Molecular Mechanisms in Vascular Endothelin B Receptor Up-Regulation

Cardiovascular diseases are characterized by changes in morphology and function of the blood vessels. Different signalling molecules are involved in these changes. Endothelin-1 (ET-1) is one of the most potent signalling molecules found in man. Its actions are mediated by two receptors, the ETA and the ETB receptors. The ETA receptor is located on the smooth muscle cells and mediates potent contractile effects throughout the vascular system. The ETB receptor, when located on the endothelial cell mediates vasodilatation. When located on the smooth muscle cell the ETB receptor mediates vasoconstriction. In cardiovascular diseases there are changes in the smooth muscle phenotype and the contractile ETB receptor is up-regulated. In this thesis an organ culture model was used to examine the variation, the time-course and the intracellular pathways responsible for the ETB receptor up-regulation. Following organ culture, the ETB receptor was up-regulated in rat mesenteric arteries, the femoral artery and in the distal part of the caudal artery, but not in the aorta or the proximal caudal artery. The up-regulation was stronger in smaller arteries, in the mesenteric system and in veins. The up-regulation process in the mesenteric artery was preceded by mRNA transcription. The maximum pharmacological efficacy was reached after 24 h and the up-regulation was complete after 48 h. In acute experiments, non-specific inhibitors of protein kinase C (PKC) decreased the ETB receptor mediated contraction, but a specific PKC inhibitor, Ro31-7549, was ineffective at blocking contraction. In long-term experiments, PKC inhibitors abolished ETB receptor upregulation after 24 h of organ culture. Thus, PKC has an important role in ETB receptor upregulation and contraction. The ERK1/2 MAP kinase was activated following 3 h of organ culture. After blocking the ERK1/2 pathway with inhibitors of raf and MEK1/2, the ETB receptor up-regulation was inhibited, showing that this pathway is crucial for the ETB receptor induction. Cytokines are important regulators of inflammation and acute phase responses. After organ culture with the cytokines IL-1β and TNF-α the ETB receptor mediated contractions were increased. There was no increase in potency and the ETB receptor mRNA was not increased beyond that of incubated control arteries. These findings suggest an effect of IL-1β and TNF-α on the contractile machinery per se. To summarize, organ culture can be used for the study of the ETB receptor upregulation. This up-regulation is pronounced in small arteries and veins. PKC and the ERK1/2 and MAP kinase system are important for the induction of this up-regulation

Protein kinase C inhibitors decrease endothelin ET(B) receptor mRNA expression and contraction during organ culture of rat mesenteric artery.

The effect of protein kinase C (PKC) inhibitors on the induction of endothelin ET(B) receptors during organ culture was examined in isolated segments of the rat mesenteric artery. After 24 h of organ culture, the endothelin ET(B) receptor agonist sarafotoxin 6c (S6c) induced a strong contraction compared to fresh segments. The contractile response after 24-h organ culture to S6c was studied in presence (30-min preincubation) or absence, after 24-h treatment, of the PKC inhibitors staurosporine, K252a and Ro31-7549. Exposure to staurosporine or K252a in presence and after 24-h treatment reduced the S6c contraction. In contrast, presence of 2-1[1-3(aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (Ro31-7549), did not affect the S6c-induced contraction, whereas 24-h treatment abolished the increase of contraction. The PKA inhibitor N-(2-[bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H89) did not affect the S6c responses. The mRNA expressions of endothelin ET(B) receptors (analysed with real-time PCR) were abolished after 24-h treatment with the PKC inhibitors. These results suggest that PKC is involved in the endothelin ET(B) receptor upregulation following organ culture

Deteriorated function of cutaneous microcirculation in chronic congestive heart failure

Background Chronic congestive heart failure is a complex condition that leads to dysfunction in the peripheral microcirculation. We have previously shown that vascular reactivity is reduced with increasing age. In this study, we examined a group of very old patients with severe chronic heart failure to test the hypothesis that vascular function is further compromised by a combination of heart failure and aging. Methods Cutaneous forearm blood flow was measured by laser Doppler flowmetry and compared among three groups: Group 1 (n = 20, mean +/- SE: 85.5 +/- 4 years), heart failure patients with New York Heart Association class IV (NYHA IV) and with a NT-proBNP level >= 5000 ng/L; Group 2 (n = 15, mean +/- SE: 76.5 +/- 2 years), heart failure patients with NYHA II and NT-proBNP <= 2000 ng/L, and Group 3 (n = 10, mean +/- SE: 67.6 +/- 3.0 years), healthy controls with no clinical signs of heart failure. The vasodilator response to the iontophoretic administration of acetylcholine (ACh), acting via an endothelial mechanism, and sodium nitroprusside (SNP), acting via a smooth muscle cell mechanism, were studied. Results All patients with heart failure had significantly reduced vascular reactivity independent of the mode of stimulation (ACh, SNP or heat) when compared to healthy controls. However, the responses did not differ between the two groups of heart failure patients. Conclusions Cutaneous vascular reactivity is reduced in heart failure patients and does not correlate with the severity of the condition or age of patients

Cytokines induce increased endothelin ET(B) receptor-mediated contraction

The effect of cytokines on the induction of contractile endothelin ET(B) receptors during organ culture was examined. Ring segments of rat superior mesenteric artery were used fresh or incubated for 24 h in Dulbecco's modified Eagle's medium alone, or with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or interleukin-2. In fresh arterial segments there was no endothelin ET(B) receptor-induced contraction. After incubation, the selective endothelin ET(B) receptor agonist sarafotoxin 6c evoked a contraction of 22 +/- 6% relative to that induced by 60 mM K+. The endothelin ET(B) receptor-induced contraction was further increased to 125 +/- 25% and 157 +/- 29% by interleukin-1beta and TNF-alpha, respectively, while interleukin-2 did not alter the endothelin ET(B) receptor-induced contraction. The identity of the contractile receptor was confirmed as the endothelin ET(B) receptor by the use of an additional specific endothelin ET(B) receptor agonist, IRL 1620, and by antagonist experiments with FR 139317 and IRL 2500. The endothelin-1-induced contraction was not altered by either of the cytokines. Reverse transcriptase-polymerase chain reaction revealed increased levels of endothelin ET(B) mRNA, relative to endothelin ET(A) mRNA following organ culture, suggesting that contractile endothelin ET(B) receptors appear via de novo transcription. None of the cytokines changed the ratio of endothelin ET(A) and endothelin ET(B) receptor mRNA, indicating that the further increased sarafotoxin 6c-induced contraction is mediated through an enhancement of intracellular signalling mechanisms

Brain natriuretic peptide is a potent vasodilator in aged human microcirculation and shows a blunted response in heart failure patients.

Brain natriuretic peptide (BNP) is normally present in low levels in the circulation, but it is elevated in parallel with the degree of congestion in heart failure subjects (CHF). BNP has natriuretic effects and is a potent vasodilator. It is suggested that BNP could be a therapeutic alternative in CHF. However, we postulated that the high levels of circulating BNP in CHF may downregulate the response of microvascular natriuretic receptors. This was tested by comparing 15 CHF patients (BNP > 3000 ng/L) with 10 matched, healthy controls

Regional variation in appearance of vascular contractile endothelin-B receptors following organ culture

OBJECTIVE: The aim of this study was to investigate the appearance of contractile endothelin (ET)-B receptors following organ culture in different vascular regions. METHOD: The contractile responses of vascular smooth muscle induced by ET-1 and the selective ETB receptor agonist sarafotoxin 6c (S6c) were investigated in circular segments representing eight vascular regions in the rat (aorta, femoral artery, mesenteric artery, branch of the mesenteric artery, proximal and distal parts of the caudal artery, femoral and mesenteric veins). To allow the ETB receptor to be expressed, the segments were placed in organ culture for 1 to 5 days. Pharmacological characterisation of the ET receptors was performed in mesenteric arterial segments. All contractile responses were measured in percentage of K(+)-induced contraction. RESULTS: ET-1 induced strong concentration-dependent contractions of all fresh (not cultured) segments. S6c had negligible effects on all fresh vessels with the exception of the mesenteric vein, where a small contraction was seen. After 1 day of organ culture all tested segments, with the exception of aorta and the proximal part of the caudal artery, showed concentration-dependent contractile responses to S6c which were further augmented after 5 days of culture. The ET-1-induced responses were only slightly affected by organ culture. Contractions induced by S6c were more enhanced in small arteries and veins than in larger arteries. Furthermore, the S6c-induced response was more pronounced in the mesenteric region as compared to the hindlimb. In fresh mesenteric arterial segments FR139317 (ETA receptor antagonist) and bosentan (ETA/ETB receptor antagonist) but not IRL 2500 (ETB receptor antagonist) shifted the ET-1-induced concentration-response curve in parallel to the right. In contrast, after organ culture the S6c-induced concentration-response curves were shifted parallel to the right in the following potency order: IRL 2500 > bosentan > FR139317. CONCLUSION: During normal conditions, the ETA receptor is the dominating mediator of endothelin-induced contraction in eight different vascular regions. Furthermore, this study indicates that most of the vessels have the ability to develop contractile ETB receptors and that this plasticity differs in vascular regions

Cerebral ischemia upregulates vascular endothelin ETB receptors in rat

Background and Purpose-Elevated levels of endothelin 1 (ET 1) have been reported in cerebral ischemia A role for ET may prove more important if the vascular receptors were changed We addressed whether there is any change in ET receptor expression in cerebral ischemia Methods-The right middle cerebral artery (MCA) was occluded in male Wistar rats for 2 hours with the intraluminal filament method The basilar artery and both MCAs were removed after 46 hours of recirculation The contractile responses to ET 1 a combined ETA and ETB receptor agonist and sarafotoxin 6c (S6c) a selective ETB receptor agonist were examined in vitro and ET receptor mRNA was quantified by real time polymerase chain reaction Results-S6c which had no contractile effect per se on fresh or sham operated rat cerebral arteries induced a marked contraction in the occluded MCA (E-m [maximum contraction calculated as percentage of the contractile capacity of 63 5 mmol/L K+] =68+/-68% P<0 0001) while there was no difference in the responses to ET 1 after cerebral ischemia Real time polymerase chain reaction revealed a significant upregulation of both the ETA and ETB receptors (both P<0 05) in the occluded MCA compared with the nonoccluded MCA from the same rats Conclusions-Focal cerebral ischemia in rat induces increased transcription of both ETA and ETB receptors which results in the appearance of a contractile response to the ETB receptor agonist S6c These results suggest a role for ET receptors in the pathogenesis of a vascular component after cerebral ischemia