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(Stroke. 1998;29:1439-1444.)
© 1998 American Heart Association, Inc.

Original Contributions

Activation of Protease-Activated Receptor-2 (PAR-2) Elicits Nitric Oxide–Dependent Dilatation of the Basilar Artery In Vivo

Christopher G. Sobey, PhD; Thomas M. Cocks, PhD

From the Department of Pharmacology, The University of Melbourne, Parkville, Victoria, Australia.

Correspondence to Christopher G. Sobey, PhD, Department of Pharmacology, the University of Melbourne, Parkville, Victoria 3052, Australia. E-mail c.sobey{at}pharmacology.unimelb.edu.au

	Abstract

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Background and Purpose—Protease-activated receptors (PARs) are a family of G-protein–coupled receptors activated by a tethered ligand amino acid sequence within the amino terminal that is revealed by site-specific proteolysis. In the vascular endothelium, activation of PAR-2 by treatment with trypsin or by using the amino acid ligand sequence (SLIGRL) produces endothelium-dependent relaxation of isolated noncerebral vascular segments. In this study, we first tested whether PAR-2 activation produces cerebral vasodilatation in vivo and then examined whether PAR-2–mediated vasodilatation is dependent on the production of nitric oxide.

Methods—Concentration-dependent vasodilator effects of the PAR-2 agonist peptide SLIGRL and trypsin were examined on the basilar artery using a cranial window in anesthetized rats. In addition, the vasodilator effects of SLIGRL, acetylcholine (ACh), and sodium nitroprusside (SNP) were examined in the absence and presence of N^G-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase.

Results—Baseline diameter of the basilar artery averaged 239±4 µm. Under control conditions, SLIGRL (10^–6 to 10^–4 mol/L) and trypsin (0.01 to 10 U/mL) produced concentration-dependent vasodilator responses. In time-control experiments, SLIGRL (3x10^–6 and 10^–5 mol/L), ACh (10^–6 and 10^–5 mol/L), and SNP (10^–8 and 10^–7 mol/L) elicited reproducible dilatation of the basilar artery. In another group of rats, L-NNA (10^–4 mol/L) markedly inhibited dilator responses to both SLIGRL (13±3% versus 1±1% and 39±7% versus 11±2%; both P<0.05) and ACh (8±1% versus 0±0% and 13±2% versus 3±1%; both P<0.05). By contrast, responses to SNP were significantly augmented after treatment with L-NNA (P<0.05 versus control), indicating that inhibitory effects of L-NNA were specific for responses mediated by endogenous nitric oxide. Furthermore, in another group ODQ (10^-5 mol/L) inhibited responses to SLIGRL to a degree similar to that seen with L-NNA, consistent with a mechanism of PAR-2–mediated vasodilatation that involves activation of guanylate cyclase by nitric oxide.

Conclusions—To the best of our knowledge, this study is the first to examine whether PAR-2–mediated vasodilatation is functional in cerebral arteries and is also the first to directly assess the effects of PAR-2 activation on vascular tone in vivo. The results suggest that activation of PAR-2 is an effective and powerful vasodilator mechanism in cerebral arteries in vivo. Cerebral vasodilator responses to PAR-2 activation are mediated by nitric oxide and are likely to be endothelium dependent.

Key Words: basilar artery • endothelium • nitric oxide • vasodilation • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Thrombin is a serine protease known to exert effects on the tone of cerebral arteries, including endothelium-dependent relaxation and direct contraction of cerebral vascular muscle.^{1 2} The thrombin receptor was the first cloned G protein–coupled receptor reported to be activated by proteolytic cleavage of its extracellular amino terminus.³ This receptor is now referred to as PAR-1. The proteolytic action of thrombin unmasks a new amino terminus that serves as a tethered peptide ligand, binding intramolecularly to other receptor domains to activate the receptor.⁴

PAR-2 is the second member to be cloned of this novel subtype of "tethered ligand" receptors, and has a protein sequence 30% identical to that of PAR-1.⁵ PAR-2 can be activated by site-specific proteolytic cleavage by trypsin of its extracellular amino terminus, to form an amino terminal peptide that acts as the PAR-2–tethered ligand. PAR-2 may also contribute to regulation of vascular tone, since PAR-2 mRNA is present in highly vascularized tissues⁵ and application of trypsin or the PAR-2 agonist peptide produces endothelium-dependent relaxation of peripheral arteries in vitro.^{6 7 8 9} Furthermore, in anesthetized rats hypotension is produced when the agonist peptide corresponding to the tethered ligand sequence in the mouse and the rat (SLIGRL) is injected intravenously,^{8 9} possibly indicating that PAR-2–mediated vasodilatation also occurs in vivo.

To the best of our knowledge, no effects of PAR-2 activation on cerebral artery tone have been reported. In addition, no study has thus far directly examined vasodilator responses of any vessel to PAR-2 activation in vivo. Therefore the purpose of the present study was to examine whether PAR-2–mediated vasodilatation is functional in cerebral arteries in vivo. We used a cranial window preparation in anesthetized rats to examine whether the PAR-2–activating compounds SLIGRL and trypsin elicit vasodilator responses of the basilar artery and whether these responses are dependent on endogenous synthesis of nitric oxide.

	Materials and Methods

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Procedures used in these experiments were approved by the University of Melbourne Animal Experimentation Ethics Committee. Experiments were performed in 29 male Sprague-Dawley rats (230 to 350 g). Animals were anesthetized with pentobarbital sodium (50 mg/kg IP) supplemented at 10 to 20 mg · kg^–1 · h^–1 (IV). A tracheostomy was performed, and the animals were mechanically ventilated with room air and supplemental oxygen. A catheter was placed into the right femoral artery to measure systemic pressure and to obtain arterial blood. The right femoral vein was cannulated for infusion of supplemental anesthetic. Arterial blood gases were monitored and maintained within normal levels throughout the experiment. Body temperature was maintained at 37°C to 38°C with a heating pad.

A craniotomy was performed over the ventral brain stem as described in detail previously.¹⁰ The cranial window was suffused with artificial CSF (temperature, 37°C to 38°C) at 3 mL/min, and a portion of the dura mater was opened. In CSF sampled from the craniotomies, PCO₂ was 38±1 mm Hg, PO₂ 102±2 mm Hg, and pH 7.38±0.01. Diameter of the basilar artery was monitored using a microscope equipped with a television camera coupled to a video monitor, and was continuously measured using a computer-based tracking program (Diamtrak; Montech).

Experimental Protocols
At the start of each experiment, diameter of the basilar artery was measured under control conditions and during continuous topical application of acetylcholine (10^–5 mol/L). Acetylcholine was used to examine reactivity of the preparation and when the response to acetylcholine had stabilized (after 1 to 2 minutes), diameter was measured. After administration of acetylcholine, the cranial window was suffused with artificial CSF for 30 minutes. Vessel diameter returned to control levels within a few minutes. The experiment was then continued according to 1 of the 5 protocols described below.

In one group of rats (SLIGRL concentration-response; n=9), we examined effects of topical application of the PAR-2 agonist peptide SLIGRL (10⁶ to 10⁴ mol/L) on diameter of the basilar artery. Aliquots of 10^–2 mol/L stock solution of SLIGRL were prepared in distilled H₂O and stored at -20°C. For each experiment, the stock solution was thawed, kept on ice, and then diluted in saline immediately before use. SLIGRL was then mixed in artificial CSF and applied to the cranial window. Concentrations of SLIGRL were applied in a cumulative fashion. The purpose of these experiments was to establish the concentration range over which SLIGRL elicits vasodilator responses of the basilar artery. Preliminary studies indicated that responses of the basilar artery were stable within 3 minutes of beginning the application of SLIGRL.

In a second group of rats (trypsin concentration-response; n=5), we examined effects of topical application of trypsin (0.01 to 10 U/mL) on diameter of the basilar artery. Aliquots of 1000 U/mL stock solution of trypsin were prepared in distilled H₂O and stored at -20°C. For each experiment, the stock solution was thawed, kept on ice, and then diluted in saline immediately before use. Trypsin was then mixed in artificial CSF and applied to the cranial window. Concentrations of trypsin were applied in a cumulative fashion. The purpose of these experiments was to determine whether trypsin elicits vasodilator responses of the basilar artery. Responses of the basilar artery were stable within 3 minutes of beginning the application of trypsin.

In a third group of rats (time control; n=5), vasodilator responses were measured in response to SLIGRL (3x10^–6 and 10^–5 mol/L), acetylcholine (10^–6 and 10^–5 mol/L), and sodium nitroprusside (10^–8 and 10^–7 mol/L). Vasodilators were tested in random order. For each vasodilator, 2 concentrations were applied topically to the basilar artery in a cumulative manner. Diameter of the basilar artery was recorded under basal conditions and during application of each concentration of agonist. Between applications of vasodilators, a recovery period of at least 15 minutes was allowed after the diameter had returned to the basal level. When each vasodilator had been tested once, a period of at least 30 minutes was allowed before re-examining responses in the same manner. The purpose of these experiments was to determine whether responses of the basilar artery were reproducible for each of the vasodilators studied.

In a fourth group of rats (L-NNA–treated; n=5), we examined responses of the basilar artery to the application of SLIGRL, acetylcholine, and sodium nitroprusside using a protocol similar to that used in time-control studies except that the second application of agonists was given during treatment of the cranial window with L-NNA (10^–4 mol/L). The cranial window was treated with L-NNA for at least 20 minutes prior to application of vasodilators. The purpose of these experiments was to determine whether L-NNA inhibits vasodilator responses of the basilar artery to the PAR-2 agonist peptide in a fashion similar to that seen with acetylcholine (which is known to stimulate endothelial nitric oxide release).

In a fifth group of rats (ODQ-treated; n=5), we examined responses of the basilar artery to the application of SLIGRL, acetylcholine, and sodium nitroprusside using a protocol similar to that used in time-control studies except that the second application of agonists was given during treatment of the cranial window with ODQ (10^–5 mol/L). The cranial window was treated with ODQ for at least 10 minutes before application of vasodilators. The purpose of these experiments was to determine whether ODQ inhibits vasodilator responses of the basilar artery to the PAR-2 agonist peptide in a fashion similar to that seen with acetylcholine or a nitric oxide donor (nitroprusside).

Drugs
Acetylcholine chloride, L-NNA, and sodium nitroprusside were obtained from Sigma Chemical Co. Rat PAR-2 agonist peptide SLIGRL-NH₂ (molecular weight, 657) was obtained from Auspep. Trypsin (bovine pancreas) was obtained from Worthington Biochemical Corp. ODQ was obtained from Sapphire, dissolved in dimethyl sulfoxide at a stock concentration of 3x10^–2 mol/L, and diluted in saline. All other drugs were dissolved in distilled H₂O as concentrated stock solutions and diluted in saline. Vehicle solutions had no effect on basilar artery diameter.

Statistics
Vascular responses are presented as percent change in diameter of the basilar artery, and are expressed as mean±SE. Single comparisons were made using Student's paired or unpaired t test, as appropriate. A value of P<0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Arterial blood gas values and pH were maintained at normal levels during the study (pH, 7.37±0.01; PCO₂, 37±1 mm Hg; PO₂, 167±10 mm Hg). In all experiments arterial blood pressure averaged 101±2 mm Hg under control conditions. Arterial pressure was not affected by application of vasodilators in the cranial window. Basilar artery diameter averaged 239±4 µm under control conditions.

Concentration-Dependent Effects of the PAR-2 Agonist Peptide SLIGRL on Basilar Artery Diameter
Application of SLIGRL to the basilar artery produced concentration-dependent vasodilator responses (n=4 to 9, Figure 1). The increase in artery diameter in response to the peptide reached a steady level within 1 to 2 minutes of application, and was maintained while the peptide perfusion was continued. The threshold concentration for vasodilator responses to SLIGRL was approximately 1 to 3x10^–6 mol/L. The highest concentration studied, 10^–4 mol/L, produced profound dilatation of the basilar artery (by approximately 50%), which appeared to be a near-maximum response to the peptide (Figure 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. Concentration-dependent effects of the PAR-2 agonist peptide SLIGRL on diameter of basilar artery (n=4 to 9). Baseline diameter of the basilar artery was 240±6 µm. All values are mean±SE.

Concentration-Dependent Effects of Trypsin
The application of trypsin to the basilar artery produced concentration-dependent vasodilator responses that reached steady levels within 3 to 5 minutes of application (n=5, Figure 2). Similar to our finding with the peptide SLIGRL, vasodilatation in response to trypsin was maintained for as long as the application of enzyme continued (approximately 5 to 6 minutes). The threshold concentration for vasodilator responses to trypsin was approximately 0.01 to 0.1 U/mL. Responses did not appear to reach maximum even at the highest concentration of trypsin studied, 10 U/mL (Figure 2).

View larger version (12K): [in this window] [in a new window]   Figure 2. Concentration-dependent effects of the trypsin on diameter of basilar artery (n=5). Baseline diameter of the basilar artery was 251±9 µm. All values are mean±SE.

Time-Control Experiments
In time-control studies (n=5), diameter of the basilar artery was stable under baseline conditions throughout each experiment, and averaged 229±5 µm during the first application of agonists and 225±8 µm during the second application of agonists.

Dilator responses of the basilar artery to SLIGRL (3x10^–6 and 10^–5 mol/L), acetylcholine (10^–6 and 10^–5 mol/L), and sodium nitroprusside (10^–8 and 10^–7 mol/L) each reached a steady state within 1 to 2 minutes of beginning the drug application, and were reproducible within 60 minutes (Figures 3, 4, and 5).

View larger version (18K): [in this window] [in a new window]   Figure 3. Change in diameter of rat basilar artery in response to SLIGRL. In time-control experiments, vasodilator responses to SLIGRL were reproducible (left; n=4). Treatment with L-NNA (10–4 mol/L) inhibited vasodilator responses to SLIGRL (right; n=5). Baseline diameter of the basilar artery was as follows: time control, 1st=225±5 µm, 2nd=226±7 µm; L-NNA study, control=240±8 µm, L-NNA–treated=185±6 µm*. All values are mean±SE. *P<0.05 vs control.

View larger version (18K): [in this window] [in a new window]   Figure 4. Change in diameter of rat basilar artery in response to acetylcholine. In time-control experiments, vasodilator responses to acetylcholine were reproducible (left; n=5). Treatment with L-NNA (10–4 mol/L) inhibited vasodilator responses to acetylcholine (right; n=5). Baseline diameter of the basilar artery was as follows: time control, 1st=229±5 µm, 2nd=225±8 µm; L-NNA study, control=240±8 µm, L-NNA–treated=185±6 µm*. All values are mean±SE. *P<0.05 vs control.

View larger version (18K): [in this window] [in a new window]   Figure 5. Change in diameter of rat basilar artery in response to nitroprusside. In time-control experiments, vasodilator responses to nitroprusside were reproducible (left; n=5). Treatment with L-NNA (10–4 mol/L) augmented vasodilator responses to nitroprusside (right; n=5). Baseline diameter of the basilar artery was as follows: time control, 1st=229±5 µm, 2nd=225±8 µm; L-NNA study, control=240±8 µm, L-NNA–treated=185±6 µm*. All values are mean±SE. *P<0.05 vs control.

Effect of L-NNA on Vasodilator Responses
Treatment with L-NNA (10^–4 mol/L), an inhibitor of nitric oxide synthase, decreased the diameter of the basilar artery by about 23%, from 240±8 to 185±6 µm (n=5; P<0.05). Vasoconstriction in response to L-NNA is thought to reflect the basal vasodilator influence of tonic release of nitric oxide on the basilar artery under resting conditions. L-NNA markedly inhibited vasodilator responses to SLIGRL (by 75% to 95%; Figure 3; P<0.05). L-NNA also profoundly inhibited vasodilator responses to acetylcholine (Figure 4). Vasodilator responses to sodium nitroprusside were augmented by treatment with L-NNA (Figure 5).

Effect of ODQ on Vasodilator Responses
Treatment with ODQ (10^–5 mol/L), an inhibitor of soluble guanylate cyclase, decreased the diameter of the basilar artery by about 16%, from 225±9 to 188±17 µm (n=5; P<0.05). As with L-NNA, vasoconstriction in response to ODQ probably reflects the basal vasodilator influence of nitric oxide–stimulated production of cGMP in the basilar artery. ODQ markedly inhibited vasodilator responses to SLIGRL (by 80% to 100%; Figure 6; P<0.05). L-NNA also profoundly inhibited vasodilator responses to sodium nitroprusside (Figure 6) and acetylcholine (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 6. Changes in diameter of rat basilar artery in response to SLIGRL and nitroprusside. Treatment with ODQ (10–5 mol/L) inhibited vasodilator responses to SLIGRL (left; n=5) and nitroprusside (right; n=5). Baseline diameter of the basilar artery was as follows: control conditions, 225±9 µm; ODQ-treated conditions, 188±17 µm*. All values are mean±SE. *P<0.05 vs control. Units of drug concentration are mol/L.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
To the best of our knowledge, this is the first study to examine the effects of PAR-2 activation on cerebral vascular tone and also the first study to directly assess vascular responses to PAR-2 activation in vivo. The major new finding is that PAR-2 activation is a powerful mechanism of vasodilatation in cerebral arteries. A second finding is that the cerebral vasodilator response to PAR-2 activation is mediated by nitric oxide, most likely released from the endothelium.

Functional Importance of PAR-2
We have known for more than a decade that trypsin stimulates endothelium-dependent vascular relaxation.¹¹ Also, more recent findings indicate that endothelial cells express PAR-2^{12 13 14} and that activation of PAR-2 by SLIGRL and trypsin in isolated segments of peripheral arteries also produces endothelium-dependent vascular relaxation.^{6 7 8 9 12} The peptide sequence SLIGRL is identical to that of the native tethered ligand of the new amino terminus of PAR-2 in the mouse and the rat, which is exposed on proteolytic cleavage of the PAR-2 exodomain.⁵ Therefore, SLIGRL is likely to cause full and specific activation of the receptor, and as such the concentration-dependent vasodilator responses of the basilar artery to SLIGRL observed here provide strong evidence that PAR-2 receptors mediate dilatation of cerebral arteries in vivo. Although there are presently no PAR-2 antagonists available to substantiate an involvement of this receptor, our conclusion is further supported by the ability of trypsin to induce concentration-dependent vasodilator responses in the basilar artery.

Previous indirect studies have indicated that PAR-2 activation may produce vasodilatation in vivo. The hypotensive effect of intravenous injection of SLIGRL was reported to be accompanied by baroreflex-mediated tachycardia and sympathetic nerve activation,^{8 9} suggesting that the decrease in arterial pressure was associated with a decrease in total peripheral resistance rather than in cardiac output. The present findings, in which topical application of PAR-2 activators produced increases in basilar artery diameter in the absence of any change in systemic arterial blood pressure, confirm directly that PAR-2–mediated vasodilatation occurs in vivo in the cerebral circulation.

Role of Nitric Oxide
Topical application of acetylcholine produces endothelium-dependent, nitric oxide–mediated dilatation of cerebral arteries in vivo.^{15 16} In a similar manner, topical applications of SLIGRL and trypsin are likely to have elicited vasodilatation via the stimulation of PAR-2 on vascular endothelial cells, leading to the release of endothelium-derived nitric oxide as has been reported to occur in isolated vessel studies.^{6 7 8 9 11} However, SLIGRL was a more effective vasodilator than trypsin, which produced only submaximal dilatation even at high concentrations. By contrast, in isolated vascular preparations in which the endothelium is directly accessible to the vasodilators, low concentrations of both SLIGRL and trypsin typically produce near-complete relaxation. Thus, the slower and relatively weaker vasodilator effect of trypsin may be due to slower or less efficient diffusion of trypsin through the vessel wall to the endothelium.

Prolonged exposure (for up to 20 minutes) of isolated arteries to SLIGRL or trypsin produces desensitization of PAR-2, thus inhibiting relaxant responses to subsequent applications of either agent.⁸ When the present protocol was used, there was no evidence of desensitization of PAR-2 because the cumulative application of 2 concentrations of SLIGRL (3x10^–6 and 10^–5 mol/L, applied for 5 minutes each) produced vasodilator responses that were fully reproducible within 60 minutes. Therefore, it is unlikely that desensitization contributed to the attenuation of SLIGRL responses after treatment with L-NNA or ODQ.

Nitric oxide activates soluble guanylate cyclase, resulting in the accumulation of cGMP and the activation of a cGMP-dependent protein kinase. This mechanism can stimulate vasorelaxation through several mechanisms that decrease intracellular Ca²⁺ levels. As demonstrated in this and previous studies,^{17 18} inhibition of endogenous nitric oxide synthesis caused dilator responses of the basilar artery to the nitric oxide donor sodium nitroprusside to be significantly augmented. Augmented vasodilator responses to sodium nitroprusside suggest that the inhibitory effects of L-NNA on responses to SLIGRL and acetylcholine were specific.

L-NNA, a nitric oxide synthase inhibitor, and ODQ, a soluble guanylate cyclase inhibitor,^{19 20} constricted the basilar artery under basal conditions, confirming previous findings^{16 17 18} that basal release of nitric oxide exerts a marked dilator influence in this artery under control conditions. Also consistent with previous findings,^{16 17 18} L-NNA inhibited dilator responses of the basilar artery to the endothelium-dependent agonist acetylcholine, indicating that this response is mediated via endogenous synthesis of nitric oxide. The new finding that vasodilator responses of the basilar artery to SLIGRL are markedly inhibited by L-NNA suggests that PAR-2 activation similarly elicits nitric oxide–mediated cerebral vasodilatation in vivo. Consistent with this conclusion, an additional new finding of ours was that ODQ profoundly inhibited dilator responses of the basilar artery to SLIGRL and sodium nitroprusside. Furthermore, the data suggest that dilatation of the basilar artery in response to nitric oxide occurs largely, and perhaps exclusively, via cGMP generation.

Although it seems likely that dilator responses of the basilar artery to PAR-2 activators are mediated by endothelium-derived nitric oxide, we cannot rule out the possibility that these agents also stimulated the release of nitric oxide from nitrergic nerves in the vascular wall. However, we are not aware of any data that clearly show functional activation of these nerves in cerebral arteries in vivo. Furthermore, SLIGRL-induced relaxation of the gastric fundus in the mouse does not appear to involve stimulation of nitric oxide release from nitrergic nerves (T.M. Cocks, unpublished data, 1998). Therefore we do not anticipate that such a mechanism is involved in PAR-2–mediated relaxation of cerebral vascular muscle.

In summary, the present findings suggest that activation of PAR-2 is an effective and powerful vasodilator mechanism in cerebral arteries in vivo, which involves production of nitric oxide probably by vascular endothelium. While these findings raise the possibility that PAR-2–mediated vasodilatation is important in the regulation of cerebrovascular tone in vivo, several aspects of this mechanism remain to be elucidated. These include identification of the endogenous activator or activators of PAR-2, the physiological role of PAR-2 in cerebral blood flow regulation, and the evaluation of possible underlying changes to PAR-2–mediated vasodilator function in cerebrovascular disease states associated with endothelial dysfunction or inflammation.²¹

	Selected Abbreviations and Acronyms

 

CSF = cerebrospinal fluid L-NNA = NG-nitro-L-arginine ODQ = 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one PAR-1 = protease-activated receptor-1 PAR-2 = protease-activated receptor-2 SLIGRL = PAR-2 amino acid ligand sequence

	Acknowledgments

 
Dr Sobey is a Senior Research Officer of the National Health and Medical Research Council of Australia (NHMRC). Dr Cocks is a Senior Research Fellow of the NHMRC. These studies were supported by funds from Dr Sobey's NHMRC C.J. Martin Fellowship and Dr Cocks' NHMRC project grant. We thank Dr Frank Faraci and Mr Justin Hamilton for their helpful advice.

Received November 18, 1997; revision received March 17, 1998; accepted April 13, 1998.

	References

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Editorial Comment

Zvonimir S. Katusic, MD, PhD, Guest Editor

Anesthesia Research Mayo Clinic, Rochester, Minnesota

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
The proteinase-activated receptor 2 (PAR-2), identified by molecular cloning techniques, belongs to the family of 7 transmembrane region receptors.^{1 2} Previous studies demonstrated that the effects of thrombin are mediated by activation of proteinase-activated receptor 1 (PAR-1).³ Both PAR-1 and PAR-2 are activated by proteolytic cleavage of their extracellular amino terminus. Subsequent intramolecular binding of tethered peptides leads to activation of these receptors. Thrombin is an endogenous activator of PAR-1 receptors, whereas the endogenous substance responsible for activation of PAR-2 has not yet been identified. Results of the Sobey and Cocks study provide strong evidence that activation of PAR-2 receptors by synthetic agonist SLIGRL peptide is coupled with formation of nitric oxide and vasodilatation of cerebral arteries.

What are the clinical implications of this finding? At the present time, our knowledge of the PAR-2 pathway signaling is very limited. Identification of the chemical nature of endogenous ligands is needed before we can speculate about the importance of these receptors for regulation of cerebrovascular vasomotor reactivity. However, studies by Nystedt et al⁴ demonstrated that inflammatory cytokines, tumor necrosis factor , interleukin-1, or bacterial lypopolysaccharide elevate expression of PAR-2 mRNA in cultured human umbilical vein endothelial cells. In contrast, expression of thrombin receptor PAR-1 gene was not affected by any of these inflammatory mediators. These findings suggest that expression of PAR-2 may play an important role in regulation of nitric oxide biosynthesis in vascular endothelial cells during acute inflammatory response. Further studies are needed to determine whether PAR-2 receptors are involved in the control of cerebral arterial tone during inflammation.

	Selected Abbreviations and Acronyms

 

CSF = cerebrospinal fluid L-NNA = NG-nitro-L-arginine ODQ = 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one PAR-1 = protease-activated receptor-1 PAR-2 = protease-activated receptor-2 SLIGRL = PAR-2 amino acid ligand sequence

Received November 18, 1997; revision received March 17, 1998; accepted April 13, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Nystedt S, Emilsson K, Larsson AK, Strombeck B, Sundelin J. Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem. 1995;232:84–89.[Medline] [Order article via Infotrieve]

2. Nystedt S, Larsson AK, Aberg H, Sundelin J. The mouse proteinase-activated receptor-2 cDNA and gene: molecular cloning and functional expression. J Biol Chem. 1995;270:5950–5955.[Abstract/Free Full Text]

3. Vu T-KH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991;64:1057–1068.

4. Nystedt S, Ramakrishnan V, Sundelin J. The proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells: comparison with the thrombin receptor. J Biol Chem. 1996;271:14910–14915.

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