Endothelin-1 as a putative modulator of gene expression and cellular physiology in cultured human corporal smooth muscle cells

PURPOSE: Increases in cytosolic calcium levels trigger smooth muscle contraction while nuclear calcium increases are thought to regulate gene expression. Endothelin-1 (ET-1) affects both. The goal of these studies was to further investigate the importance of ET-1 to corporal physiology by examining its actions on proliferation and immediate early gene (IEG) expression in cultured human corporal smooth muscle cells. MATERIALS & METHODS: Early passage (1-3) smooth muscle cells were grown in culture and exposed to either phenylephrine (PE) or ET-1 in the absence and presence of serum, the ET(A) or ET(B) selective antagonist BQ123 or IRL1038, or the L-type Ca2+ channel blocker, verapamil. Cell proliferation was assessed with a hemocytometer. The effects of ET-1 on c-myc and c-fos were evaluated using Northern blot analysis. Parametric or nonparametric statistics were used as appropriate. RESULTS: Addition of ET-1 (100 nM) to serum-starved cultured corporal smooth muscle cells was associated with a nearly 2-fold increase in cell number, as well as 2 to 6-fold increases in c-myc and c-fos levels. Cellular proliferation was inhibited by ET(A)- or ET(B)-receptor subtype blockade with BQ123 (1 microM) or IRL1038 (1 microM), respectively, or blockade of Ca2+ channels with verapamil (10 microM). PE (3 microM) had no detectable effect on smooth muscle proliferation. CONCLUSIONS: Cell proliferation was mediated by activation of the ET(A) and ET(B) receptor subtypes, dependent on transmembrane Ca2+ flux, and correlated with significant increases in c-myc and c-fos mRNA levels. These studies extend previous observations to indicate the potential pleotropic actions of this peptide in the regulation of human corporal smooth muscle physiology in vivo.     

Activation of phospholipase C and guanylyl cyclase by endothelins in human trabecular meshwork cells

PURPOSE: To characterize effects of endothelins on activities of phospholipase C (PLC) and nucleotide cyclases in human trabecular meshwork (TM) cells. METHODS: Cultured simian virus 40-transformed human TM (HTM-3) or non-transformed (HTM-16) cells were used. Changes in the PLC activity were determined by assaying the production of [3H] inositol phosphates. Accumulation of cyclic GMP or cyclic AMP in cell lysate was measured by radioimmunoassay. RESULTS: Endothelin-1 (ET-1; 1 microM) stimulated PLC in HTM-16 cells, but Sarafotoxin S6c (SRTX), an ET(B) receptor subtype-selective agonist (1 microM), did not. Similar results were obtained in HTM-3 cells: ET-1, but not ET-3 or SRTX, activated PLC in a dose-dependent manner, with a calculated EC50 of 646 pM. The peptide also stimulated the accumulation of cGMP in a concentration-dependent manner with an EC50 of 37.2 pM. ET-3 or SRTX was not effective except at much higher concentrations. Both the PLC and guanylyl cyclase stimulation induced by ET-1 (10 nM) were completely inhibited by pretreating the cells with BQ-123 (<10 microM), an ET(A) receptor selective antagonist, but not by BQ-788 (10 microM), an ET(B) receptor subtype-specific antagonist. Neither ET-1 nor ET-3 stimulated adenylyl cyclase activity in HTM-3 cells at concentration as high as 1 microM. CONCLUSION: ET-1 activates PLC and guanylyl cyclase in TM cells. Potency profiles of ET receptor agonists and antagonists suggest that the ET(A) receptor subtype is involved in both actions of ET-1. The effects of the ET peptides in TM cells are interesting and could be part of the mechanism of their IOP-lowering effect.     

Effects of thrombin and thrombin receptor activating peptides on rat aortic vascular smooth muscle

The role of thrombin receptor activation in isolated rat aortic rings was examined. The human thrombin receptor activating peptides (TRAPs) SFLLRNPNDKYEPF (TRAP1-14), SFLLRNP (TRAP1-7) and rat TRAP1-7 (SFFLRNP) all caused concentration-related (0.1-100 microM) contractions of endothelium-rubbed rat aortic rings. Reversal of the first two amino acids in TRAP1-14 ("reverse TRAP1-14") resulted in total loss of activity. The contractions caused by the TRAPs were reduced substantially in endothelium-intact rings due to endothelium-derived relaxing factor release because the reduced contractions were reversed by N omega-nitro-L-arginine or methylene blue. Contractions were significantly but only slightly enhanced by alpha receptor blockade and were not affected by thromboxane- or endothelin-receptor blockade or by cyclooxygenase inhibition. TRAP1-7 had no effect on contractile responses to norepinephrine, serotonin, angiotensin II or endothelin-1; however, pretreatment with nifedipine or removal of extracellular Ca++ markedly inhibited the contraction. Neither human nor rat alpha-thrombin had any contractile effect on rat aortic rings. In cultured rat aortic smooth muscle cells, alpha-thrombin (EC50 = 1.9 +/- 0.7 nM), TRAP1-14 (EC50 = 30 +/- 4 microM) and TRAP1-7 (EC50 = 20 +/- 9 microM) caused concentration-dependent increases in intracellular calcium [Ca++]i, whereas reverse TRAP1-14 was ineffective. The effect of thrombin on [Ca++]i was abolished by the thrombin inhibitor MD-805, whereas the responses to TRAP were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Teratocarcinosarcoma of the paranasal sinuses: a clinicopathologic and immunohistochemical study

We observed endothelin (ET)-induced contractile responses on prostatic and epididymal segments, as well as the facilitation of an electrically stimulated tone on prostatic segments of isolated rat vas deferens. In both segments, the selective ET(B)-receptor agonists, IRL 1620 and sarafotoxin S6c, produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of contraction potencies (pD2 value) was ET-1 = ET-2 > ET-3 > sarafotoxin S6c = IRL 1620. The maximum responses of ET-induced contractions in the prostatic segments were larger than those in the epididymal segments. The contractile response to ET-3 was antagonized by pretreatment for 30 min with BQ-123 (10 nM), a selective ET(A) receptor antagonist, and BQ-788 (1 microM), a selective ET(B) receptor antagonist. The contractile responses to ET-1 were antagonized by pretreatment with BQ-123 (10 microM), but not with BQ-788 (1 microM). The ET-3-induced facilitation on the twitch response to electrical stimulation in the prostatic segment of the vas deferens was antagonized by BQ-123 (0.1 microM) and BQ-788 (1 microM). The ET-1-induced facilitation was antagonized by pretreatment with BQ-123 (3 microM), but not with BQ-788 (10 microM). These results suggest that in rat vas deferens the ET(A) receptors are divided into BQ-123-sensitive ET(A1) and BQ-123-insensitive ET(A2) subtypes, and the production of a contractile response of smooth muscle as well as the facilitation of neurotransmission are accomplished through mediation by ET(A1)- and ET(A2)-subtypes.     

Glaucoma, capillaries and pericytes. 3. Peptide hormone binding and influence on pericytes

To test the potential for vasoactive neuropeptide receptors to affect capillary resistance, we have begun to study the plausibility that pericytes might be equipped to respond to a representative peptide vasoconstrictor and a representative peptide vasodilator. Pericytes cultured from the bovine retinal vasculature specifically bind the angiotensin II (Ang II) antagonist saralasin (1 nM125I-saralasin bound at 2.2 +/- 0.41 fmol/mg protein) and 125I-vasoactive intestinal peptide (VIP; Kd of 0.5 nM with a population of 30 fmol/mg protein). Incubation with 100 microM Ang II induced minimal cAMP synthesis, while VIP (1 microM, 10 microM) did not induce any change in cAMP concentration. Ang II (10 microM and 100 microM) caused contraction of pericytes cultured on an elastic silicone surface. Circulating or locally produced vasoactive neuropeptides might affect pericyte contractile tone via several intracellular pathways, moderated by indirect effects of these peptides through endothelial stimulation, with the net effect on local blood flow resulting from the effects on arteries and veins as well as capillaries.     

Non-ionophoretic elevation of intracellular Ca2+ by Lonidamine

Lonidamine is an antispermatogenic and anticancer drug that is believed to act by inhibition of energy metabolism. In this study, the effects of Lonidamine on the concentration of intracellular free Ca2+ of several tumor cell lines were assessed because of the important role that cytosolic Ca2+ plays in cell viability and proliferation. The presence of 300 microM Lonidamine resulted in large elevations of cytosolic Ca2+ (> 100 nM) in AS-30D rat ascites hepatoma cells and in cultured EMT6 murine mammary adenocarcinoma cells but had little effect on cultured NCI-H345 human small cell lung cancer cells. The apparent EC50 for Lonidamine was approximately 175 microM. The source of elevated cytosolic Ca2+ was primarily intracellular stores, and the effects of Lonidamine on Ca2+ efflux from these stores did not appear to be due to an ionophoretic action of this compound or to a decline in the level of cellular ATP. These results indicate that the Ca2+ homeostasis of certain lines of tumor cells is specifically altered by Lonidamine at concentrations known to affect cell proliferation.     

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.     

In vitro antitumor activity of the novel marine agent, ecteinascidin-743 (ET-743, NSC-648766) against human tumors explanted from patients

BACKGROUND: Ecteinascidin-743 (ET-743), a member of the ecteinascidin family selected for clinical development, is a tetrahydroisoquinolone alkaloid isolated from the marine ascidian, Ecteinascidia turbinata. This novel compound is a minor groove binding, guanine-specific alkylating agent which also interacts with the microtubule network and blocks cell cycle progression at late S/G2. MATERIALS AND METHODS: A soft agar cloning assay was used to determine the in vitro effects of ET-743 against primary human tumor specimens taken directly from patients. A total of 93 evaluable specimens were exposed to ET-743 for one-hour (n = 25) and/or 14-day continuous exposure (n = 92) at concentrations ranging from 0.1 nM to 1 microM. In vitro responses were defined as an inhibition > or = 50% of human tumor colony forming units at a given concentration. RESULTS: One-hour exposure to ET-743 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 1 microM induced in vitro responses in 0% (0/17), 6% (1/17), 16% (4/25), 13% (1/8), and 25% (2/8) of specimens, respectively. Continuous exposure to ET-743 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 1 microM, inhibited 0% (0/16), 13% (2/16), 49% (44/90), 62% (47/76), and 77% (58/75) of tumor specimens, respectively. Tumor-specific responses and concentration-dependent relationships were observed with a continuous exposure to ET-743. At 100 nM, the compound inhibited 79% (11/14) breast, 69% (9/13) non-small-cell lung, 58% (7/12) ovary, and 88% (7/8) melanoma specimens. At 1 microM, ET-743 inhibited 100% (14/14) breast specimens, 85% (11/13) non-small-cell lung, 67% (8/12) ovary and 86% (6/7) melanoma specimens. Activity of ET-743 at and above 10 nM was also observed against sarcoma and kidney tumors. At 10 nM concentration and continuous exposure ET-743 demonstrated incomplete cross-resistance with paclitaxel, alkylating agents, doxorubicin and cisplatin. CONCLUSIONS: Our data from the cloning assay indicate that the duration of exposure to ET-743 is an important factor in human tumors. Therefore, long-term exposure to ET-743 may be preferred in future clinical trials. The activity of ET-743 in breast, non-small-cell lung, and ovarian cancers as well as in melanoma may deserve further clinical evaluations. The potential of ET-743 in sarcoma and renal tumors might also be considered. In addition, our data indicate that a plasma concentration of 100 nM of ET-743 must be considered as a target during the clinical development of the compound; also the concept of continuous/protracted exposure in clinical trials with ET-743 has to be taken into account.     

Pharmacological characterization of endothelin-induced contraction in the guinea-pig oesophageal muscularis mucosae

1. In the oesophageal muscularis mucosae, we examined the effects of endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3) and sarafotoxin S6c (SX6c) as agonists, and FR139317, BQ-123 and RES-701-1 as endothelin receptor antagonists. 2. All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration-dependent manner. The order of potency (-log EC50) was ET-1 (8.61)=SX6c (8.65)>ET-2 (8.40)>ET-3 (8.18). 3. FR139317 (1-3 microM) and BQ-123 (1 microM) caused parallel rightward shifts of the concentration-response curve to ET-1, but at higher concentrations caused no further shift. RES-701-1 (3 microM) caused a rightward shift of the concentration-response curve to ET-1, while RES-701-1 (10 microM) had no additional effect. RES-701-1 (0.1-1 microM) concentration-dependently caused a rightward shift of the concentration-response curve to SX6c. The contraction to ET-1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with FR139317 (10 microM). 4. Modulation of the Ca2+ concentration in the Krebs solution caused the concentration-response curve to ET-1 or SX6c to shift to the right and downward as external Ca2+ concentrations decreased. Verapamil (30 microM) abolished rhythmic motility induced by ET-1 or SX6c. Ni2+ (0.1 mM) weakly inhibited ET-1- or SX6c-induced tonic contraction. SK&F 96365 (60 microM) completely inhibited ET-1-induced contractions. 5. We conclude that there are two types of ET-receptors, excitatory ET(A)- and ET(B)-receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor-operated Ca2+ channels (ROCs) and partly by opening T-type Ca2+ channels, and mediate rhythmic motility by opening L-type Ca2+ channels.     

Endothelin-1 stimulates deoxyribonucleic acid synthesis and contraction in testicular peritubular myoid cells

In the testis, endothelin-1 (ET-1) is produced by Sertoli cells, and it has been proposed to be a paracrine factor participating in the regulation of tubular and interstitial function. The response of purified testicular peritubular myoid cells (TPMC) to ET-1 was investigated in the present study. TPMC expressed a single class of high-affinity receptors that were shown by competitive binding experiments with sarafotoxin-6c to belong to the ETA subtype. The binding of ET-1 to TPMC was followed by rapid internalization of the receptor-ligand complex. ET-1 induced a prompt rise in intracellular Ca2+ concentration that was blunted in Ca(2+)-free medium and in the presence of Mn2+ or of voltage-operated-calcium-channel (VOC) blockers, indicating that both Ca2+ mobilization from intracellular stores and extracellular Ca2+ influx were involved. Thymidine uptake was promoted by ET-1 in a time-dependent manner, and the use of cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp] (BQ123) reduced the incorporation of thymidine. Protein kinase C (PKC) inhibition (100 nM calphostin C) abolished the ET-1 mitogenic effect. ET-1 also promoted TPMC contraction, as evaluated in collagen lattices, in a dose-related manner, with the half-maximal response observed at 1 nM. As in the case of mitogenesis, BQ123 blunted ET-1-induced contraction. PKC inhibition abolished ET-1-induced contraction. These findings indicate that ET-1 promotes DNA synthesis and contraction of TPMC and that both effects are mediated by PKC; they suggest as well that ET-1 may have a physiological role in the interaction between Sertoli cells and TPMC.     

Cloning and tissue distribution of two new potassium channel alpha-subunits from rat brain

The release of excitatory amino acids from Schwann cell cultures in the rat was monitored using high-performance liquid chromatography. The basal concentration of glutamate and aspartate was 33 +/- 4 nM (mean +/- S.E.M., n = 12) and 8 +/- 1 nM (mean +/- S.E.M., n = 12), respectively. ATP (100 microM) caused a receptor-mediated increase in release of glutamate and aspartate from Schwann cell cultures. Bath application of adenosine (100 microM) was without effect on release of excitatory amino acids suggesting involvement of P2 receptors. Suramin, a competitive antagonist at P2 receptors, prevented the response to ATP. The release of excitatory amino acids evoked by ATP was not abolished in calcium-depleted saline. Pretreatment of the Schwann cultures with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetracetic acid-acetoxymethyl ester (BAPTA-AM) abolished the effect of ATP. ATP-evoked release of glutamate from cultured Schwann cells was significantly reduced by thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase of the Ca2+ pump of internal stores. U73122, a selective inhibitor of receptor-coupled phospholipase C-dependent processes, abolished stimulatory effect of ATP suggesting that ATP's action is mediated through an inositol 1,4,5-triphosphate-sensitive calcium store. The action of ATP was not blocked by L-trans-pyrrolidine-2,4-dicarboxylate, an inhibitor of the electrogenic glutamate transporter, nor was it blocked in Na(+)-free medium, and glutamate release was not stimulated by a depolarizing stimulus, suggesting that ATP-evoked release of glutamate from Schwann cells is not due to the reversal of the glutamate uptake. An anion transport blocker, furosemide, reduced ATP-induced glutamate release. These results suggest that ATP-stimulated glutamate and aspartate release from Schwann cells may be through a calcium-dependent furosemide-sensitive mechanism.     

Hybrid peptides constructed from RES-701-1, an endothelin B receptor antagonist, and endothelin; binding selectivity for endothelin receptors and their pharmacological activity

Hybrid peptides were constructed from endothelin B receptor (ET(B)) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ETB as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ETB but for ETA. Although all analogues had antagonistic effects on ETA, some analogues had proved to function as agonist on ETB confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ETB-selective agonist with weak ETA antagonism (3, KT7421); (2) ETB-selective antagonist with weak ETA antagonism (29, KT7539); (3) ETB agonist with potent ETA antagonism (27, KT7538); and (4) non-selective ETA/ETB antagonist (26, KT7540).     

Use of D-myo inositol 1,2,6 trisphosphate to inhibit contractile activity in rat ventricular cardiomyocytes induced by neuropeptide Y and other cardioactive peptides through phospholipase C

1. D-Myo inositol 1,2,6 trisphosphate (alpha-trinositol, pp56), an isomer of the second messenger substance, inositol 1,4,5 trisphosphate, has an interesting pharmacological profile that includes antagonism of a number of neuropeptide Y (NPY)-mediated cellular processes. The ability of pp56 to inhibit selectively the myocardial contraction mediated by NPY in relation to the responses to other cardioactive peptides, including endothelin-1, calcitonin gene-related peptide (CGRP), secretin and vasoactive intestinal peptide (VIP), was assessed. In order to investigate the possible interaction of pp56 with mechanisms of inositol phosphate signalling generated in heart muscle cells by activation of the beta-isoenzyme of phospholipase C (PLC beta), noradrenaline was used as a positive control, and isoprenaline and forskolin were included as negative controls. 2. Ventricular cardiomyocytes, isolated from the hearts of adult rats, were stimulated to contract at 0.5 Hz in the presence of calcium ion (2 mM). The concentrations of agonists used were in the region of their maximally effective concentrations for myocyte contraction and the concentration of pp56 was in the range of 1-100 microM. Contractile activity was monitored by video microscopy and maximum shortening determined by image analysis. 3. In the absence of agonist, contractile amplitudes following 20 min preincubation with pp56 were not different from that observed in the absence of pp56. Pp56 (1-100 microM) inhibited significantly the positive contractile response to noradrenaline (5 microM) in the presence of propranolol (500 nM), such that the response was almost completely attenuated at the highest concentration of the inhibitor. Pp56 did not inhibit the positive contractile responses to forskolin (40 microM) or isoprenaline (100 nM). 4. NPY alone does not influence the basal level of contraction of cardiomyocytes, but can attenuate isoprenaline-stimulated contraction and can increase contractile amplitude from basal when the transient outward current is blocked with 4-aminopyridine. In the presence of isoprenaline (100 nM), the negative response to NPY (100 nM) was attenuated significantly by pp56 (1-100 microM). With 4-aminopyridine, the positive contractile response to NPY (200 nM) was decreased by pp56, although this was not statistically significant. 5. Pp56 inhibited the positive contractile responses to CGRP (1 nM) and endothelin-1 (20 nM) completely, but did not affect the responses to secretin (20 nM) or VIP (20 nM). 6. In conclusion, these data challenge the previously obtained selectivity of pp56 as an antagonist of NPY-mediated cellular processes, since responses to CGRP and endothelin-1 were at least equally sensitive. Furthermore, as pp56 discriminated clearly in its inhibition of responses to alpha-adrenoceptor by comparison with beta-adrenoceptor/adenylate cyclase stimulation, it appears that pp56 may be a useful pharmacological agent with which to distinguish between PLC beta-dependent and PLC beta-independent coupling mechanisms. On this basis, further evidence has been obtained that, in rat cardiomyocytes, the contractile responses to NPY, CGRP and endothelin-1 are attributable to the activation of PLC beta-dependent pathways, whereas the responses to secretin and VIP are mediated by PLC beta-independent pathways.     

Endothelin converting enzyme (ECE) activity in human vascular smooth muscle

1. We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium-denuded human umbilical vein preparation. 2. Endothelin-1 (ET-1) and ET-2 were potent constrictors of umbilical vein with EC50 values of 9.2 nM and 29.6 nM, respectively. ET-1 was at least 30 times more potent than ET-3 suggesting the presence of constrictor ETA receptors. Little or no response was obtained to the ETB-selective agonist sarafotoxin 6c. These data suggest that endothelin-mediated vasoconstriction is via ETA receptors in this preparation. 3. Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ETA receptor in the media of umbilical vein. High density of binding was obtained with the ETA selective [125I]-PD151242, with much lower levels detected with the ETB selective [125I]-BQ3020. 4. Big ET-1 (EC50 = 42.7 nM) and big ET-2(1-38) (EC50 = 99.0 nM) were less potent than ET-1 and ET-2, respectively. Big ET-2(1-38) was more potent than its isoform big ET-2(1-37) with concentration-response curves to big ET-2(1-37) incomplete at 300 nM. No response was obtained to big ET-3 at concentrations up to 700 nM. The C-terminal fragments, big ET-1(22-38) and big ET-2(22-38) were inactive. 5. Responses to ET-1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10(-5) M) or by the dual NEP/ECE inhibitor phosphoramidon (10(-5) M). Big ET-1 was also unaffected by thiorphan but antagonized in a concentration-dependent manner by phosphoramidon (10(-5) M and 10(-4) M). 6. Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET-IR in the bathing medium. Therefore, although big ET-3 was functionally inactive this reflects the low potency of ET-3 at the ETA receptor rather than the lack of ability of this smooth muscle ECE to convert big ET-3 to ET-3. 7. To conclude we have demonstrated the presence of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2(1-37), big ET-2(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.     

Effects of semotiadil fumarate (SD-3211) and its enantiomer, SD-3212, on the changes in cytosolic Ca2+ and tension caused by KCl and norepinephrine in isolated rat aortas

Semotiadil fumarate (SD-3211), a Ca2+ channel blocker of benzothiazine derivative and its (S)-(-)-enantiomer (SD-3212), inhibited K(+)- and norepinephrine (NE)-induced contractions in isolated rat aortas. Inhibition of NE contraction induced by both drugs was greater than that induced by diltiazem or bepridil, whereas inhibition of K(+)-contraction was similar to that induced by diltiazem or bepridil. Semotiadil and SD-3212 (10 microM) inhibited the increase in cytosolic Ca2+ ([Ca2+]i) induced by 65.4 mM K+ in fura-2-loaded preparations as well as diltiazem and bepridil (10 microM). On the other hand, semotiadil and SD-3212 (10 microM) inhibited only the early phase of increase in [Ca2+]i induced by 1 microM NE. After 5 min, no significant effect on [Ca2+]i was observed with these compounds despite the significant decrease in the contraction. In contrast to these compounds, diltiazem and bepridil 10 microM affected neither the increase in [Ca2+]i nor the contraction induced by NE. Semotiadil and SD-3212 inhibited the transient contraction induced by 1 microM NE in the absence of external Ca2+. Both compounds partially but significantly inhibited the NE-induced contraction in nifedipine-treated muscles. These results suggest that semotiadil and SD-3212 inhibit contractions of vascular smooth muscle (VSM) not only through blockade of voltage-dependent Ca2+ channels but also through other mechanisms, such as inhibition of Ca2+ release from Ca2+ stores or decrease in sensitivity of the contractile elements to Ca2+.     

Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules

We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn234) with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glycosylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD8+ CTL has implications for the design of antiviral vaccines.