenosine increasing vascular conductance in the presence of methoxamine (a selective x-agonist). Our results showed an enhanced effect of adenosine induc- ing hypotension and increasing vascular conductance and cardiac index in the presence of methoxamine, indicating a total reversibility of the vasoconstrictor effect of methoxamine by adenosine infusion. How- ever, since L-NAME administration resulted in a lower vascular conductance than methoxamine infu- sion, we could expect a greater response to adenosine infusion in the presence of L-NAME instead of the attenuated effect seen in the present work. Thus, this discrepancy supports the idea that part of the haemo- dynamic effects of adenosine can bc mediated by nitric oxide.

In summary, our observations suggest an inter- action between the vasorelaxant effect of adenosine and inhibition of NO synthesis by L-NAME, indi- cating that part of the increase in vascular conduc- tance induced by adenosine could be mediated by a mechanism nitric oxide-dependent. Finally, it is ap- parent that proper assessment of the involvement of L-NAME in the response to adenosine, must involve measurement of haemodynamic parameters such as cardiac index and vascular conductance, since moni- toring mean arterial blood pressure alone would give misleading results.

SUMMARY

The purpose of the present study was to evaluate the role of NO in the cardiovascular effects of adenosine in conscious rats. Cardiac index was deter- minated by thermodilution. In a group of rats, three doses of adenosine was infused (i.v.) at a rate of 150, 300 and 450pg/kg/min in the absence and in the presence of L-NAME. Because the administration of L-NAME induced a rise in blood pressure, we com- pared the adenosine effects in the presence of methox- amine at doses that increase blood pressure at the same level as L-NAME. Thus, in a second group of rats, the experimental protocol was the same as that in the first group, except an infusion of methoxamine (50 g/kg/min) was given during the second adenosine administration, instead of L-NAME. A similar increase in mean arterial pressure was achieved with L-NAME and methoxamine, although L-NAME in- duced a greater decrease in cardiac index and vascu- lar conductance than methoxamine. In the absence of L-NAME or methoxamine, adenosine had a dose- dependent decrease in mean arterial pressure and increase in vascular conductance although adenosine did not affect cardiac index. L-NAME administration attenuated the decreasing effect on mean arterial pressure in response to the two lower doses of adenosine and significantly attenuated the adenosine- induced increase in vascular conductance. Methox- amine infusion induced a enhanced response to adenosine infusion. In the presence of methoxamine, adenosine induced a significantly greater decrease in mean arterial pressure, and a greater increase in cardiac index and vascular conductance. These re- sults suggest an interaction between the vasorelaxant effect of adenosine and inhibition of NO synthesis by L-NAME, indicating that part of the increase in vascular conductance induced by adenosine could be mediated by a nitric oxide-dependent mechanism.

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