Differences in the yields of dicentrics and reciprocal translocations observed in the chromosomes of irradiated human skin fibroblasts and blood lymphocytes from the same healthy individuals

The modulatory role of locally produced cyclooxygenase products and endothelium-derived nitric oxide in controlling vascular tone was investigated in bovine intra-mammary artery. Vascular reactivity initiated by vasoactive compounds, endothelin-1 (ET-1), bradykinin (BK), and substance P (SP) was measured isometrically in an isolated tissue bath. The effects of a cyclooxygenase inhibitor, indomethacin (10(-5) M) and an inhibitor of nitric oxide production, N omega-Nitro L-Arginine (L-NNA: 3 x 10(-4) M) were determined during agonist-mediated responses. Indomethacin alone markedly enhanced vascular contraction produced by ET-1, while L-NNA did not. Inhibition of endothelium-derived nitric oxide synthesis by L-NNA, however, significantly attenuated BK- and SP-induced vascular relaxations, whereas indomethacin had slight influence. The potentiation between indomethacin and L-NNA in regulating vasomotor tone was not observed in this vascular bed. Thus, it appeared that both the cyclooxygenase and endothelium-derived nitric oxide pathways participated in modifying vascular reactivity. Domination of one pathway over the other depended upon the agonist used to stimulate vascular tissue.     

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

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

Vasodilator responses of coronary resistance arteries of exercise-trained pigs

BACKGROUND: The purpose of this study was to test the hypothesis that vasodilator responses of porcine coronary resistance arteries are increased by exercise training. METHODS AND RESULTS: Yucatan miniature swine were randomly divided into groups of exercise-trained (ET) and sedentary (SED) control pigs. ET pigs were placed on a progressive treadmill training program lasting 16 to 20 weeks, and SED pigs remained inactive during the same time period. Coronary resistance arteries 64 to 157 microns in diameter were isolated for in vitro evaluation of relaxation responses to the endothelium-independent dilators sodium nitroprusside (1 x 10(-10) to 1 x 10(-4) mol/L) and adenosine (1 x 10(-10) to 1 x 10(-5) mol/L) and to bradykinin (1 x 10(-13) to 3 x 10(-7) mol/L), an endothelium-dependent agent. Relaxation responses to adenosine and sodium nitroprusside were not altered by exercise training. Endothelium-dependent relaxation to bradykinin was enhanced in coronary resistance arteries from ET pigs (IC50: ET, 0.07 +/- 0.02 nmol/L; SED, 1.59 +/- 0.09 nmol/L). To determine whether prostanoids and/or the nitric oxide synthase pathway were involved in the ET-induced changes in bradykinin-induced vasodilation, responses to bradykinin were examined in coronary resistance arteries from both ET and SED pigs in the presence of indomethacin and in the presence of nitro-monomethyl L-arginine (L-NMMA). Both indomethacin and L-NMMA produced significant inhibition of the bradykinin-induced relaxation in vessels from both groups. Despite decreased bradykinin-induced relaxation after indomethacin, bradykinin-induced vasodilation was still enhanced in vessels from the ET group. L-NMMA caused greater inhibition of the bradykinin-induced relaxation in coronary resistance arteries from ET pigs relative to arteries from SED pigs and eliminated the training-induced enhancement of the bradykinin responses. CONCLUSIONS: These results suggest that exercise training enhances bradykinin-induced vasodilation through increased endothelium-derived relaxing factor/nitric oxide production by the L-arginine/nitric oxide synthase pathway.     

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.     

Selective and non-selective ET antagonists reveal an ETB receptors mediated ET-1-induced behavioural effect in conscious rats

The injection of endothelin-1 (ET-1) (1 pmol/rat) into the dorsolateral periaqueductal gray (PAG) area of freely moving rats induced rotation along the long axis of the body (barrel-rolling). Preinjection (10 min before) of BQ-788 (an ETB receptor selective antagonist; 5 nmol) or bosentan (an ETA/ETB receptor non-selective antagonist; 10 nmol) to the PAG reduced the behavioral response to ET-1. In contrast, pretreatment with FR139317 (an ETA receptor selective antagonist; 5 nmol) did not affect the ET-1-induced barrel-rolling. These results suggest that barrel-rolling induced by microinjection of ET-1 into the PAG area is predominantly mediated via ETB-like receptors.     

Endothelin-A receptors mediate vasoconstriction of capillaries in the spiral ligament

The purpose of this study was to determine whether endothelin-1 (ET-1), endothelin-2 (ET-2) or endothelin-3 (ET-3) alter the vascular diameter of capillaries in the spiral ligament. Changes in vascular tone were measured in capillaries from the isolated spiral ligament in vitro. Capillaries were occluded on one end and opened on the other end. Red blood cells trapped in the capillaries served as markers for a luminal volume defined by the red cell itself, the capillary wall and the occluder. Movement of the red cell toward the open end was taken as evidence for vasoconstriction and movement of the red cell toward the occluder was taken as evidence for vasodilation. The inner diameter of the capillaries was 7.0 microm and decreased maximally by a factor of 0.8 in response to ET-1 and ET-2 (both 10(-8) M). Vasoconstriction induced by ET-1 and ET-2 was concentration-dependent in the range between 10(-12) and 10(-8) M whereas ET-3 (10(-8) M) had no effect. The EC50s for ET-1 and ET-2 were 1.2 x 10(-10) M and 1.4 x 10(-9) M, respectively. Thus, the potency order was ET-1 > ET-2 > ET-3. Vasoconstriction induced by ET-1 and ET-2 was completely inhibited by the competitive antagonist 10(-6) M BQ-123 (cyclic D-Asp-L-Pro-D-Val-L-Leu-D-Trp). Vasoconstriction induced by ET-1 or ET-2 continued for more than 1 min after removal of agonist from the perfusate. Rapid vasodilation of capillaries preconstricted by ET-1 was observed in response to 10(-3) M sodium nitroprusside. Sodium nitroprusside, however, had no significant effect on the vascular diameter of resting capillaries. These results demonstrate that capillaries in the spiral ligament can constrict and the endothelin-mediated vasoconstriction occurs via ET(A) receptors.     

Mechanism of rat uterine smooth muscle contraction induced by endothelin-1

1. Endothelin (ET)-1 has been demonstrated to cause contraction of uterine smooth muscle. We investigated the role of ET receptor subtypes (ETA and ETB receptors) in ET-1-induced contraction of rat uterine smooth muscle by using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. 2. ET-1 caused a contraction with superimposed oscillations of the rat isolated uterus suspended in Krebs-Ringer solution; both the amplitude of contraction as well as the oscillation frequency increased in a dose-dependent manner (10(-11)-10(-7)M). 3. BQ-123 (10(-6)M) markedly shifted the dose-response curve of ET-1 for both contractile effects and oscillation frequency to the right. 4. BQ-3020 (10(-11)-3 x 10(-7) M) did not cause uterine contraction; neither did it affect the dose-response curve of ET-1 for either the contractile effect or the increase in oscillation frequency. Thus, stimulation of ETB receptors is not involved in these responses. 5. The present findings suggest that ET-1-induced contractile responses and the increase in oscillation frequency in rat uterine smooth muscle is mediated through ETA receptors, and that ETB receptors play no role in these responses.     

Hemorrhagic bullous lesions in Henoch-Sch?nlein purpura

A microinjection of endothelin-1 (ET-1; 10 pmol) into the superior colliculus (SC) of anaesthetised rats caused a decrease in blood pressure. Microinjection into the same nucleus of Nomega-nitro-L-arginine methyl ester (L-NAME; 0.1, 0.5, 1 micromol), an L-arginine analogue and a potent inhibitor of nitric oxide (NO) synthase, increased the basal mean arterial blood pressure. Treatment of SC with L-NAME (1 micromol) did not affect the depressor response induced by injection of ET-1 (10 pmol), into the SC, at the peak of the pressor response to L-NAME. In addition, L-arginine L-ARG) (1 micromol), the substrate for NO synthase, microinjected into the superior colliculus prior to ET-1 did not significantly increase the depressor response to ET-1. These findings, therefore, suggest that within the SC the nitric oxide synthesis does not affect depressor responses induced by ET-1.(c) 1998 The Italian Pharmacological Society     

Endothelin-1 infusion inhibits plasma insulin responsiveness in normal men

OBJECTIVES: Elevated plasma endothelin (ET)-1 levels have been described in insulin-resistant states such as syndrome X, obesity, non-insulin-dependent diabetes mellitus, and in some studies in essential hypertension. To investigate whether increases in circulating ET-1 to levels observed in insulin-resistant states can modulate insulin levels and/or insulin sensitivity in humans, we assessed these variables during low, non-pressor-dose ET-1 compared with placebo infusion. DESIGN: In a randomized, single blind, crossover design, 10 lean normotensive male subjects received either an intravenous infusion of subpressor doses of ET-1 dissolved in polygeline or a control infusion of polygeline only (placebo). Using dynamic assessment by the minimal model approach with the modified frequent sampling intravenous glucose tolerance test (FSIGT) the following and other parameters were measured: insulin sensitivity; acute insulin response to glucose (AIR(G)) calculated as the average of the three peak values between 2 and 5 min after injection of glucose from which the basal insulin levels were subtracted; the initial area under the curve (AUC(1-19)) from insulin values between time 0 and 19 min and the first-phase insulin secretion (phi1) from insulin kinetics parameters. RESULTS: ET-1 infusion reduced AIR(G) (to 34.85 +/- 4.27 compared with 49.3 +/- 6.9 microU/ml during placebo, P=0.017) and the acute C-peptide response to glucose (to 2.33 +/- 0.41 compared with 3.1 +/- 0.44 ng/ml, P=0.018), decreased plasma insulin levels during the FSIGT compared with placebo (analysis of variance P<0.0001) and decreased the AUC(1-19) (to 2.1 +/- 0.2 compared with 2.9 +/- 0.3 U/l per 20 min, P<0.01) while phi1 tended to be lower. S1 measured during ET-1 infusion was unaltered (11.11 +/- 1.91 x 10(-4) versus 10.88 +/- 2.11 10(-4)/min per mU per l, NS). CONCLUSIONS: These findings demonstrate that an increase in circulating ET-1 to levels observed in insulin-resistant states acutely diminishes the insulin secretory response but does not significantly modify insulin sensitivity.     

Effect of endothelin-1 on spontaneous contractions and effects of nimodipine and isradipine on endothelin-1-induced contractions in myometrial strips isolated from normal pregnant and preeclamptic women

BACKGROUND: To examine the effect of endothelin-1 (ET-1) on spontaneous contractile activity and the effects of nimodipine and isradipine on ET-1-induced contractile responses in myometrial strips isolated from normal pregnant and preeclamptic women. MATERIAL AND METHODS:. Isolated myometrial strips were obtained from seven normal pregnant and seven preeclamptic women undergoing elective cesarean section and the strips were mounted in organ baths for recording of isometric tension. The effect of increasing concentration of ET-1 on spontaneous contractions and effects of increasing concentration of nimodipine and isradipine on ET-1-induced contractions were recorded. RESULTS: ET-1 dose-dependently (10(-11)-10(-8) M) increased the amplitude and frequency of spontaneous contractions in all myometrial strips obtained from normal pregnant and preeclamptic women. The increase in the amplitude of contractions in preeclamptic strips was significantly higher than that of normal strips. The increase in amplitude of contractions in normal and preeclamptic strips reached statistical significance beginning from the concentrations of 10(-9) M and 10(-11) M, respectively. ET-1 (10(-8) M) also increased the basal tone of all myometrial strips isolated from normal pregnant and preeclamptic women. When ET-1 (10(-8) M)-contracted myoinetrial strips were exposed to increasing concentrations of nimodipine (10(-6)-3x10(-5)M) and isradipine (10(-5)-3x10(-4) M), nimodipine and isradipine dose-dependently decreased the amplitude and frequency of contractions. CONCLUSIONS: The increase in contractile response with ET-1 was significantly higher in myometrial strips isolated from preeclamptic women compared to those of myometrial strips isolated from normal pregnant women. These increases in contractile response are at least in part mediated by dihydropyridine-sensitive calcium channels, since they were significantly reduced in the presence of increasing concentrations of nimodipine and isradipine.     

Development of endothelin receptors in perinatal rabbit pulmonary resistance arteries

1. Contractile responses to endothelin-1 (ET-1) and sarafotoxin S6c (S6c) were studied in pulmonary resistance arteries (approximately 320 microm i.d.) from fetal, 0-24 h, 4 day and 7 day rabbits. The effects of the ET(A)-selective antagonist FR139317, the selective ET(B) receptor antagonist BQ-788 and the non-selective ET(A)/ ET(B) receptor antagonist SB 209670, on these responses, were determined. Acetylcholine-induced vasodilation and noradrenaline-evoked contractions were also examined. 2. ET-1 potency was in the following order (pEC50 values): fetal (8.7) = 0-24 h (8.8) = 4 day (8.6) > 7 day (8.0). The order of potency for S6c was 7 days (11.1) = 4 days (10.8) > 0-24 h (9.7) > fetal (8.6). Hence, S6c and ET-1 were equipotent in the fetus but S6c was increasingly more potent than ET-1 with increasing age, being some 1000 times more potent by 7 days. By 7 days, responses to ET-1 were also resistant to both FR139317 and BQ-788. FR139317 inhibited responses to ET-1 in vessels from 0-24 h and 4 day, but not fetal, rabbits (pKb: 6.4 in 4 day rabbits). BQ-788 inhibited responses to ET-1 at all age points except for 7 days (pKb: 6.7 at 0-24 h; 6.2 at 4 days). BQ-788 inhibited responses to S6c at all age points (pKb: 8.5 at 4 days). SB 209670 inhibited responses to ET-1 and S6c at 0-24 h and 4 days (pKb for ET-1: 8.3 and 8.0 respectively; pKb for S6c: 9.2 and 10.2 respectively). 3. Acetylcholine (1 microM) induced vasodilation at all age points (inhibited by 100 microM L-N(omega)-nitroarginine methylester) although the degree of vasodilation was significantly reduced (approximately 75%) at 0-24 h. Noradrenaline induced contraction at all age points except 7 days and its response was significantly enhanced at 0-24 h. 4. Over the first week of life, the potency of S6c increases whilst that to ET-1 decreases suggesting differential development of responses to ET-1 and S6c and heterogeneity of ET(A)- or 'ET(B)-like' receptor-mediated responses. There is no synergism between ET(A) and ET(B) receptors at birth but this is established by 7 days. Immediately after birth rabbit Pulmonary Resistance Arteries are hyperresponsive to ET-1 and noradrenaline but exhibit impaired nitric-oxide dependent vasodilation.     

Mediation of endothelin-1-induced inhibition of platelet aggregation via the ETB receptor

1. The effects of FR139317 (ETA antagonist) or PD145065 (non-selective ETA/ETB antagonist) on endothelin-1 (ET-1)-induced changes in blood pressure and inhibition of ex vivo platelet aggregation were investigated in the anaesthetized rabbit. 2. ET-1 (1 nmol kg-1, i.a. bolus) caused a sustained increase in mean arterial pressure (MAP) (peak increase 47 +/- 5 mmHg, n = 8). Intravenous infusion of FR139317 at 0.2 (n = 4) or 0.6 mg kg-1 min-1 (n = 4) inhibited the ET-1 pressor response by 83 or 89%, respectively. Infusion of PD145065 at 0.2 (n = 4) or 0.6 mg kg-1 min-1 (n = 4) inhibited the ET-1-induced increase in MAP by 79 or 75%, respectively. 3. The transient depressor response (-16 +/- 3 mmHg) which preceded the rise in blood pressure induced by ET-1 (1 nmol kg-1, i.a., n = 8) was enhanced by an intravenous infusion of FR139317 (0.6 mg kg-1 min-1) to -35 +/- 5 mmHg (P < 0.05, n = 4). This enhancement was abolished by indomethacin (5 mg kg-1, i.v.) pretreatment (-17 +/- 1 mmHg, n = 4). PD145065 (0.2 mg kg-1 min-1, i.v.) attenuated the ET-1-induced fall in blood pressure to -9 +/- 1 mmHg (n = 4), while a higher dose of this antagonist (0.6 mg kg-1 min-1, i.v.) completely abolished the ET-1-mediated depressor response. 4. ET-1 (1 nmol kg-1, n = 8) inhibited ex vivo platelet aggregation by 96% at 5 min after injection of the peptide. FR139317 (0.2 or 0.6 mg kg-1 min-1, i.v.) or PD145065 (0.2mg kg-1 min-1, i.v.) did not affect the inhibition of ex vivo platelet aggregation in response to ET-1. In contrast, intravenous infusion of PD145065 (0.6 mg kg-1 min-1) abolished the anti-aggregatory effects of ET-1.5. Thus, FR139317 inhibits the pressor, but not the depressor actions of ET-1 and has no effect on the ET-l-induced inhibition of ex vivo platelet aggregation. In contrast, PD145065 antagonizes the pressor and depressor responses to ET-1 and abolishes the anti-aggregatory effects of the peptide.6. These results strongly suggest that ET-1-induced vasoconstriction in the anaesthetized rabbit is primarily mediated via the ETA receptor while the depressor and antiaggregatory actions of ET-1 are due to activation of the ETB receptor.     

Endothelin-1 inhibits L-type Ca2+ current enhanced by isoprenaline in rat atrial myocytes

Endothelin-1 (ET-1) was shown to exert direct cardiac effects by complex signaling pathways and to interact with neurotransmitter regulation of cardiac activity. The effect of ET-1 was investigated on the beta-adrenergic stimulation of cardiac L-type Ca2+ current (ICaL) on isolated rat atrial myocytes by using the patch-clamp technique. ET-1 (5 x 10(-8) M) reversed the increase in ICaL induced by isoprenaline (10(-6) M) but had no effect on basal ICaL and on (-) Bay K 8644-increased ICaL (10(-6) M); so ET-1 might exert an effect only when the Ca2+ channels are phosphorylated. The antiadrenergic action of ET-1, blocked by BQ-123 (10(-6) M) and unaffected by IRL 1038 (3.5 x 10(-8) M) should be mediated by ET-A receptors. The inhibitory action of ET-1 was still observed when ICaL was previously increased by forskolin (3 x 10(-6) M), 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP; 200 microM), or cAMP (100 microM) in presence of isobutyl methyl xanthine (IBMX; 10(-6) M), suggesting that the antiadrenergic action of ET-1 on ICaL was exerted independent of the cAMP-dependent phosphorylation pathway. ET-1 is known to be an activator of phosphoinositide hydrolysis, resulting in an increased production of IP3 and diacylglycerol (DAG). A Ca(2+)-dependent inhibition of ICaL consequently to an elevation of the intracellular Ca2+ pool via IP3 might be excluded in the action of ET-1, because of the presence of EGTA in the intrapipette medium. ET-1 reversed the isoprenaline-induced increase in ICaL in the presence of protein kinase C inhibitor [PKC(19-31); 100 microM), making unlikely the involvement of a DAG-dependent activation of PKC. Therefore the antiadrenergic action of ET-1 might also be independent on the phosphoinositide pathway.     

The effects of topical doxepin on responses to histamine, substance P and prostaglandin E2 in human skin

The tricyclic antidepressant, doxepin, is known to have H1 and H2 antihistaminic effects. Recently, 5% doxepin cream has been marketed in the U.S.A. for treatment of eczematous dermatoses. We investigated the effects of topical doxepin treatment on histamine-, substance P- and prostaglandin E2- (PGE2) induced responses in the skin of normal and atopic subjects. We compared the effects of topical doxepin with those of the oral antihistamine terfenadine. The weal volume and flare area responses to histamine were significantly reduced by treatment with topical doxepin or oral terfenadine in both normal and atopic subjects (P < 0.05). The mean +/- SEM percentage reduction in flare area for 10 micrograms/site of histamine in non-atopics and atopics was 48 +/- 8% and 60 +/- 17% with terfenadine, and 54 +/- 12% and 81 +/- 4% with topical doxepin, respectively. The mean percentage reduction in weal volume for the same dose of histamine in non-atopics and atopics was 70 +/- 9% and 63 +/- 16% with terfenadine, and 96 +/- 2% and 89 +/- 6% with topical doxepin, respectively. The flare but not the weal response to substance P was inhibited by both treatments in all subjects (P < 0.05). The mean +/- SEM percentage reduction in flare area for 200 pmol/site of substance P in non-atopics and atopics was 53 +/- 10% and 73 +/- 4% with terfenadine, and 74 +/- 7% and 75 +/- 4% with topical doxepin, respectively. The cutaneous responses to PGE2 were not affected by either drug. The inhibitory effects of doxepin were as great as those of terfenadine, and doxepin had a significantly greater effect than terfenadine in inhibiting the weal response to histamine and flare response to substance P in normal volunteers (P < 0.05). There was no significant difference between atopics and non-atopics in the percentage reduction of cutaneous responses by oral terfenadine or topical doxepin. Marked sedation occurred in three of the first 10 subjects treated with topical doxepin, necessitating a reduction in dosage for the remaining six subjects. In summary, topical doxepin was as effective as, and sometimes more effective than, a standard dose of oral terfenadine in the inhibition of histamine-induced and axon-reflex-mediated cutaneous responses. The marked sedative effect may limit its clinical use in some patients.     

Ketamine relaxes airway smooth muscle contracted by endothelin

Endothelins (ETs) are synthesized not only in vascular endothelial cells but also in airway epithelial cells. Increased ET-1 has been demonstrated in bronchial epithelium of asthmatic patients, and, in severe asthma attacks, ET-1 increases in plasma and bronchoalveolar lavage fluid. In this study, we investigated whether ketamine (KET) relaxes ET-induced tracheal contractions. Female guinea pigs were killed with an overdose of pentobarbital. The trachea was removed and cut spirally into two strips that were mounted in an organ bath filled with Krebs-bicarbonate buffer. The response of each strip to 10(-7) M carbachol was taken as 100% contraction to which the response to ET was referred. The contribution of the epithelium to the relaxant effect of KET was studied in denuded tracheae or in the presence of 5 x 10(-5) M indomethacin. ET-1 (3 x 10(-8) M) induced contractions that were 76 +/- 3% of those induced by carbachol. KET reversed the response to ET-1 in a dose-dependent fashion. Similarly, ET-2 (3 x 10(-8) M) induced contractions that were 74 +/- 5% of those induced by carbachol, and KET also reversed this response in a dose-dependent manner. In epithelium-denuded strips, ET-1 induced contractions that were 104 +/- 3% of those induced by carbachol, and KET still reversed this response. The tonic phase of the response to ET-1 was equal (100 +/- 6%) to the response to carbachol, and KET did not affect it significantly. In the presence of ryanodine, KET reduced the ET-1-induced contraction from 67 +/- 2% to 36 +/- 3.%, P < 0.01. In the presence of nicardipine, KET also inhibited the ET-1-induced contraction. We conclude that KET relaxes the tracheal smooth muscle contracted by ETs via a mechanism that is independent of the tracheal epithelium. The relaxant effect of KET on the ET-induced contraction of the trachealis muscle is not dependent upon blockade of 1) sarcolemma influx of Ca2+ through the dihydropyridine Ca2+ channel or 2) the release of intracellular Ca2+ through the ryanodine-sensitive intracellular Ca2+ channel. It is likely that the action of KET relaxing ET-induced tracheal contractions is at some point of the inositol 1,4,5-trisphosphate signaling pathway.     

Effect of endothelin-1 on corticosteroid secretion by the frog adrenal gland is mediated by an endothelinA receptor

We have previously reported that endothelin-1 (ET-1) stimulates the in vitro secretion of corticosterone and aldosterone from the adrenal gland of the frog Rana ridibunda. The aim of the present study was to investigate the pharmacological profile of the endothelin receptor subtype involved in the corticotropic effect of ET-1. The mixed ET(A)/ET(B) receptor antagonist Ro 47-0203 (10(-5) M) totally blocked the stimulatory effect of ET-1 (5 x 10(-9) M) on corticosterone and aldosterone secretion. The action of ET-1 was also inhibited by the selective ET(A) receptor antagonist BQ-485 (10(-7) M). In contrast, the selective ET(B) receptor antagonist IRL 1038 (10(-6) M) did not affect the response of the frog adrenal gland to ET-1. In addition, the selective ET(B) receptor agonist IRL 1620 (10(-6) M) did not mimic the stimulatory effect of ET-1. The high affinity ET(C) receptor agonist endothelin-3 (ET-3) stimulated corticosteroid secretion, but was 400 times less potent than ET-1. Moreover, the action of ET-3 was also blocked by BQ-485 (10(-7) M). These data indicate that the stimulatory effects of ET-1 and ET-3 on corticosteroid secretion by the frog adrenal gland are mediated by an ET(A) receptor subtype.     

ACE inhibition: postsynaptic adrenergic sympatholytic action in men

BACKGROUND: The mechanisms by which ACE inhibitors produce a sustained clinical benefit are not entirely clear but may involve the sympathetic nervous system. We compared the effect of local brachial artery infusions of an ACE inhibitor (perindoprilat) with the effect of placebo (0.9% NaCl) on endogenously mediated (lower body negative pressure [LBNP]) and exogenously mediated (brachial artery infusions of norepinephrine) sympathetic vasoconstriction. METHODS AND RESULTS: Eight healthy, normotensive male volunteers (20 to 32 years) were studied on one occasion. Forearm blood flow (FABF; mL x dL forearm(-1) x min(-1)) responses to LBNP (-20 cm H2O) and increasing increments of norepinephrine (60, 120, and 240 pmol/min) were compared when coinfused with placebo and perindoprilat (5 nmol/mL). FABF was measured simultaneously in both arms by venous occlusion plethysmography with mercury-in-Silastic strain gauges with drugs infused locally at the left brachial artery. The right arm served as a control. Baseline FABFs did not differ between the infused and control arms (3.04+/-0.52 versus 3.05+/-0.42 mL x dL forearm(-1) x min(-1); P=.98). Perindoprilat did not alter FABF when infused alone, but the FABF response to LBNP in the infused arm was attenuated during the perindoprilat infusion compared with placebo (-17.8+/-4.3% versus -33.8+/-3.1%, respectively; P=.015). The FABF response to the maximum dose of norepinephrine was also attenuated during the perindoprilat infusion compared with placebo (-28.3+/-1.4% versus -36.9+/-2.8%, respectively; P=.015). The mean slope of the FABF (log transformed) versus norepinephrine dose-response curve was significantly attenuated by perindoprilat compared with placebo (-0.11+/-0.019 versus -0.02+/-0.02; P=.001). CONCLUSIONS: We conclude that ACE inhibition has a significant postsynaptic sympatholytic effect in the forearm circulation of men.     

Role of nitric oxide in coronary arterial vasomotion and the influence of coronary atherosclerosis and its risks

Healthy coronary vascular endothelium releases nitric oxide to modulate resting and dynamic coronary arterial tone. We studied the impact of atherosclerosis and/or its risks on endothelial nitric oxide release in response to metabolic stimuli by evaluating coronary vasomotor responses to atrial pacing before and after the inhibition of nitric oxide production by intracoronary NG-monomethyl-L-arginine (L-NMMA) (20 micromol/min) infusion. The study includes 34 patients (15 with coronary disease, 11 with normal coronary arteries and > or =1 risk factor, and 8 with normal coronary arteries and no risks). Coronary blood flow was derived from Doppler flow velocity (0.018-inch Doppler wire) and coronary diameter. L-NMMA infusion reduced coronary blood flow by 18 +/- 16% and coronary diameter by 10 +/- 9%. Responses were identical in all subgroups. Coronary blood flow responses to pacing were similar in all subgroups and were unaffected by L-NMMA (11 +/- 11 vs 13 +/- 9 ml/min; p = 0.26). Epicardial coronary vasodilation to control pacing occurred in patients with normal coronary arteries with (4.0 +/- 5.2%, p = 0.01) or without (8.0 +/- 5.2%, p = 0.03) risks, but not in patients with coronary disease (2.8 +/- 5.9%). L-NMMA abolished pacing-induced epicardial vasodilation in patients without coronary artery disease, producing a 1.8 +/- 5.1% response, which was similar in all subgroups. We conclude that microvascular responses to rapid atrial pacing are not mediated by nitric oxide. Flow-mediated epicardial coronary arterial responses may be nitric oxide dependent.     

Endothelin-1 in the superior colliculus of rats induces depressor responses due to peripheral hemodynamic changes

Microinjection of endothelin-1 (ET-1; 10 pmol) into the superficial layer of the superior colliculus caused systemic and regional hemodynamic changes, as measured by injection of radioactive microspheres at the peak of the hypotensive effect of endothelin-1. Endothelin-1 decreased total peripheral resistance by 39 +/- 2% (n=5); the vascular resistances were decreased in the spleen, the mesentery, the large intestine and the small intestine. Moreover, we found that in consequence of the increased fraction of cardiac output received by the above organs, decreases in vascular resistances were associated with increases in blood flows in them. Interestingly, ET-1 also decreased the vascular resistances and increased the total blood flows in the kidneys. The haemodynamic changes induced by injection of endothelin-1 to the superior colliculus were associated with significant decreases in the mean arterial blood pressure (37 +/- 4 mmHg, n=6) and no changes in heart rate. Exogenous ET-1, therefore, within the SC decreases blood pressure due to peripheral hemodynamic changes.     

Ethanol inhibits mitogen activated protein kinase activity and growth of vascular smooth muscle cells in vitro

The intrathecal (i.t.) injection of endothelins to conscious rats was found to cause respiratory arrest. To gain some insights into this central phenomenon, peripheral vascular permeability and lung oedema were measured after i.t. and i.v. injections of these peptides. When injected at T-8 spinal cord level, endothelin-1 (65 and 650 pmol) and endothelin-3 (650 pmol) enhanced vascular permeability in the lungs by 22-fold and 7-fold, respectively, and caused sudden death at the highest dose. Less prominent increases (between 1.4- and 2.2-fold) of vascular permeability were observed in other tissues (trachea, kidney, ears, skin of hind paws and back skin) with endothelin-1. Endothelin-1 (650 pmol) caused a similar increase (27-fold) in lung vascular permeability when injected at T-2, although the response was significantly less (P < 0.05) if injected at the L-4 (15-fold) spinal cord level. Only endothelin-1 produced lung oedema when injected at the T-2 or T-8 level. In contrast, intravenous injection of endothelins-1 and -3 (650 pmol) did not produce lung oedema and the lung vascular permeability was increased by only 1.4-1.6-fold and all rats survived. The prior i.t. injection of 6.5 nmol BQ-123 (cyclo[D-Trp, D-Asp, L-Pro, D-Val, L-Leu]), a selective endothelin ET(A) receptor antagonist, prevented the increases of lung vascular permeability and oedema and the mortality induced by i.t. endothelin-1 (650 pmol). Whereas i.v. treatment with phentolamine (2 mg/kg) or pentolinium (25 mg/kg + 50 mg/kg per h x 15 min) abolished the lung vascular permeability changes evoked by endothelin-1 (650) pmol), atropine (1 mg/kg), NG-nitro-L-arginine (50 mg/kg) or indomethacin (5 mg/kg) had no effect. Moreover, the effects of endothelin-1 were attenuated in capsaicin pretreated rats (125 mg/kg, 10 days earlier) and almost abolished in rats subjected to sympathectomy with 6-hydroxydopamine (100 mg/kg, 24-48 h earlier). All these treatments except atropine and NG-nitro-L-arginine prevented the endothelin-1-induced lung oedema and reduced the lethality by around 50%. These results suggest that the increases of pulmonary vascular permeability and oedema induced by i.t. endothelin-1 are due to an intense pulmonary vasoconstriction mediated by alpha-adrenoceptors following the release of catecholamines in response to the activation of endothelin ET(A) receptor in the spinal cord. This central phenomenon seems to be reflexogenic, including the involvement of primary afferent C-fibers and spinal cord ascending fibers to the brain. Thus, endothelin-1 could play a role in neurogenic pulmonary oedema through a central mechanism.