The effects of halothane and isoflurane on intracellular Ca2+ regulation in cultured cells with characteristics of vascular smooth muscle.

The Ca(2+)-sensitive photoprotein aequorin was used to monitor changes in intracellular [Ca2+] within cultured cells with characteristics of vascular smooth muscle. Two cell lines were investigated: they were A10 cells, which are transformed cells originally derived from rat aorta, and BC3H1 cells obtained from mouse brain neoplasm. Transient increases in intracellular [Ca2+] were induced following exposure to two different volatile anaesthetics (halothane and isoflurane) and various vasoactive substances (acetylcholine, endothelin, histamine, serotonin and vasopressin). The amplitude of the transients induced by isoflurane were more dependent on the presence of extracellular Ca2+ than those induced by halothane, thus the modes and/or locations of action of these two anesthetics are somewhat different. The response of the two cell lines to the vasoactive substances are unique. Receptor activated changes in [Ca2+]i by various agonists were diminished in the presence and absence of either anesthetic. These data suggest that, although the receptor populations within each cell line were slightly different, the prior application of a volatile anesthetic in a clinically-relevant dose induced a transient increase in [Ca2+]i that could subsequently diminish agonist responses.     

Cytokines suppress the shear stress-stimulated release of vasoactive peptides from human endothelial cells

Endothelial cells from human umbilical vein perfused at 0.5 ml/min released vasopressin, endothelin, and substance P. Upon perfusion of the cells at 3.0 ml/min, the release of endothelia and vasopressin was significantly increased whereas the release of substance P was significantly decreased. Endothelial cells precultured for 24 h with interleukin-I (IL-1) and interferon-γ (IFN-γ) released more endothelin and less substance P at low flow and there was no further increase in release at high flow rate. These results suggest that cytokines suppress the normal responses of endothelial cells to increased fluid shear stress.     

Chronic hypoxia changes the ratio of endothelin to ATP release from rat aortic endothelial cells exposed to high flow.

Endothelial cells isolated from the thoracic aorta of normoxic and chronically hypoxic rats were perfused (0.5 ml min-1) and stimulated by increased flow rate (3.0 ml min-1). The release of ATP, endothelin and vasopressin was investigated. During periods of high flow rate, endothelial cells isolated from normoxic rats increased their release of ATP and endothelin. In comparison, in hypoxic rats, ATP release during the period of high flow rate was less, whereas endothelin release was greater. Vasopressin release was not increased during periods of stimulation in either group of animals. These results suggest that, under conditions of reduced arterial oxygen tension, a dynamic balance between ATP and endothelin release could regulate the response of vessels to shear stress.     

Cerebral endothelial cell culture. I. The presence of beta 2 and alpha 2-adrenergic receptors linked to adenylate cyclase activity

Cultured endothelial cells derived from cerebral microvessels separated from 2-day-old rat brain contain a specific beta 2 and alpha 2-adrenergic sensitive adenylate cyclase (AC). Among the various tested hormones, PGE1 and PGE2 were found to be the most potent activators, while adenosine, angiotensin I and II, gamma-aminobutyric acid and vasoactive intestinal peptide inhibited the enzyme activity. However, acetylcholine, histamine, serotonin, glycine, glutamine, bradykinin, neurotensin and vasopressin (Lysine and Arginine) had no effect on the adenylate cyclase activity in this model. The susceptibility of the cerebrovascular endothelial AC system to the vasoactive substances as well as presence of beta 2 and alpha 2-type adrenergic receptors in the cultured endothelium provides additional support for the proposed endothelial involvement in the regulation of cerebrovascular permeability and blood flow.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Endothelin and sarafotoxin produce dissimilar effects on renal blood flow, but both block the antidiuretic effects of vasopressin

Human endothelin, a 21-residue peptide produced by vascular endothelial cells, was infused intravenously into trained conscious dogs at a rate of 20 ng.kg-1.min-1 for 1 hr. Endothelin produced a renal vasoconstriction that persisted during a 40-minute recovery period. Sarafotoxin S6b, a closely related 21-residue peptide that has been isolated from the venom of the burrowing asp, was also infused into the same conscious dogs at 20 ng.kg-1.min-1. Sarafotoxin produced a renal vasodilation that persisted throughout the infusion; when the infusion ended, however, renal blood flow decreased rapidly to below control levels. Both endothelin and sarafotoxin produced marked decreases in urine osmolality even though plasma vasopressin remained normal, thus indicating that these peptides inhibit the antidiuretic effects of vasopressin. These results imply that a broad spectrum of structure-activity relationships may exist among analogues of this unique group of 21-residue peptides.     

Plasminogen activator activity by cultured bovine aortic endothelial cells

This communication reports the observations that bovine aortic endothelial cells (EC) in culture during their log phase of growth secrete plasminogen activator. Hydrocortisone, dibutyryl cAMP, theophylline, colchicine and cycloheximide, dependent upon concentration, inhibit plasminogen activator activity. Several substances associated with inflammation and thrombosis, such as thrombin, serotonin,catecholomines, histamine, vasopressin, endotoxin, and indomethacin, at the concentrations tested, did not significantly alter plasminogen activator activity when compared with controls.     

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.     

Endothelial cells stimulate ANF secretion from atrial myocytes in co-culture

Using a novel in vitro co-culture system, we investigated the possible influence of vascular endothelial cells on the secretion of atrial natriuretic factor (ANF) from atrial myocytes. Co-culture of bovine aortic endothelial cells grown on Cytodex-3 microcarrier beads with primary monolayer cultures of neonatal rat myocytes induced a 2.1-fold increase in immunoreactive ANF (irANF) in the medium, compared with irANF in medium from atrial cultures alone. This increase did not appear to be the result of processing of prohormone to more immunoreactive species, and could be inhibited by 47% with 10 microM acetylcholine. The endothelium-derived vasoconstrictor peptide, endothelin, elicited a dose-dependent increase in ANF secretion from atrial cultures, but, contrary to vasopressin, was incapable of further stimulating release from atrial-endothelial co-cultures. These experiments suggest that endothelium stimulates the release of ANF from myocytes, possibly by the action of the peptide endothelin.     

Vasopressin V1a receptor signaling in a rat choroid plexus cell line

A new cell line was derived from primary culture of rat choroid plexus (RCP) by immortalization with the TSOri minus adenovirus. The selected clone expressed vasopressin V1a receptors at a density of 64,000 sites per cell, and a K(d) of 7.2 nM. Addition of vasopressin to the RCP cells induced a transient calcium peak comparable to V1a receptor signalling in different expression systems. This [Ca(2+)](i) increase was dose-dependent with an EC(50) of 22 nM vasopressin. Similar [Ca(2+)](i) increase was elicited by addition of serotonin, angiotensin II, endothelin-1, and bradykinin. Heterologous desensitization of V1a receptor was observed in RCP cells exposed to the phorbol ester PMA or following stimulation of other receptors coupled to the phosphoinositide pathway. Positive immunolabelling with Factor VIII, Flt1 and CD 34 antibodies suggests that this new RCP cell line originated from endothelial cells of rat choroid plexus.     

Modulation of secretion of vasoactive materials from human and bovine endothelial cells by cyclic strain

The effects of cyclical expansion and elaxation of the vessel wall on endothelial cell metabolism have been modeled using a uniaxial strain device and cultured endothelial cell monolayers. Also, the effects of stopping and then restarting cyclic strain on metabolite secreation rates were determined. Secretion rates of prostacyclin (PGI(2)), endothelin, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-type 1 (PaI-1) by endothelial cells were constant over24-h periods The secreation of both PGI(2) and endothelin was enhanced in cells exposed to high physiological levels of cyclical strain (10% at 1Hz) compared with controls, while tPA production was unaltered. These results were true for both human and bovine endothelial cells. Characterization of the response of human endothelial cells to cyclical strain made evaluation of stretch effects on PAl-1 secretion possible. A nearly twofold increase in PAl-1 secretion by cells exposed to arterial levels of strain was observed. Endothelin secretion remained elevated even after strain was stopped for 12 h, while PGl(2) secretion returned to control values upon cessation of cyclic stretch. These results indicate that physiological levels of cyclic mechanical strain ca significantly modulate secretion of vasoactive metabolited form endothelial cells. The changes sen secretion are, in some cases, quite different from those caused by arterial levels of fluid shear stress exposure. (c) 1994 John Wiley & Sons, Inc.     

Inositol phosphate release and steroidogenesis in rat adrenal glomerulosa cells. Comparison of the effects of endothelin, angiotensin II and vasopressin.

Endothelin has steroidogenic activity in adrenal glomerulosa cells, as do two other vasoconstrictor peptides, angiotensin II and vasopressin. The steroidogenic activities of angiotensin II and vasopressin are probably mediated via the phosphatidylinositol-turnover pathway and associated changes in cytosolic Ca2+ concentration. Endothelin caused a steroidogenic response, which was small compared with that to angiotensin II and quantitatively similar to the vasopressin response. Cytosolic free Ca2+ responses were similarly higher to angiotensin II than to either of the other two peptides. However, total inositol phosphate responses to endothelin and angiotensin II were similar when these were measured over 20 min, and were quantitatively greater than the vasopressin response. A detailed study has been made of the phosphatidylinositol-turnover response to endothelin in comparison with responses to angiotensin II and vasopressin. Each of the three peptides produced a rapid and transient rise in Ins(1,4,5)P3 (max. 5-15 s), followed by a slow sustained rise. Ins(1,4,5)P3 was metabolized by both dephosphorylation and phosphorylation pathways, but the relative importance of the two metabolic pathways was different under stimulation by each of the three peptides. These findings show that adrenal glomerulosa cells can distinguish between the stimulation of phosphatidylinositol turnover by three different effectors. These differences in the pathway may be associated with the observed different steroidogenic and Ca2+ responses to the three peptides.     

Localization of vasopressin,serotonin and angiotensin II in endothelial cells of the renal and mesenteric arteries of the rat

Summary The localization of vasopressin, serotonin and angiotensin II in the endothelial cells of renal and mesenteric arteries was investigated using the pre-embedding peroxidase-antiperoxidase technique for electron microscopy. Vasopressin-and serotonin-positive endothelial cells were present in both renal and mesenteric arteries while angiotensin II-positive cells were observed in the mesenteric artery exclusively. Both arteries showed less than 10% immunoreactive cells. The lack of angiotensin II in the endothelial cells of the renal artery suggests that there may be subtle physiological differences between the renal and mesenteric arteries with respect to the local control of blood flow.     

Parasite-induced processes for adenosine permeation in mouse erythrocytes infected with the malarial parasite Plasmodium yoelii.

In fura-2-loaded A10 vascular smooth-muscle cells, 1 nM-vasopressin and 200 nM-endothelin evoked a rapid transient rise in intracellular free Ca2+ concentration [( Ca2+]i), which was then followed by a maintained elevation of [Ca2+]i. The maintained elevation of [Ca2+]i was only partially inhibited by 5 microM-nifedipine, but completely abolished in the presence of 1 mM-EGTA. When extracellular Ca2+ was replaced with 1 mM-Mn2+ (Mn2+ quenches fura-2 fluorescence), both endothelin and vasopressin evoked an Mn2+ quench of the fluorescence from the intracellularly trapped fura-2, even in the presence of 5 microM-nifedipine. These data suggest that both vasopressin and endothelin promote a bivalent-cation influx and provide further evidence for receptor-mediated Ca2+ entry in vascular smooth muscle.     

Cytokine stimulated endothelin release from endothelial cells

Endothelin release from bovine endothelial cells of the aorta, pulmonary artery, and retinal microvessels was measured in response to various cytokines. Transforming growth factor beta (0.05-5 ng/ml) was found to be a potent stimulator (3-4 fold increase) of endothelin secretion in all three cell types. Tumour necrosis factor alpha (0.1-10 ng/ml) and interferon gamma (8-800 U/ml) had a small (1.5-2 fold increase) but significant effect on endothelin secretion from endothelial cells of large vessels but not the retinal microvessels. Interleukin-1 beta, Interleukin-6 and interleukin-8 at various doses did not affect endothelin secretion. These effects were observed at various time points from 6-24 hrs and indicate that of the cytokines tested, only transforming growth factor beta has a potent effect on endothelin release from endothelial cells of different organs.     

剪切应力下内皮细胞内皮素及其mRNA的表达

应用Ｎｏｒｔｈｅｒｎ印迹方法研究高血压（ＳＨＲ）和正常（ＷＫＹ）大鼠脑微血管培养内皮细胞,在剪切应力０、０．５、１、２Ｐａ作用下２４ｈ后检测内皮素及其基因ｍＲＮＡ表达上的区别。结果表明,在剪切应力０、０．５、１Ｐａ下,ＷＫＹ大鼠的内皮细胞随着剪切应力加大,其内皮素水平及其基因的ｍＲＮＡ的表达均比ＷＫＹ相应组的为高。在剪切应力２Ｐａ时,ＷＫＹ和ＳＨＲ大鼠的内皮细胞内皮素及其基因的ｍＲＮＡ表达水平不同     

Traumatic shock causes elevation of the serotonin 5-HT2B receptor mRNA level in rat aorta

Previously we have shown that at traumatic shock in rats the force of contraction of isolated aorta in response to angiotensin II, vasopressin, endothelin 1, or norepinephrine is decreased. On the contrary, vasoconstriction caused by serotonin is increased. A possible reason of the alterations of neuroendocrine regulation of vascular tone in shock may be a change in the expression of the receptors of these agonists in blood vessels. In the present study, using real-time PCR, we demonstrated that a day after injury the contents of mRNA encoding receptors V1A for vasopressin, ETA for endothelin 1, and AT1 for angiotensin II are not changed in aorta. There was a slight increase of the serotonin 5-HT2A receptor mRNA (36 ± 16%; p = 0.41). The level of the 5-HT2B receptor mRNA in aorta, initially low (2% of the content of the mRNA of receptors 5-HT2A), after the injury increased 15.8 ± 0.3 times (p < 0.01). However, at traumatic shock there was no contraction of aorta in response to 5-HT2B receptor agonist BW723C86, while vasodilation of the isolated aorta preconstricted with norepinephrine in response to BW723C86 was similar to that of the vessel isolated from control rats. The data obtained suggest that the observed 5-HT2B receptor overexpression is not related to the increased serotonin-induced vasoconstriction and might cause other vascular pathological changes at traumatic shock.     

ACTH release in pituitary cell cultures. Effect of neurogenic peptides and neurotransmitter substances on ACTH release induced by hypothalamic corticotropin releasing factor (CRF).

The effects of various neurogenic peptides and neurotransmitter substances on the release of ACTH induced by hypothalamic corticotropin releasing factor (HY-CRF) were investigated using monolayer cultured anterior pituitary cells. Test substances were given in combination with 0.05-0.1 hypothalamic extract (HE)/ml, because HE evoked a significant ACTH release and a linear dose response relationship was demonstrated sequentially between 0.0165 HE/ml and 0.5 HE/ml. Relative high doses of lysine-vasopressin showed a slight additive effect on the release of ACTH induced by 0.1 HE/ml. Leu-enkephalin, dopamine, prostaglandin E1 and E2 slightly reduced the release of ACTH induced by HY-CRF, but the inhibitory effect of these substances were not dose-related. Other tested substances including luteinizing hormone releasing hormone, thyrotropin releasing hormone, somatostatin, melanocyte stimulating hormone release inhibiting factor, beta-endorphin, neurotensin, substance P, vasoactive intestinal polypeptide, angiotensin II, norepinephrine, serotonin, acetylcholine, histamine and gamma-amino butyric acid showed neither agonistic nor antagonistic effect on the release of ACTH induced by HY-CRF. These results indicate that the release of ACTH is controlled specifically by HY-CRF and corticosterone, and modified slightly by some other substances such as vasopressin and prostaglandins, and that the effect of most other neurogenic peptides and neurotransmitter substances is negligible or non-physiological at the pituitary level.     

Inhibition by angiotensin converting enzyme inhibitors of endothelin secretion from cultured human endothelial cells.

We conducted a study to determine whether angiotensin converting enzyme inhibitors (ACEIs) inhibit endothelin secretion from cultured human endothelial cells. Confluent umbilical vein endothelial cells were incubated in multi-well plates with culture medium containing either captopril (10(-6), 10(-5), 10(-4) M) or enalaprilat (10(-7), 10(-6), 10(-5) M) for 6 hours. Immunoreactive endothelin in the medium was measured by radioimmunoassay. Calf serum (CS) stimulated endothelin release in a concentration-dependent manner, and both ACEIs inhibited 5% CS-stimulated endothelin release in a concentration-dependent manner. To explore the mechanisms of ACEI-induced suppression of endothelin release, the effects of angiotensin II (10(-8), 10(-7), 10(-6) M), angiotensin converting enzyme (0.1, 1, 10 mU/ml), bradykinin (10(-8), 10(-7), 10(-6) M), and sodium nitroprusside (10(-6), 10(-5), 10(-4) M) on endothelin release were also examined. Although angiotensin II and angiotensin converting enzyme had no significant effect on endothelin release, concentration-dependent suppression occurred with bradykinin and sodium nitroprusside. These results indicate that ACEIs inhibit the stimulated release of endothelin from human endothelial cells, and provide indirect evidence that ACEI-induced ET suppression may be mediated via potentiation of autacoid formation from the cells.