Nitric oxide modulates basal and endothelin-induced coronary artery vascular smooth muscle cell proliferation and collagen levels

The mechanisms governing the pathological accumulation of collagen in the extracellular matrix following angioplasty are complex, but may involve interactions between endothelium-derived paracrine agents and vascular cellular components. We tested the hypothesis that nitric oxide (NO) directly decreases collagen levels and decreases endothelin (ET-1)-stimulated increases in levels of specific collagen subtypes in coronary vascular smooth muscle cells (VSMC). Cultured VSMC were incubated for 48 h with the NO donor CAS 754 (10(-4) M), ET-1 (10(-8) M), or ET-1 plus CAS 754. In some experiments, angiotensin II (Ang II; 10(-8) M) was utilized in place of ET-1. Soluble collagen types I and III were quantitated with an ELISA method, and cell counts were performed. CAS 754 significantly inhibited cell proliferation (-17+/-2% v control), basal total protein synthesis (-65+/-7% v control), and basal collagen type I levels (-39+/-6% v control), but not collagen type III levels. ET-1 and Ang II both significantly stimulated cell proliferation (26+/-5% v control), total protein synthesis (169+/-6% v control), and collagen type I levels (200+/-11% v control). Ang II, but not ET-1, significantly increased collagen type III levels. Co-incubations of ET-1 and CAS 754 resulted in a significant decrease in cell proliferation, protein synthesis, and collagen levels (-23+/-2% v control, 90+/-5% v control, and 63+/-3% v control, respectively) compared to ET-1 alone. In contrast, co-incubation of Ang II and CAS 754 had no significant effect on cell proliferation, protein synthesis, and collagen levels seen with Ang II alone. These results demonstrate that NO inhibits basal collagen levels and cell division. Additionally, NO alters ET-1 stimulation of VSMC proliferation, protein synthesis, and production of extracellular matrix components. Thus, an imbalance in key endothelium-derived compounds could significantly impact upon extracellular matrix deposition following mechanical revascularization.     

Decreased type II/type I TGF-beta receptor ratio in cells derived from human atherosclerotic lesions. Conversion from an antiproliferative to profibrotic response to TGF-beta1

Atherosclerosis and postangioplasty restenosis may result from abnormal wound healing. The present studies report that normal human smooth muscle cells are growth inhibited by TGF-beta1, a potent wound healing agent, and show little induction of collagen synthesis to TGF-beta1, yet cells grown from human vascular lesions are growth stimulated by TGF-beta1 and markedly increase collagen synthesis. Both cell types increase plasminogen activator inhibitor-1 production, switch actin phenotypes in response to TGF-beta1, and produce similar levels of TGF-beta activity. Membrane cross-linking of 125I-TGF-beta1 indicates that normal human smooth muscle cells express type I, II, and III receptors. The type II receptor is strikingly decreased in lesion cells, with little change in the type I or III receptors. RT-PCR confirmed that the type II TGF-beta1 receptor mRNA is reduced in lesion cells. Transfection of the type II receptor into lesion cells restores the growth inhibitory response to TGF-beta1, implying that signaling remains responsive. Because TGF-beta1 is overexpressed in fibroproliferative vascular lesions, receptor-variant cells would be allowed to grow in a slow, but uncontrolled fashion, while overproducing extracellular matrix components. This TGF-beta1 receptor dysfunction may be relevant for atherosclerosis, restenosis and related fibroproliferative diseases.     

The significance of eating patterns: an elderly Greek case study

BACKGROUND: Endothelin-1 (ET-1), a novel vasoconstrictor, possibly plays a role in the mediation of ischemia/reperfusion (I/R) injury. Tacrolimus (FK506) and cyclosporin A (CsA) were reported to maintain tissue microcirculation of the liver subjected to I/R. This study investigated the effects of these immunosuppressants on intestinal I/R in terms of intestinal tissue microcirculation associated with ET-1. METHODS AND RESULTS: Male S-D rats were pretreated twice with FK506 (0.2 mg/kg), CsA (10 mg/kg) or only saline solution (0.5 mL). The tissue microcirculation in the control was reduced after I/R (29% +/- 10%) accompanied by hypotension, increased tissue ET-1 expression (25.0% +/- 6.4% to 67.9% +/- 5.0% 60 minutes after reperfusion), and increased ET-1 level in the portal blood (3.4 +/- 0.9 to 23.6 +/- 6.1 pg/mL). FK506 suppressed ET-1 expression (27.3% +/- 5.2%, 4.1 +/- 2.2 pg/mL), maintained microcirculation (96% +/- 16%), and blood pressure, reduced histologic damage, and improved survival. CsA had a similar but weaker effect compared with FK506. An additional experiment was performed with BQ485Na (BQ), an ETA receptor antagonist, to evaluate the genuine role of ET-1. BQ showed almost the same effects as FK506. CONCLUSIONS: FK506 and CsA, particularly the former, maintain microcirculation and protect the tissue from I/R injury by suppressing the production and release of ET-1.     

Endothelin receptor subtypes and their functional relevance in human small coronary arteries

1. The potent constrictor peptide endothelin (ET) has been implicated in various cardiovascular disorders including myocardial infarction and atherosclerosis. We have investigated the nature of ET receptor subtypes present on human small coronary arteries. 2. Small coronary arteries were mounted in a wire-myograph for in vitro pharmacology. To investigate the ET receptor subtypes present in different segments of the coronary vascular tree, arteries were grouped according to internal diameter. Responses in arteries with small internal diameters (mean 316.7+/-7.9 microm; Group B) were compared to those in larger arteries (mean 586.2+/-23.1 microm; Group A). 3. ET-1 consistently and potently contracted arteries from Group A and B, with EC50 values of 1.7 (0.9-3.2) nM (n=15) and 2.3 (1.4-4.2) nM (n=14), respectively. No correlation was observed between ET-1 potency and internal diameter. The response to ET-1 was potently antagonized by the selective ET(A) receptor antagonist PD156707 in both Group A and Group B, yielding pA2 values of 8.60+/-0.12 (n=4-6) and 8.38+/-0.17 (n=4-6), respectively. Slopes from Schild regression were not significantly different from unity. 4. In contrast to ET-1, individual responses to ET-3 were variable. While all arteries from Group A responded to ET-3 (EC50 approximately 69 (23-210) nM) (n=12), no response was obtained in 5 of the 14 tested in Group B. Of those responding, many failed to reach a maximum at concentrations up to 1 microM. ET-1 was more potent than ET-3 in all arteries tested. A biphasic ET-3 response was observed in 8 arteries suggesting that a small ET(B) population was also present in some patients. The selective ET(B) receptor agonist sarafotoxin S6c had little or no effect up to 10 nM (n=4-6). 5. Responses to ET-1 and ET-3 were unaffected by removal of the endothelium in arteries from both groups suggesting a lack of functional, relaxant ET(B) receptors on endothelial cells (n=5). 6. Using autoradiography, specific high density binding of the non-selective, ET(A)/ET(B) ligand [125I]-ET-1 and selective ET(A) ligand [125I]-PD151242 was detected on the vascular smooth muscle layer of small intramyocardial coronary arteries (n=5). In contrast, little or no binding of the selective ET(B) receptor ligand [125I]-BQ3020 was observed (n=5). Similarly, [125I]-ET-1 binding to vascular smooth muscle was absent in the presence of the selective ET(A) receptor antagonist PD156707. 7. We conclude that human small epi- and intramyocardial coronary arteries express predominantly ET(A) receptors and it is these receptors which mediate ET-induced contractions. A constrictor ET(B) receptor population may exist in some patients. However, these receptors may have a limited role as contractions to ET-1 can be blocked fully by the selective ET(A) receptor antagonist PD156707.     

Selective ET(A) receptor antagonism reduces neointimal hyperplasia in a porcine coronary stent model

BACKGROUND: As endothelin binds to ET(A) receptors, it stimulates vascular smooth muscle cell proliferation and may thus be pivotally involved in the pathogenesis of restenosis. This study assessed the ability of a potent and selective ET(A) antagonist to reduce neointimal hyperplasia in a porcine coronary artery stented injury model. METHODS AND RESULTS: Fifty-five pigs were randomized to receive placebo or the oral ET(A)-selective antagonist ABT147627 twice daily for 28 days in one of three doses: 0.75 mg/kg (low), 3.75 mg/kg (mid), and 10.0 mg/kg (high). Each underwent oversized stent deployment in two randomly assigned major epicardial coronary arteries. Three animals (5.5%) died as a consequence of stent thrombosis within 24 hours of the procedure. The remaining 52 animals (13 pigs per group) survived without complication until predetermined euthanasia at 28 days. In the placebo group, mean injury score was 1.73+/-0.80, with a mean neointimal response of 0.45+/-0.24 mm. By comparison, the low-dose group had a similar mean injury score of 1.79+/-0.75 with reduced neointimal response, 0.36+/-0.22 mm (P<0.01). Mean injury score in the mid-dose animals was significantly greater than in the placebo group (1.94+/-0.92; P<0.05). The neointimal hyperplasia associated with this injury was less than with placebo, although the difference did not reach statistical significance (0.40+/-0.25 mm; P=0.05). In the high-dose pigs, mean injury score was also significantly greater than in the placebo arm (1.93+/-0.73; P<0.05). Despite this, neointimal response was also significantly less (0.37+/-0.37 mm; P<0.01). CONCLUSIONS: Oral, selective ET(A) receptor antagonism significantly reduced neointimal hyperplasia forming over porcine coronary stented injuries in the first 28 days. This strategy may have clinical potential for the limitation and treatment of coronary restenosis after percutaneous revascularization.     

Central pathology review in clinical trials for patients with malignant glioma. A Report of Radiation Therapy Oncology Group 83-02

We previously described delayed pressor response (DPR) 3 h after endothelin (ET)-1 injection in normotensive rats. In the current study, we examined effects of the ETA receptor antagonist BQ123 (0.01 mumol/kg/min intravenously, i.v.), phosphoramidon (100 mumol/kg i.v.), the neutral endopeptidase inhibitor SQ28603 (112 mumol/kg + 0.04 mumol/kg/min i.v.), the angiotensin-converting enzyme inhibitor enalaprilat (10 mumol/kg i.v.), and the thromboxane receptor antagonist, SQ29548 (0.5 mumol/kg + 0.5 mumol/kg/h i.v.) on DPR. Vehicle and ET-1 (1.0 nmol/kg i.v.) were administered on day 1; vehicle or drug and ET-1 were administered on day 2. BQ123 inhibited DPR 36% (vehicle 44 +/- 5, BQ123 28 +/- 3 mm Hg); phosphoramidon inhibited DPR 56% (vehicle 45 +/- 4, and phosphoramidon 20 +/- 5 mm Hg). DPR was unchanged after SQ28603 (vehicle 39 +/- 2 and SQ28603 44 +/- 2 mm Hg), enalaprilat (vehicle 39 +/- 2 and enalaprilat 38 +/- 7 mm Hg), or SQ29548 (vehicle 46 +/- 6 and SQ29548 43 +/- 3 mm Hg). The results suggest that DPR 3 h after ET-1 injection in rats is mediated in part through ETA receptors. DPR does not appear to involve thromboxane or synthesis of angiotensin II (AII), but may be related to synthesis of ET-1.     

Upregulation of IGF-I and collagen I mRNA in human atherosclerotic tissue is not accompanied by changes in type 1 IGF receptor or collagen III mRNA: an in situ hybridization study

BACKGROUND: Immunoreactive insulin-like growth factor-I (IGF-I) has recently been localized to vascular smooth muscle cells in coronary atherectomy plaques, but it remains unclear whether these cells are the source of this growth factor. We therefore investigated the gene expression of this factor, and the expression of the genes for its receptor and two types of collagen known to be regulated by IGF-I, in vascular tissue samples from patients with atherosclerosis or restenosis. METHODS: Gene expression and localization were investigated by in situ hybridization, using 35S-labelled complementary RNA probes specific for IGF-I, its type a receptor, collagen I, and collagen III. The cellular composition of the tissue samples was determined by immunohistochemistry using antibodies specific for smooth muscle cells and macrophages. RESULTS: IGF-I and collagen I messenger RNAs were found in areas containing smooth muscle cells and macrophages, but collagen III and type 1 IGF receptor gene expression could not be detected in any tissue samples. CONCLUSION: IGF-I appears to be involved in the progression of the atherosclerotic plaque, even at an advanced stage, but preliminary data from two restenotic plaques indicate that it may not be involved in the later stages of restenosis.     

Endothelin-1 regulates tone of isolated small arteries in the rat: effect of hyperendothelinemia

Chronic elevation of plasma endothelin-1 (ET-1) levels has been reported in several pathological conditions. To investigate the consequences of increased circulating ET-1 on vascular responsiveness, Sprague-Dawley rats (n=16) were chronically instrumented with a minipump delivering ET-1 at a constant dose for 7 days. Plasma ET-1 levels were more than doubled in treated (0.98+/-0.09 pmol/L; P<.05) versus untreated sham-operated rats (0.43+/-0.04 pmol/L), whereas systolic arterial blood pressure increased (139+/-5 versus 128+/-4 mm Hg in untreated rats; P<.05). After rats were killed, segments of middle cerebral (MCA) and mesenteric (MES) arteries were mounted on an isometric myograph. ET-induced contraction was shifted to the right in ET-1-treated animals and not modified by BQ123 (an ET(A) receptor antagonist); bosentan (ET(A/B) receptor antagonist) prevented ET-1-induced contraction in both groups. After inhibition of nitric oxide synthase with N(omega)-nitro-L-arginine (L-NNA), both phenylephrine and oxymetazoline (an alpha2-adrenoceptor agonist) induced MCA contraction. The sensitivity to phenylephrine was decreased in ET-1-treated compared with control rats (P<.05). Sensitivity to phenylephrine-induced contraction was decreased by BQ123 in control rats only. In contrast, L-NNA revealed greater oxymetazoline-induced contractions in treated compared with control MCA rings (P<.05); this potentiation was blunted by bosentan but unaffected by BQ123. Removal of the endothelium revealed a direct constrictor effect of oxymetazoline that was insensitive to L-NNA alone or combined with bosentan; however, oxymetazoline induced significantly lower constriction in treated rat MCA segments. Responses to oxymetazoline were also blunted in treated compared with untreated denuded MES arteries. In conclusion, chronic elevated plasmatic ET-1 decreases smooth muscle cell sensitivity to contractile agonists both in MCA and MES rings. In cerebral vessels, endothelial alpha2-adrenoceptor-dependent stimulation induced greater contractile responses in treated rats which were sensitive to bosentan, suggesting that oxymetazoline stimulates ET-1 release from the endothelium. This may represent a compensatory mechanism for the loss of smooth muscle sensitivity.     

EndothelinB receptors in rabbit pulmonary resistance arteries: effect of left ventricular dysfunction

The endothelin (ET) receptor that mediates vasoconstriction of the isolated rabbit pulmonary resistance artery was characterized using selective ET receptor agonists and antagonists. We also examined changes in ET-induced vasoconstriction brought about by left ventricular dysfunction using the rabbit coronary ligation model. The rank order of potency for contraction was sarafotoxin S6c (S6c) > ET-1 = ET-3, which is characteristic of an ETB-like receptor. The combined ETA/ETB receptor antagonist SB209670 (1 microM) antagonized responses to ET-1 and S6c with estimated pKb values of 6.8 +/- 0.2 and 7.8 +/- 0.2, respectively. BQ788 (1 microM) antagonized responses to S6c and ET-3 (but not ET-1) with estimated pKb values of 7.1 +/- 0.2 and 6.6 +/- 0.1, respectively. The ETA receptor antagonist FR139317 (1 microM), either alone or in combination with BQ788, did not inhibit responses to ET-1. The profile of the ET-1 response was not altered by left ventricular dysfunction. In control rabbits, the inhibitor of nitric oxide synthase N omega-nitro-L-arginine methyl ester (100 microM) had no significant effect on the potency of either ET-1 or S6c. In the coronary-ligated rabbits, however, it significantly increased the potency (10-15-fold) of both ET-1 and S6c. These results suggest that the ET receptor that mediates contraction in rabbit pulmonary resistance arteries has the characteristics of an ETB-like receptor. The responses to ET-1 are not altered by LVD but may be modified by increased release of nitric oxide.     

The mechanism of myometrial contractions induced by endothelin-1 in rat

Experiments were performed to characterize endothelin-1-induced contractions and the role of endothelin (ET) receptor subtypes in rat myometrium. The binding sites of [(125)I]-ET-1 were saturable with high affinity. Scatchard plot analysis revealed that ET-1 binding sites in the myometrium constituted a single population. The dissociation equilibrium constant (Kd) and the maximum binding sites (Bmax) were determined to be 48.9+/-3.0 pM and 1364.0+/-210.3 fmol/mg protein respectively. Specific [(125)I]-ET-1 binding was inhibited completely by unlabelled ET-1 and Ro 46-2005 (mixed-type ET receptor antagonist), but not fully (90.7+/-1.4%) by BQ 123 (a selective ETA receptor antagonist), and not at all by RES 701-1 (a selective ETB receptor antagonist). ET-1 induced myometrial contractions were composed of two types, an increase in resting tone and rhythmic contractions. These contractions were inhibited by BQ 123 and Ro 46-2005, but not by RES 701-1. ET-1-induced contractions were greatly reduced in Ca2+-free Krebs' solution. Nifedipine abolished the rhythmic contractions without affecting the increase in resting tone. These results suggest that ETA receptors are predominantly localized in rat myometrium and that excitation of ETA receptors evokes two types of contractions by increasing the cytoplasmic Ca2+ concentration.     

The structure of HLA-DM, the peptide exchange catalyst that loads antigen onto class II MHC molecules during antigen presentation

Endothelial cells produce C-type natriuretic peptide (CNP), which has been proposed as an endothelium-derived hyperpolarizing factor. In porcine coronary arteries, we investigated the vasodilatory effects of CNP and compared them with endothelium-dependent relaxations and hyperpolarizations to bradykinin. Isolated epicardial porcine coronary arteries were studied in organ chambers, and concentration-response curves to CNP and bradykinin were obtained. Membrane potential was measured in endothelial cells and smooth muscle of intact porcine coronary arteries during stimulation with CNP or bradykinin. In precontracted porcine coronary arteries with or without endothelium, CNP (10[-10]-10[-6] M) evoked relaxations (maximum, 42 +/- 4%) smaller than those evoked by bradykinin (100 +/- 1%), blunted in preparations contracted by KCl instead of U46619 (9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2alpha; p < 0.05) and unaffected by inhibition of NO synthase (NS). CNP evoked hyperpolarization of vascular smooth muscle of similar magnitude in endothelium-intact (-4.4 +/- 1 mV) and endothelium-denuded (-4.6 +/- 1 mV) porcine coronary arteries. Bradykinin (10[-10]-10[-6] M) evoked concentration-dependent relaxations in preparations with endothelium only. Although atrial natriuretic peptide-receptor antagonist HS-142-1 (25 microM) slightly reduced the sensitivity to bradykinin (log shift at IC50, twofold; p < 0.05), it had no effect on the maximal response to bradykinin. Inhibition of NO synthase partially attenuated, whereas high potassium chloride (30 mM) markedly inhibited relaxations to bradykinin (p < 0.05). Hyperpolarization to bradykinin was much more pronounced than that to CNP (-17 +/- 3 mV; p < 0.05 vs. CNP) and was observed in endothelium-intact preparations only and unaffected by HS-142-1. In conclusion, in contrast to bradykinin, CNP induces endothelium-independent and weaker relaxation and hyperpolarization of coronary artery vascular smooth muscle, suggesting that CNP is an unlikely mediator of endothelium-dependent hyperpolarization of porcine coronary arteries.     

Binding of the nonpeptide antagonist, SB 209670, to endothelin receptors on cultured neurons

We studied the binding characteristics of a novel, nonpeptide endothelin antagonist, SB 209670, to two subtypes of endothelin (ET) receptor in cultured rat cerebellar granule cell neurons. Displacement binding studies of [125I]ET-1 performed in the presence of the ETB receptor-selective agonist, sarafotoxin 6c (S6c), allowed us to measure a Ki of 4.0 +/- 1.5 nM for (+/-)SB 209670 at the ETA receptor (n = 4). Similarly, binding studies in the presence of the ETA receptor-selective antagonist, BQ123, allowed us to measure a Ki of 46 +/- 14 nM for (+/-)SB 209670 at the ETB receptor (n = 4). These studies indicate that the novel endothelin antagonist, SB 209670, has high affinity for both types of neuronal endothelin receptor.     

Survey of echinococcosis in Hokkaido and measure against it

Exposure to hyperoxia results in lung injury and a decrease in lung collagen. Retinol is known to influence collagen gene expression, and retinol deficiency has been shown to potentiate hyperoxic lung injury. To investigate the combined effects of retinol deficiency and hyperoxia on lung collagen expression, retinol-deficient rats were exposed to acute hyperoxia, and expression of the alpha-1 chains of type I procollagen [pro alpha 1 (I)] and type III procollagen [pro alpha 1 (III)] were determined using Northern hybridization analyses and immunohistochemical staining. Hyperoxia alone reduced pro alpha 1 (I) mRNA by 60 +/- 4% (p < .05) and pro alpha 1 (III) mRNA by 30 +/- 5% (p < .05), and retinol deficiency alone reduced pro alpha 1 (I) mRNA abundance by 49 +/- 8.8% (p < .05) and pro alpha 1 (III) mRNA abundance by 14 +/- 7.5% (p = not significant), respectively. Retinol deficiency plus hyperoxia did not cause any further reduction in procollagen mRNA than that seen with oxygen exposure alone. Immunohistochemical staining demonstrated decreased staining for type I collagen in retinol-deficient animals. Hyperoxic exposure resulted in decreased connective tissue staining and increased alveolar wall staining for type I collagen. Retinol deficiency and hyperoxia together resulted in a marked increase in alveolar exudates staining for type I collagen. No changes in type III collagen staining were seen. These findings demonstrate that while retinol deficiency does not potentiate hyperoxia-induced reductions in procollagen mRNA, it is associated with alterations in collagen staining in distal lung and immunohistologic evidence of collagen fragments in alveolar exudates.     

Endothelin production links superoxide generation to altered opioid-induced pial artery vasodilation after brain injury in pigs

BACKGROUND AND PURPOSE: Traumatic brain injury conveys significant morbidity and mortality to infants and children. In the newborn pig, opioids contribute to pial artery vasconstriction after fluid percussion injury (FPI). FPI attenuates vasodilation and cGMP production by methionine enkephalin (Met) and leucine enkephalin (Leu) and reverse dynorphin (Dyn) from a dilator to a constrictor. Superoxide anion (O2-) production contributes to altered cerebral hemodynamics after FPI, and O2- scavengers partially restore decreased dilator responses after FPI. Endothelin-1 (ET-1), a purported mediator of cerebral vasospasm, has been suggested to alter nitric oxide function and cGMP concentration. The present study was designed to determine the contribution of ET-1 to altered opioid-induced dilation after FPI and the role of O2- in such altered responses. METHODS: Injury of moderate severity (1.9 to 2.3 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O2- generation. RESULTS: FPI increased cerebrospinal fluid ET-1 from 20 +/- 2 to 93 +/- 6 pg/mL (approximately 10(-10) mol/L). Topical ET-1 (10(-10) mol/L) increased SOD-inhibitable NBT reduction from 1 +/- 1 to 16 +/- 3 pmol/mm2, similar to previously reported NBT reduction after FPI (14 +/- 2 pmol/mm2). BQ123 (10(-6) mol/L), an ET-1 antagonist, blunted the NBT reduction observed after FPI (4 +/- 1 pmol/mm2). Met produced pial vasodilation that was attenuated by FPI and partially restored by BQ123 pretreatment (7 +/- 1%, 11 +/- 1%, and 17 +/- 1% versus 3 +/- 1%, 6 +/- 1%, and 9 +/- 2% versus 5 +/- 1%, 9 +/- 1%, and 14 +/- 2% for 10(-10), 10(-8), and 10(-6) mol/L Met during control conditions, after FPI, and after FPI pretreated with BQ123, respectively). Met-induced dilation was associated with increased cerebrospinal fluid cGMP, and these biochemical changes were likewise blunted by FPI and partially restored by BQ123 (357 +/- 12, 455 +/- 15, 500 +/- 19, and 632 +/- 11 versus 264 +/- 4, 267 +/- 4, 295 +/- 12, and 305 +/- 15 versus 309 +/- 19, 432 +/- 11, 529 +/- 10, and 593 +/- 4 pg/mL for resting conditions, 10(-10), 10(-8), and 10(-6) mol/L Met during control conditions, after FPI, and after FPI pretreated with BQ123, respectively). Similar partial restoration of vascular and biochemical parameters was observed for Leu and Dyn. CONCLUSIONS: These data show that ET-1, in concentrations similar to that present in cerebrospinal fluid after FPI, increases O2- production. These data also indicate the opioid-induced vasodilation and cGMP production are partially restored after FPI by ET-1 receptor blockade. These data suggest that ET-1 contributes to altered cerebral hemodynamics after FPI, at least in part, through elevated O2- production.     

Comparable potent coronary constrictor effects of endothelin-1 and big endothelin-1 in humans

BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictor produced from the precursor big ET-1 in endothelial cells. The coronary effects of these peptides in humans in vivo are unknown. Therefore, the effects of ET-1 and big ET-1 on coronary blood flow in relation to plasma ET-1 and big ET-1 levels were compared in healthy subjects. METHODS AND RESULTS: The peptides were infused intravenously at the rates of 0.2, 1, and 8 pmol/kg per minute. Each dose administered for 20 minutes except the highest dose of ET-1, which was administered for 10 minutes. ET-1 and big ET-1 evoked dose-related increases in mean arterial blood pressure from 93 +/- 4 to 107 +/- 4 mm Hg and from 89 +/- 2 to 122 +/- 5 mm Hg, respectively, at the highest dose. ET-1 and big ET-1 reduced coronary sinus blood flow, measured with thermodilution by a maximum of 25 +/- 4% and 28 +/- 8% and increased coronary vascular resistance by 50 +/- 9% and 107 +/- 26%, respectively. Coronary sinus, but not arterial, oxygen saturation was reduced in parallel with the coronary sinus blood flow. The effects of ET-1 and big ET-1 were similar at corresponding time points. During infusion of ET-1, a 19 +/- 5% extraction of ET-1 was observed over the coronary vascular bed (P < .05). Administration of big ET-1 elevated arterial plasma ET-1 levels by 2.4-fold, and after correction for the local extraction of ET-1, a myocardial production of ET-1 was observed. CONCLUSIONS: ET-1 and big ET-1 induce comparable increases in blood pressure and coronary constriction in humans in vivo. The results also suggest a net local removal of circulating ET-1 and big ET-1 and a local conversion of big ET-1 into ET-1 within the coronary vascular bed.     

Activation of BK(Ca) channel via endothelin ET(A) receptors in porcine coronary artery smooth muscle cells

It has been demonstrated previously that endothelin-1 stimulates the Ca2+-activated K+ (BK(Ca)) channel activity in porcine coronary artery smooth muscle cells. The purpose of the present study was to delineate the endothelin receptor subtype involved in this action. In receptor binding studies, [125I]endothelin-1 was shown to bind to the homogenate of porcine primary coronary artery smooth muscle cells in a single class of binding sites with K(D) and Bmax values of 73 pM and 99 fmol/mg protein, respectively. Furthermore, endothelin-1 and endothelin-3 displaced the binding of [125I]endothelin-1 to these cells with respective IC50 values of 70 and 17000 pM, a 240-fold difference in potency. The effects of endothelin-3 on the activity of the BK(Ca) channel in porcine coronary artery smooth muscle cells were examined using the cell-attached patch-clamp technique. Similar to endothelin-1, endothelin-3 also exhibited a bell-shaped concentration-response curve. A maximal increase of 95% in channel open-state probability (Po) was induced by 100 nM endothelin-3 as compared with the 320% increase in Po by 1 nM endothelin-1. Thus, endothelin-1 was about 100-fold more potent and 3.4-fold more efficacious than endothelin-3 in this action. Both the receptor binding and the electrophysiological results suggest that the effects of endothelins on the BK(Ca) channel are mediated through the endothelin ET(A) receptor subtype.     

Differential effects of IFN-beta1b on the proliferation of human vascular smooth muscle and endothelial cells

The effect of human interferon (IFN)-beta1b (Betaseron) on the proliferation of cultured human vascular smooth muscle and endothelial cells was tested in vitro. IFN-beta1b inhibited thymidine incorporation and growth of primary cultures of human aortic and coronary artery smooth muscle in a concentration-dependent manner. The same concentrations of IFN-beta1b did not inhibit thymidine incorporation or growth of primary cultures of human aortic or coronary artery endothelial cells. IFN-beta1b induced the expression of MxA (an antiviral protein induced by type I IFNs) in both smooth muscle and endothelial cells, suggesting that both cell types express receptors for type I IFNs. The growth-inhibitory effect of IFN-beta1b could be mimicked by commercially available human IFN-beta, but not by IFN-alpha2 or IFN-alpha8. The effect of IFN-beta1b was species specific, as it did not inhibit thymidine incorporation in aortic smooth muscle cells derived from pig, rabbit, rat, or mouse. The action of IFN-beta1b on smooth muscle cells persisted for at least 4 days following a 24 h preincubation with IFN-beta1b. Human vascular smooth muscle cells treated with IFN-beta1b did not release lactate dehydrogenase, nor did they show any morphologic change, suggesting that IFN-beta1b was not toxic to the human vascular smooth muscle cells. IFN-beta1b inhibited vascular smooth muscle growth while having no growth-inhibitory effect on endothelial cells obtained from the same blood vessel, making it a potential candidate for treating pathologic conditions where abnormal vascular smooth muscle proliferation is implicated, such as restenosis following balloon angioplasty or smooth muscle proliferation following vascular stenting.