Rabbit Pulmonary Artery Research Articles

Dual effect of blocking agents on Ca2+-activated Cl(-) currents in rabbit pulmonary artery smooth muscle cells.

The effects of the Cl- channel antagonists, niflumic acid (NFA), dichloro-diphenylamine 2-carboxylic acid (DCDPC) and diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) on Ca2+-activated Cl- current (I(Cl(Ca))) evoked by adding fixed intracellular calcium concentrations ([Ca2+]i) to the pipette solution were studied in rabbit pulmonary artery myocytes. With 250 and 500 nM [Ca2+]i bath application of NFA (100 microM) increased inward current at negative potentials, but inhibited outward current at positive potentials. On wash out of NFA, I(Cl(Ca)) was greatly enhanced at all potentials. When external Na+ ions were replaced by N-methyl-D-glucamine (NMDG+) NFA still enhanced I(Cl(Ca)) at negative potentials but the increase of I(Cl(Ca)) on wash out was blocked. When the mean reversal potential (E(r)) of I(Cl(Ca)) was shifted to negative potentials by replacing external Cl- with SCN-, NFA increased inward current but blocked outward current suggesting that the effect of NFA is dependent on current flow. Inclusion of NFA in the pipette solution had no effect on I(Cl(Ca)). Voltage jump experiments indicated that I(Cl(Ca)) displayed characteristic outward current relaxations at +70 mV and inward current relaxations at -80 mV that were abolished by NFA. DCDPC (100 microM) produced similar effects to NFA but 1 mM DIDS produced inhibition of I(Cl(Ca)) at both positive and negative potentials and there was no increase in current on wash out of DIDS. These results suggest that NFA and DCDPC, but not DIDS, simultaneously enhance and block I(Cl(Ca)) by binding to an external site, probably close to the mouth of the chloride channel.

Pharmacological profile of the novel mammalian tachykinin, hemokinin 1.

1. The effects of the novel mammalian tachykinin, hemokinin 1 (HEK-1), have been investigated by radioligand binding and functional in vitro and in vivo experiments. 2. Similar to SP (K(i)=0.13 nM), HEK-1 inhibited in a concentration-dependent manner and with high affinity [(3)H]-substance P (SP) binding to human NK(1) receptor (K(i)=0.175 nM) while its affinity for [(125)I]-neurokinin A (NKA) binding at human NK(2) receptor was markedly lower (K(i)=560 nM). 3. In isolated bioassays HEK-1 was a full agonist at tachykinin NK(1), NK(2) and NK(3) receptors. In the rat urinary bladder (RUB) HEK-1 was about 3 fold less potent than SP. In the rabbit pulmonary artery (RPA) HEK-1 and in the guinea-pig ileum (GPI), HEK-1 was about 500 fold less potent than NKA and NKB, respectively. 4. The responses to HEK-1 were antagonized by GR 82334 in RUB (pK(B)=5.6+/-0.07), by nepadutant in RPA (pK(B)=8.6+/-0.04) and by SR 142801 in GPI (pK(B)=9.0+/-0.2) with apparent affinities comparable to that measured against tachykinin NK(1), NK(2) and NK(3) receptor-selective agonists, respectively. 5. Intravenous HEK-1 produced dose-related decrease of blood pressure in anaesthetized guinea-pigs (ED(50)=0.1 nmol kg(-1)) and salivary secretion in anaesthetized rats (ED(50)=6 nmol kg(-1)) with potencies similar to that of SP. All these effects were blocked by the selective tachykinin NK(1) receptor antagonist, SR 140333. 6. We conclude that HEK-1 is a full agonist at tachykinin NK(1), NK(2) and NK(3) receptors, possesses a remarkable selectivity for NK(1) as compared to NK(2) or NK(3) receptors and acts in vivo experiments with potency similar to that of SP.

Peculiarities of adrenergic regulation of smooth muscles in rabbit pulmonary arteries.

Muscle tension recording was used to study adrenergic contractile reactions of pulmonary artery smooth muscles in rabbits. Stimulation of alpha-adrenoceptors induced contraction of pulmonary artery smooth muscle cells. Endothelium partially inhibited the contractile effect of alpha-adrenoceptor agonists. Activation of smooth muscle beta-adrenoceptors in pulmonary arteries produced a dual dose-dependent effect (relaxation at agonist concentrations of 10 nM-1 muM and contraction at concentrations above 10 muM). The contractile effect of beta-adrenoceptor activation in smooth muscle cells is a specific feature of pulmonary arteries probably related to peculiarities of cAMP-signaling in these cells.

Differential regulation of Ca(2+)-activated Cl(-) currents in rabbit arterial and portal vein smooth muscle cells by Ca(2+)-calmodulin-dependent kinase.

1. Ca(2+)-activated chloride currents (I(Cl(Ca))) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca(2+)-calmodulin-dependent kinase II (CaMKII) were compared between the three cell types. 2. In PA and CA myocytes dialysed and superfused with K+ -free media, pipette solutions containing fixed levels of free Ca(2+) in the range of 250 nM to 1 microM evoked well sustained, outwardly rectifying I(Cl(Ca)) currents in about 90 % of cells. The CaMKII inhibitor KN-93 (5 microM) increased the amplitude of I(Cl(Ca)) in PA and CA myocytes. However, the threshold intracellular Ca(2+) concentration for detecting this effect was different in the two arterial cell types. KN-93 also enhanced the rate of activation of the time-dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady-state activation curve. 3. In PA myocytes, the effects of KN-93 were not mirrored by its inactive analogue KN-92 but were reproduced by the inclusion of autocamtide-2-related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nM) significantly reduced I(Cl(Ca)) evoked by 500 nM Ca(2+). 4. In PV myocytes, I(Cl(Ca)) was evoked by pipette solutions containing up to 1 microM free Ca(2+) in less than 40 % of cells. Application of KN-93 to cells where I(Cl(Ca)) was sustained produced a small inhibition (approximately 25%) of the current in 70 % of the cells. 5. The present study shows that regulation of Ca(2+)-dependent Cl(-) channels by CaMKII differs between arterial and portal vein myocytes.

Effect of progesterone on the contractile response of isolated pulmonary artery in rabbits

The purpose of this study was to assess the direct effect of progesterone on rabbit pulmonary arteries and to examine the mechanism of its action. Rings of pulmonary artery from male rabbits were suspended in organ baths containing Krebs solution, and isometric tension was measured. The response to progesterone was investigated in arterial rings contracted with noradrenaline (NA), KCl, and CaCl2. The effects of endothelium, nitric oxide (NO), prostaglandins, cyclic GMP (cGMP), and the adrenergic beta-receptor on progesterone-induced relaxation were also assessed. Progesterone inhibited the vasocontractivity to NA, KCl, and CaCl2, and relaxed rabbit pulmonary artery. The relaxing response of progesterone in pulmonary artery was significantly reduced by removal of endothelium, inhibitors of nitric oxide synthase and guanylate cyclase, but not by prostaglandin synthase inhibitor and blockage of the adrenergic beta-receptor. In Ca2+-free (0.1 mM EGTA) Krebs solution, progesterone inhibited NA-induced contraction that was intracellular Ca2+-dependent, but didn't affect the contraction of extracellular Ca2+-dependent component. Our results suggest that progesterone induces relaxation of isolated rabbit pulmonary arteries partially via NO and cGMP. Progesterone may also inhibit Ca2+ influx through potential-dependent calcium channels (PDCs) and Ca2+ release from intracellular stores.

Pharmacologic characterization of S-1255, a highly potent and orally active endothelin A receptor antagonist.

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.

Endothelin-1-induced potentiation of adrenergic responses in the rabbit pulmonary artery: role of thromboxane A 2

To examine whether low concentrations of endothelin-1 potentiate the vasocontrictor response to adrenergic stimulation, we recorded the isometric response of rings of rabbit pulmonary artery to electrical stimulation and noradrenaline. Endothelin-1 (10 −10 M) potentiated the contractions induced by electrical stimulation and noradrenaline. The endothelin ET B receptor antagonist (2,6-dimethylpiperidinecarbonyl-γ-methyl-Leu- N in-[Methoxycarbonyl]- d-Trp- d-Nle) (BQ-788, 10 −6 M), but not the endothelin ET A receptor antagonist cyclo( d-Asp-Pro- d-Val-Leu- d-TRP) (BQ-123, 10 −6 M), inhibited the potentiating effects of endothelin-1. Pretreatment with the cyclooxygenase inhibitor indomethacin, the thromboxane synthase inhibitor furegrelate and the thromboxane receptor antagonist [1 S-[1α,2α( Z),3α,4α]]-7-[3-[[[[(1-oxoheptyl)amino]acetyl]amino] methyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-5-heptenoic acid (SQ-30741) (all at 10 −5 M) prevented the potentiation induced by endothelin-1 on adrenergic stimulation. The Ca 2+ channel antagonist nifedipine (10 −6 M) did not affect the potentiation induced by endothelin-1. The results indicate that endothelin-1 potentiates the responses to electrical stimulation and noradrenaline by activating endothelin ET B receptors. This potentiation depends on the production of cyclooxygenase-generated factors, probably thromboxane A 2.

Histaminergic regulation of smooth muscles in rabbit pulmonary arteries.

Histaminergic contractile reactions of smooth muscles in rabbit lobar pulmonary arteries were studied using in vitro mechanographic technique. Histamine via activation of H-receptors induced a dose-dependent constriction of smooth muscle segments, while endothelium inhibited histamine-induced contractile reactions. Histamine increased sensitivity of smooth muscle cells in pulmonary artery to sodium nitroprusside.

Fetal rabbit pulmonary artery smooth muscle cell response to ryanodine is developmentally regulated.

To study developmental changes in intracellular calcium handling in pulmonary artery smooth muscle cells (PASMCs), cells were isolated from distal and proximal pulmonary arteries from rabbits at different developmental stages: juvenile (4-6 wk old), newborn (<48 h), and full-term fetal. Isolated PASMCs were studied using the calcium-sensitive dye fura 2. Cells from each age group responded to caffeine with an increase in calcium; however, ryanodine (50 microM) only increased calcium in fetal distal PASMCs. The ryanodine-induced increase was due to influx of extracellular calcium because it was blocked by removal of extracellular calcium or by diltiazem. The calcium-sensitive potassium (K(Ca)) channel blocker iberiotoxin produced a transient increase in calcium in the fetal distal PASMCs, which could be inhibited by prior application of ryanodine. Conversely, the ryanodine response was inhibited if iberiotoxin was given first. With the use of electrophysiology and confocal microscopy, fetal PASMCs were shown to exhibit spontaneous transient outward currents and calcium sparks, respectively. These observations suggest that ryanodine-sensitive release of calcium from the sarcoplasmic reticulum and K(Ca) channels act together to control intracellular calcium only in fetal distal PASMCs.

Hypoxic pulmonary vasoconstriction is modified by P-450 metabolites.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A (CYP4A) metabolite of arachidonic acid (AA) in human and rabbit lung microsomes and is a dilator of isolated human pulmonary arteries (PA). However, little is known regarding the contribution of P-450 metabolites to pulmonary vascular tone. We examined 1) the effect of two mechanistically distinct omega- and omega1-hydroxylase inhibitors on perfusion pressures in isolated rabbit lungs ventilated with normoxic or hypoxic gases, 2) changes in rabbit PA ring tone elicited by 20-HETE or omega- and omega1-hydroxylase inhibitors, and 3) expression of CYP4A protein in lung tissue. A modest increase in perfusion pressure (55 +/- 11% above normoxic conditions) was observed in isolated perfused lungs during ventilation with hypoxic gas (FI(O(2)) = 0.05). Inhibitors of 20-HETE synthesis, 17-oxydecanoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), increased baseline perfusion pressure above that of vehicle and amplified hypoxia-induced increases in perfusion pressures by 92 +/- 11% and 105 +/- 11% over baseline pressures, respectively. 20-HETE relaxed phenylephrine (PE)-constricted PA rings. Treatment with 17-ODYA enhanced PE-induced contraction of PA rings, consistent with inhibition of a product that promotes arterial relaxation, whereas 6-(20-propargyloxyphenyl)hexanoic acid (PPOH), an epoxygenase inhibitor, blunted contraction to PE. Conversion of AA into 20-HETE was blocked by 17-ODYA, DDMS, and hypoxia. CYP4A immunospecific protein confirms expression of CYP4A in male rabbit lung tissue. Our data suggest that endogenously produced 20-HETE could modify rabbit pulmonary vascular tone, particularly under hypoxic conditions.

Species-specific pharmacological properties of human alpha(2A)-adrenoceptors.

On the basis of data obtained in rabbits, the imidazoline receptor ligand rilmenidine has been suggested to decrease blood pressure in humans by activating central alpha(2A)-adrenoceptors. A prerequisite for this hypothesis was the unproved assumption that rabbit and human alpha(2A)-adrenoceptors are equally activated by rilmenidine. Because alpha(2A)-adrenoceptors in the brain and on cardiovascular sympathetic nerve terminals are identical, the latter were used as a model for the former to confirm or disprove this assumption. Human atrial appendages and rabbit pulmonary arteries were used to determine the potencies of alpha(2)-adrenoceptor agonists in inhibiting the electrically (2 Hz) evoked [(3)H]norepinephrine release and of antagonists in counteracting the alpha(2)-adrenoceptor-mediated inhibition induced by moxonidine. In the rabbit pulmonary artery, rilmenidine and oxymetazoline are potent full agonists, whereas in the human atrial appendages they are antagonists at the alpha(2)-autoreceptors, sharing this property with rauwolscine, phentolamine, and idazoxan. In contrast, prazosin is ineffective. In addition, a partial nucleotide and amino acid sequence of the rabbit alpha(2A)-adrenoceptor (a region known to substantially influence the pharmacological characteristics of the alpha(2)-adrenoceptor) revealed marked differences between the rabbit and the human alpha(2A)-adrenoceptor. The sympathetic nerves of both the human atrial appendages and rabbit pulmonary artery are endowed with alpha(2A)-autoreceptors, at which, however, both rilmenidine and oxymetazoline exhibit different properties (antagonism and agonism, respectively). The antagonistic property of rilmenidine at human alpha(2A)-adrenoceptors indicates that in contrast to the suggestion based on rabbit data, the hypotensive property of the drug in humans is not due to activation of alpha(2A)-adrenoceptors but other, presumably I(1)-imidazoline receptors, are probably involved.

Peculiarities of cholinergic regulation of smooth muscles in rabbit pulmonary arteries.

Cholinergic contractile reactions of smooth muscles in rabbit pulmonary arteries were studied by mechanography. The muscarinic receptor agonist pilocarpine induced biphasic relaxation of mesatone- or potassium-precontracted segments from lobar pulmonary arteries with intact endothelium. The low-threshold endothelium-dependent component of pilocarpine-induced relaxation was suppressed by denudation or nitric oxide synthase blocker L-NAME.

Peculiarities of cholinergic regulation of smooth muscles in rabbit pulmonary arteries

Peculiarities of cholinergic regulation of smooth muscles in rabbit pulmonary arteries

Endothelin receptor subtype antagonist activity of S-0139 in various isolated rabbit and canine arteries

Vascular responses to endothelin peptides have been proposed to be mainly mediated via subtypes of the endothelin receptor, endothelin ET A1, endothelin ET B1, and endothelin ET B2. The antagonist activity of 27- O-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]acryloyloxy myricerone, sodium salt (S-0139) at these endothelin receptor subtypes was evaluated using isolated rabbit femoral, pulmonary, and mesenteric arteries. S-0139 competitively antagonized the endothelin-1-induced contraction mediated by the endothelin ET A1 receptor in endothelium-denuded rabbit femoral arteries with a p A 2 value of 8.6±0.1. Endothelin ET B2 receptor-mediated contraction induced by sarafotoxin S6c in endothelium-denuded rabbit pulmonary arteries was also inhibited by S-0139 with a p A 2 value of 5.6±0.1. The p A 2 value of S-0139 for the endothelin ET B1 receptor, evaluated from the endothelin-3-induced relaxant response in endothelium-intact rabbit mesenteric arteries, was 6.2±0.2. In isolated canine basilar, coronary, mesenteric and renal arteries, endothelin-1 caused concentration-dependent contractions with EC 50 values of 0.49±0.07, 0.61±0.25, 0.92±0.21 and 1.18±0.24 nM, respectively. S-0139 antagonized the endothelin-1-induced contraction in these arteries with p A 2 values of 8.0±0.1, 7.6±0.2, 7.6±0.2 and 7.6±0.1, respectively. These results suggest that S-0139 is a potent and selective endothelin ET A1 receptor antagonist, and that the contractions induced by endothelin-1 in canine basilar, coronary, mesenteric and renal arteries are mediated mainly via the endothelin ET A1 receptor subtype.

Epoxyeicosatrienoic acids constrict isolated pressurized rabbit pulmonary arteries.

Little information is available regarding the vasoactive effects of epoxyeicosatrienoic acids (EETs) in the lung. We demonstrate that 5, 6-, 8,9-, 11,12-, and 14,15-EETs contract pressurized rabbit pulmonary arteries in a concentration-dependent manner. Constriction to 5,6-EET methyl ester or 14,15-EET is blocked by indomethacin or ibuprofen (10(-5) M), SQ-29548, endothelial denuding, or submaximal preconstriction with the thromboxane mimetic U-46619. Constriction of pulmonary artery rings to phenylephrine is blunted by treatment with the epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide. Pulmonary arteries and peripheral lung microsomes metabolize arachidonate to products that comigrate on reverse-phrase HPLC with authentic regioisomers of 5,6-, 8,9-, 11,12-, and 14,15-EETs, but no cyclooxygenase products of EETs could be demonstrated. Proteins of the CYP2B, CYP2E, CYP2J, CYP1A, and CYP2C subfamilies are present in pulmonary artery and peripheral lung microsomes. Constriction of isolated rabbit pulmonary arteries to EETs is nonregioselective and depends on intact endothelium and cyclooxygenase, consistent with the formation of a pressor prostanoid compound. These data raise the possibility that EETs may contribute to regulation of pulmonary vascular tone.

Differential participation of herbimycin A-sensitive tyrosine phosphorylation in the stretch-induced contraction of rabbit cerebral and pulmonary arteries

Differential participation of herbimycin A-sensitive tyrosine phosphorylation in the stretch-induced contraction of rabbit cerebral and pulmonary arteries

Mechanisms underlying the relaxation caused by the sesquiterpene polygodial in vessels from rabbit and guinea-pig

The sesquiterpene polygodial produces graded relaxation in rings of rabbit pulmonary artery or thoracic aorta and guinea-pig pulmonary artery with endothelium. In rings with rubbed endothelium its vasorelaxant action was largely reduced. The N ω-nitro- l-arginine ( l-NOARG), N G-nitro- l-arginine methyl ester ( l-NAME), 6-anilino-5,8-quinolinedione (LY 83583) and 1 H-[1,2,4]Oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), inhibited the endothelium-dependent vasorelaxant action of polygodial. In contrast, N ω-nitro- d-arginine ( d-NOARG), indomethacin, N 2-[(4 R)-4-hydroxy-1-(1methyl-1 H-indol-3yl)carbonyl- l-prolyl]- N-methyl- N-phenylmethyl-3-(2-naphthyl)- l-alaninamide (FK 888), ( S)- N-methyl- N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR 48968), (8 R,9 S,11 S)-(−)-9-hydroxy-9- n-hexyloxy-carbonyl-8-methyl-2,3,9,20-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7 b,11 a-triaqzadibenzo[ a, g]cycloocta[ c, d, e]-trinden-1-one (KT 5720), calcitocin gene-related peptide receptor antagonist (CGRP-(8-37), apamin, charybdotoxin and 4-aminopyridine had no effect on polygodial action. However, glibenclamide inhibited partially, but significantly, its relaxant responses. These results demonstrate that the vasorelaxation of polygodial is partly dependent on the release of nitric oxide (NO )or an NO-derived substance from the vascular endothelium through an activation of a guanylyl cyclase-dependent mechanism. Finally, results demonstrate that the polygodial vasorelaxant action is not related with the opening of potassium (K +) channels, release of prostacyclin, substance P, or with the activation of adenylyl cyclase-dependent mechanisms.

Developmental change in magnesium sulfate-induced relaxation of rabbit pulmonary arteries.

Magnesium causes a variety of vascular smooth muscle to relax. The present study was designed to determine whether there is a developmental change in the magnesium-induced response of pulmonary vasculature. Isolated pulmonary arteries (PA) of newborn (1- to 3-day-old) and juvenile (4- to 6-wk-old) rabbits were suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O(2)-5% CO(2), 37.0 degrees C), and their isometric tension was recorded. In arteries preconstricted with endothelin-1 to a similar tension level, MgSO(4) caused greater relaxation of juvenile rabbit PA than that of the newborn rabbit PA. Verapamil, a voltage-dependent Ca(2+) channel blocker, attenuated magnesium-induced relaxation in juvenile rabbit PA but not in newborn PA. The uptake of Ca(2+) of juvenile rabbit PA was inhibited by MgSO(4), and the inhibition was attenuated by verapamil. The uptake of Ca(2+) of newborn rabbit PA was smaller than that of the juvenile PA and was not significantly affected by MgSO(4) and verapamil. These results demonstrate that there is a developmental increase in the dilator effect of MgSO(4) in rabbit PA. In newborn rabbit PA, an incomplete maturation of the voltage-dependent Ca(2+) channels may contribute to the smaller vasodilation induced by MgSO(4).

A search for presynaptic imidazoline receptors at rabbit and rat noradrenergic neurones in the absence of alpha 2-autoinhibition.

Presynaptic, release-inhibiting imidazoline receptors have hitherto been detected mainly under conditions of ongoing alpha 2-autoinhibition. We tried to find them under alpha2-autoinhibition-free conditions, in the majority of experiments in the rabbit pulmonary artery but in a few experiments also in rabbit atria, rabbit brain cortex and rat brain cortex. Tissue segments were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Ten compounds, some thought to inhibit noradrenaline release through activation of presynaptic imidazoline receptors, some thought to act through alpha 2-adrenoceptors, were tested. Rauwolscine and 6-chloro-N-methyl-2,3,4,5-tetrahydro-1-H-3-benzazepine (SKF 86466) were used as antagonists. In rabbit pulmonary artery segments stimulated by trains of 20 pulses/50 Hz (alpha 2-autoinhibition-free conditions), all ten "agonists" [medetomidine, aganodine, 4-chloro-2-guanidyl-isoindoline (BDF 7579), 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline (BDF 6143), 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), alpha-methylnoradrenaline, clonidine, moxonidine, cirazoline and idazoxan] reduced the stimulation-evoked overflow of tritium, with potency decreasing in that order and with maximal inhibition by 59% (idazoxan) to 95% (moxonidine). Rauwolscine competitively antagonized the effects of all ten "agonists" with similar potency, the pKd-values lying between 7.6 and 8.2. Similarly, SKF 86466 competitively antagonized the effects of clonidine and moxonidine with the same potency (pKd 7.6). Cirazoline was also studied in the other three tissues. In rabbit atrial segments stimulated by 20 pulses/50 Hz and rabbit brain cortex slices stimulated by 4 pulses/100 Hz (both autoinhibition-free), cirazoline reduced the evoked overflow of tritium with concentration-inhibition curves similar to the curve in the pulmonary artery. In the brain cortex, the pKd-value of rauwolscine against cirazoline was 7.7 (pulmonary artery: 7.6). In rat brain cortex slices stimulated by 3 pulses/100 Hz (autoinhibition-free), cirazoline failed to change the evoked overflow of tritium but antagonized the inhibitory effect of UK 14304, pKd of cirazoline against UK 14304 6.9. In rat brain cortex slices stimulated by trains of 36 pulses/3 Hz, finally (marked alpha 2-autoinhibition), cirazoline increased the evoked overflow of tritium. In the rabbit pulmonary artery, rauwolscine and SKF 86466 acted with the same potency against typical presynaptic imidazoline receptor agonists (such as clonidine) and typical alpha 2-adrenoceptor agonists (such as moxonidine). Hence, presynaptic imidazoline receptors were not detectable, in a tissue that is prototypical for presynaptic imidazoline receptors, in the absence of alpha 2-autoinhibition, i.e. under conditions usually thought to be optimal for presynaptic receptor characterization. The pKd-values of rauwolscine and SKF 86466 indicate that all ten agonists activated the alpha 2A-autoreceptors of the pulmonary artery. Cirazoline behaved as an alpha 2(A)-autoreceptor agonist in rabbit tissues but as an alpha 2(D)-autoreceptor antagonist in rat tissues. Perhaps cirazoline generally possesses greater intrinsic activity at (human, rabbit) alpha 2A-adrenoceptors than the orthologous (rodent) alpha 2D-adrenoceptors.

Transcriptional down-regulation of the rabbit pulmonary artery endothelin B receptor during phenotypic modulation.

1. We confirmed that endothelium-independent contraction of the rabbit pulmonary artery (RPA) is mediated through both an endothelin A (ET(A)R) and endothelin B (ET(B2)R) receptor. 2. The response of endothelium-denuded RPA rings to endothelin-1 (ET-1, pD2 = 7.84 +/- 0.03) was only partially inhibited by BQ123 (10 microM), an ET(A)R antagonist. 3. Pretreatment with 1 nM sarafotoxin S6c (S6c), an ET(B)R agonist, desensitized the ET(B2)R and significantly attenuated the response to ET-3 (pD2 = 7.40 +/- 0.02 before, <6.50 after S6c). 4. Pretreatment with S6c had little effect on the response to ET-1, but BQ123 (10 microM) caused a parallel shift to the right of the residual ETAR-mediated response to ET-1 (pD2 = 7.84 +/- 0.03 before S6c, 7.93 +/- 0.03 after S6c, 6.81 +/- 0.05 after BQ123). 5. Binding of radiolabelled ET-1 to early passage cultures of RPA vascular smooth muscle cells (VSMC) displayed two patterns of competitive displacement characteristic of the ET(A)R (BQ123 pIC50 = 8.73 +/- 0.05) or ET(B2)R (S6c pIC50 = 10.15). 6. Competitive displacement experiments using membranes from late passage VSMC confirmed only the presence of the ET(A)R (ET-1 pIC50 = 9.3, BQ123 pIC50 = 8.0, S6c pIC50 < 6.0). 7. The ET(A)R was functionally active and coupled to rises in intracellular calcium which exhibited prolonged homologous desensitization. 8. Using a reverse transcriptase polymerase chain reaction for the rabbit ET(B2)R, we demonstrated the absence of mRNA expression in phenotypically modified VSMC. 9. We conclude that the ET(B2)R expressed by VSMC which mediates contraction of RPA is rapidly down-regulated at the transcriptional level during phenotypic modulation in vitro.