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Background: Inflammasomes are cytosolic multiprotein complexes which, upon assembly, activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischaemic stroke remains controversial. Our aims were to determine the role of NLRP3 in brain ischaemia, and explore the mechanism involved in the potential protective effect of the neurovascular unit., Methods: WT and NLRP3 knock-out mice were subjected to ischaemia by middle cerebral artery occlusion (60 min) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining., Results: We identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β and TNF-α), NLRP3 inflammasome components (NLRP3 and pro-caspase-1), protease expression (MMP9), and endothelial adhesion molecules (ICAM and VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junction proteins (ZO-1 and Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration., Conclusions: These findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischaemia with potential clinical translation., (© 2021 The British Pharmacological Society.)

1 Urocortin is a vasodilator peptide related to corticotrophin-releasing factor, which may protect endothelial function during coronary ischemia-reperfusion (I-R). The aim of this study was to study the mechanisms of this protective effect. 2 Hearts from Sprague-Dawley rats were isolated and perfused at constant flow and then exposed to 15 min global zero-flow ischemia, followed by 15 min reperfusion. The relaxation to acetylcholine (10 nM-10 microM) was recorded after pre-constriction of the coronary vasculature with U46619 (100-300 nM) in ischemic-reperfused or time-control hearts. 3 After I-R, the coronary relaxation to acetylcholine was reduced and this reduction was attenuated by treatment with urocortin (10 pM), administered before ischemia and during reperfusion. 4 This urocortin-induced improvement of the relaxation to acetylcholine was not modified by tetraethylammonium (10 mM), blocker of Ca2+ dependent-potassium channels; glibenclamide (10 microM), blocker of K(ATP) channels; N(w)-nitro-L-arginine methyl ester (L-NAME, 100 microM), blocker of nitric oxide synthesis; or meclofenamate (10 microM), blocker of cyclooxygenase, but it was abolished by chelerythrine (3 microM), blocker of protein kinase C (PKC). 5 These results suggest that urocortin may protect coronary endothelial function during I-R by activation of PKC.

Urocortin is a peptide recently identified, which is structurally related to the corticotropin-releasing factor (CRF). To analyze the mechanisms that could be involved in its effect on renal arteries from male and female rats, the response to urocortin was studied in isolated segments, 2 mm long, of renal arteries from male and female rats. In renal artery segments precontracted with endothelin-1 (1 nm), urocortin (1 pm-10 nm) produced concentration-dependent relaxation, which was similar in the arteries from male and female rats. This relaxation was reduced by the antagonists of urocortin receptors astressin (1 microM) and alpha-helical CRF(9-41) (1 microM) in arteries from both male and female rats. In renal arteries from female rats, the relaxation to urocortin was reduced by the inhibitor of adenyl cyclase SQ22536 (300 microM), by 8-bromo-cyclic-ADP-ribose (cADPR; 30 microM), an antagonist of the endogenous activator of sarcoplasmic Ca2+ channel cADPR and by ryanodine (1 microM), which produces depletion of sarcoplasmic Ca2+. In renal arteries from male rats, the relaxation to urocortin was increased by ryanodine, and was not modified by SQ22536 or 8-bromo-cADPR. These results suggest that the mechanisms involved in the relaxation to urocortin in renal arteries differ between female and male rats. In female rats, this relaxation may be mediated by the production of cyclic AMP (cAMP), synthesis of cADPR and release of sarcoplasmic Ca2+, whereas in male rats it is not mediated by cAMP.

Urocortin, an endogenous peptide structurally related to corticotropin-releasing factor (CRF), has potent cardiovascular effects, suggesting that it may be of significance in cardiovascular regulation. The objective of this study was to analyse the effects of urocortin and its action mechanisms on human blood vessels. To this, 3 mm long segments from human saphenous veins were prepared for isometric tension recording in an organ bath. In the segments at basal resting tone, urocortin did not produce any effect, but in the segments precontracted with endothelin-1 (1 - 10 nM), urocortin (1 pM - 10 nM) produced concentration-dependent relaxation. This relaxation was not modified by the inhibitor of nitric oxide synthase NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), but it was potentiated by the cyclo-oxygenase inhibitor meclofenamate (10 microM) and it was reduced by the inhibitors of high-conductance Ca2+-dependent potassium channels tetraethylammonium (TEA, 10 mM) and charybdotoxin (100 nM). These results indicate that human saphenous veins are very sensitive to urocortin, which produces vascular relaxation by a mechanism independent of nitric oxide and dependent of high-conductance Ca2+-dependent potassium channels, and that it may be opposed by the release of vasoconstrictor prostanoids. Therefore, urocortin may be of significance for regulation of the venous circulation in humans.

To examine the role of vasopressin V(1) and V(2) receptors, nitric oxide and prostanoids in the cerebrovascular effects of arginine vasopressin, cerebral blood flow was electromagnetically measured in awake goats. In 16 animals, vasopressin (0.03 - 1 microg), injected into the cerebral circulation, caused increments of resting cerebrovascular resistance which ranged from 18% (0.03 microg, P<0.01) to 79% (1 microg, P<0.01). Desmopressin (0.03 - 1 microg, four goats) did not affect significantly cerebrovascular resistance. The cerebrovascular resistance increases by vasopressin were reduced significantly by the antagonist for vasopressin V(1) receptors d(CH(2))(5)Tyr(Me)-AVP in a rate depending way (five (six goats) and 15 (four goats) microg min(-1)), and by the mixed antagonist for vasopressin V(1) and V(2) receptors desGly-d(CH(2))(5)-D-Tyr(Et)Val-AVP (5 microg min(-1), four goats), and they were not significantly affected by the antagonist for vasopressin V(2) receptors d(CH(2))(5), D-Ile(2), Ile(4)-AVP (5 microg min(-1), four goats). The inhibitor of nitric oxide synthesis N(w)-nitro-L-arginine methyl ester (L-NAME, 47 mg kg(-1) i.v., five goats) augmented cerebrovascular resistance by 130% (P<0.01), and for 24 h after this treatment the cerebrovascular effects of vasopressin were potentiated. The inhibitor of cyclo-oxygenase meclofenamate (6 mg kg(-1) i.v., five goats) did not modify significantly resting haemodynamic variables measured or the cerebrovascular effects of vasopressin. Therefore, the vasopressin-induced cerebral vasoconstriction may be mediated by vasopressin V(1) receptors, without involvement of vasopressin V(2) receptors, and may be modulated by nitric oxide but not by prostanoids.

1. The isometric response to arginine-vasopressin (10(-10)-10(-7)M) was studied in 2 mm long rabbit arterial segments isolated from several vascular beds (cutaneous, pial, renal, coronary, muscular, mesenteric and pulmonary). 2. Vasopressin induced contraction in central ear (cutaneous), basilar (pial), renal, coronary and saphenous (muscular) arteries, but had no effect in mesenteric and pulmonary arteries; the order of potency for the contraction was: ear > basilar > renal > coronary > saphenous arteries. 3. Treatment with the blocker of nitric oxide synthesis NG-nitro-L-arginine methyl ester (L-NAME; 10(-6)-10(-4) M) increased significantly (P < 0.05) the contraction to vasopressin in ear (148% of control), basilar (150% of control), renal (304% of control), coronary (437% of control) and saphenous (235% of control) arteries. Removal of the endothelium increased significantly (P < 0.05) the contraction to vasopressin in basilar (138% of control), renal (253% of control), coronary (637% of control) and saphenous (662% of control) arteries, but not in ear artery. Mesenteric and pulmonary arteries in the presence of L-NAME or after endothelium removal did not respond to vasopressin, as occurred in control conditions. 4. The specific antagonist for V1 vasopressin receptors d(CH2)5Tyr(Me)AVP (3 x 10(-9)-10(-7) M) was more potent (pA2 = 9.3-10.1) than the antagonist for both V1 and V2 vasopressin receptors desGly-d(CH2)5-D-Tyr(Et)ValAVP (10(-7)-10(-6) M) (pA2 = 7.4-8.4) to block the contraction to vasopressin of ear, basilar, renal and coronary arteries. 5. The specific V2 vasopressin agonist [deamino-Cys1, D-Arg8]-vasopressin (desmopressin) (10(-10)-10(-7) M) did not produce any effect in any effect in any of the arteries studied, with or without endothelium. 6. In arteries precontracted with endothelin-1, vasopressin or desmopressin did not produce relaxation. 7. These results suggest: (a) most arterial beds studied (5 of 7) exhibit contraction to vasopressin with different intensity; (b) the vasoconstriction to this peptide is mediated mainly by stimulation of V1 vasopressin receptors, and (c) endothelial nitric oxide may inhibit the vasoconstriction to this peptide, especially in coronary and renal vasculatures.