Arginine vasopressin (AVP) is a peptide released from the posterior pituitary gland and known to play an important role in body fluid and cardiovascular homeostasis (1). This peptide has an antidiuretic activity by interacting with V2 receptors linked with the adenylate cyclase (cyclic adenosine monophosphate; cAMP) system (2). It also causes a potent vasoconstriction by stimulating V1 receptors located on vascular smooth-muscle cells and coupled with the phosphoinositol system (3). The pressor response to AVP is markedly attenuated by a concomitant decrease in cardiac output, which may be caused by a direct effect on myocytes or a potentiation of the baroreceptor reflex or both (4,5).
The main effect of AVP in intact mammals is vasoconstrictor, as revealed by the use of selective V1 vasopressin antagonists (6), but the availability of such antagonists unmasked a vasodilatory effect of vasopressin, both in dogs and in rats (7,8). The vasorelaxation produced by AVP appears to be endothelium dependent. This was suggested by experiments performed in isolated vascular preparations (9-11), as well as in intact dogs (12). It is, however, still controversial whether the endothelium-mediated vasodilatation induced by AVP is triggered by the activation of V1 (9-11) or V2 receptors (12). There exist, however, differences in the ability of arteries to vasodilate in response to AVP. For example, the arteries composing the circle of Willis are more sensitive to AVP, in terms of vasorelaxation, than are other intracranial and extracranial arteries, as demonstrated angiographically in dogs (13). There is, however, still no information on the in vivo comparative response to AVP and other vasoconstrictors in the precerebral vasculature.
This study was undertaken in intact anesthetized rats to assess the effects of equipressor doses of various agonists on the diameter of the right carotid artery. These diameter measurements were performed noninvasively by using a high-precision ultrasonic echo-tracking device. The test agonists were the α1-adrenoceptor stimulant methoxamine, lysine vasopressin (LVP), and angiotensin II (Ang II).
Male Wistar rats weighing between 240 and 280 g were obtained from Iffa-Credo (Lyon, France). Animal care, surgical preparation, and experimental procedures were approved by the Government Review Committee for animal experiments. On the day of the experiment, anesthesia was given and maintained with halothane/O2 (Halothane BP, 1.5%; Anovet AG,) under spontaneous ventilation. The left common carotid artery and the right femoral vein were cannulated with a PE-50 (Portex) and a PE-10 catheter, respectively. The catheters contained heparin (1 U/ml 0.9% NaCl). Intraarterial pressure and heart rate were continuously monitored with the use of a computerized data-acquisition system, as described previously (14). The venous catheter was used to administer the various test substances, which consisted of either LVP (Vasopressin-Sandoz; Sandoz-Wander Pharma AG, Bern, Switzerland), angiotensin II (Hypertensin-Ciba; Ciba-Geigy Limited, Basel, Switzerland), methoxamine (Vasoxine; The Wellcome Foundation Ltd, London, England), or vehicle (0.9% NaCl). In some rats, the selective V1-antagonist d(CH2)5Tyr(Me)AVP was used (15). This compound was dissolved in 0.9% NaCl to achieve the concentration of 100 μg/ml. A syringe pump was used for the infusions.
The internal diameter of the right common carotid artery was measured with an A-mode ultrasonic echo-tracking device (Diarad; Asulab) that was already used in rats (16,17). This apparatus allows measurement of variations in the diameter of the carotid artery with a precision close to 1 μm. The high resolution reached with this device is made possible by oversampling (5,000 arterial diameter measurements per second) and averaging 16 consecutive values. A 10-MHz localized transducer is placed perpendicularly to the arterial axis by using Doppler mode, and an ultrasonic gel is used for signal transduction.
The carotid artery diameter was determined in eight groups of rats. The animals were allocated to the different study groups in a random fashion. After initial instrumentation, the rats were left to stabilize the blood pressure and heart rate for 30 min. They were then infused for 60 min with either angiotensin II, 10 ng/min (n = 8, group I); methoxamine, 5 μg/min (n = 8, group II); LVP, 5 mU/min (n = 9, group III); LVP, 1 mU/min (n = 8, group IV); or vehicle, 10 μl/min (n = 8, group V). In other rats, the LVP infusion was started 10 min after administration of d(CH2)5Tyr(Me)AVP, 10 μg, i.v., as a bolus. In 16 and 11 rats pretreated with the V1-receptor antagonist, LVP was infused at a rate of 5 mU/min (group VI) or 1 mU/min (group VII), respectively. Additional rats received the V1-antagonist d(CH2)5Tyr(Me)AVP, 10 μg, i.v., as a bolus, followed by an infusion of 0.9% NaCl (n = 8, group VIII). All rats were killed at the end of the experiment with a lethal dose (90 mg/kg, i.v.) of pentobarbital (Hospital Pharmacy).
The results are expressed as means ± SEM. The statistical significance of differences was evaluated by two-way analysis of variance (ANOVA) followed by a Fisher's least significant test. A p value of <0.05 was considered significant. Both within- and between-group comparisons were performed.
There was no significant difference in mean blood pressure between the various study groups 10 min before (t−10) as well as immediately before (t0) the start of the 60-min infusion (Table 1). Angiotensin II (group I), methoxamine (group II), and the 5 mU/min dose of LVP (group III) significantly increased mean blood pressure, as assessed by ANOVA (p < 0.05), whereas no significant change was observed in 0.9% NaCl-infused rats (group V). The blood pressure increase was of similar magnitude with the three agonists. When compared with control rats (group V), blood pressure was significantly higher at times t10, t20, and t40 in rats of groups I-III. The smallest dose of LVP (group IV) had no significant blood pressure effect. The V1-receptor antagonist per se did not change blood pressure (group VIII). It prevented, however, the blood pressure increase induced by the highest dose of LVP (group VI).
The values of the internal diameter of the common carotid artery measured at mean blood pressure are shown in Table 2. No significant difference was found between groups during baseline evaluation. Angiotensin II, methoxamine, and the two doses of LVP significantly increased the internal carotid diameter (groups I-IV). The diameter measured during the high dose of LVP was significantly greater (p < 0.05) than that obtained with methoxamine or Ang II despite the equal blood pressure increase. In rats infused with 0.9% NaCl (group V), as well as in all rats having received the V1-receptor antagonist, a significant decrease in the carotid artery diameter was observed during the course of the study (groups VI-VIII), with no significant difference, however, between these four groups.
Figure 1 shows the relation between mean blood pressure and arterial diameter (measured at mean blood pressure) at times 10, 20, 40, and 60 min after the start of the angiotensin II, methoxamine, LVP, or 0.9% NaCl infusion (groups I-V). It appears that LVP-treated rats exhibit, at any blood pressure, higher values of arterial diameter than all other groups of rats.
No significant change in heart rate was observed in comparison with 0.9% NaCl-infused rats (group V) except in rats infused with the highest dose of LVP (group III). In those animals, the increase in blood pressure was associated with a significant slowing of heart rate (Table 3).
Angiotensin II (Ang II), methoxamine, and LVP are potent vasoconstrictors. Their pressor effect is due to an enhanced arteriolar tone and, thereby, an increase in peripheral resistance. These agonists act by stimulating receptors located on vascular smooth-muscle cells (i.e., the AT1-receptor for Ang II, the α1-receptor for methoxamine, and the V1 receptor for LVP). These three receptors are present not only in the resistance vasculature but also in muscular and elastic conduit arteries. Not surprisingly, therefore, activation of AT1-, α1-, and V1 receptors causes contraction of medium and large arteries studied in vitro. There is, however, still little information on the contractile response of conduit arteries in vivo. This is mainly because the technical tools to measure in vivo small changes in arterial diameter were missing until recently.
In this study, a high-resolution ultrasonic echo-tracking device (16,17) was used to explore the changes in diameter occurring at the carotid artery while blood pressure was increased by infusion of vasoconstrictors. The experiments were performed in intact halothane-anesthetized normotensive rats. Equipressor doses of Ang II, methoxamine, and LVP were first infused for 60 min. A striking feature was that the increase in blood pressure was associated with an increase rather than a decrease in the arterial diameter. Such an arterial response may reflect a passive distention consecutive to the increased blood pressure. Conceptually, this distention could even occur if the tone of the blood vessel were at the same time increased. Unfortunately, the exact contribution of the counteracting distending and contractile forces to the regulation of the diameter of a large elastic vessel like the carotid artery cannot be evaluated with our method. The rats were anesthetized with halothane, which may also modulate the changes in arterial diameter in response to changes in blood pressure. This might explain why the parallelism between arterial and pressure changes becomes looser at the end of the 60-min observation period.
Unexpectedly, LVP caused a clearly more pronounced increase in arteriolar diameter for a pressor effect of magnitude similar to that of Ang II and methoxamine. Moreover, LVP increased the arterial diameter even when it was infused at a dose lacking any effect on systemic blood pressure. These observations point to an active dilatation of the carotid artery during V1-receptor stimulation. In this blood vessel, vasopressin seems therefore to have a predominantly vasodilating effect.
How could an active vasodilatation during LVP infusion be explained? One possibility is an endothelium-dependent mechanism. Endothelial cells release vasodilatory substances such as nitric oxide (NO) and prostacyclin (18). Functional vasopressin receptors are present on endothelium, as demonstrated in cultured endothelial cells (19). The vasorelaxant activity of vasopressin seems to depend on the release of NO rather than prostacyclin. Thus in isolated canine brainstem arteries, vasopressin induces a vasodilatation despite concurrent inhibition of cyclooxygenase activity, whereas blockade of NO synthesis prevents the relaxation (9,11). In rat renal, mesenteric, and hindquarters vascular beds, however, both the NO- and the cyclooxygenase-dependent vasodilator mechanisms seem to oppose the vasoconstrictor effects of vasopressin (20).
Another important question is whether the relaxation induced by vasopressin is due to the activation of V1- or V2-vasopressinergic receptors. In our hands, the selective V1 antagonist d(CH2)5Tyr(Me)AVP completely abolished the change in arterial diameter mediated by LVP infusion, whether administered at pressor or nonpressor doses. This is a very strong argument in support of a V1-mediated mechanism, which is in agreement with previous observations made by using d(CH2)5Tyr(Me)AVP in isolated canine basilar arteries (9) and in intact rats (8). In the latter experiments, rats were infused with a pressor dose of vasopressin. A vasodilatory effect of this peptide was suggested by the finding of a decrease in blood pressure and total peripheral resistance in response to the V1 antagonist to levels below those preceding the start of the vasopressin infusion. In contrast, investigations carried out in conscious dogs showed that V2 agonists may cause a vasodilatation, as reflected by an increased total systemic conductance, and that this vasodilatation can be blunted by an inhibitor of NO synthase (12).
Interestingly, vasopressin infusion in conscious rats has been reported to decrease carotid blood flow and to increase carotid vascular resistance (21). This effect was reversed by administering a V1-receptor antagonist, whereas the stimulation of the V2 receptor by a specific agonist had no effect on carotid blood flow and resistance. These findings point therefore to an action of vasopressin on V1 receptors in the carotid circulation.
The release of NO from the endothelium can also be triggered by shear stress. It is obviously impossible to assess whether endothelial cells are exposed to differential shear stress during infusion of Ang II, methoxamine, and LVP. In fact, the low dose of LVP had no effect on systemic blood pressure, which is presumably associated with a smaller shear stress than when blood pressure is increased, but this low dose of LVP was still able to cause a vasodilatation.
That vasopressin can cause an endothelium-dependent relaxation, as shown here at the level of the rat carotid artery, does not apply necessarily to all blood vessels. Regional differences have been reported in canine cerebral arteries in vivo, the arteries composing the circle of Willis being more sensitive to vasopressin than are other intracranial or extracranial arteries (13). Some peripheral arteries, like the femoral artery of the dog, may even not vasodilate in response to vasopressin (9). It is still largely unknown how V1-vasopressinergic receptors are distributed in the different vascular beds of the mammalian organisms.
In summary, our study in intact rats demonstrates that the diameter of the carotid artery increases during infusion of pressor doses of Ang II, methoxamine, and LVP. It appears, however, that LVP causes an active dilatation, even when infused at a nonpressor dose. This vasorelaxation is mediated by V1-vasopressinergic receptors and most likely involves endothelium-dependent mechanisms.
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