Sarafotoxin 6b Research Articles

Pharmacological Heterogeneity of Both Endothelin ETA- and ETB-Receptors in the Human Saphenous Vein

To study endothelin receptor subtypes that mediate the smooth muscle contraction of human saphenous vein, effects of some endothelin-receptor agonists and antagonists were examined. Endothelin (ET)-l and sarafotoxin 6b (S6b) elicited potent concentration-dependent contractions with similar pD2 values and similar maximal responses. Selective ETB-receptor agonists, sarafotoxin 6c (S6c) and IRL1620 (Suc-[Glu9, Ala11,15]-endothelin-1(8-21)), also caused contractions, but their maximal responses were about one third of that of ET-1. ET-3 showed a biphasic concentration-response curve. An ETA-receptor antagonist, BQ-123 (cyclo(-D-Asp-L-Pro-D-Val-L-Leu-D-Trp-)), an ETA/ETB-receptor antagonist, PD142893 (Ac-D-Dip-Leu-Asp-Ile-Ile-Trp), or the combination of these two antagonists hardly affected the contractile effect of ET-1, while each of them markedly antagonized the effects of higher concentrations of ET-3 and S6b. Contractions induced by lower concentrations of ET-3 and S6b were resistant to these antagonists. The concentration-response curves for S6c and IRL1620 were not affected by BQ-123. The effect of IRL1620 was markedly inhibited by PD142893, while S6c-induced contractions were much more resistant to PD142893. These different sensitivities to antagonists suggested heterogeneity of both ETA- and ETB-receptors [ETA1 (sensitive to BQ-123), ETA2 (resistant to BQ-123), ETB1 (sensitive to PD142893) and ETB2 (resistant to PD 142893)] in the human saphenous vein, although contractions mediated by ETB-subtypes have smaller maximal responses than those mediated by the ETA-subtypes.

Two Different Endothelin B Receptor Subtypes Mediate Contraction of the Rabbit Saphenous Vein

To study endothelin receptor subtypes that mediate venous smooth muscle contraction, effects of some endothelin receptor agonists and antagonists on the rabbit lateral saphenous vein were examined and compared with those on the saphenous artery. In the artery, endothelin (ET)-l elicited concentration-dependent contractions, while selective ETB-receptor agonists, IRLI620 (Suc-[Glu9,Ala11,15]ET-1(8-21)) and sarafotoxin 6c (S6c) had almost no effect. The ET-1-induced responses shifted in parallel to the right by BQ-123 (cyclo (-D-Trp-D-Asp-Pro-D-Val-Leu-)), an ETA-receptor antagonist, or PD 142893 (Ac-D-Dip-Leu-Asp-Ile-Ile-Trp), an ETA/ETB-receptor antagonist, indicating the involvement of the ETA receptor in this response. In the saphenous vein, not only ET-1 and ET-3, but also ETB-receptor agonists, IRL1620, S6c and [Glu9]sarafotoxin 6b ([Glu9]S6b), produced concentration-dependent, BQ-123-insensitive contractions. PD142893 did not affect the ΕΤ-1-induced contraction, but it shifted greatly the IRL1620-induced concentration-response curve in parallel to the right. The major components of ET-3-, S6c- and [Glu9]S6b-induced contractions were resistant to PD142893. These results indicate that two different vasoconstrictive ETB-receptor subtypes, ETB1 (sensitive to IRLI620 and PD 142893) and ETB2 (insensitive to IRL1620 and PD142893), are located on the smooth muscle of the saphenous vein.

Endothelin ET A receptor antagonist, BQ-123, blocks the vasoconstriction induced by sarafotoxin 6b in the heart but not in other vascular beds

1. 1. The cardiovascular effects of SRT6b in control and BQ-123, a specific ET A receptor antagonist, pretreated rats were determined in anesthetized rats using a radioactive microsphere technique. 2. 2. Infusion of SRT6b produced an increase in blood pressure and total peripheral resistance, decrease in cardiac output and stroke volume, and no change in heart rate of control or BQ-123 treated rats. 3. 3. SRT6b induced a decrease in blood flow to the heart which was completely blocked by BQ-123 pretreatment. The decrease in blood flow to other organs by SRT6b was not affected by BQ-123 pretreatment. 4. 4. This study indicates that ET receptors in the coronary blood vessels are of a different type (neither ET A nor ET B) to those in other vascular beds.

Vasoconstrictor endothelin receptors characterized in human renal artery and vein in vitro

1. We have identified the endothelin receptors present in the media of human main stem renal artery and vein and characterized the subtypes mediating vasoconstriction in these blood vessels in vitro. 2. Messenger RNA encoding both ETA and ETB receptors was identified in the smooth muscle layer of human renal artery and vein by reverse transcriptase-polymerase chain reaction assay. In cryostat-cut cross-sections of both vessels autoradiographical visualisation suggested a majority of ETA receptors. Intense binding was obtained to the non-selective ligand [125I]-ET-1 and the ETA-selective [125I]-PD151242 but only weak labelling of sites by the ETB-selective [125I]-BQ3020. 3. ET-1 potently constricted renal artery and vein preparations with EC50 values of 4.06 nM and 1.00 nM, respectively. Sarafotoxin 6b was approximately ten times less potent than ET-1 with EC50 values of 36.3 nM and 13.8 nM respectively. In the renal artery, ET-3 and sarafotoxin 6c showed little or no activity up to 300 nM. Responses to these peptides were more variable in the renal vein. Preparations from three individuals did not respond to ET-3 but in three further cases, although ET-3 was much less potent than ET-1, full dose-response curves were obtained. S6c elicited dose-related contractions in vein preparations from 5/6 individuals and although more potent than ET-1, the maximum response was 30-60% of that obtained to ET-1. 4. ET-1-induced vasoconstriction of renal artery and vein was antagonized by the ETA-selective, BQ123 (3-10 microM). The dose-response curves to ET-1 were displaced in a parallel rightward fashion with no attenuation of the maximum responses. pA2 values were estimated to be 6.8 +/- 0.1 and 6.8 +/- 0.4 for artery and vein respectively.5. These data suggest that mRNA encoding both ETA and ETB receptors is present in the media of human main stem renal artery and vein. However, autoradiographical studies indicate that the majority of ET receptors expressed are of the ETA subtype. The relative potencies of ET-1 and ET-3 as vasoconstrictors of renal blood vessels in vitro is consistent with this being an ETA-mediated response,and therefore whilst responses to S6c indicate that constrictor ETB receptors may be present in renal veins from some individuals these are likely to be of less importance in these blood vessels.

Endothelin-1 stimulation of aldosterone and zona glomerulosa ouabain-sensitive sodium/potassium-ATPase

Endothelin stimulates the cells of the zona glomerulosa of the adrenal gland and releases aldosterone. While it is a less potent aldosterone secretagogue than angiotensin II endothelin also potentiates the effects of angiotensin II on aldosterone biosynthesis. Two endothelin receptors have been cloned and are expressed in the adrenal zona glomerulosa. Intravenous infusion of endothelin at a rate of 80 ng/kg/min for 30 min into rats produced increases in blood pressure, adrenal content of aldosterone and stimulated the ouabain-sensitive sodium potassium ATPase in the zona glomerulosa, but not in the zona fasciculata, of the adrenal. The simultaneous infusion of the isopeptide specific endothelin receptor A (ET A) antagonist BQ-123 blocked the pressor effects of endothelin, but did not alter the increase in aldosterone content of the zona glomerulosa or the ouabain-sensitive sodium potassium ATPase activity. Infusion of Sarafotoxin 6b, an ET B agonist, also increased the aldosterone content of the adrenal and stimulated the ouabain-sensitive sodium potassium ATPase in the zona glomerulosa, further indicating that the effect of endothelin is probably mediated by ET B or isopeptide non-specific endothelin receptor. The mechanism by which endothelin stimulates the sodium potassium ATPase is unclear as is the relation between a stimulated sodium potassium ATPase and the potentiation of angiotensin II effect on the adrenal.

Endothelin-stimulated secretion of natriuretic peptides by rat atrial myocytes is mediated by endothelin A receptors.

Endothelin (ET), a potent vasoconstrictor peptide, is known to enhance the secretion of atrial natriuretic factor (ANF) by the heart. In the present study, we investigated the potency of ET isopeptides to stimulate ANF and brain natriuretic peptide (BNP) secretion in primary cultures of neonatal atrial myocytes, and we characterized the receptor mediating these effects. All ET isopeptides caused a twofold increase of ANF and BNP secretion with the following order of potency: ET-1 approximately ET-2 > sarafotoxin 6b >> ET-3. Secretion of the natriuretic peptides was blocked by BQ-123, an ETA-receptor antagonist, but was not affected by either IRL-1620 or [Ala1,3,11,15]ET-1, two ETB-receptor agonists. ET receptors were localized by autoradiography on the surface of atrial myocytes, indicating that contaminating cells were not responsible for 125I-ET-1 binding. Competition binding analyses were then used to assess the ET-receptor subtype on atrial myocyte membrane preparations. A high-affinity (100 pmol/L) binding site with high density (approximately 1500 fmol/mg) was found to preferentially bind the ET isopeptides in the following order: ET-1 > or = ET-2 > or = sarafotoxin 6b > ET-3. Binding was totally displaced by BQ-123 but not by IRL-1620. The ET binding site therefore had the characteristics of an ETA-like receptor. Analysis by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that it possessed a molecular mass of approximately 50 kD. Northern blot analysis of both ETA- and ETB-receptor mRNAs allowed only the detection of the former, indicating that the ETB receptor may be expressed in very small amounts. These results demonstrate that ANF and BNP secretion by atrial myocytes is enhanced by ET via binding to an ETA-like receptor.

Evidence from receptor antagonists of an important role for ETB receptor‐mediated vasoconstrictor effects of endothelin‐1 in the rat kidney

1. To characterize the receptor subtype(s) mediating the renal vasoconstrictor effects of the endothelin (ET) and sarafotoxin (SX) peptides in the isolated perfused kidney of the rat, we have examined the effects of endothelin-1 (ET-1), sarafotoxin 6b (SX6b) and sarafotoxin 6c (SX6c) as agonists, BQ-123 and FR 139317 as selective ETA receptor antagonists, and PD 145065 as a non-selective (ETA and ETB) receptor antagonist. We have also compared in the anaesthetized rat the systemic pressor and renal vasoconstrictor effects of ET-1 and SX6c alone or after pretreatment with PD 145065. 2. In the isolated perfused kidney, ET-1, SX6b and SX6c all gave similar concentration-dependent increases in perfusion pressure. The ETA receptor selective antagonists, BQ-123 and FR 139317, both partially blocked the increase in perfusion pressure induced by ET-1. In contrast, PD 145065 completely blocked the increase in perfusion pressure caused by ET-1. 3. Indomethacin (10 microM) had no effect on the ET-1-induced increases in perfusion pressure but significantly reduced the vasoconstriction induced by low concentrations of SX6c, without affecting responses to high concentrations. In the anaesthetized rat, indomethacin (5 mg kg-1) did not modify the systemic pressor or renal vasoconstrictor effects of ET-1 or SX6c. 4. In anaesthetized rats, bolus intravenous injections of ET-1 or SX6c (0.1, 0.25, 0.5 or 1.0 nmol kg-1) produced initial transient depressor responses followed by sustained and dose-dependent increases in mean arterial pressure (MAP). Both peptides caused an equipotent fall in renal blood flow (RBF).PD 145065 (5 mg kg-1) partially antagonized the systemic pressor effects of ET-1 and SX6c but completely blocked the fall in RBF and rise in renal vascular resistance (RVR) induced by ET-1 and SX6c. PD 145065 also antagonized the transient depressor effect following the bolus administration of either ET-1 or SX6c.5. These results indicate that ET/SX induced renal vasoconstriction is mediated via ETA and ETB-like receptors with ETB receptors having a predominant role in vivo. This may be of therapeutic relevance for an ETA receptor-selective antagonist may offer only limited protection against the deleterious renal effects of endogenous ETs.

Characterization of endothelin receptors mediating the effects of the endothelin/sarafotoxin peptides on autonomic neurotransmission in the rat vas deferens and guinea-pig ileum.

1. To characterize the receptors mediating the effects of the endothelin/sarafotoxin family of peptides on the responses to electrical stimulation of the rat vas deferens (RVD) and guinea-pig ileum (GPI) we have used endothelin-1 (ET-1), ET-3, sarafotoxin 6b (SX6b) and SX6c as agonists and the endothelin-receptor antagonists BQ-123 (ETA receptor selective) and PD 142893 (non-selective). 2. In the RVD, ET-1 and SX6b increased the twitches induced by field stimulation starting at a threshold concentration of 10(-10) M while the threshold concentration for ET-3 was 3 x 10(-9) M. SX6c (up to 3 x 10(-8) M) did not potentiate the twitches. SX6b produced significantly (P < 0.05) greater potentiations than ET-1 at concentrations of 3 x 10(-9) M and higher, and 10(-7) M ET-3 also produced a significantly greater effect than ET-1 at the same concentration. Thus, at threshold the rank order of peptides was ET-1 = SX6b > ET-3 >>> SX6c, and at concentrations of 3 x 10(-8) M and higher, SX6b > ET-3 > ET-1 >>> SX6c. 3. In the presence of BQ-123 or PD 142893 (10(-5) M) the threshold concentrations for ET-1 to augment the twitches were increased 30 fold. In the same conditions neither SX6b nor ET-3 potentiated the responses. The relative activities of the endothelin/sarafotoxin peptides and the effectiveness of the endothelin receptor antagonists are consistent with postjunctional ETA receptors mediating these effects. 4. In the transmurally stimulated GPI the endothelin/sarafotoxin peptides produced two effects; an increase in the basal tension of the tissues and an inhibition of the twitch responses. To increase the basal tension the peptides had the order of potency ET-1 > SX6b>> ET-3 = SX6c. These direct effects of ET-1 or SX6b were strongly antagonized (100 fold) by either BQ-123 (10-5M) or PD 142893(10-5 M). Thus, ETA receptors mediate contractions of the GPI induced by these peptides.5. The endothelin/sarafotoxin peptides were approximately equipotent at depressing twitches of the GPI in response to transmural stimulation (EC50s, 4 x 10-11 to 1.5 x 10-10 M). The depressions induced byET-1 were unaffected by either BQ-123 (10-5 M) or PD 142893 (10-5 M). BQ-123 produced a small(three fold) antagonism of the inhibitory effects of ET-3 or SX6c. These results indicate that a receptor of the ETB type mediates the inhibitory effects of the endothelin/sarafotoxin peptides on neurotransmission in the GPI.6. Thus, both ETA receptors and ETB receptors mediate the effects of the endothelin/sarafotoxinpeptides on neurotransmission.

Functional evidence for different endothelin receptors in the lung

The present study was undertaken to compare and contrast the characteristics of the pulmonary and systemic vascular responses to endothelin (ET) isoforms in the intact spontaneously breathing cat under conditions of constant pulmonary blood flow and left atrial pressure. When pulmonary vasomotor tone (PVT) was actively increased by intralobar infusion of U-46619, intralobar arterial bolus injections of 1 microgram ET-1, 1 microgram ET-2, or 3 micrograms ET-3 markedly decreased lobar arterial pressure, systemic arterial pressure, and systemic vascular resistance. After seven repeated injections of ET-1 or ET-2 to separate groups of cats, pulmonary and systemic responses were largely reversed from vasodilation to vasoconstriction. In contrast, the pulmonary vasodilator response to ET-3 remained intact after multiple ET-3 injections, whereas its systemic vasodilator response was lost. Repeated intralobar arterial bolus injections of ET-1, ET-2, or ET-3 also caused the loss of pulmonary vasodilation to subsequent doses of ET-1, ET-2, or sarafotoxin 6b but not to ET-3. The present data suggest that the pulmonary and systemic vasodilator responses to ET-1 and ET-2 undergo tachyphylaxis and cross-tachyphylaxis. In contrast, the pulmonary vasodilator response to ET-3, unlike its systemic vasodilator response, is resistant to tachyphylaxis and cross-tachyphylaxis. The present data provide a functional correlate for the existence of at least two ET receptor subtypes, ETA-like and ETC-like receptors, in the adult pulmonary vascular bed.

Endothelins-induced inositol phosphate release in smooth muscle of stem villi vessels of human term placenta: Are et a receptors involved

The cardiovascular and pulmonary responses to vasoactive intestinal contractor (VIC) were compared with those of endothelin (ET)-1, ET-2, ET-3 and sarafotoxin 6b (S6b) and the mechanism by which ET-1 alters vascular resistance was investigated in the hindquarters vascular bed of the cat. In a manner similar to ET-1 and ET-2, VIC at a dose of 0.3 nmol/kg i.v. produced increases in pulmonary arterial pressure (PAP) and biphasic changes in systemic arterial pressure (AP), systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR). The biphasic changes were characterized by initial decreases followed by increases. In contrast, ET-3 and S6b at doses of 0.3 nmol/kg i.v. produced mainly decreases in AP and SVR, increases in PAP, and biphasic changes in PVR. A monocyclic ET-1 analog and the ET-1 C-terminal hexapeptide fragment produced no effect on AP, SVR, PAP and PVR at doses of 30–100 nmol/kg i.v. ET-1 at a dose of 0.3 nmol i.a. produced a biphasic change in hindquarters perfusion pressure. The initial vasodilation and secondary vasoconstriction were not modified by a variety of blocking agents, whereas the vasoconstrictor response was significantly reduced by infusion of nimodipine, a calcium entry blocking agent. Results of the present study indicate that VIC, a peptide specific to the mouse gastrointestinal tract, elicits cardiovascular responses that are similar to those elicited by ET-1 and ET-2. The present results indicate that responses to these novel peptides are complex and while the mechanism of action remains uncertain, these data indicate that structural differences among the peptides confer differences in biological activity.

Mediation via different receptors of the vasoconstrictor effects of endothelins and sarafotoxins in the systemic circulation and renal vasculature of the anaesthetized rat.

1. Using endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6b (SX6b) and sarafotoxin 6c (SX6c) as agonists and BQ-123 as a selective ETA receptor antagonist, we have examined the endothelin receptor subtypes mediating the systemic pressor and renal vasoconstrictor effects of the ET/SX family of peptides. 2. In anaesthetized rats, bolus intravenous injections of ET-1, ET-3, SX6b or SX6c (0.1, 0.25 and 0.50 nmol kg-1) produced initial transient depressor responses followed by sustained and dose-dependent increases in mean arterial pressure (MAP) with the following rank order of potency: SX6b > ET-1 >> SX6c > ET-3. In contrast, in the renal vasculature these peptides caused equipotent dose-dependent falls in renal blood flow (RBF) (ET-1 = ET-3 = SX6b = SX6c). 3. BQ-123 (1 mg kg-1, i.v. bolus) significantly reduced the systemic pressor effects of all the peptides but was largely ineffective against the renal vasoconstrictions. 4. These results indicate that although the systemic pressor effects of the ET/SX peptides are mediated via ETA receptors, the vasoconstriction in the kidney in vivo may be mediated predominantly via ETB-like receptors. This may be of therapeutic relevance, for an ETA-receptor-selective antagonist could offer only poor protection of the renal circulation from the deleterious effects of endogenously produced members of this peptide family.

Effects of endothelins on the human megakaryoblastic cell line MEG-01

Some effects of endothelin-1 (ET-1) were studied on the megakaryoblastic cell line MEG-01. ET-1 induced an elevation of the intracellular levels of Ca 2+ ([Ca 2+] 1) as measured with the fluorescent indicator indo-1, which consists of an initial transient increase and an ensuing sustained plateau. The plateau phase was abolished by removal extracellular Ca 2+. In addition, ET-1 induced a rapid (within 5 s) increase in the accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P 1) and more delayed increases in Ins(1,3,4)P 3 and Ins(1,3,4,5)P 4, but did not modify cAMP levels. ET-1 homodogues (ET-2, ET-3, sarafotoxin ϱ 1 and vasointestinal constrictor) also induced biphasic effects on [Ca 2+] 1. The Ca 2+ elevation was concentration dependent, the order of potency being sarafotoxin 6b > ET-1 > ET-2 = vasointestinal constrictor > ET-3. The actions of ET analogs in raising [Ca 2+], were mutually exclusive, suggesting that they act through the same mechanism. These results suggest that cells of the megakaryoblast/megakaryocyte lineage are targets for endothelins.

Heterogeneity of endothelin-sarafotoxin receptors mediating contraction of pig coronary artery.

The contractile effects of endothelin-1, endothelin-3, sarafotoxin 6b and sarafotoxin 6c were studied in endothelium-denuded rings of pig coronary artery. Endothelin-1, sarafotoxin 6b and sarafotoxin 6c produced monophasic concentration-response curves (mean EC50 values 6.7, 14.8 and 1.6 nM), whereas the concentration-response curve to endothelin-3 was biphasic (mean EC50 values 9.6 nM and 0.32 microM). The maximal effect of sarafotoxin 6c was about one third of that reached by the other peptides. The higher sensitivity component of the curve to endothelin-3 was abolished in the presence of sarafotoxin 6c (0.3 microM), while the EC50 value for the other component remained unchanged. Sarafotoxin 6c (0.3 microM) failed to alter the EC50 values of endothelin-1 and sarafotoxin 6b. These data strongly suggest the presence of at least two endothelin-sarafotoxin receptors mediating contraction of pig coronary artery, one with the profile of the endothelin ETA receptor subtype, the other recognizing sarafotoxin 6c and endothelin-3, but not endothelin-1 and sarafotoxin 6b, being thus different from the ETB receptor subtype.

Characteristics of endothelin receptors in the central nervous system of spontaneously hypertensive rats

The binding of [ 125I]sarafotoxin 6b (SRT 6b) and [ 125I]endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared, from regions of the brain and spinal cord of 8 week-old, spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. Spontaneously hypertensive rats had significantly higher blood pressure as compared to WKY rats. Heart rate was similar in SHR and WKY rats. [ 125I]SRT 6b and 125I]ET-1 bound to the membranes of the cerebral cortex, hypothalamus, ventrolateral medulla, dorsomedial medulla and spinal cord at a single, high affinity site. The K d and B max values of the binding of [ 125I]SRT 6b were found to be similar to binding of [ 125I]ET-1 in all the regions. The concentration-dependent inhibition of binding of [ 125I]ET-1 by unlabeled ET-1, in spinal cord membranes showed an IC 50 value of 2.48 ± 0.72 nM and a K i , value of 2.35 ± 0.68 nM in WKY rats and an IC 50 value of 1.77 ± 0.49 nM and a K i value of 1.68 ± 0.46 nM in SHR rats. On the other hand, the concentration-dependent inhibition of the binding of [ 125I]SRT 6b by unlabeled ET-1, in spinal cord membranes showed an IC 50 value of 12.86 ± 3.09 pM and a K i value of 9.54 ± 2.18 pM in WKY rats, while SHR rats showed an IC 50 value of 10.30 ± 3.26 pM and a K i value of 7.46 ± 2.36 pM. The binding of [ 125I]SRT 6b and [ 125I]ET-1 in the cerebral cortex, dorsomedial medulla and spinal cord membranes was found to be similar in SHR and WKY rats. In the hypothalamus and ventrolateral medulla, the binding of both [ 125I]SRT 6b and [ 125I]ET-1 were decreased in SHR, as compared to WKY rats. The decreased binding of [ 125I]SRT 6b was due to 27.1% and 39.2% decreases in the B max values in hypothalamus and ventrolateral medulla, respectively. The decreased binding of [ 125I]ET-1 was due to 49.8% and 47.5% decreases in the B max values in hypothalamus and ventrolateral medulla, respectively. The K d values were similar in SHR and WKY rats. The endothelin receptors in the hypothalamus and ventrolateral medulla of SHR rats were down-regulated as compared to WKY rats and may be contributing to the regulation of blood pressure.

Activation of endothelin receptors by sarafotoxin regulates Ca2+ homeostasis in cerebellar astrocytes

We carried out experiments designed to investigate the effects of sarafotoxin-6B (SFTx) on [Ca2+]i in cerebellar astrocytes using the Ca2+ indicator fura-2. Both endothelin-1 and sarafotoxin-6B increased [Ca2+]i in individual cerebellar astrocytes in cell culture. The shape of the response was variable but usually consisted of an initial peak of [Ca2+]i followed by an extended plateau increase in [Ca2+]i. In Ca(2+)-free medium only the initial peak was observed. If Ca2+ was subsequently readmitted to the external medium a plateau was now formed. When external Ca2+ was removed during a plateau, [Ca2+]i rapidly declined; replacing the external Ca2+ reversed this decline. The plateau was also reversibly reduced by addition of Ni2+ (5 mM) to the external medium. Addition of 50 mM K+ produced a small increase in [Ca2+]i in most cells. This response was blocked by nimodipine. However, nimodipine only slightly blocked the plateau increase in [Ca2+]i that was formed following activation of endothelin receptors. Furthermore, perfusion of cells with 50 mM K+ during the plateau portion of a response to SFTx reduced [Ca2+]i. In some cells addition of a phorbol ester produced a sustained increase in [Ca2+]i that was blocked by nimodipine. In conclusion, activation of endothelin receptors by SFTx in cerebellar astrocytes produces both Ca2+ mobilization and Ca2+ influx. The pathway for Ca2+ influx is predominantly a non-voltage-dependent one, although some entry through a dihydropyridine-sensitive pathway also appears to occur. Furthermore, activation of protein kinase C in cerebellar astrocytes activates voltage-sensitive Ca2+ channels.

Characteristics of endothelin binding sites in the spinal cord of spontaneously hypertensive rats

The binding of [ 125I]sarafotoxin 6b (SRT 6b) and [ 125I]endothelin-l (ET-1) to endothelin (ET) receptors of neuronal membranes prepared from cerebral cortex and spinal cord of 8-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) was determined. SHR had significantly higher blood pressure as compared to WKY. Heart rate was similar in SHR and WKY. [ 125I]SRT 6b and [ 125I] ET-1 bound to the membranes of cerebral cortex and spinal cord at a single, high affinity site. The binding of [ 125I]SRT 6b and [ 125I]ET-l in the cerebral cortex and spinal cord membranes was found to be similar in SHR and WKY. Concentration-dependent inhibition of [ 125I]ET-l binding in spinal cord membranes by unlabeled ET-1, ET-2 and ET-3 was performed. The K i values were found to be 2.35 ± 0.68, 0.29 ± 0.08 and 24.10 ± 5.90 nM for ET-1, ET-2 and ET-3, respectively in WKY. The K i values of ET-1 and ET-2 were found to be similar in WKY and SHR. However, the K i value of ET-3 were found to be significantly lower (P < 0.004) in SHR as compared to WKY. Concentration-dependent inhibition of [ 125I]SRT 6b binding in spinal cord membranes by unlabeled ET-1, ET-2 and ET-3 were also performed. The K i values were found to be 9.50 ± 2.10 pM, 0.17 ± 0.04 nM and 31.20 ± 6.00 nM for ET-1, ET-2 and ET-3, respectively in WKY. The K i values of ET-1 and ET-2 were found to be similar in WKY and SHR. However, the K i value of ET-3 were found to be significantly lower (P < 0.01) in SHR as compared to WKY. In the cerebral cortex inhibition of [ 125I]SRT 6b and [ 125I]ET-l binding by ET-3 was similar in WKY and SHR. It is concluded that in spinal cord ET receptors are of ET A type. It appears that there are two subtypes of ET A receptors in the spinal cord, ET A which are more selective to ET-1 as compared to ET-2 and ET A2 which are more selective to ET-2 as compared to ET-1. In SHR these receptors are more sensitive to ET-3 as compared to WKY and may be involved in the regulation of blood pressure.

Analysis of responses to endothelins 1, 2, and 3 and sarafotoxin 6b in airways of the cat

Airway responses to endothelin (ET) 1, ET-2, ET-3, and sarafotoxin 6b (S6b) were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of ET-1 (0.1-1 nmol/kg) increased transpulmonary pressure (Ptp) and lung resistance (RL) and decreased dynamic compliance (Cdyn) in a dose-related manner. Airway responses to ET-1 were decreased significantly by sodium meclofenamate, a cyclooxygenase inhibitor, and by SKF 96148, a thromboxane receptor blocking agent. In terms of relative bronchoconstrictor activity, the thromboxane mimic, U-46619, was threefold more potent than ET-1 on a molar basis in increasing Ptp. ET-1 and ET-3 had similar bronchoconstrictor activity, and these peptides were less potent than ET-2 and S6b. Bronchoconstrictor responses to ET-2, ET-3, and S6b were also decreased significantly by meclofenamate and by thromboxane receptor blocking agents. The ET-1 precursor ET-1-(1-38) (big ET-1) caused a significant slowly developing increase in Ptp, RL, and aortic pressure (PAO) and a decrease in Cdyn, whereas a monocyclic ET-1 analogue and ET-1-(16-21) hexapeptide fragment had little or no activity in the airways. The present data indicate that ET-1, ET-2, ET-3, and S6b have significant bronchoconstrictor activity in the cat and that responses are dependent in part on the release of arachidonic acid and the formation of thromboxane A2. These data also suggest that big ET-1 is converted into a mature peptide in the cat and that ET-1-(16-21) hexapeptide fragment and a monocyclic ET-1 analogue have little if any bronchoconstrictor activity in the anesthetized cat.

Regional extraction of endothelins and conversion of big endothelin to endothelin‐1 in the pig

Endothelin-like immunoreactivity (ET-LI), mean arterial blood pressure (MABP) and vascular resistance in the spleen, kidney and femoral vascular bed were measured during intravenous infusions (20 pmol.kg-1.min) of endothelin-2 (ET-2), endothelin-3 (ET-3), big endothelin-1 (big ET) and sarafotoxin 6b in the pig. All peptides (especially endothelin-2 and sarafotoxin 6b) caused vasoconstrictor effects in the kidney. Endothelin-2, endothelin-3 and sarafotoxin 6b also evoked significant increases in splenic and femoral vascular resistance. The relative vasoconstrictor response to endothelin-2 was larger in the kidney and spleen than in the femoral vascular bed whereas the opposite was observed for endothelin-3. A high degree of plasma clearance for endothelin-like immunoreactivity was observed. Thus, for ET-2 and ET-3 about 70% of arterial endothelin-like immunoreactivity was removed over the kidney while over the spleen and femoral vascular bed an extraction of 50% for plasma endothelin-2 and 30-40% for endothelin-3 was observed. Big endothelin-1 was only extracted by 34% over the kidney and not at all in the splenic or femoral vascular bed. The metabolic plasma half-lifes of endothelin-2 and endothelin-3 in vivo were in the same range, 1-2 minutes, whereas the half-life of big endothelin-1 was 9 minutes. HPLC-characterization of the plasma endothelin-like immunoreactivity during and after big endothelin-1 infusion as well as measurements using a specific endothelin-1 antiserum revealed formation of endothelin-1 from circulating big endothelin-1 in vivo but not in plasma in vitro. It is concluded that there exists specificity concerning the vasoconstrictor effects and the removal of endothelin-peptides from the circulation, both mechanisms being most prominent in the kidney. Big endothelin-1 has a much longer metabolic half-life, less regional clearance and poor vasoconstrictor activity compared with endothelin-1. Furthermore, endothelin-1 is formed from circulating big endothelin-1 probably by an endothelin-converting enzyme.

Comparative responses to endothelin-2 and sarafotoxin 6b in the pulmonary vascular bed of the cat

Pulmonary vascular responses to endothelin-2 and sarafotoxin 6b were investigated in the feline pulmonary vascular bed under natural flow and constant flow conditions. Injections of endothelin-2 and sarafotoxin 6b in a dose of 0.3 nmol/kg iv increased pulmonary arterial and left atrial pressures and cardiac output, and caused a biphasic change in calculated pulmonary vascular resistance. Endothelin-2 caused a biphasic change in systemic arterial pressure, while sarafotoxin 6b only decreased arterial pressure. Under constant flow conditions in the intact-chest cat, injections of endothelin-2 and sarafotoxin 6b in doses of 0.1-1 nmol into the perfused lobar artery increased lobar arterial pressure in a dose-related manner but were less potent than the thromboxane A2 mimic, U46619. An ET analog with only the Cys1-Cys15 disulfide bond and an amidated carboxy terminus had no significant activity in the pulmonary vascular bed. The present data show that endothelin-2 and sarafotoxin 6b have significant vasoconstrictor activity in the pulmonary vascular bed of the cat.

Localization and characterization of endothelin receptor binding sites in the rat brain visualized by in vitro autoradiography4

Endothelin binding sites in rat brain were mapped by quantitative in vitro autoradiography employing [ 125I]endothelin-l as radioligand. [ 125I]Endothelin-1 bound with high affinity and specificity to rat cerebellar sections and was potently displaced by unlabelled endothelins (endothelin-1 > endothelin-2 = endothelin-3) and sarafotoxin 6B. The highest densities of endothelin binding sites were found in the cerebellum (especially Purkinje cell layer), choroid plexus and median eminence. High densities were found in the supraoptic and paraventricular hypothalamic nuclei, anterior hypothalamic area, ventromedial hypothalamic nucleus, mammillary nuclei and glomerular layer of olfactory bulb. Moderate densities were found in many thalamic nuclei, the pretectal region, interpeduncular nucleus, suprachiasmatic nucleus, raphe nuclei, tegmental nuclei, olfactory ventricle, red nucleus, subthalamic nucleus, central gray, reticular nuclei, vestibular nuclei, oculomotor and trochlear nuclei, hypoglossal nucleus, motor trigeminal nucleus, nucleus of the trapezoid body and lateral cerebellar nucleus. Low but detectable densities of endothelin binding sites were found in medial geniculate nucleus, fields of Ammon's horn, caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra, anterior commissure, internal capsule, anterior pituitary, median preoptic nucleus, septohypothalamic nucleus, superior colliculus and area postrema. These patterns were completely abolished by 1 μM unlabelled endothelin-1, -2 and -3 and sarafotoxin S6B. Brain endothelin binding sites show high affinity for endothelin-1, -2 and -3 and sarafotoxin 6B with highest affinity for endothelin-1. Endothelin binding sites show a non-vascular pattern of distribution in the brain, suggesting that the peptide may have widespread functions as a modulator of neuronal function.