s6c-sarafotoxin and Hypertension

In humans, the endothelins (ETs) comprise a family of three 21-amino-acid peptides, ET-1, ET-2 and ET-3. ET-1 is synthesised from a biologically inactive precursor, Big ET-1, by an unusual hydrolysis of the Trp21 -Val22 bond by the endothelin converting enzyme (ECE-1). In humans, there are four isoforms (ECE-1a-d) derived from a single gene by the action of alternative promoters. Structurally, they differ only in the amino acid sequence of the extreme N-terminus. A second enzyme, ECE-2, also exists as four isoforms and differs from ECE-1 in requiring an acidic pH for optimal activity. Human chymase can also cleave Big ET-1 to ET-1, which is cleaved, in turn, to the mature peptide as an alternative pathway. ET-1 is the principal isoform in the human cardiovascular system and remains one of the most potent constrictors of human vessels discovered. ET-1 is unusual in being released from a dual secretory pathway. The peptide is continuously released from vascular endothelial cells by the constitutive pathway, producing intense constriction of the underlying smooth muscle and contributing to the maintenance of endogenous vascular tone. ET-1 is also released from endothelial cell-specific storage granules (Weibel-Palade bodies) in response to external stimuli. ETs mediate their action by activating two G protein-coupled receptor sub-types, ETA and ET(B). Two therapeutic strategies have emerged to oppose the actions of ET-1, namely inhibition of the synthetic enzyme by combined ECE/neutral endopeptidase inhibitors such as SLV306, and receptor antagonists such as bosentan. The ET system is up-regulated in atherosclerosis, and ET antagonists may be of benefit in reducing blood pressure in essential hypertension. Bosentan, the first ET antagonist approved for clinical use, represents a significant new therapeutic strategy in the treatment of pulmonary arterial hypertension (PAH).

Topics: Amino Acid Sequence; Animals; Antihypertensive Agents; Aspartic Acid Endopeptidases; Atherosclerosis; Binding Sites; Bosentan; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Humans; Hypertension; Hypertension, Pulmonary; Metalloendopeptidases; Molecular Sequence Data; Muscle, Smooth, Vascular; Receptors, Endothelin; Signal Transduction; Sulfonamides; Vasoconstrictor Agents; Viper Venoms

Stroke is one of the leading causes of mortality and morbidity worldwide, and few therapeutic treatments have shown beneficial effect clinically. One reason for this could be the lack of risk factors incorporated into the preclinical stroke research. We have previously demonstrated phenotypic receptor changes to be one of the injurious mechanisms occurring after stroke but mostly in healthy rats. The aim of this study was to investigate if hypertension has an effect on vasoconstrictive receptor responses to endothelin 1, sarafotoxin 6c and angiotensin II after stroke by inducing transient middle cerebral artery occlusion in spontaneously hypertensive rats and Wistar-Kyoto rats using the wire-myograph. We demonstrated an increased contractile response to endothelin 1 and extracellular potassium as well as an increased carbachol-induced dilator response in the middle cerebral arteries from hypertensive rats after stroke. This study demonstrates the importance of including risk factors in experimental stroke research.

Topics: Angiotensin II; Animals; Blood Pressure; Carbachol; Endothelin-1; Hypertension; Infarction, Middle Cerebral Artery; Male; Middle Cerebral Artery; Potassium; Rats, Inbred SHR; Rats, Inbred WKY; Vasodilation; Viper Venoms

We hypothesized that angiotensin (Ang) II hypertensive rats have impaired natriuresis after renal medullary endothelin (ET) B receptor stimulation that would be more evident in male versus female rats. Acute intramedullary infusion of the ET(B) agonist sarafotoxin 6c in normotensive male rats increased sodium excretion from 0.51±0.11 μmol/min during baseline to 1.64±0.19 μmol/min (P<0.05) after S6c. After 2 weeks of Ang II infusion (260 ng/kg per minute SC), male rats had an attenuated natriuretic response to S6c of 0.62±0.16 μmol/min during baseline versus 0.95±0.07 μmol/min after S6c. In contrast, ET(B)-dependent natriuresis was similar in female hypertensive rats (0.48±0.07 versus 1.5±0.18 μmol/min; P<0.05) compared with normotensive controls (1.05±0.07 versus 2.14±0.24 μmol/min; P<0.05). Because ET(A) receptors also mediate natriuresis in normotensive female rats, we examined ET(A) receptor function in female Ang II hypertensive rats. Intramedullary infusion of ET-1 increased sodium excretion in both hypertensive and normotensive female rats, which was partially blocked by the ET(A) antagonist BQ-123. Maximum ET(B) receptor binding in inner medullary membrane preparations was comparable between vehicle and Ang II hypertensive females; however, maximum ET(B) binding was significantly lower in male hypertensive rats (1952±251 versus 985±176 fmol/mg; P<0.05). These results indicate that renal ET(B) function is impaired in male Ang II hypertension attributed, at least in part, to a reduced number of ET(B) binding sites. Furthermore, renal ET receptor function is preserved in female rats during chronic Ang II infusion, suggesting that renal ET receptor function could serve to limit hypertension in females compared with males.

Topics: Angiotensin II; Animals; Blood Pressure; Female; Hypertension; Kidney Medulla; Male; Natriuresis; Rats; Receptors, Endothelin; Sex Characteristics; Viper Venoms

Endothelin (ET) type B receptors (ET(B)R) are expressed in multiple tissues and perform different functions depending on their location. ET(B)R mediate endothelium-dependent vasodilation, clearance of circulating ET, and diuretic effects; all of these should produce a fall in arterial blood pressure. However, we recently showed that chronic activation of ET(B)R in rats with the selective agonist sarafotoxin 6c (S6c) causes sustained hypertension. We have proposed that one mechanism of this effect is constriction of capacitance vessels. The current study was performed to determine whether S6c hypertension is caused by increased generation of reactive oxygen species (ROS) and/or activation of the sympathetic nervous system. The model used was continuous 5-day infusion of S6c into male Sprague-Dawley rats. No changes in superoxide anion levels in arteries and veins were found in hypertensive S6c-treated rats. However, superoxide levels were increased in sympathetic ganglia from S6c-treated rats. In addition, superoxide levels in ganglia increased progressively the longer the animals received S6c. Treatment with the antioxidant tempol impaired S6c-induced hypertension and decreased superoxide levels in ganglia. Acute ganglion blockade lowered blood pressure more in S6c-treated rats than in vehicle-treated rats. Although plasma norepinephrine levels were not increased in S6c hypertension, surgical ablation of the celiac ganglion plexus, which provides most of the sympathetic innervation to the splanchnic organs, significantly attenuated hypertension development. The results suggest that S6c-induced hypertension is partially mediated by sympathoexcitation to the splanchnic organs driven by increased oxidative stress in prevertebral sympathetic ganglia.

Topics: Acridines; Animals; Denervation; Ethidium; Fluorescent Dyes; Ganglia, Sympathetic; Ganglionic Blockers; Hypertension; Luminescence; Male; Muscle Tonus; Muscle, Smooth, Vascular; Norepinephrine; Rats; Rats, Sprague-Dawley; Superoxides; Sympathetic Nervous System; Trimethaphan; Vasoconstrictor Agents; Viper Venoms

Topics: Aged; Female; Humans; Hypertension; Male; Mammary Arteries; Receptor, Endothelin A; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation; Vasoconstriction; Viper Venoms

Endothelin (ET) exerts powerful pressor actions primarily through activation of the ET(A) receptor subtype. The ET(B) receptor (ET(B)R) subtype, on the other hand, is generally thought to initiate physiological actions that decrease arterial pressure. Such actions include clearing ET from the bloodstream, initiating endothelium-mediated vasodilation, and facilitating renal sodium and water excretion. The effect of long-term activation of the ET(B)R on arterial pressure, however, never has been directly tested. In this study we evaluated cardiovascular responses to chronic (5-day) activation of ET(B)R in male rats using continuous intravenous infusion of the selective agonist sarafotoxin 6c. Surprisingly, we found that sarafotoxin 6c caused a sustained increase in arterial pressure that rapidly reversed on termination of infusion. The hypertension was associated with increased renal excretion of sodium and water and decreased plasma volume. Alterations in daily sodium intake did not affect the magnitude of the hypertension. Hemodynamic studies revealed a decreased cardiac output and increased total peripheral resistance during sarafotoxin 6c infusion. Infusion of sarafotoxin 6c caused a small increase in plasma ET levels. Nevertheless, the hypertension was not affected by coadministration of a selective ET(A) receptor antagonist (atrasentan) but was completely prevented by treatment with a combined ET(A) receptor and ET(B)R antagonist (A186280). These experiments reveal for the first time that chronic activation of ET(B)R in rats causes sustained hypertension.

Topics: Animals; Atrasentan; Blood Pressure; Blood Volume; Cardiac Output; Disease Models, Animal; Diuresis; Drug Administration Schedule; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Heart Rate; Hypertension; Male; Natriuresis; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Stroke Volume; Sulfonamides; Vascular Resistance; Viper Venoms

We characterized vascular reactivity to endothelin-1 (ET-1) in mesenteric vessels from DOCA-salt hypertensive and SHAM control mice and assessed the effect that endothelial-derived vasodilators have on ET-1-induced vasoconstriction. Changes in the diameter of unpressurized small mesenteric arteries and veins (100- to 300-microm outside diameter) were measured in vitro using computer-assisted video microscopy. Veins were more sensitive than arteries to the contractile effects of ET-1. There was a decrease in arterial maximal responses (E(max)) compared to veins, this effect was larger in DOCA-salt arteries. The selective ET(B) receptor agonist, sarafotoxin 6c (S6c), contracted DOCA-salt and SHAM veins but did not contract arteries. The ET(B) receptor antagonist, BQ-788 (100 nM), but not the ET(A) receptor antagonist, BQ-610 (100 nM), blocked S6c responses. BQ-610 partially inhibited responses to ET-1 in mesenteric veins from DOCA-salt and SHAM mice while BQ-788 did not affect responses to ET-1. Co-administration of both antagonists inhibited responses to ET-1 to a greater extent than BQ-610 alone suggesting a possible functional interaction between ET(A) and ET(B) receptors. Responses to ET-1 in mesenteric arteries were completely inhibited by BQ-610 while BQ-788 did not affect arterial responses. Nitric oxide synthase inhibition potentiated ET-1 responses in veins from SHAM but not DOCA-salt mice. There was a prominent role for ET-mediated nitric oxide release in DOCA-salt but not SHAM arteries. In summary, these studies showed a differential regulation of ET-1 contractile mechanisms between murine mesenteric arteries and veins.

Topics: Algorithms; Animals; Cyclooxygenase Inhibitors; Desoxycorticosterone; Endothelin Receptor Antagonists; Endothelin-1; Heart; Hypertension; Indomethacin; Kidney; Male; Mesenteric Arteries; Mesenteric Veins; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocardium; Oligopeptides; Organ Size; Piperidines; Receptors, Endothelin; Vasodilator Agents; Viper Venoms

Superoxide anion (O2*-) production is elevated in the vasculature of hypertensive animals but it is not known if O2*- production is also elevated in the sympathetic nervous system. We measured O2*- levels in prevertebral sympathetic ganglia of deoxycorticosterone acetate (DOCA)-salt hypertensive rats using the dihydroethidine (DHE) fluorescence method. O2*- was elevated in ganglia from DOCA-salt rats compared with normotensive sham rats. Treatment of ganglia with endothelin (ET)-1 (3x10(-8) mol/L) resulted in a 200% increase in fluorescence intensity in neurons, which was attenuated by the ET(B) receptor antagonist BQ788 (10(-7) mol/L). ET-1 also increased the O2*- induced fluorescence in dissociated sympathetic neurons and PC-12 cells via activation of ET(B) receptors, but not ET(A) receptors. To evaluate whether elevated ET-1 levels in the ganglia might contribute to the elevated O2*- found in ganglia we measured the amount of ET-1 using an ELISA assay. ET-1 levels in sham rat celiac ganglia were 695.6+/-40.9 picogram per gram; they were not different than ET-1 levels in ganglia from DOCA-salt rats. We then compared ET(B) receptor levels in ganglia from sham and DOCA-salt animals. ET(B) receptor mRNA levels were 32% higher and ET(B) receptor protein levels were 20% higher in celiac ganglia from DOCA-salt rats than from sham rats separately. In conclusion, O2*- is elevated in prevertebral sympathetic ganglia in DOCA-salt hypertension, and ET-1 is a potent stimulus for the elevation of O2*- levels in sympathetic ganglia, an effect that may be mediated by the upregulation of ET(B) receptors.

Topics: Animals; Desoxycorticosterone; Endothelin A Receptor Antagonists; Endothelin-1; Ganglia, Sympathetic; Hypertension; Male; Nerve Growth Factor; Oligopeptides; Oxidative Stress; PC12 Cells; Piperidines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptor, Endothelin B; Sodium Chloride, Dietary; Superoxides; Sympathetic Fibers, Postganglionic; Up-Regulation; Viper Venoms

Deoxycorticosterone acetate (DOCA)-salt hypertension is characterized by low renin/angiotensin but increased arterial superoxide levels. We have recently reported that the arterial endothelin-1 (ET-1) level is increased, resulting in NADPH oxidase activation and superoxide generation. However, the effect of ET-1 on venous superoxide production and its relation to venoconstriction are unknown. The present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and superoxide via its ET(A) receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Treatment with ET-1 (0.01 to 1 nmol/L), but not the selective ET(B) receptor agonist sarafotoxin s6c, of vena cavas of normal rats concentration-dependently increased superoxide levels, an effect that was abolished by the selective ET(A) receptor antagonist ABT-627. Although the ET-1 level was not increased in the vena cava and plasma, both venous NADPH oxidase activity and superoxide levels were significantly higher in DOCA-salt compared with sham rats. Moreover, ET-1 treatment (10(-9) mol/L, 10 minutes) of isolated vena cavas further elevated superoxide levels in DOCA-salt rats only but not sham rats, an effect that was abrogated by the superoxide scavenger tempol. Similarly, ET-1-induced contractions of isolated vena cavas of DOCA-salt but not sham rats were significantly inhibited by tempol. The NADPH oxidase inhibitor apocynin significantly reduced superoxide levels in vena cavas of DOCA-salt rats and in ET-1-treated vena cavas of normal rats. Finally, in vivo ET(A) receptor blockade by ABT-627 significantly lowered venous superoxide levels and blood pressure in DOCA-salt but not sham rats. These results suggest that superoxide contributes to ET-1-induced venoconstriction through an elevated venous NADPH oxidase activity in mineralocorticoid hypertension.

Topics: Acetophenones; Allopurinol; Animals; Atrasentan; Desoxycorticosterone; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Hypertension; In Vitro Techniques; Male; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Mineralocorticoid; Superoxides; Vasoconstriction; Vasoconstrictor Agents; Venae Cavae; Viper Venoms; Xanthine Oxidase

The importance of endothelin-1 (ET-1) in the pathophysiology of essential hypertension is unclear. We therefore compared the effects of endothelin ET(A) receptor blockade and the stimulation of ET(A) and ET(B) receptors, and their interaction with the sympathetic nervous system, in the forearm resistance vessels of patients with essential hypertension and healthy control subjects. A total of 27 untreated patients with essential hypertension (blood pressure >160/100 mmHg) and 25 normotensive (blood pressure <140/90 mmHg) age- and sex-matched control subjects participated in these studies. A total of 10 patients and 10 controls took part in each phase. Locally active doses of study drugs were infused into the non-dominant brachial artery, while forearm blood flow was measured by venous occlusion plethysmography. A 60 min infusion of BQ-123 (an ET(A) receptor antagonist; 100 nmol/min) significantly increased forearm blood flow by 40+/-8% in hypertensive patients and by 35+/-5% in controls, with no difference between groups (P=0.49). Forearm vasoconstriction to ET-1 (an ET(A) and ET(B) receptor agonist; 5 pmol/min) for 90 min was significantly blunted in hypertensive patients (21+/-4%) compared with control subjects (37+/-3%; P=0.0001). Forearm vasoconstriction to sarafotoxin S6c (an ET(B) receptor agonist; 10 pmol/min) for 90 min was similar in hypertensive patients (44+/-5%) and control subjects (48+/-4%; P=0.95). Sympathetically mediated vasoconstriction produced by lower-body negative pressure was not different in hypertensive patients compared with controls, and was not affected by infusion of ET-1 or sarafotoxin S6c. There were no differences in the observed increase in forearm blood flow with a control vasodilator (sodium nitroprusside) or the observed decrease in forearm blood flow with a control vasoconstrictor (noradrenaline) between hypertensive patients and control subjects. BQ-123 produced a significant increase in forearm blood flow in hypertensive patients, consistent with the anti-hypertensive actions of this agent. In conclusion, forearm vasoconstriction to ET-1, but not to sarafotoxin S6c, was reduced in patients with essential hypertension, consistent with possible down-regulation of the ET(A) receptor in this condition.

Topics: Acetylcholine; Brachial Artery; Case-Control Studies; Endothelin Receptor Antagonists; Endothelin-1; Female; Humans; Hypertension; Male; Middle Aged; Nitroprusside; Norepinephrine; Peptides, Cyclic; Receptor, Endothelin A; Receptors, Endothelin; Regional Blood Flow; Vasoconstrictor Agents; Vasodilator Agents; Viper Venoms

To identify the receptors by which endothelin-1 (ET-1) increases venomotor tone in hypertension.. Vascular reactivity to ET-1 and the selective endothelin receptor subtype B (ET(B)) agonist, sarafotoxin 6c (S6c), was studied in mesenteric blood vessels from deoxycorticosterone acetate (DOCA-salt) hypertensive and normotensive control rats. The diameter of small (< or = 280 microm) mesenteric arteries and veins was monitored in vitro using computer-assisted video microscopy. Contractions of mesenteric arteries (< or= 250 microm diameter) were also studied, using a myograph. ET-1 mRNA levels were measured in mesenteric arteries and veins using real-time RT-PCR techniques.. ET-1-induced contractions were reduced in arteries of DOCA-salt hypertensive rats compared with those of normotensive control rats; S6c produced negligible contractions in arteries from both groups. ET-1 concentration-responses curves in arteries measured using video microscopy or a myograph were similar. ET-1 and S6c caused veins to contract, and there were no differences between responses to these agonists in tissues from DOCA-salt hypertensive rats or normotensive control rats. Studies using ET(A) and ET(B) receptor antagonists indicated that ET-1-induced venoconstriction was mediated by ET(A) receptors. Potassium chloride concentration-response curves were similar in arteries and veins from normotensive control rats and DOCA-salt hypertensive rats. ET-1 mRNA levels in DOCA-salt hypertensive rat arteries or veins were not different from those in normotensive control rat arteries and veins.. These data indicate that ET-1 reactivity is maintained in mesenteric veins, but not arteries, in DOCA-salt hypertension. Therefore, the sustained increase in venomotor tone mediated by ET(A) receptors that is known to occur in vivo in DOCA-salt hypertensive rats is not caused by direct venoconstriction.

Topics: Animals; Desoxycorticosterone; Endothelin Receptor Antagonists; Endothelin-1; Hypertension; In Vitro Techniques; Male; Mesenteric Arteries; Mesenteric Veins; Oligopeptides; Piperidines; Potassium Chloride; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Sodium, Dietary; Vasoconstriction; Vasoconstrictor Agents; Viper Venoms

The pharmacologic properties of a novel nonpeptide endothelin (ET) receptor antagonist, S-1255 ([R]-[+]-2-[benzo(1,3)dioxol-5-yl]-6-isopropyl-4-[4-methoxyphenyl]-2H-chromene-3-carboxylic acid), was studied. [3H]S-1255 specifically bound to porcine aortic smooth muscle membranes expressing only ET(A) receptors with a Kd value of 0.39 nM. [3H]S-1255 binding was potently inhibited by ET-1 and selective ET(A) or ET(A)/ET(B) receptor antagonists, such as L-749329, SB209670, bosentan, and BQ-123, but the inhibitory effect of ET-3 and the selective ET(B) receptor antagonist, BQ-788, on the binding was weak. These inhibitory effects on [3H]S-1255 binding correlated well with those on [125I]ET-1 binding. S-1255 inhibited ET(A) receptor- and ET(B) receptor-mediated contractions in isolated rabbit femoral and pulmonary arteries with pA2 values of 8.8 and 6.3, respectively. The pA2 value of S-1255 for ET(B) receptor-mediated relaxation in isolated rabbit mesenteric artery was 7.4. Oral administration of S-1255 (0.3-10 mg/kg) caused dose-dependent inhibition of the pressor response to exogenous ET-1 (0.1 nmol/kg) in conscious normotensive rats, which was similar to that produced by intravenous administration (1 and 3 mg/kg). S-1255 (10 and 30 mg/kg, p.o.) significantly reduced blood pressure in deoxycorticosterone acetate-salt hypertensive rats from 6 h after administration, and the hypotensive effects were sustained up to 24-48 h. These results suggest that S-1255 is a highly potent and orally active ET(A) receptor antagonist.

Topics: Animals; Aorta; Benzopyrans; Blood Pressure; CHO Cells; Cricetinae; Endothelin Receptor Antagonists; Endothelins; Endothelium, Vascular; Humans; Hypertension; In Vitro Techniques; Male; Molecular Structure; Muscle, Smooth, Vascular; Rabbits; Radioligand Assay; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptor, Endothelin A; Swine; Vasoconstrictor Agents; Viper Venoms

Polymorphism of the dopamine receptor type-2 (D(2)) gene is associated with essential hypertension. To assess whether D(2) receptors participate in regulation of blood pressure (BP), we studied mice in which the D(2) receptor was disrupted. In anesthetized mice, systolic and diastolic BPs (in millimeters of mercury) were higher in D(2) homozygous and heterozygous mutant mice than in D(2)+/+ littermates. BP after alpha-adrenergic blockade decreased to a greater extent in D(2)-/- mice than in D(2)+/+ mice. Epinephrine excretion was greater in D(2)-/- mice than in D(2)+/+ mice, and acute adrenalectomy decreased BP to a similar level in D(2)-/- and D(2)+/+ mice. An endothelin B (ET[B]) receptor blocker for both ET(B1) and ET(B2) receptors decreased, whereas a selective ET(B1) blocker increased, BP in D(2)-/- mice but not D(2)+/+ mice. ET(B) receptor expression was greater in D(2)-/- mice than in D(2)+/+ mice. In contrast, blockade of ET(A) and V(1) vasopressin receptors had no effect on BP in either D(2)-/- or D(2)+/+ mice. The hypotensive effect of an AT(1) antagonist was also similar in D(2)-/- and D(2)+/+ mice. Basal Na(+),K(+)-ATPase activities in renal cortex and medulla were higher in D(2)+/+ mice than in D(2)-/- mice. Urine flow and sodium excretion were higher in D(2)-/- mice than in D(2)+/+ mice before and after acute saline loading. Thus, complete loss of the D(2) receptor results in hypertension that is not due to impairment of sodium excretion. Instead, enhanced vascular reactivity in the D(2) mutant mice may be caused by increased sympathetic and ET(B) receptor activities.

Topics: Adrenergic alpha-Antagonists; Angiotensin Receptor Antagonists; Animals; Antidiuretic Hormone Receptor Antagonists; Antihypertensive Agents; Blood Pressure; Body Weight; Catechols; Endothelin Receptor Antagonists; Endothelin-1; Female; Genotype; Hypertension; Losartan; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Oligopeptides; Phentolamine; Piperidines; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Adrenergic; Receptors, Dopamine D2; Receptors, Endothelin; Sodium; Sodium-Potassium-Exchanging ATPase; Urodynamics; Viper Venoms

To assess whether primary changes in endothelin-1 (ET-1) receptor responsiveness or secondary vessel functional modifications could characterize the effects evoked by ET-1 in the mesenteric vascular bed (MVB) of prehypertensive 5-week-old and 12-week-old spontaneously hypertensive rats (SHRs).. We used male 5-week-old and 12-week-old SHRs and sex- and age-matched Wistar-Kyoto (WKY) rats as controls. ET-1 receptor responsiveness was evaluated by ET-1 (0.04-2 micromol/l) concentration-response curves and repeated with indomethacin and BQ-123 (0.1-0.5 micromol/l), the latter a selective ETA receptor antagonist. ETB receptor responsiveness was tested by sarafotoxin S6c (1-100 nmol/l) and IRL-1620 (0.1-10 nmol/l) concentration-response curves, obtained in the noradrenaline-precontracted MVB.. At 5 weeks of age, ET-1 induced a similar concentration-dependent contraction in SHRs and WKY rats, with an overlapping BQ-123 pA2 value (negative common logarithm of the antagonist that produces an agonist dose ratio of 2) in the two strains. Indomethacin was ineffective in both groups. Sarafotoxin S6c and IRL-1620 both evoked an ETB-mediated, significant relaxation, only in WKY rats. In 12-week-old SHRs, ET-1 evoked a markedly increased maximal effect compared with the response in WKY rats (P< 0.01); this was prevented by treatment with indomethacin. The BQ-123 pA2 value was higher in SHRs than in WKY rats (P< 0.01). Both sarafotoxin S6c and IRL-1620 evoked a significant concentration-dependent relaxation in WKY rats, which was not detected in SHR preparations.. Our results could suggest that the different responses evoked by ET-1 in the MVB of SHRs during the onset of hypertension may be related partially to primary alterations in the ET-1 receptorial pattern and partially to the onset of high blood pressure, leading to an impairment in the haemodynamic balance.

Topics: Aging; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antihypertensive Agents; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Hypertension; Indomethacin; Mesenteric Arteries; Norepinephrine; Peptide Fragments; Peptides, Cyclic; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Endothelin A; Receptors, Endothelin; Vascular Resistance; Vasoconstrictor Agents; Viper Venoms

We investigated the endothelin production and endothelin receptor activity of pericardial mesothelial cells obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. The pericardial mesothelial cells were maintained in vitro and the production of endothelin-1 by these cells was evaluated by using a sensitive sandwich-type enzyme immunoassay for endothelin-1 and big endothelin-1. Endothelin receptor subtypes were pharmacologically analyzed by measuring the changes of intracellular Ca2+ concentration ([Ca2+]i) in pericardial mesothelial cells. Mesothelial cells from both strains produced more immunoreactive endothelin-1 than big endothelin-1. The production of immunoreactive endothelin-1 progressively increased in a culture time-dependent manner. The amount of immunoreactive endothelin-1 detected after 72 h in pericardial mesothelial cells of SHR was significantly (P < 0.05) higher than that in the cells of WKY rats (SHR: 196.7 +/- 19.1 pg/10(6) cells; WKY: 122.7 +/- 10.6 pg/10(6) cells). Endothelin-1 and endothelin-3 induced elevation of [Ca2+]i in pericardial mesothelial cells. The selective agonist of the endothelin ETB receptor, sarafotoxin S6c, also induced elevation of [Ca2+]i. The endothelin- and sarafotoxin S6c-induced elevations of [Ca2+]i in pericardial mesothelial cells from SHR were greater than those in mesothelial cells from WKY rats. The endothelin ETB receptor antagonist, IRL 1038 ([Cys11,Cys15]endothelin-1-(11-21)), significantly inhibited the endothelin-1- and endothelin-3-induced changes in [Ca2+]i. The endothelin ETA receptor antagonist, FR 1393171 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]ammino -4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indoyl)]propio nyl]amino-3-(2-pyridyl)propionic acid), did not affect these changes. From these results, pericardial mesothelial cells from both SHR and WKY rats shared the ability to produce endothelin-1 spontaneously in culture, although consistently greater production was detected in cultures of SHR origin. Moreover, pericardial mesothelial cells of SHR origin may have increased numbers of endothelin ETB receptors and/or a more active signal transduction system compared with those of WKY rats. These results suggest that endothelin-1 may play an important role in the pericardium in an autocrine and/or paracrine fashion.

Topics: Animals; Calcium; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-3; Epithelium; Hypertension; Pericardium; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Vasoconstrictor Agents; Viper Venoms

We used Ro 46-8443, non-peptidic antagonist selective of endothelin ETB receptors, to study the role of ETB receptors in rat hypertension models. In normotensive rats, Ro 46-8443 decreased blood pressure, but in SHR and DOCA rats, it induced a pressor effect, due to blockade of ETB-mediated release of nitric oxide since L-NAME prevented it. In rats rendered hypertensive by chronic L-NAME, Ro 46-8443 did not induce a pressor but depressor effect. Thus, in DOCA rats and SHR, Ro 46-8443 reveals a predominant influence of endothelial 'vasorelaxant' ETB receptors, while in normotensive rats the prevailing role of ETB receptors seems to be in mediating a vasoconstrictor tone.

Topics: Animals; Arginine; Blood Pressure; Endothelin Receptor Antagonists; Hypertension; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peptides, Cyclic; Pyrimidines; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Endothelin B; Receptors, Endothelin; Sulfonamides; Viper Venoms

Renal effects of endothelin (ET)-3 have been described in normotensive but not spontaneously hypertensive rats (SHR). Infusion (170 ng/kg/min) of the ETB receptor selective agonists ET-3 and sarafotoxin S6c (SS6c) was used to investigate ETB receptor modulation of renal function in SHR. ET-3 decreased glomerular filtration rate (GFR) and renal plasma flow (RPF) by approximately 95% (0.1 +/- 0.01 and 0.1 +/- 0.02 ml/min, respectively) versus vehicle (1.3 +/- 0.08 and 3.6 +/- 0.23, respectively) in SHR. ET-3 exerted a biphasic effect on urine flow (UV); an initial increase and then a decrease (vehicle, 4.2 +/- 0.55; ET-3, 0.2 +/- 0.09 microliter/min). ET-3 increased mean arterial pressure (vehicle, 159 +/- 4.1; ET-3, 174 +/- 3.1 mm Hg). SS6c decreased GFR and RPF by approximately 60% (0.8 +/- 0.12 and 2.0 +/- 0.18 ml/min, respectively) versus vehicle (2.0 +/- 0.19 and 5.2 +/- 0.45, respectively). UV did not change. Depressor effects of SS6c were observed (vehicle, 154 +/- 1.5; SS6c, 127 +/- 3.1 mm Hg). The ETB receptor selective agonists ET-3 and SS6c markedly decreased GFR and RPF in SHR, suggesting that endogenous ET-3 may modulate renal function through ETB receptors in SHR.

Topics: Animals; Blood Pressure; Endothelin-3; Glomerular Filtration Rate; Hypertension; Kidney; Male; Rats; Rats, Inbred SHR; Receptor, Endothelin B; Receptors, Endothelin; Regional Blood Flow; Vasoconstrictor Agents; Viper Venoms

Endothelin-1 elicits vasoconstrictor responses through endothelin subtype A receptors, which are located on vascular smooth muscle cells, and vasodilator responses through endothelin subtype B receptors, which occur predominantly on endothelial cells. Endothelin subtype B receptors also may be present on vascular smooth muscle cells, in which they may mediate vasoconstriction. The aims of this study were to determine the presence of vascular smooth muscle vasoconstrictor endothelin subtype B receptors in mesenteric resistance arteries and to assess whether endothelin subtype B receptor-mediated responses differ between spontaneously hypertensive rats and Wistar-Kyoto rats. Contractile responses to the endothelin subtype B receptor agonist sarafotoxin S6c and endothelin-1 were measured simultaneously with [Ca2+]i in endothelium-denuded mesenteric resistance arteries from adult spontaneously hypertensive rats and Wistar-Kyoto rats. To simulate in vivo conditions matched as closely as possible to in vitro conditions, vessels were mounted in a vessel flow chamber in which intraluminal pressure was maintained at 60 mm Hg. Contraction was determined by video imaging to record lumen diameter, and [Ca2+]i was measured by the fura 2 method. Basal [Ca2+]i was significantly higher (P < .01) in hypertensive (170 +/- 4 nmol/L) compared with normotensive rats (134 +/- nmol/L). The endothelin subtype B receptor agonist sarafotoxin S6c increased [Ca2+]i in a concentration-dependent manner. Sarafotoxin S6c-induced [Ca2+]i and contractile responses were significantly lower in hypertensive compared with normotensive rats. These data demonstrate that endothelin subtype B receptors are present in vascular smooth muscle of small arteries and that endothelin subtype B receptor-mediated vasoconstriction occurs through intracellular calcium signaling pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Pressure; Calcium; Data Interpretation, Statistical; Fluorescence; Hypertension; In Vitro Techniques; Mesenteric Arteries; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Endothelin B; Receptors, Endothelin; Signal Transduction; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Viper Venoms

Both glucocorticoids and mineralocorticoids are involved in circulatory homoeostasis and blood pressure control. In recent years direct effects of both steroid classes on vascular smooth muscle cells (VSMC) have been reported. We have thus examined the effects of RU 28362, a pure glucocorticoid agonist, and aldosterone, the physiologic mineralocorticoid, on the binding to VSMC from spontaneously hypertensive rats (SHR) of two key vasoactive peptides, endothelin-1 and angiotensin II. Binding of angiotensin II rose, and that of endothelin-1 declined, in a time- and dose-dependent fashion with maximal effects observed at 24 h and half-maximal effects for each at 2-3 nM RU 28362. Scatchard analysis showed that for both endothelin-1 and angiotensin II, RU 28362 alters receptor number but not affinity; competition studies with receptor-selective ligands (BQ123, S6C, DuP753 and PD123319) show that glucocorticoids specifically elevate (X2) AT-1 receptors and specifically lower (to approximately 30%) levels of ETA receptors. Treatment of VSMC with the antiglucocorticoid RU 38486 reversed the effect of glucocorticoids on endothelin-1 and angiotensin II binding, confirming the Type II (glucocorticoid) receptor mediated effect of the glucocorticoids. Aldosterone (100 nM) also lowers endothelin-1 binding and increases angiotensin II binding in VSMC; that this effect reflects aldosterone occupancy of classical glucocorticoid receptors is shown by the blockade of the aldosterone effect by an equal concentration (100 nM) of RU 38486--i.e. there is no evidence for an action of aldosterone via mineralocorticoid receptors. We interpret our results as evidence for a complex modulation of receptors for vasoactive peptides in VSMC by glucocorticoid but not mineralocorticoid hormones.

Topics: Aldosterone; Androstanols; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Biphenyl Compounds; Cells, Cultured; Endothelin Receptor Antagonists; Endothelins; Glucocorticoids; Hypertension; Imidazoles; Losartan; Male; Mifepristone; Muscle, Smooth, Vascular; Peptides, Cyclic; Rats; Rats, Inbred SHR; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Receptors, Glucocorticoid; Receptors, Mineralocorticoid; Tetrazoles; Viper Venoms

Experiments were designed to compare the distribution and physiological roles of endothelin (ET) receptor subtypes, ETA and ETB, in the kidneys of normotensive Sprague-Dawley (SD) and spontaneously hypertensive (SH) rats. Using [125I] ET-1 and subtype-selective ligands sarafotoxin 6c (S6c, ETB-selective agonist) and BQ123 (ETA-selective antagonist), the distribution of ETA and ETB receptors in SD rat kidney cortex, outer medulla and papilla was calculated to be 50:50, 30:70 and 10:90, respectively. The ET receptor subtypes in outer medulla and papilla of age-matched SH rats were similar to those of SD. However, in the cortex of SH rats, the ratio of ETA to ETB was 25:75 compared to 50:50 in SD rats. In addition, the affinity of the ET receptors was also higher in SH rats (117 pM vs. 235 pM). In the conscious SD rats, bolus i.v. injections of ET-1 and S6c elicited similar dose-dependent decrease in renal blood flow (RBF), which were unaffected by the infusion of the selective ETA receptor antagonist, BQ123. The SH rats were more sensitive to the renal vasoconstrictor effect of S6c and ET-1. Also, the dose-response curve to S6c was shifted to the left when compared to ET-1; however, BQ123 infusion abolished this difference. In renal clearance studies, BQ123 infusions decreased RBF and glomerular filtration rate (GFR) only in SH rats, and the fractional excretion of sodium only in SD rats. The combined data indicate that the distribution and functional roles of ETA and ETB receptor subtypes are altered in the kidneys of SH rats.

Topics: Animals; Blood Pressure; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelins; Hypertension; Kidney; Male; Peptides, Cyclic; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Receptors, Endothelin; Renal Circulation; Vasoconstrictor Agents; Viper Venoms

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 Kd and Bmax 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 IC50 value of 2.66 +/- 0.59 nM and a Ki value of 2.35 +/- 0.52 nM in WKY rats and an IC50 value of 2.82 +/- 0.76 nM and a Ki value of 2.43 +/- 0.70 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 IC50 value of 10.31 +/- 1.82 pM and a Ki value of 8.44 +/- 1.41 pM in WKY rats, while SHR rats showed an IC50 value of 10.28 +/- 1.94 pM and a Ki value of 8.89 +/- 2.00 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.(ABSTRACT TRUNCATED AT 250 WORDS) [corrected]

Topics: Animals; Blood Pressure; Central Nervous System; Endothelins; Heart Rate; Hypertension; In Vitro Techniques; Iodine Radioisotopes; Kinetics; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Cell Surface; Receptors, Endothelin; Viper Venoms

The binding of [125I] sarafotoxin 6b (SRT 6b) and [125I] endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared from cerebral cortex and ventrolateral medulla of 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [125I] SRT 6b bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [125I] SRT 6b in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [125I] SRT 6b binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due to decrease (48%) in the Bmax values in SHR rats as compared to WKY rats. The Kd values were similar in SHR and WKY rats. [125I] ET-1 also bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [125I] ET-1 in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [125I] ET-1 binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due to 36% decrease in the Bmax values in SHR rats as compared to WKY rats. The Kd values were similar in SHR and WKY rats. It is concluded that the population of ET receptors is less in the ventrolateral medulla of SHR rats and may be contributing to the regulation of blood pressure.

Topics: Animals; Binding Sites; Blood Pressure; Cell Membrane; Cerebral Cortex; Down-Regulation; Endothelins; Heart Rate; Hypertension; Iodine Radioisotopes; Kinetics; Medulla Oblongata; Neurons; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Cell Surface; Receptors, Endothelin; Viper Venoms

Sarafotoxins (SRTa, SRTb and SRTc) and ET-1 produced a potent vasodilator effect in spontaneously hypertensive rats in vivo and in rat isolated perfused mesenteries in vitro. Among these peptides SRTc demonstrated the most potent vasodilator activity, and was three times more active than SRTa in both preparations. These peptides induced endothelium-dependent vasodilatation in vitro and pretreatment with methylene blue inhibited this effect, while exposure to the antagonists of other vasodilators did not. In contrast, [nitrophenylsulfenylated Trp21]SRTc, SRTc(1-18) and reduced and S-carboxymethylated SRTc caused no vasodilatation in either animal model; the vasodilator effect of acetylated SRTc was less potent than that of SRTc. These results suggest that (i) the vasodilatations of these peptides may be exerted through the release of endothelium derived relaxing factor; (ii) the C-terminal Trp21 and disulfide bonds are essential; and (iii) the N-terminal amino group plays an important role in vasodilator activity.

Topics: Amino Acid Sequence; Animals; Blood Pressure; Endothelins; Hypertension; In Vitro Techniques; Mesenteric Arteries; Mesenteric Veins; Molecular Sequence Data; Perfusion; Rats; Rats, Inbred SHR; Sequence Alignment; Structure-Activity Relationship; Vasodilation; Viper Venoms