Thomas M. Cocks

Endothelium-dependent hyperpolarization: a role in the control of vascular tone

Endothelial-dependent relaxation of vascular smooth muscle cells evoked by a number of agonists, including cholinomimetics and substance P, is often accompanied by an increase (repolarization and/or hyperpolarization) in the membrane potential. This change in membrane potential appears predominantly to reflect the action of an endothelial-derived hyperpolarizing factor (EDHF), which is distinct from NO (or endothelial-derived relaxing factor), and is discussed in this article by Chris Garland and colleagues. In large conducting arteries, EDHF may provide a secondary system to NO, which assumes primary importance in some disease states such as pulmonary hypertension and atherosclerosis. However, in small resistance arteries (100-300 microns), EDHF appears to be a major determinant of vascular calibre under normal conditions, and may therefore be of primary importance in the regulation of vascular resistance.

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.

Purinergic innervation of the guinea-pig urinary bladder.

1 A number of criteria for considering adenosine 5′‐triphosphate (ATP) as a neurotransmitter in the guinea‐pig urinary bladder have been examined. In addition, the effect of tachyphylaxis to ATP on the response to non‐adrenergic, non‐cholinergic nerve stimulation has been re‐examined. 2 Quinacrine fluorescence histochemistry revealed a population of nerve fibres, ganglion cells, and nerve bundles in the bladder which were not seen in either the iris or vas deferens, where adrenergic and cholinergic nerves predominate. The distribution and morphology of the quinacrine‐positive nerves in the bladder were different from those observed with catecholamine fluorescence and cholinesterase histochemistry, and were unaffected by chemical sympathectomy. 3 Release of ATP from the bladder during stimulation of intramural excitatory nerves, in the presence of atropine and guanethidine increased to 3–12 times prestimulation levels. Tetrodotoxin abolished both the contractile response and the increase in ATP release resulting from intramural nerve stimulation. There was no increase in ATP release during contraction resulting from direct muscle stimulation following nerve paralysis with tetrodotoxin. 4 Sympathectomy with 6‐hydroxydopamine did not affect release of ATP in response to intramural nerve stimulation. 5 Release of ATP was dependent on the concentration of calcium ion in the medium. 6 Contractions in response to non‐adrenergic, non‐cholinergic intramural nerve stimulation were closely mimicked by ATP, but not by acetylcholine or histamine. 7 Adenosine and dipyridamole reduced the contractions to both ATP and non‐cholinergic nerve stimulation. 8 2–2′‐Pyridylisatogen was not a specific blocker of either ATP or intramural nerve stimulation in the guinea‐pig bladder. 2‐Substituted imidazolines initiated spontaneous activity making it impossible to assess any blocking action that they may have had. 9 Prostaglandins (E1 E2 and F2a) gave weak, slow contractions and an increase in spontaneous activity. Both the response to ATP and non‐adrenergic, non‐cholinergic nerve stimulation were greatly potentiated in the presence of prostaglandins. 10 In the presence of indomethacin the response to non‐adrenergic, non‐cholinergic nerve stimulation was virtually abolished following desensitization to ATP.

Direct evidence for ATP release from non-adrenergic, non-cholinergic ("purinergic") nerves in the guinea-pig taenia coli and bladder.

Demonstration of release of ATP from smooth muscle preparations during stimulation of purinergic nerves is complicated by the difficulty in showing whether it comes from nerve or muscle. ATP released during relaxation of the guinea-pig taenia coli and contraction of bladder strips in response to purinergic nerve stimulation was measured in the superfusate using the luciferin-luciferase ATP assay method. The amount of ATP increased 2-6 fold during isometric responses to purinergic nerve stimulation. This release was blocked by tetrodotoxin but not by adrenergic nerve destruction with 6-hydroxydopamine. No significant release of ATP was detected during comparable responses elicited by direct muscle stimulation. These results provide further support for the purinergic nerve hypothesis.

EFFECTS OF ADENOSINE 5′‐TRIPHOSPHATE (ATP) AND β‐γ‐METHYLENE ATP ON THE RAT URINARY BLADDER

1 High concentrations of adenosine 5′‐triphosphate (ATP, 100 to 1000 μm) were required to cause contraction of the rat urinary bladder, while adenosine and adenosine 5′‐monophosphate (AMP, 1 to 50/im) produced relaxation. 2 One hundred fold lower concentrations of β‐γ‐methylene ATP, which is resistant to degradation to AMP and adenosine, caused dose‐dependent, phasic contractions which mimicked atropine‐resistant responses to nerve stimulation. 3 Adenosine and AMP caused dose‐dependent inhibition of carbachol‐induced contractions; theophylline competitively antagonized this inhibition but not the contractile responses to β‐γ‐methylene ATP, ATP or atropine‐resistant nerve stimulation. 4 These results suggest that the insensitivity of the rat bladder to ATP is due to its rapid degradation to AMP and adenosine and support the hypothesis that the bladder receives a purinergic excitatory innervation.

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

BACKGROUND & AIMS Protease-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. METHODS Responses 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. RESULTS In 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. CONCLUSIONS The 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.

Reactivity of endothelin-1 on human and canine large veins compared with large arteries in vitro.

In comparison with snake venom sarafotoxins S6, the novel, 21-amino acid peptide, endothelin may have selective coronary artery vasoconstrictor actions. We examined endothelin-1 (ET-1) in vitro in five pairs of large arteries and veins from the greyhound dog; (coronary, internal mammary, mesenteric, renal and femoral) as well as the human forearm vein and internal mammary artery and vein. ET-1 caused concentration-dependent, tonic contractions in each pair of vessels, with EC50s significantly lower (5-10 times more sensitive) in each vein compared with the corresponding artery. The coronary artery did not show selective sensitivity to ET-1. For all veins the maximal contraction to ET-1 was approximately 100% that of the maximal contraction (Fmax) achieved with K+ depolarization. In the arteries, however, the Fmax for ET-1 ranged from only 25 to 80% of K+. The contraction responses to ET-1 in all arteries and veins were well maintained after repeated washing with ET-1-free medium. In the dog coronary artery the contraction curve to ET-1 (0.1-30 nM) was endothelium-independent. At the higher concentrations (10-100 nM), however, the peptide often induced transient, endothelium-dependent relaxations prior to the development of the tonic contractions. These results demonstrate that ET-1 is a more potent and efficacious constrictor of large veins than arteries and at high concentrations can release endothelium-derived relaxing factor-like activity from large arteries.

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

BACKGROUND AND PURPOSE Protease-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. METHODS We 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. RESULTS Increases 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. CONCLUSIONS These 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.

Atypical Protease-Activated Receptor Mediates Endothelium-Dependent Relaxation of Human Coronary Arteries

Protease-activated receptors (PARs) are a family of G protein-coupled receptors activated by a tethered ligand sequence within the amino terminal that are revealed by site-specific proteolysis. The thrombin-sensitive PAR-1 and trypsin-activated PAR-2 mediate endothelium-dependent vascular relaxation in a number of species. Because both thrombin and trypsin-like enzymes have been implicated in coronary artery disease, the purpose of this study was to investigate whether similar receptors are present in human coronary arteries. Thrombin (0.001 to 0.1 U/mL) and trypsin (0.001 to 1 U/mL) caused concentration- and endothelium-dependent relaxations of human coronary artery ring segments suspended in organ chambers for isometric tension recording and contracted with the thromboxane A2 mimetic U46619. These relaxations were dependent on the catalytic activity of each enzyme and were inhibited by the NO synthase inhibitor NG-nitro-L-arginine (100 micromol/L) and the NO scavenger oxyhemoglobin (20 micromol/L). The synthetic PAR-1 tethered ligand sequence SFLLRN-NH2 (0.01 to 10 micromol/L) also caused endothelium-dependent relaxation of U46619-contracted human coronary arteries; however, the equivalent PAR-2 ligand SLIGKV-NH2 caused almost no relaxation. In addition, desensitization to either thrombin or trypsin resulted in cross-desensitization to the other enzyme but had only a minimal affect on the response to SFLLRN-NH2. Therefore, we conclude that human coronary artery endothelial cells possess a PAR-1-like receptor that is potently activated by thrombin, trypsin, and SFLLRN-NH2 to cause NO-mediated vascular relaxation. Once cleaved, this receptor is recycled in a truncated form, able to respond to exogenous application of only its tethered ligand sequence, suggesting the presence of another endogenous activator possibly acting independently of receptor cleavage.

Oxyhaemoglobin increases the production of endothelin-1 by endothelial cells in culture

Cultured endothelial cells from bovine thoracic aortas conditioned with serum-free culture media released an endothelin-1 (ET)-1-like substance. Concentrations of ET-1-like material were determined by bioassay as contractions of isolated ring segments of dog internal mammary vein and by radioimmunoassay. ET-1-like immunoreactivity (ET-1-IR) increased progressively over a 24 h conditioning period and correlated with the bioassay for the first 12 h. Oxyhaemoglobin (1-3 microM) caused a significant two-fold increase in the concentration of ET-1-IR in the medium at 6, 8 and 12 h incubation. Methaemoglobin also caused an approximate doubling of the amount of ET-1-IR at eight h of incubation. NG-Monomethyl-L-arginine (L-NMMA), a blocker of the production of endothelium-derived relaxing factor (EDRF), had no effect on the time-dependent increase in ET-1-IR in the conditioned medium. These results may have important implications for the mechanisms underlying vascular smooth muscle hyperreactivity such as cerebral spasm following subarachnoid haemorrhage.