James D Moffatt

A protective role for protease-activated receptors in the airways.

The protection of cells in the upper intestine against digestion by pancreatic trypsin depends on the prostanoid prostaglandin E2 (PGE2) and is mediated by protease-activated receptors in the epithelium,. As the airway epithelium is morphologically similar and also expresses one of these receptors, PAR2 (ref. 3), and is a major source of PGE2 (ref. 4), we reasoned that bronchial epithelial PAR2 might also participate in prostanoid-dependent cytoprotection in the airways. Here we show that activation of PAR2, which co-localizes immunohistochemically with trypsin(ogen) in airway epithelium, causes the relaxation of airway preparations from mouse, rat, guinea-pig and humans by the release of a cyclooxygenase product from the epithelium. This physiological protective response in isolated airways also occurred in anaesthetized rats, where activation of PAR2 caused a marked and prolonged inhibition of bronchoconstriction. After desensitization of PAR2, the response to trypsin recovered rapidly by mechanisms dependent on de novo synthesis and trafficking of proteins. Our results indicate that trypsin released from the epithelium can initiate powerful bronchoprotection in the airways by activation of epithelial PAR2.

Protease-activated receptors: sentries for inflammation?

Cell-surface protease-activated receptors (PARs) appear to have evolved to detect extracellular enzymatically active serine proteases such as trypsin and thrombin. The predominant location of PARs on endothelia and epithelia and the discovery of enzymes such as trypsin within these tissues, together with the linkage of PARs to cytoprotective pathways, provide new information on autocrine and paracrine signalling within these critical barriers. In this article, the ways in which the distribution and function of PARs could be harnessed by pharmacologists as novel anti-inflammatory therapeutic strategies are discussed.

Protease-Activated Receptors Mediate Apamin-Sensitive Relaxation of Mouse and Guinea Pig Gastrointestinal Smooth Muscle

BACKGROUND & AIMSnProtease-activated receptor (PAR)-1 and PAR-2 are expressed on gastrointestinal smooth muscle, but knowledge of their functionality is limited. The aim of this study was to determine if PAR-1 and PAR-2 mediate gastrointestinal smooth muscle relaxation and to clarify the underlying mechanisms.nnnMETHODSnResponses to PAR activation using the serine proteases thrombin and trypsin and the peptide agonists for PAR-1 and PAR-2, SFLLRN-NH2 and SLIGRL-NH2, respectively, were investigated in submaximally contracted longitudinal strips of mouse gastric fundus and guinea pig taenia coli.nnnRESULTSnIn mouse gastric fundus, both thrombin and trypsin caused relaxations followed by contractions. SFLLRN-NH2 and SLIGRL-NH2 caused similar biphasic responses, the relaxation components of which were eliminated by apamin or ryanodine. For SFLLRN-NH2, apamin and ryanodine revealed contractions. Nifedipine inhibited both relaxations and contractions to each peptide. In guinea-pig taenia coli, thrombin but not trypsin caused relaxation, whereas SFLLRN-NH2 and SLIGRL-NH2 caused concentration-dependent relaxations that were eliminated by apamin but were unaffected by ryanodine.nnnCONCLUSIONSnThe mouse gastric fundus and guinea pig taenia coli contain functional PAR-1 and PAR-2 that mediate relaxations via ryanodine-sensitive and -insensitive activation of small-conductance, Ca2+-activated K+ channels. We propose that smooth muscle PARs act as sensors for inflammatory signals in gut and respond by inhibiting gut motility during peritoneal infections or tissue damage.

Evidence for Selective Effects of Chronic Hypertension on Cerebral Artery Vasodilatation to Protease-Activated Receptor-2 Activation

BACKGROUND AND PURPOSEnProtease-activated receptor-2 (PAR-2) can be activated after proteolysis of the amino terminal of the receptor by trypsin or by synthetic peptides with a sequence corresponding to the endogenous tethered ligand exposed by trypsin (eg, SLIGRL-NH(2)). PAR-2 mediates nitric oxide (NO)-dependent dilatation in cerebral arteries, but it is unknown whether PAR-2 function is altered in cardiovascular diseases. Since hypertension selectively impairs NO-mediated cerebral vasodilatation in response to acetylcholine and bradykinin, we sought to determine whether PAR-2-mediated vasodilatation is similarly adversely affected by this disease state.nnnMETHODSnWe studied basilar artery responses in Wistar-Kyoto rats (WKY) (normotensive) and spontaneously hypertensive rats (SHR) in vivo (cranial window preparation) and in vitro (isolated arterial rings). The vasodilator effects of acetylcholine, sodium nitroprusside, and activators of PAR-2 and protease-activated receptor-1 (PAR-1) were compared in WKY versus SHR. Immunohistochemical localization of PAR-2 was also assessed in the basilar artery.nnnRESULTSnIncreases in basilar artery diameter in response to acetylcholine were 65% to 85% smaller in SHR versus WKY, whereas responses to sodium nitroprusside were not different. In contrast to acetylcholine, vasodilatation in vivo to SLIGRL-NH(2) was largely preserved in SHR, and SLIGRL-NH(2) was approximately 3-fold more potent in causing vasorelaxation in SHR versus WKY in vitro. In both strains, responses to SLIGRL-NH(2) were abolished by N(G)-nitro-L-arginine, an inhibitor of NO synthesis. Activators of PAR-1 had little or no effect on the rat basilar artery. PAR-2-like immunoreactivity was observed in both the endothelial and smooth muscle cells of the basilar artery in both strains of rat.nnnCONCLUSIONSnThese data indicate that NO-mediated vasodilatation to PAR-2 activation is selectively preserved or augmented in SHR and may suggest protective roles for PAR-2 in the cerebral circulation during chronic hypertension.

Effect of protease-activated receptor (PAR)-1, -2 and -4-activating peptides, thrombin and trypsin in rat isolated airways

Mechanisms of relaxation and contraction to protease‐activated receptor‐ (PAR) tethered ligand peptides (SFLLRN/TFLLR, SLIGRL and GYPGKF (all C‐terminally amidated) for PAR1, PAR2 and PAR4, respectively) and enzymes (thrombin and trypsin) were investigated in isolated segments of rat trachea, main and first order intrapulmonary bronchi. In airway segments previously exposed to SLIGRL, SFLLRN caused contractions that were potentiated by indomethacin, but were independent of mast cell degranulation. Contractions to TFLLR in the intrapulmonary bronchi were similarly potentiated by indomethacin. SLIGRL caused epithelium‐dependent relaxations which were unaffected by NG‐nitro‐L‐arginine, 1‐H‐oxodiazol‐[1,2,4]‐[4,3‐a]quinoxaline‐1‐one or zinc‐protoporphyrin‐IX but were abolished by haemoglobin in all three regions of the airways. Relaxations to SLIGRL were markedly attenuated by indomethacin only in the main and intrapulmonary bronchi. GYPGKF caused epithelium‐dependent relaxations in all three regions of the airway which were only significantly inhibited by indomethacin in the intrapulmonary bronchi. In general, thrombin and trypsin failed to cause any response in the airways tested. Intense PAR2‐immunoreactivity was observed on airway epithelium. PAR1‐immunoreactivity was faint on airway epithelium and smooth muscle, but was prevalent in mast cells. These findings indicate that PAR2 and possibly PAR4 present on rat airway epithelia mediate smooth muscle relaxation via cyclo‐oxygenase‐dependent and ‐independent mechanisms. PAR1‐mediated contractions were most likely due to activation of smooth muscle receptors. The general failure of thrombin and trypsin to cause responses which may have been due to endogenous protease inhibitors, highlights the need for caution in assessing pathophysiological roles for PARs if only enzymes are used to activate PARs.

Endothelium‐dependent and ‐independent responses to protease‐activated receptor‐2 (PAR‐2) activation in mouse isolated renal arteries

Protease‐activated receptors (PARs) are receptors which require proteolytic cleavage to be self‐activated by newly exposed N‐terminal ‘tethered ligands’, and hence serve as sensors for protelytic enzymes. While both the thrombin receptor (PAR‐1) and PAR‐2 (activated by tryptic enzymes) have been shown to mediate endothelium‐dependent vasorelaxation, only PAR‐1 has been shown to cause direct vascular smooth muscle contraction. In this study, we report that trypsin and the PAR‐2 selective peptide ligand SLIGRL‐NH2 not only caused endothelium‐dependent relaxation of mouse renal arteries but also direct smooth muscle contraction if endothelial nitric oxide synthase was inhibited or if the endothelium was removed.

Enzymatic activation of endothelial protease‐activated receptors is dependent on artery diameter in human and porcine isolated coronary arteries

Protease‐activated receptor (PAR)‐mediated vascular relaxations have been compared in coronary arteries of different diameters isolated from both humans and pigs. Thrombin, trypsin, and the PAR1‐activating peptide, TFLLR, all caused concentration‐dependent relaxation of both large (epicardial; ∼2u2003mm internal diameter) and small (intramyocardial; ∼200u2003μm internal diameter) human coronary arteries. EC50 values for thrombin (0.006u2003uu2003ml−1 in epicardial, 1.69u2003uu2003ml−1 in intramyocardial) and trypsin (0.02u2003uu2003ml−1 in epicardial, 1.05u2003uu2003ml−1 in intramyocardial) were significantly (P<0.01) greater in intramyocardial arteries. By contrast, EC50 values for TFLLR were not different between epicardial (0.35u2003μM) and intramyocardial (0.43u2003μM) arteries. In porcine coronary arteries, EC50 values for relaxations to thrombin (0.03u2003uu2003ml−1 in epicardial 0.17u2003uu2003ml−1 in intramyocardial) were also significantly (P<0.01) greater in the smaller arteries. EC50 values for both TFLLR and the PAR2‐activating peptide, SLIGKV, were not different between the two different‐sized pig coronary arteries. PAR1‐immunoreactivity was localized to the endothelium of human epicardial and intramyocardial arteries and both PAR1‐ and PAR2‐immunoreactivity was observed in endothelial cells of equivalent porcine arteries. These findings indicate that enzymatic activation of endothelial cell PARs in human (PAR1) and porcine (PAR1 and PAR2) coronary arteries is markedly reduced in intramyocardial arteries when compared with epicardial arteries, suggesting increased regulation of PAR‐mediated vascular responses in resistance‐type arteries.

Storage of Radial Artery Grafts in Blood Increases Vessel Reactivity to Vasoconstrictors In Vitro

BACKGROUNDnAlthough prone to spasm, the radial artery (RA) is commonly used as a graft in coronary artery bypass surgery (CABG). Successful use of the RA as a graft is dependent on techniques to manage vasospasm during operation. We routinely store the RA in a papaverine blood solution after harvesting, a procedure which might damage the endothelium and predispose the RA to postoperative spasm. The aim of the present study was to evaluate the vasodilator and vasoconstrictor responsiveness in freshly obtained and stored segments of RA.nnnMETHODSnDiscarded segments of RA were obtained at operation from patients undergoing CABG and mounted as 3-mm rings in organ baths for isometric recording of changes in smooth muscle force production. Responses to cumulative additions of acetylcholine, noradrenaline, serotonin, angiotensin II, and the thromboxane A2 mimetic U46619 were normalized to contractions induced by a high potassium solution.nnnRESULTSnEndothelium-dependent relaxation to acetylcholine was not different between preparations from freshly-obtained and blood-stored RA segments. However, maximum contractions to all vasoconstrictors studied were markedly increased in preparations from stored arteries. The sensitivity (pEC50) of stored arteries to U46619, noradrenaline, and angiotensin were also enhanced when compared to preparations from freshly-obtained segments.nnnCONCLUSIONSnPapaverine blood solutions do not damage the endothelium of the RA. The observed heightened vasoconstrictor reactivity of stored arteries, most likely mediated by elements of the blood, indicates that asangineous storage solutions should be explored.

Contribution of β‐adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wildtype and β3‐adrenoceptor knockout mice

The smooth muscle relaxant responses to the mixed β3‐, putative β4‐adrenoceptor agonist, (−)‐CGP 12177 in rat colon are partially resistant to blockade by the β3‐adrenoceptor antagonist SR59230A suggesting involvement of β3‐ and putative β4‐adrenoceptors. We now investigated the function of the putative β4‐adrenoceptor and other β‐adrenoceptor subtypes in the colon, oesophagus and ureter of wildtype (WT) and β3‐adrenoceptor knockout (β3KO) mice. (−)‐Noradrenaline and (−)‐adrenaline relaxed KCl (30u2003mM)‐precontracted colon mostly through β1‐and β3‐adrenoceptors to a similar extent and to a minor extent through β2‐adrenoceptors. In colon from β3KO mice, (−)‐noradrenaline was as potent as in WT mice but the effects were mediated entirely through β1‐adrenoceptors. (−)‐CGP 12177 relaxed colon from β3KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (−)‐noradrenaline and increase for (−)‐CGP 12177 indicate compensatory increases in β1‐ and putative β4‐adrenoceptor function in β3KO mice. In oesophagi precontracted with 1u2003μM carbachol, (−)‐noradrenaline caused relaxation mainly through β1‐and β3‐adrenoceptors. (−)‐CGP 12177 (2u2003μM) relaxed oesophagi from WT by 61.4±5.1% and β3KO by 67.3±10.1% of the (−)‐isoprenaline‐evoked relaxation, consistent with mediation through putative β4‐adrenoceptors. In ureter, (−)‐CGP 12177 (2u2003μM) reduced pacemaker activity by 31.1±2.3% in WT and 31.3±7.5% in β3KO, consistent with mediation through putative β4‐adrenoceptors. Relaxation of mouse colon and oesophagus by catecholamines are mediated through β1‐ and β3‐adrenoceptors in WT. The putative β4‐adrenoceptor, which presumably is an atypical state of the β1‐adrenoceptor, mediates the effects of (−)‐CGP 12177 in colon, oesophagus and ureter.

Non-adrenergic, non-cholinergic neurons innervating the guinea-pig trachea are located in the oesophagus : evidence from retrograde neuronal tracing

The possibility that non-adrenergic, non-cholinergic (NANC) neurons innervating the guinea-pig trachea may be located within the oesophagus has been investigated using an in vitro retrograde tracing technique. The cervical trachea and oesophagus were excised from guinea-pigs and Dil was applied to a 5 mm region of the trachealis muscle. These preparations were maintained in organotypic culture for 3 days and processed for immunohistochemistry. A mean of 44 (4 neural cell bodies in the oesophageal myenteric plexus were found to be labelled by Dil. The vast majority of these neurons contained nitric oxide synthase, vasoactive intestinal polypeptide and neuropeptide Y. It is suggested that the population of neurons identified in this study are postganglionic parasympathetic neurons mediating NANC relaxation of the trachealis muscle in this species.