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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 6, 2001 as doi:10.1096/fj.00-0633fje. |
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* Dipartimento di Farmacologia Sperimentale and
Dipartimento di Chimica Farmaceutica, Università degli Studi di Napoli ‘Federico II’, via D. Montesano, 49 80131 Naples, Italy
2Correspondence: Dipartimento di Farmacologia Sperimentale, Via Domenico Montesano 49, 80131 Naples, Italy. E-mail: cicala{at}unina.it
SPECIFIC AIMS
To clarify the role played by protease activated receptors 1 and 2 (PAR-1 and PAR-2) in the control of blood pressure, we have evaluated changes in blood pressure induced by a peptide activating the receptor PAR-1 (PAR-1AP, SFLLRNPND) and a peptide activating the receptor PAR-2 (PAR-2AP, SLIGRL) in naive and ganglion-blocked anesthetized rats. To investigate the role played by nitric oxide (NO), the effect of PAR-1AP and PAR-2AP in ganglion-blocked rats was evaluated before and after L-NAME administration.
PRINCIPAL FINDINGS
1. Both PAR-1AP and PAR-2AP cause a biphasic change in MABP in rats
Intravenous (i.v.) injection of PAR-1AP (1 mg/kg) into naive
anesthetized rats caused a biphasic response characterized by
hypotension (13±2 mmHg; n=6), immediately followed by
hypertension (21±3 mmHg; n=6), returning to baseline value
in 3 min; i.v. injection of PAR-2AP (1 mg/kg) caused a hypotension
(28±5 mmHg; n=5) lasting about 2 min. Hypotension was
followed by a slowly developing hypertension, reaching its peak value
(19±4 mmHg; n=5) after 
5–6 min and steadily decreasing
to baseline. Repeated administration of PAR-1AP or PAR-2AP did not
cause desensitization to the effects observed. Control peptides (1
mg/kg i.v.) were both without effect.
2. Ganglion-blocking treatment increases PAR-1AP-induced
hypertension and abolishes PAR-2AP-induced hypertension
In rats treated with the irreversible ganglion-blocking agent
chlorisondamine (2.5 mg/kg) intraperitoneal (i.p.), PAR-1AP-induced
hypertension was significantly increased compared with values obtained
in naive rats (Fig. 1A
). Hypotension was not modified.
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Hypertension induced by PAR-2AP was abolished after ganglion blockade
(Fig. 1B
).
PAR-2AP-induced hypotension evaluated as a maximum decrease was not modified by the ganglion-blocking agent, but the endurance, evaluated as area under the curve (AUC) percentage change below baseline over the time, was prolonged (AUC 150±14% n=6; vs. 62±2%, n=5; P<0.01).
3. NO accounts for the endurance of hypotension induced by PAR-2AP
in ganglion-blocked rats
In ganglion-blocked rats, infusion of L-NAME reduced the endurance
of hypotension induced by PAR-2AP to naive rat values. Nonetheless,
L-NAME increased the maximum peak of hypotension induced by PAR-2AP
(Fig. 2A
). To discriminate between effects of L-NAME specifically
dependent on NO inhibition and those dependent on the increase in
vascular tone caused by L-NAME, we also performed experiments in
ganglion-blocked rats in which blood pressure was restored by infusing
angiotensin II (ANG II); responses to PAR-1AP or PAR-2AP (1 mg/kg i.v.)
were evaluated before and after the infusion. ANG II did not reduce the
endurance of PAR-2AP-induced hypotension; however, similar to
L-NAME, it increased the maximum peak of PAR-2AP-induced
hypotension (Fig. 2B
). Neither L-NAME nor ANG II modified
hypotension induced by PAR-1AP.
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4. NO modulates hypertension induced by PAR-1AP and PAR-2AP
After L-NAME infusion in ganglion-blocked rats, hypertension
induced by PAR-1AP was still higher than values obtained in naive
animals. This effect was abolished by L-arginine, but not D-arginine.
On the contrary, when ANG II was infused in ganglion-blocked rats,
hypertension induced by PAR-1AP returned to naive rat values (Fig. 1A
).
It is worth noting that L-NAME restored hypertension induced by
PAR-2AP; this effect was abolished after the infusion of L-arginine,
but not when D-arginine was infused. ANG II infusion did not restore
hypertension induced by PAR-2AP (Fig. 1B
and Fig. 2
).
CONCLUSIONS
Our study shows for the first time a hypertensive response after
activation of PAR-2 in anesthetized rats. In contrast to what was
observed after PAR-1AP administration, this hypertensive response is
delayed in its appearance, reaching the maximum value after 5 min.
In addition, since it is abolished in ganglion-blocked rats, it is
likely secondary to a sympathetic reflex. This latter finding agrees
with previous studies that show an increase in sympathetic nerve
activity after PAR-2 activation in rats.
Several studies describing the effect of L-NAME on changes in MABP induced by PAR-APs have been performed in naive animals. However, in naive animals the substantial increase in MABP and vascular resistance above physiological values, due to NO inhibition by L-NAME, might activate a reflex response masking the real effect given by peptides activating PARs. For this reason, we performed experiments on ganglion-blocked rats in an attempt to clarify the role of NO on changes in blood pressure induced by peptides activating PAR-1 or PAR-2. We found that in ganglion-blocked rats, the endurance of hypotension induced by PAR-2AP was greatly increased and that this increase was completely dependent on NO release, since it was abolished by L-NAME. These findings agree with previous studies where it has been suggested that NO released after PAR-2 activation affects only the time of recovery of blood pressure to baseline. Nonetheless, after L-NAME infusion, the maximum peak of PAR-2AP-induced hypotension was increased, and a similar increase was observed after angiotensin II infusion. This result implies that in vivo PAR-2 activation causes an NO-dependent and NO-independent vasorelaxation, the latter appears to be dependent on the basal vascular tone. Our results agree with in vitro data showing that PAR-2 activation on porcine coronary artery causes NO-dependent and NO-independent, EDHF-like, relaxation. L-NAME infusion in ganglion-blocked rats restored the hypertensive effect of PAR-2AP, suggesting that in the absence of sympathetic reflexes, the hypertensive effect of PAR-2AP is completely masked by NO. This finding is supported by the observation that when NO synthesis was restored by infusing L-arginine, the hypertensive effect of PAR-2AP was abolished. The effect of L-NAME observed appeared to be specifically linked to NO inhibition, ruling out the possibility that it was dependent on the increase in MABP caused by L-NAME, since it was absent in angiotensin II infused rats.
In contrast to what observed for PAR-2AP, hypertension after PAR-1AP administration was strongly increased after ganglion blockade. Similarly, in vitro there is evidence for a direct contractile effect of PAR-1APs on vascular preparations in the absence of a functional endothelium. In vivo, hypertension in pithed rats due to PAR-1 activation has been shown to be strongly augmented. Therefore, it appears that the hypertensive effect of PAR-1AP increases as the basal vascular tone decreases. However, in ganglion-blocked rats in which blood pressure was restored by L-NAME, hypertension induced by PAR-1AP was significantly higher than values obtained in naive animals. Conversely, when blood pressure in ganglion-blocked rats was restored by angiotensin II infusion, hypertension induced by PAR-1AP returned to values obtained in naive rats. This finding suggests that hypertension induced by PAR-1AP is dependent on vascular tone and modulated by NO. To confirm this modulator role for NO on hypertension induced by PAR-1AP, we demonstrated that when NO tone was restored by infusing L-arginine, hypertension induced by PAR-1AP was significantly reduced despite the low vascular tone. It has been demonstrated in vivo that in an endothelium-intact aortic ring preparation, the contractile effect given by PAR-1 activation becomes visible only after NO synthase inhibition by L-NAME. Similarly, it has been described that L-NAME reveals hypertension induced by PAR-1 activation in mice in vivo.
Our data confirm that NO is not involved in hypotension induced by PAR-1 activation. Furthermore, the observation that hypotension was neither followed by the activation of a compensatory reflex mechanism nor modulated through changes in vascular tone induced by either L-NAME or angiotensin II suggests that it is not due to a vascular peripheral action.
In contrast, hypotension induced by PAR-2AP deals with a peripheral mechanism, since it was followed by a reflex hypertension and was increased by increasing the vascular tone, as described for several known peripheral agents. This hypotensive response is relying on both an NO-dependent and -independent mechanism.
In addition, we have characterized an in vivo hypertensive effect of PAR-2AP secondary to a sympathetic reflex that has not been evidenced before. When sympathetic reflex is abolished by ganglion blockade, the hypertensive effect is unmasked by inhibition of nitric oxide.
In conclusion, by dissecting vascular responses to PAR-1AP and PAR-2AP
under different basal conditions, we define a key role for PAR-1 and
PAR-2 in the physiopathological control of vascular resistance,
pointing to PAR-1 and PAR-2 as new therapeutic targets in the
development of drugs for the control of cardiovascular diseases
(Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0633fje ; to cite this article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0633fje. This work was financed by P.R.I.N./M.U.R.S.T. 2000. 
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