Presence and location of endothelin and endothelin-binding sites in human nasal mucosa under normal and metaplastic conditions

The presence and site of production of endothelin-1 (ET-1) was investigated in biopsies obtained from the nasal mucosa of 10 healthy human subjects and 10 patients affected by chronic rhinitis. The presence and localization of receptors for ET-1 was also investigated. Bioptic fragments were examined by scanning electron microscopy. ET-1 was present in the vessels and in the respiratory epithelium of normal subjects, whereas in patients affected by epithelial metaplasia induced by chronic rhinitis, it was absent in the metaplastic epithelium and present in the endothelium and vascular wall. Receptors for ET (A- and B-receptor subtypes) were localized in the vessels of the nasal mucosa, both in normal and in pathological subjects. In particular, A-receptors were identified in the vascular wall, whereas B-receptors were mainly distributed in the endothelium. We suggest that ET-1 is involved in the homeostasis of nasal blood flow (shunting the blood toward the deep cavernous plexus and inducing mucosal swelling) by an autocrine and/or paracrine mechanism. Normal epithelium seems to be important in this mechanism, since it is able to produce ET. However, when pathologic conditions induce squamous or cuboidal metaplasia, the epithelium is no longer able to play this role.     

Interleukin 1 increases the production of endothelin-1 by cultured endothelial cells

We examined the effect of human recombinant interleukin 1 (IL-1) on the production of endothelin-1 by cultured porcine endothelial cells. The induction of endothelin-1 mRNA began within 1 hr of exposure to IL-1, showed twin peaks at 4 and 24 hr, and declined thereafter. Enzyme-linked immunosorbent assay revealed that the amount of endothelin-1 peptide in conditioned media was also increased by IL-1 in a dose- and time-dependent manner. Our results suggested that IL-1, a macrophage-derived cytokine, may affect the contraction and proliferation of vascular smooth muscle cells by stimulating the production of endothelin by endothelial cells.     

Adrenomedullin level in the nasal discharge from allergic rhinitis cohort

Adrenomedullin (AM) is a potent hypotensive and vasodilatory peptide. AM may exert protective actions against the development of many diseases by modulating the blood circulation and body fluid balance. In addition to these functions, it has recently been reported to play important roles in the development of allergy and infections. The purpose of the present study was to demonstrate the existence of AM in the human nasal mucosa and to discuss whether AM might contribute to the pathogenesis of nasal congestion. We measured the total AM concentrations in the nasal discharge. The total AM concentration in the nasal discharge was significantly higher in the non-allergy group (72.1 ± 55.5 fmol/ml) than in the allergy group (37.1 ± 44.2 fmol/ml). By immunohistochemical examination, we identified AM-containing cells in the nasal mucosa from both subjects with and without nasal allergy, and also in nasal polyps. Moreover, those cells were positive for anti-tryptase antibody which recognizes mast cells. In nasal allergy, vasodilatation and increase in vascular permeability are characteristic features of the immediate phase response. Reduced AM levels in the nasal discharge may be associated with attenuation of both of these factors. On the other hand, immunohistochemical analysis demonstrated AM-immunoreactive cells in the chronic phase of rhinosinusitis. In the late and inflammatory phase, mast cells produce AM, which possibly acts as an inhibitor of inflammatory cell migration. In conclusion, AM may be actively secreted into the nasal discharge. AM in the nasal discharge may have protective and anti-inflammatory effects in the nasal mucosa.     

Insulin-like growth factor-I in wound healing of rat skin

Endothelin-1 is involved in physiology and pathophysiology of the alimentary tract. The peptide modulates blood flow in the gastrointestinal microvasculature and regulates contractility of smooth muscles and, when present in excess, may be an important factor contributing to pathogenesis of various forms of mucosal injury and peristaltic disorders. Mechanisms that regulate endothelin concentration in the gastrointestinal tissues are unknown. Therefore, the aim of our study was to identify and characterize endothelin inactivating peptidases in the rat gastrointestinal mucosa and smooth muscle cells. We have found three high affinity and efficient endothelin-1 inactivating peptidases. The acidic (pH optimum 5.5), membrane-bound, thiorphan- (ED(50) 1.2+/-0.2 nM) and phosphoramidon (ED(50) 150+/-25 pM) sensitive, endothelin-1 inactivating peptidase (K(M) 0.12+/-0.03 microM) was present in the mucosal cells of duodenum and small intestine. The enzyme exhibited high molecular weight (>100 kDa) and characteristics similar to that of the rat and human kidney, acidic metalloendopeptidase that was recently described. Two forms of the unique, low molecular weight (100>MW>30 kDa), alkaline (pH optimum 8.5), specific (K(M) 0.5+/-0.2 microM), thiorphan- and phosphoramidon insensitive, 1,10 phenanthroline inhibitable (ED(50) 0.65+/-0.20 mM, mean+/-S.E.M.) endothelin-1 inactivating peptidase were present exclusively in the duodenal mucosal cells; soluble form in cytosol and membrane-bound form exhibiting an abundance ratio 5:1, respectively. Mucosa of the stomach and large intestine, and gastrointestinal smooth muscle cells do not contain the specific endothelin-1 inactivating peptidases. The enzymes may play a crucial role in regulation of endothelin concentration in the gastrointestinal tissues. Whether impairment of activity of the mucosal endothelin inactivating peptidases, resulting in the increase of concentration of endothelin peptides in gastrointestinal tissues, occurs in various pathological conditions is actually studied in our laboratory.     

Endothelin-1 directly modulates its own secretion: studies utilising the cell immunoblot technique

Endothelin-1 is an important factor in vasoregulation and circulating levels of the peptide are increased in a number of cardiovascular disorders. However, control of endothelin-1 secretion is only sketchily understood. The possibility that endothelin-1 influences its own release was investigated. A cell immunoblot method, which can detect local secretion of peptide from individual human vascular endothelial cells, was employed. Cells were dispersed onto a protein-binding membrane. Endothelin-1 in cells or secreted and adhering to the protein-binding membrane outside the cells was detected using immunohistochemical techniques. The numbers of cells that contained endothelin-1 and secreted endothelin-1 were counted after the cells had been incubated in control conditions, or with added endothelin-1, angiotensin-II, or endothelin receptor antagonists, bosentan and BQ788. Endothelin-1 and angiotensin-II increased the numbers of cells that secreted endothelin-1. On the other hand, bosentan and BQ788 caused a reduction in the numbers of endothelin-1-secreting cells. These results indicate that human endothelial cells contain a pathway by which endothelin-1 induces its own release. The receptor antagonists, bosentan and BQ788, inhibited basal secretion of endothelin-1.     

Endothelin and endothelin antagonists: Potential role in cardiovascular and renal disease

Endothelin-1 is a recently discovered peptide mainly released from endothelial cells. Hypoxia and ischemia as well as numerous factors such as angiotensin 11, thrombin and transforming growth factor ₁ stimulate the fomation of the peptide. On the other hand the synthesis of endothelin is inhibited by nitric oxide and atrial natriuretic peptide via the formation of cyclic guanosine monophosphate. Released from endothelial cells endothelin-1 mediates transient vasodilation followed by a profound and longlasting vasoconstriction. Endothelin is also a mitogen for smooth muscle proliferation. Endothelins exert their biological effects via activation of specific receptors. Two different receptors have been cloned from mammalian tissues (ET_A and ET_B receptors). On vascular smooth muscle cells both receptors mediate contractions. Endothelial cells only express ET_B receptors linked to the formation of nitric oxide and/or prostacyclin formation. Increased plasma concentrations of endothelin-1 have been described in a variety of diseases such as pulmonary hypertension, arteriosclerosis, renal failure, acute coronary syndromes, heart failure, migraine and vascular diseases.Recently an increasing number of endothelin receptor antagonists have been synthetized, which have been shown to inhibit endothelin-mediated vasoconstriction. Clinical studies are now ongoing to elucidate the pathophysiologic role of endothelin and the potential benefit of the blockade of the system in different disease states.     

Identification of G protein-coupled endothelin receptors in cultured bovine endothelial cells.

Autocrine and paracrine regulation of vascular endothelial cells by endothelins has been postulated and has been the target of many recent investigations. In the present study, we demonstrated, by affinity labeling, the presence of endothelin-1- and endothelin-3-specific receptors on cultured bovine endothelial cells that secrete endothelin; the endothelin-3 receptor was the major form. Analysis using the GTP analogue guanosine 5'-O- (thiotriphosphate) indicated that these receptors are coupled to G proteins.     

Immunohistochemical localization of endothelin in human vascular endothelial cells

The cellular localization of endothelin (ET), a novel vasoconstrictor peptide, was studied in human vascular tissues by immunohistochemistry. Distinct and diffuse staining for ET-like immunoreactivity was demonstrated in the cytoplasm of vascular endothelial cells, but not in smooth muscle cells or adventitial fibroblasts. The specificity was confirmed by the negative results following immunoabsorption. These findings suggest that human vascular endothelial cells function as an endocrine and/or paracrine cells for ET secretion.     

Endothelin-converting enzyme in human tissues

Our aim was to determine whether the expression of endothelin-converting enzyme in human tissues would correlate with the distribution of its substrate, big endothelin-1, and its product, the mature peptide. Site-directed antisera raised against the conserved C-terminus of the mammalian enzyme were used to measure the immunoreactive enzyme in microsomal fractions prepared from tissue homogenates and to localize staining to the endothelial cells lining large conduit and smaller resistance vessels within cardiac, adrenal, respiratory and brain tissue. The activity of endothelin-converting enzyme was measured and characterized in isolated endothelial cells. This pattern of staining in the vascular endothelium paralleled that of mature endothelin and big endothelin-1, and these peptides were detectable by radioimmunoassay in all tissues examined. Immunoreactive endothelin-converting enzyme localized to other cell types, including bronchial epithelial cells, and to fibres within the glial limitans, neuronal processes and cell bodies of the cerebral cortex. Although perivascular astrocytes in the subcortical white matter displayed intense endothelin-converting enzyme-like immunoreactivity, endothelin staining was not detected. The results suggest that endothelin-converting enzyme has a ubiquitous distribution within the human vascular endothelium and is positioned to catalyse the conversion of big endothelin-1 to the biologically active endothelin-1, which on release may contribute to the maintenance of basal tone in humans. Endothelin-converting enzyme localized to epithelial cells in peripheral tissues or astrocytes within the brain may be upregulated in pathophysiological conditions in which endothelin levels are increased and could represent a further target for therapeutic intervention by enzyme inhibitors. © 1998 Chapman & Hall     

Homeostasis in the gastric mucosa and blood circulation. Part 1. Mechanisms of the adequate blood flow maintenance in the gastric mucosa]

The increase of mucosal blood flow in response to food entrance into stomach or different irritant action is the component of gastric mucosal defence barrier. Sufficient blood flow ensures normal acid-bicarbonate balance in gastric mucosa, supports the healing process and prevents superficial damages from developing into deep ones. Capsaicin-sensitive afferent nerve fibers play the large role in the blood flow regulation. The influence of these nerve fibers on the gastric blood flow is mediated by the calcitonin-gene related peptide. This peptide released from peripheral afferent terminals improves microcirculation in stomach walls. Moreover nerve impulses from afferent neurons modulate parasympathetic activity that in turn induces the increase of gastric mucosal blood flow through both choilinergic and noncholinergic mechanisms. The gastric mucosal blood flow may be also regulated by humoral and paracrine metabolites. Nitric oxide and prostaglandines are the most important low molecular weight compounds. They play the main role in the maintenance of the basal gastric mucosal blood flow and in the development of hyperemic responses to harmful agents.     

Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1

The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extra- and intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone.Australian Peptide Conference Issue.     

Expression and localization of the thromboxane A2 receptor in human nasal mucosa

Thromboxane A2 (TXA2) has been thought a potent mediator involved in allergic rhinitis, because TXA2 was recovered from the nasal lavage fluid of allergic rhinitis patients after allergen provocation and TXA2 receptor antagonists relief nasal allergic symptoms. In order to clarify the expression of TXA2 receptor in human nasal mucosa, we investigated TXA2 receptor mRNA expression and its protein localization by polymerase chain reaction (PCR) and immunohistochemistry, respectively. Human turbinates were obtained after turbinectomy from 10 patients with nasal obstruction refractory to medical therapy. RT-PCR analysis of total RNA from nasal mucosa demonstrated the expression of TXA2 receptor alpha mRNA. The immunohistochemical studies revealed that anti-TXA2 receptor alpha antibody labeled vascular smooth muscle cells, vascular endothelial cells, epithelial cells and submucosal glands in the nasal mucosa. The results may have an important clinical implication for understanding the role of TXA2 receptor on upper airway diseases such as allergic rhinitis and non-allergic rhinitis.     

Electron-microscopic immunolabelling of vasoactive substances in human umbilical endothelial cells and their actions in early and late pregnancy

Human umbilical vessels are devoid of nerves and therefore endothelial cells may play an important role in the control of feto-placental blood flow. The pharmacological effects of 5-hydroxytryptamine, histamine and endothelin were examined in umbilical arteries and veins from legal terminations (gestational age 8–17 weeks, n=12) and normal term vaginal deliveries (gestational age 38–41, n=12). Immunocytochemistry of human unbilical vessels indicated that 5-hydroxytryptamine, histamine and endothelin were localised in subpopulations of endothelial cells of both artery and vein in late, but not early, pregnancy. 5-Hydroxytryptamine (10 nM–30 μM) caused sustained concentration-dependent contractions in all vessels from early and late pregnancy. Histamine (0.1 μM–30 mM) also caused sustained contractions in all vessels from late pregnancy but only 27% of arteries and 41% of veins from early pregnancy responded. Endothelin (10 pM–30 nM) caused slow long-lasting contractions in all vessels from early and late pregnancy. Atrial natriuretic peptide and neuropeptide Y did not alter vascular tone. The endothelium may thus play an autocrine/paracrine role, by synthesizing and releasing the above reactive substances in late pregnancy to influence feto-placental blood flow. Received: 23 May 1995 / Accepted: 13 October 1995     

Expression of human preproendothelin-1 cDNA in COS cells results in the production of mature vasoactive endothelin-1

Transient transfection of simian kidney (COS) cells with a recombinant plasmid encoding human preproendothelin-1 resulted in the production of biologically active endothelin-1. Conditioned medium from human preproendothelin-1 transfected cells demonstrated a significant increase in immunoreactive endothelin and big endothelin which co-eluted, when analyzed by reverse phase HPLC, with synthetic endothelin-1 and big endothelin-1, respectively. In addition, biological activity was confirmed by both inhibition of [125I]endothelin-1 binding to rat cerebellar and renal medullary membrane endothelin receptors and in vitro vasoconstriction of rabbit aorta. This is the first demonstration that human preproendothelin-1 is capable of being processed to a vasoactive form in a heterologous system and suggests that human preproendothelin-1 transfected COS cells may provide a useful model system for the study of endothelin biosynthesis.     

Transforming growth factor-beta stimulates the expression of endothelin mRNA by vascular endothelial cells

Endothelin is a potent vasoconstrictor peptide produced by vascular endothelial cells. Incubation of the serum-deprived confluent porcine aortic endothelial cells with 10-300 pM TGF-beta 1, resulted in a several fold increase in endothelin mRNA levels with a peak time of 2 h. An enzyme-linked immunosorbent assay revealed that the levels of endothelin in endothelial cell conditioned media was also increased by TGF-beta 1. These results suggest that TGF-beta 1, secreted by activated platelets, is involved not only in wound healing, but in the regulation of local vascular tone by stimulating endothelin production in the endothelial cells.     

Nasal wall compliance in vasomotor rhinitis.

Nasal compliance is a measure related to the blood volume in the nasal mucosa. The objective of this study was to better understand the vascular response in vasomotor rhinitis by measuring nasal cross-sectional area and nasal compliance before and after mucosal decongestion in 10 patients with vasomotor rhinitis compared with 10 healthy subjects. Nasal compliance was inferred by measuring nasal area by acoustic rhinometry at pressures ranging from atmospheric pressure to a negative pressure of -10 cmH2O. Mucosal decongestion was obtained with one puff per nostril of 0.05% oxymetazoline. At atmospheric pressure, nasal cross-sectional areas were similar in the vasomotor rhinitis group and the healthy subject group. Mucosal decongestion did not induce any decrease of nasal compliance in patients with vasomotor rhinitis in contrast with healthy subjects. Our results support the hypothesis, already proposed, of an autonomic dysfunction based on a paradoxical response of the nasal mucosa in vasomotor rhinitis. Moreover, the clearly different behavior between healthy subjects and vasomotor rhinitis subjects suggests that nasal compliance measurement may therefore represent a potential line of research to develop a diagnostic tool for vasomotor rhinitis, which remains a diagnosis of exclusion.     

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.     

Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells

Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [³H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two-to threefold) and secretion (1.8-to 2.8-fold) of VEGF reaching maximal levels between 4–8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [³H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma.     

Release of calcitonin gene-related peptide in the pig nasal mucosa by antidromic nerve stimulation and capsaicin

The overflow of calcitonin gene-related peptide like-immunoreactivity (CGRP-LI) in the nasal venous effluent upon antidromic stimulation of the maxillary division of the trigeminal nerve with 6.9 Hz for 3 min or upon capsaicin (0.3 mumol bolus injection) were analysed in the nasal mucosa of sympathectomized pentobarbital anaesthetized pigs. The overflow of CGRP-LI upon antidromic stimulation displayed a slower appearance in the venous effluent than the overflow upon bolus injection of capsaicin. The vascular effects as revealed by the arterial blood flow, the venous blood flow, the blood volume of the nasal mucosa, i.e., the filling of the capacitance vessels and the superficial mucosal blood flow as revealed by the laser-Doppler signal were also studied. Antidromic stimulation of the trigeminal nerve as well as capsaicin bolus injection induced a marked vasodilation which was parallel to the overflow of CGRP. However, capsaicin bolus injection also resulted in a marked increase in the mean arterial blood pressure which may be due to reflex activation of sympathetic fibers. In conclusion, we have demonstrated that chemical stimulation with capsaicin as well as antidromic stimulation of nasal sensory nerves in sympathectomized animals induces both vasodilation and overflow of CGRP-LI in vivo. This indicates that CGRP may contribute to the sensory regulation of the microcirculation in the nasal mucosa.