Modulation of pulmonary endothelial endothelin B receptor expression and signaling: implications for experimental hepatopulmonary syndrome

The hepatopulmonary syndrome (HPS) results from intrapulmonary vasodilation in the setting of cirrhosis and portal hypertension. In experimental HPS, pulmonary endothelial endothelin B (ET(B)) receptor overexpression and increased circulating endothelin-1 (ET-1) contribute to vasodilation through enhanced endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. In both experimental cirrhosis and prehepatic portal hypertension, ET(B) receptor overexpression correlates with increased vascular shear stress, a known modulator of ET(B) receptor expression. We investigated the mechanisms of pulmonary endothelial ET(B) receptor-mediated eNOS activation by ET-1 in vitro and in vivo. The effect of shear stress on ET(B) receptor expression was assessed in rat pulmonary microvascular endothelial cells (RPMVECs). The consequences of ET(B) receptor overexpression on ET-1-dependent ET(B) receptor-mediated eNOS activation were evaluated in RPMVECs and in prehepatic portal hypertensive animals exposed to exogenous ET-1. Laminar shear stress increased ET(B) receptor expression in RPMVECs without altering mRNA stability. Both shear-mediated and targeted overexpression of the ET(B) receptor enhanced ET-1-mediated ET(B) receptor-dependent eNOS activation in RPMVECs through Ca(2+)-mediated signaling pathways and independent of Akt activation. In prehepatic portal hypertensive animals relative to control, ET-1 administration also activated eNOS independent of Akt activation and triggered HPS. These findings support that increased pulmonary microvascular endothelial ET(B) receptor expression modulates ET-1-mediated eNOS activation, independent of Akt, and contributes to the development of HPS.     

Receptor subtype mediating the action of circulating endothelin on glucose metabolism and hemodynamics in perfused rat liver.

The subtype of endothelin receptor that mediates metabolic and hemodynamic effects of circulating endothelin was explored using perfused rat liver. Infusion of endothelin (ET)-1 or ET-3 into the portal vein at a concentration of 0.3 nM increased glucose and lactate output and decreased perfusion flow, although ET-3 was less effective than ET-1. The metabolic effects of ET-1 were observed even under costant-flow perfusion. Infusion of either sarafotoxin S6b or S6c, an ET(A)- or ET(B)-receptor agonist, mimicked the actions of ET-1 to an equal extent. The flow reduction and glucose production induced by ET-1 were partly attenuated by the ET(A)-receptor antagonist BQ485. By contrast, ET(B)-receptor antagonist BQ788 enhanced glucose production caused by ET-1 and ET-3 without affecting the hemodynamic change. The effects of ET-1 and ET-3 were almost totally inhibited by the combination of BQ485 and BQ788. These results suggest that both ET(A) and ET(B) receptors are involved in the metabolic and hemodynamic effects of circulating endothelin in rat liver, while the ET(A)-receptor-mediated action appears to be dominant.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

Production of nitric oxide from endothelial cells by 31-amino-acid-length endothelin-1, a novel vasoconstrictive product by human chymase

Human chymase selectively converts big endothelin (ET)-1 to 31-amino-acid-length ET-1 [ET-1(1-31)]. In this study we examined effect of ET-1(1-31) on endothelial function. ET-1(1-31) evoked contraction in a concentration-dependent manner at > 10(-8) M, which was about 10 times weaker than that of conventional ET-1 [ET-1(1-21)]. BQ485, an ETA receptor antagonist, completely abolished ET-1(1-31)-induced contraction, but BQ788, an ETB receptor antagonist, slightly enhanced it, suggesting that ET-1(1-31) relaxes artery via endothelium. On endothelial cells, ET-1(1-21) and ET-1(1-31) increased [Ca2+]i and produced NO, both of which were significantly inhibited by BQ788 and not by BQ485. These results indicate that ET-1(1-31) increased [Ca2+]i and produced NO in endothelial cells through ETB receptor similarly with ET-1(1-21), although slight difference in effect on smooth muscle cells.     

Role of endothelin-1 in regulation of the postnatal intestinal circulation

Newborn intestine is uniquely prone to vasoconstriction in response to a wide variety of perturbations. To test the hypothesis that endothelin (ET)-1 is an important factor in this process, we determined the effects of exogenous ET-1 administration and blockade of endogenous ET-1 in vivo and in vitro in 3- and 35-day-old swine. Intramesenteric artery administration of exogenous ET-1 to vascularly isolated in vivo gut loops (10(-9) M/kg bolus) caused vasoconstriction and reduced gut O(2) uptake similarly in these age groups. Selective blockade of ET(A) or ET(B) receptors with BQ-610 or BQ-788, respectively, in vascularly isolated in vivo gut loops had no effect on gut vascular resistance or O(2) uptake in either age group; within in vitro gut loops, BQ-610 significantly increased vasoconstriction when perfusion pressure was reduced below baseline, but only in 3-day-old animals; i.e., it impaired the autoregulatory response to perfusion pressure reduction. Exogenous ET-1 significantly decreased capillary perfusion within in vitro gut loops, as evidenced by a decrease in capillary filtration coefficient, but only in 3-day-old animals; furthermore, blockade of endogenous ET-1 activity with BQ-610 significantly enhanced capillary filtration coefficient in 3-day-old animals and increased O(2) extraction ratio. ET-1 did not depress intestinal metabolic rate, as evidenced by its effect on the O(2) uptake-blood flow relationship; it did compromise tissue oxygenation because of its effects on intestinal O(2) transport. ET-1 concentration in mesenteric venous effluent exceeded arterial concentration, but only in 3-day-old intestine, suggesting production of ET-1 by newborn intestine. We conclude that ET-1 exerts an age-dependent effect on intestinal hemodynamics in postnatal intestine, having a greater impact in 3- than in 35-day-old intestine.     

Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells.

Growing evidence suggests that a pressure-induced increase in the synthesis of endothelin (ET-1) is involved in arterial remodeling and, as a consequence, in the manifestation of chronic hypertension. To study potential stretch-induced changes in gene expression and their functional consequences, we have cultured rat aortic smooth muscle cells (raSMC) and porcine aortic endothelial cells (PAEC) on flexible elastomer membranes. The cells were periodically stretched (up to 20% elongation, 0.5 Hz, 6 h) and the expression of prepro-ET-1 and that of the endothelin A and B receptors (ET(A)-R and ET(B)-R) were analyzed by semi-quantitative RT-PCR analysis and ELISA (ET-1). In contrast to PAEC where ET-1 synthesis was up-regulated up to eightfold on exposure to cyclic stretch, ET-1 synthesis in raSMC was decreased by more than 80% under these conditions. ET(A) R -mRNA expression in stretched raSMC declined to 50% whereas ET(B) R -mRNA levels were increased up to 10-fold. One functional consequence of this apparent shift in receptor abundance was an apoptosis-promoting action of exogenous ET-1 (10 nM), as judged by the appearance of subdiploid peaks during FACS analysis, caspase-3 activation and chromatin condensation. This ET-1-induced apoptosis appeared to be ET(B)-R mediated, as it was completely suppressed by the ET(B)-R antagonist BQ 788 but not by the ET(A)-R antagonist BQ 123. Moreover, raSMC derived from homozygous spotting lethal rats, which lack a functional ET(B)-R, showed no signs of apoptosis after exposure to cyclic strain and exogenous ET-1. These findings suggest a central role for the endothelin system in the onset of hypertension-induced remodeling in conduit arteries, which may proceed via an initial stretch-induced apoptosis of the smooth muscle cells.     

Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein.

Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin 1 (ET-1) actively. Specific binding of [125I]ET-1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET-1. However, addition of phosphoramidon, an inhibitor of ET-converting enzyme, to the medium reduced the production of ET-1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET-1. Binding studies using phosphoramidon-treated HUVECs indicated the existence of a non-isopeptide-selective type (ETB) of ET receptor with a Kd of 17 pM. This receptor is thought to be involved in ET-induced vasodilation in an autocrine manner in vivo.     

Central endothelin ET(B) receptors mediate IL-1-dependent fever induced by preformed pyrogenic factor and corticotropin-releasing factor in the rat

Blockade of central endothelin ET(B) receptors inhibits fever induced by LPS in conscious rats. The contribution of ET(B) receptor-mediated mechanisms to fever triggered by intracerebroventricular IL-6, PGE2, PGF(2alpha), corticotropin-releasing factor (CRF), and preformed pyrogenic factor derived from LPS-stimulated macrophages (PFPF) was examined. The influence of natural IL-1 receptor antagonist or soluble TNF receptor I on endothelin (ET)-1-induced fever was also assessed. The selective ET(B) receptor antagonist BQ-788 (3 pmol icv) abolished fever induced by intracerebroventricular ET-1 (1 pmol) or PFPF (200 ng) and reduced that caused by ICV CRF (1 nmol) but not by IL-6 (14.6 pmol), PGE2 (1.4 nmol), or PGF(2alpha) (2 nmol). CRF-induced fever was also attenuated by bosentan (dual ET(A)/ET(B) receptor antagonist; 10 mg/kg iv) but unaffected by BQ-123 (selective ET(A) receptor antagonist; 3 pmol icv). alpha-Helical CRF(9-41) (dual CRF1/CRF2 receptor antagonist; 6.5 nmol icv) attenuated fever induced by CRF but not by ET-1. Human IL-1 receptor antagonist (9.1 pmol) markedly reduced fever to IL-1beta (180 fmol) or ET-1 and attenuated that caused by PFPF or CRF. Murine soluble TNF receptor I (23.8 pmol) reduced fever to TNF-alpha (14.7 pmol) but not to ET-1. The results of the present study suggest that PFPF and CRF recruit the brain ET system to cause ET(B) receptor-mediated IL-1-dependent fever.     

Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation.     

Endothelin promotes neurite elongation by a mechanism dependent on c-Jun N-terminal kinase

Endothelin (ET), which is known as a vasoconstrictive peptide, is associated with a lot of biological functions. Although endothelin receptors are expressed in the central nervous system (CNS), little is known about the effects of endothelin on neuronal function. In this study, we reported that endothelins elongate cortical neurites via the endothelin A receptor. All the endothelin isoforms tested, endothelin-1, endothelin-2, and endothelin-3, promoted neurite elongation. ET-1-induced neurite elongation was specifically inhibited by treatment with BQ123, an antagonist for the endothelin A receptor. In addition, inhibition of ET-1-induced c-Jun N-terminal kinase (JNK) activation by treatment with SP600125, a JNK inhibitor, also prevented the ET-1-mediated promotion of neurite elongation. Thus, endothelin induces cortical neurite elongation through the endothelin A receptor by a mechanism dependent on JNK.     

Cardiac effects of endothelin-1 (ET-1) and related C terminal peptide fragment: increased inotropy or contribution to heart failure?

The contrasting pattern of cardiac inotropy induced by human peptide endothelin-1 (ET-1) has not been satisfactorily explained. It is not clear whether ET-1 is primarily responsible for increased myocardial ET-1 expression and release with resultant inotropic effects, or for the induction of myocardial hypertrophy and heart failure. There are at least two subtypes of endothelin receptors (ET(A) and ET(B)) and the inotropic effects of ET-1 differ depending on the receptor involved. Along with some other groups, we reported significant subtype-ET(B) endothelin receptor down-regulation in human cardiac cells preincubated with endothelin agonists (Drímal et al. 1999, 2000). The present study was therefore designed to clarify the subtype-selective mechanisms underlying the inotropic response to ET-1 and to its ET(B)-selective fragment (8-21)ET-1 in the isolated rat heart. The hearts were subjected to (1-21)ET-1 and to (8-21)ET-1, or to 30 min of stop-flow ischemia followed by 40 min of reperfusion, both before and after selective blockade of endothelin receptors.The present study revealed that both peptides, ET-1 and its (8-21)ET-1 fragment, significantly reduced coronary blood flow in nmolar and higher concentrations. The concomitant negative inotropy and chronotropy were marked after ET-1, while the infusion of the ET-1(8-21) fragment produced a slight but significant positive inotropic effect. Among the four endothelin antagonists tested in continuous infusion only the non-selective PD145065 and ET(B1/B2) selective BQ788 (in molar concentrations) slightly reduced the early contractile dysfunction of the heart induced by ischemia, whereas ET(A)-selective PD155080 partially protected the rat heart on reperfusion.     

Characterization of the contractile and relaxant action of the endothelin-1 precursor, big endothelin-1, in the isolated rat basilar artery

The presence of functional endothelin converting enzyme (ECE) activity in basilar artery ring segments was investigated by measuring the contractile and relaxant effects of big endothelin (ET)-1. Under resting tension conditions cumulative application of big ET1-1 elicited a concentration-related contraction with the concentration-effect curve (CEC) shifted to the right against ET-1 by a factor of 31 and 29 in segments with the endothelium intact or mechanically removed, respectively. Preincubation with the ET(A) receptor antagonist, BQ123, induced an apparently parallel rightwards shift without affecting the maximum contraction. This shift was more pronounced for ET-1 than for big ET-1. With the putative ECE inhibitor phosphoramidon (10(-3) M) in the bath a small rightwards shift of the CEC for big ET-1 was observed in control segments and a more marked one in de-endothelialized segments. In segments precontracted with prostaglandin (PG) F(2alpha) big ET-1 induced a significant although transient relaxation whereas ET-1 did not. However, in the presence of BQ123 both ET-1 and big ET-1 elicited concentration-related relaxation with a significantly higher maximum effect obtained with big ET-1. The potency was 13 fold higher for ET-1, which is markedly less than that found for contraction. The results, therefore, suggest 1) the presence of functional ECE-activity in the rat basilar artery wall, and 2) differences in the functional ECE activity located in the endothelium and media.     

Endothelin-1-dependent leptin induction in gastric mucosal inflammatory responses to Helicobacter pylori lipopolysaccharide

Leptin, a multifunctional hormone that regulates food intake and energy expenditure, has emerged recently as an important modulator of gastric mucosal responses to Helicobacter pylori infection. We applied the animal model of H. pylori LPS-induced gastritis to investigate the role of endothelin-1 (ET-1) in the mucosal leptin production. We show that the histologic pattern of inflammation reached a maximum on the fourth day following the LPS and was reflected in a marked increase in the mucosal level of ET-1 and leptin. Therapeutic administration of phosphoramidon, an inhibitor of ECE-1 activity, led to a 61.2% decline in the mucosal ET-1 level and a 64.1% reduction in leptin, while the severity of mucosal inflammatory involvement increased by 28.6%. A drop in the level of leptin and the increase in severity of the inflammatory involvement elicited by the LPS was also attained in the presence of ET(A) receptor antagonist BQ610, but not the ET(B) receptor antagonist BQ788. Moreover, administration of ERK inhibitor, PD98059, in the presence of ET(B) receptor antagonist, but not the ET(A) receptor antagonist, caused reduction in the mucosal leptin level. Our findings are the first to implicate ET-1 as a key factor in up-regulation of gastric mucosal leptin-associated H. pylori infection. We also show that the effect of ET-1 on leptin production is a consequence of ET(A) receptor activation.     

Effects of ET-A receptor blockade on eNOS gene expression in chronic hypoxic rat lungs

We tested the hypothesis that pulmonary endothelial nitric oxide synthase (eNOS) gene expression is primarily regulated by hemodynamic factors and is thus increased in rats with chronic hypoxic pulmonary hypertension. Furthermore, we examined the role of endothelin (ET)-1 in this regulatory process, since ET-1 is able to induce eNOS via activation of the ET-B receptor. Therefore, chronic hypoxic rats (10% O(2)) were treated with the selective ET-A receptor antagonist LU-135252 (50 mg x kg(-1) x day(-1)). Right ventricular systolic pressure and cross-sectional medial vascular wall area of pulmonary arteries rose significantly, and eNOS mRNA levels increased 1.8- and 2.6-fold after 2 and 4 wk of hypoxia, respectively (each P < 0.05). Pulmonary ET-1 mRNA and ET-1 plasma levels increased significantly after 4 wk of hypoxia (each P < 0.05). LU-135252 reduced right ventricular systolic pressure, vascular remodeling, and eNOS gene expression in chronic hypoxic rats (each P < 0.05), whereas ET-1 production was not altered. We conclude that eNOS expression in chronic hypoxic rat lungs is modified predominantly by hemodynamic factors, whereas the ET-B receptor-mediated pathway and hypoxia seem to be less important.     

Transgenic mice over-expressing ET-1 in the endothelial cells develop systemic hypertension with altered vascular reactivity

Endothelin-1 (ET-1) is a potent vasoconstrictor involved in the regulation of vascular tone and implicated in hypertension. However, the role of small blood vessels endothelial ET-1 in hypertension remains unclear. The present study investigated the effect of chronic over-expression of endothelial ET-1 on arterial blood pressure and vascular reactivity using transgenic mice approach. Transgenic mice (TET-1) with endothelial ET-1 over-expression showed increased in ET-1 level in the endothelial cells of small pulmonary blood vessels. Although TET-1 mice appeared normal, they developed mild hypertension which was normalized by the ET(A) receptor (BQ123) but not by ET(B) receptor (BQ788) antagonist. Tail-cuff measurements showed a significant elevation of systolic and mean blood pressure in conscious TET-1 mice. The mice also exhibited left ventricular hypertrophy and left axis deviation in electrocardiogram, suggesting an increased peripheral resistance. The ionic concentrations in the urine and serum were normal in 8-week old TET-1 mice, indicating that the systemic hypertension was independent of renal function, although, higher serum urea levels suggested the occurrence of kidney dysfunction. The vascular reactivity of the aorta and the mesenteric artery was altered in the TET-1 mice indicating that chronic endothelial ET-1 up-regulation leads to vascular tone imbalance in both conduit and resistance arteries. These findings provide evidence for the role of spatial expression of ET-1 in the endothelium contributing to mild hypertension was mediated by ET(A) receptors. The results also suggest that chronic endothelial ET-1 over-expression affects both cardiac and vascular functions, which, at least in part, causes blood pressure elevation.     

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.     

Endothelin-1 stimulates c-fos mRNA expression in C6 glioma cells via MAP kinase pathway.

Exposure of C6 glioma cells to endothelin-1 (ET-1) caused dose-dependent (10(-11) M to 10(-7) M) increments in intracellular calcium concentration ([Ca2+]i) and c-fos mRNA expression (4.5-fold) that were abolished by the endothelinA receptor antagonist, BQ610, and by inhibition of phospholipase C with U73122. ET-1 stimulated c-fos mRNA expression was also inhibited by protein kinase C inhibition (chelerythrine) and by the MAP kinase kinase inhibitor PD98059, but not by inhibitors of tyrosine kinases, protein kinase A type I or II, calmodulin kinase II, or calcium channel blockade. C6 cells treated with ET-1 demonstrated a significant increase in MAP kinase activity as evidenced by Western blotting. These results indicate a mechanism of long-term signaling by ET-1 involving an ET(A) receptor-mediated, phospholipase C(beta)-linked pathway that is dependent on protein kinase C and MAP kinase activation.     

ENOS,ET-1 and ETB-R immunoreactivities in the porcine mesometrial lymphatics during the estrous cycle

Abstract: Immunohistochemical localization and distribution of nitric oxide synthase (eNOS), endothelin (ET-1) and endothelin beta receptor (ETB-R) were investigated in precollector and collector lymph vessels in the broad ligament of the uterus during different phases of the estrous cycle in pigs. The polyclonal antibody for ET-1 and ETB-R and monoclonal antibody for eNOS isoform were used to perform observations on the light microscopic level. Immunoreactivities to ET-1, ETB-R and eNOS were observed in the endothelium of precollector and collector lymphangions but not in smooth muscle cells of the lymphatics examined. The staining for eNOS in the endothelial cells of all studied lymphatic vessels was stronger comparing to ET-1 and ETB-R. During the estrous cycle, only eNOS showed the correlation with the particular phases of the estrous cycle. The differences between ET-1 and ETB-R immunoreactivities were very slight and rather independent of the size or type of the lymphatic lymphangions and estrous cycle. The highest immunoreactivity level for eNOS was displayed by collector lymphangions with widened lumen in the follicular phase comparing to the precollector ones. During the luteal phase, a slight decrease in the reaction intensity was observed. The immunoreactivities for ET-1 in the endothelium of the studied vessels was not comparable with the presence or with the reactivity level of ETB-R. Optically stronger immunoreaction for ETB-R was observed in the cytoplasm of collector lymphangions in the follicular phase. eNOS, ET-1 and ETB-R were also present in the cytoplasm of the lymphatic valves. These results suggest that ET-1 and eNOS can play a role in the mechanisms regulating the vascular contractile activity, promoting lymph flow during the estrous cycle in the porcine broad ligament.