Complete blockade by phenoxybenzamine of α1- but not of α2-vascular receptors in dogs and the effects of propranolol

Summary In pithed dogs pressor responses to phenylephrine were completely inhibited 1 h after phenoxybenzamine 20 mg/kg i.v., but those to norepinephrine were only partially inhibited. The pressor effects of norepinephrine in phenoxybenzamine-treated animals were inhibited by yohimbine, 2.0 mg/kg i.v., but not by prazosin, 0.5 mg/kg i.v. In animals treated with phenoxybenzamine, 20 mg/kg i.v., plus propranolol, 5.0 mg/ kg i.v., the partially restored pressor response to epinephrine, and the responses to norepinephrine, were completely inhibited by yohimbine, 2.0 mg/kg i.v., partially inhibited by corynanthine, 5.0 mg/kg i.v., but not affected by prazosin, 0.5 mg/kg i.v. In additional animals treated with phenoxybenzamine plus propranolol, yohimbine, 10, 50, 200 and 500 g/kg i.v., caused dose-related inhibition of both the partially restored pressor response to epinephrine, and the pressor responses to norepinephrine. It is concluded that: 1) phenoxybenzamine completely blocks ₁, but not ₂ vascular receptors; 2) the pressor effect of norepinephrine in phenoxybenzamine-treated animals, and the partially restored pressor effect of epinephrine in phenoxybenzamine-propranolol-treated animals, are both mediated by ₂ vascular receptors which are resistant to blockade by phenoxybenzamine.     

α1- and α2-Adrenoceptors in rat cerebral cortex: Effect of frontal lobotomy

Summary Surgical noradrenergic denervation of the cortex via frontal lobotomy was used to destroy the noradrenergic nerve endings and thus give some insight into the distribution of alpha-adrenoceptors. Frontal lobotomy caused a reduction in noradrenaline content in rat cerebral cortex (2.1±0.4 ng/mg protein for lesioned side, 6.0±0.3 mg/mg protein for nonlesioned side), indicating an effective noradrenergic denervation. The differences in ³H-clonidine and ³H-prazosin binding observed following surgery were a significant decrease in the number of ₂-adrenoceptors (115.0±4.5 to 91.7±3.2 fmol/mg protein, n=7, P<0.001) and a smaller but significant increase in the number of ₁-adrenoceptors (119.7±2.5 to 131.6±5.4 fmol/mg protein, n=7, P<0.05) in the lesioned cortex. Results of this study indicate that ₂-adrenoceptors located on presynaptic noradrenergic terminals represent only a small proportion of the total ₂-adrenoceptors in rat cerebral cortex.     

α1B- but not α1A-adrenoceptors mediate inositol phosphate generation

Summary We used novel highly subtype-selective antagonists to study whether _1A- and/or _1B-adrenoceptors mediate the stimulation of inositol phosphate generation by noradrenaline in rat cerebral cortex. Phentolamine (10 M) and prazosin (100 nM) completely abolished the stimulated inositol phosphate generation. The _1A-selective antagonists 5-methyl-urapidil (100 nM) and (+)– and (–)-niguldipine (10 nM) caused only weak inhibition or none at all although these concentrations occupied _1A-adrenoceptors almost completely. In contrast, pretreatment with the irreversible _1B-selective chloroethylclonidine reduced the noradrenaline-stimulated inositol phosphate generation by 76 ± 8%. These data demonstrate that _1B-adrenoceptors couple to inositol phosphate generation; the signal transduction system of _1A-adrenoceptors remains unclear. Send offprint requests to G. Gross at the above address     

The effect of selective α1- and α2-adrenoceptor stimulation on intraocular pressure in the conscious rabbit

Summary The influence of topically applied selective ₁- and ₂-adrenoceptor agonists on intraocular pressure and the diameter of the pupil was investigated in conscious rabbits. Selective stimulation of the ₁-subtype of receptors induced an elevation in intraocular pressure, accompanied by mydriasis, whereas stimulation of the ₂-subtype caused a marked and dose-dependent ocular hypotensive response, which was blocked by the selective ₂-adrenoceptor antagonist yohimbine. ₂-Agonists induced neither macroscopic ocular side effects, nor an effect on the pupil size. Possibly, the subclass of ₂-adrenoceptor stimulating drugs represent a group of new antiglaucomatous agents.     

Binding sites of α1- and α2-adrenoreceptors

Conclusions The models constructed for the binding sites of rat brain ₁-AR and ₂-AR satisfy all the steric requirements and energy characteristics of interaction with known ligands, cited in [5–7, 14]. The differences detected in the arrangement and orientation of the functional groups of the binding sites permit an explanation of a whole series of typical differences in the interaction of adrenoactive substances with both subtypes of-AR.Our analysis showed that the greatest contribution to the interaction with the receptor is made by ionic, donor-acceptor, and hydrophobic bonds. The role of van der Waals forces in the interactions examined is evidently extremely negligible. The most effective and specific preparations prove to be compounds that not only form the maximum number of donor-acceptor bonds with the receptor but also orient their own hydrophobic fragments in such a way that the ionic and donor-acceptor bonds formed between the molecule and the receptor are shielded from contact with the aqueous phase. The energy effects of hydrophobic interactions of this type may be rather substantial (3.9–3.12).The production of new synthetic preparations, for which the conditions of complementarity to the-AR will be most fully satisfied, can be carried out taking the requirements of structural correspondence of the topography of the binding sites into account.Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 18, No. 8, pp. 904–912, August, 1984.     

β-Adrenoceptors differentially regulate vascular tone and angiogenesis of rat aorta via ERK1/2 and p38

β-Adrenoceptors (β-ARs) modulate ERK1/2 and p38 in different cells, but little is known about the contribution of these signaling pathways to the function of β-ARs in vascular tissue. Immunoblotting analysis of rat aortic rings, primary endothelial (ECs) and smooth muscle cells (SMCs) isolated from aorta showed that β-AR stimulation with isoprenaline activated p38 in aortic rings and in both cultured cell types, whereas it had a dual effect on ERK1/2 phosphorylation, decreasing it in ECs while increasing it in SMCs. These effects were reversed by propranolol, which by itself increased p-ERK1/2 in ECs. Isoprenaline β-AR mediated vasodilation of aortic rings was potentiated by the ERK1/2 inhibitor, U0126, in the presence or absence of endothelium or l-NAME, whereas inhibition of p38 had no impact. Isoprenaline moderately decreased sprouting from aorta rings in the Matrigel angiogenesis assay; conversely propranolol not only prevented isoprenaline inhibition, but stimulated angiogenesis. ERK1/2 inhibition decreased angiogenesis, while a dramatic stimulation was observed by p38 blockade. Our results suggest that ERK1/2 activation after β-ARs stimulation in the smooth muscle hinders the vasodilator effect of isoprenaline, but in the endothelium β-ARs decreases ERK1/2 and increases p38 activity reducing therefore angiogenesis.     

Age-related alterations in α1- and β-adrenoceptors mediated responsiveness of rat aorta

Summary We have examined the responsiveness of - and -adrenoceptors in aortae from 1.5, 3, 6 and 24 month old rats. The isometric contraction to phenylephrine was antagonised competitively by prazosin with a pA₂ value of 9.45, suggesting that the receptor is an ₁-adrenoceptor. The potency of phenylephrine was significantly reduced in 24 months old as compared with all younger rats combined. The maximum contraction to phenylephrine was unaltered in 24 month old rats. The maximum contraction to potassium chloride was significantly less than that to phenylephrine only in 1.5 months old rats. In tissues contracted by potassium chloride, isoprenaline produced a marked relaxation in 1.5 months old animals, but there was a progressive loss with increasing age of the -adrenoceptor-mediated relaxation which was markedly reduced by 6 months and abolished in 24 months old. It is concluded that, in the rat aorta, there is a decrease in ₁-adrenoceptor responsiveness in senescence, and a loss of -adrenoceptor-mediated responses in maturation. Send offprint requests to J. R. Docherty at the above address     

Subclassification of presynaptic α2-adrenoceptors: α2A-autoreceptors in rabbit atria and kidney

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors of rabbit atria and kidney in terms of _2A, _2B, _2C and _2D-A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the left atrium and slices of the kidney cortex were preincubated with ³H-Noadrenaline and then superfused and stimulated electrically.In one series of experiments, tissue pieces were stimulated by relatively long pulse trains (1 or 2 min) leading to ₂-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC_30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments, tissue pieces were stimulated by brief pulse trains (0.4 s) that did not lead to ₂-autoinhibition, and concentration-inhibition curves of the ₂-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. Most of the 11 (atria) or 8 (kidney) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pEC_30% values correlated with pK_d values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC_30% values in atria correlated with pEC_30% values in the kidney (r = 0.988) and pK_d values in atria correlated with pK_d values in kidney (r = 0.923).It is concluded that the ₂-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both autoreceptors are _2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least rodent and lagomorph species.     

The preferential inhibition of α1- over β-adrenoceptor-mediated positive inotropic effect by organic calcium antagonists in the rabbit papillary muscle

Summary Experiments were carried out to elucidate the mechanism that the positive inotropic effect mediated by ₁-adrenoceptors is more susceptible to organic calcium antagonists than the -adrenoceptor-mediated effect. Verapamil and diltiazem displaced the specific binding of [³H]prazosin to the membrane fraction derived from the rabbit ventricular myocardium, verapamil being about 70 times more potent than diltiazem. Nifedipine did not displace the binding. While these compounds suppressed the positive inotropic effect mediated via _l-adrenoceptors in ₁- concentration-dependent manner, there was no correlation between the potency of the compounds to displace the [³H]prazosin binding and to inhibit the -mediated positive inotropic effect. The relative potency of three calcium antagonists to decrease the basal force of contraction and the al-mediated effect (of the same extent as compared to basal force of contraction) was consistent to each other. The positive inotropic effect mediated by -adrenoceptors was inhibited much less, and was enhanced by low concentrations of organic calcium antagonists. The differential action of calcium antagonists on the - and -mediated positive inotropic effect was mimicked by lowering the extracellular calcium concentration to 1/2, 1/4 and 1/8 of that in normal Krebs-Henseleit solution (2.5 mmol/l). These results indicate that the ₁-adrenoceptor blocking activity does not play an essential role for the preferential inhibition of -mediated positive inotropic effect by organic calcium antagonists. Difference in the subcellular mechanism involved in mobilization of intracellular Ca²⁺ subsequent to ₁-and -adrenoceptor activation may be responsible for the differential inhibitory action of calcium antagonists in the rabbit heart. Send offprint requests to M. Endoh at the above address     

The sympathetic axons innervating the sinus node of the rabbit possess presynaptic opioid к- but not μ- or δ-receptors

Summary The postganglionic sympathic nerves of rabbit isolated hearts were stimulated with pulses delivered at 5 Hz and train durations of 1–5 s. Ethylketocyclazone 0.01–1 mol/l and fentanyl 1 and 10 mol/l but not morphine 1 and 10 mol/l, Met-enkephalin 1 and 4 mol/l or d-Ala², d-Leu⁵-enkephalin 0.5 and 5 mol/l diminished the stimulation-evoked increase in heart rate. The effect of ethylketocyclazocine 0.1 mol/l was antagonized by naloxone 1 and 10 mol/l. In contrast, the effect of fentanyl was not changed by naloxone 10 mol/l. Ethylketocyclazocine 0.03 and 1 mol/l did not reduce the tachycardia elicited by exogenous noradrenaline. The results suggest that, under in vitro conditions, only presynaptic opioid - but not - or -receptors inhibit the release of noradrenaline from the sympathetic neurones innervating the sinus node.     

Participation of cardiac presynaptic α1-Adrenoceptors in the bradycardiac effects of clonidine and analogues

Summary The possible involvement of cardiac presynaptic ₂-adrenoceptors in the acute negative chronotropic effect of intravenous clonidine and four selected analogues was tested in pentobarbitone-anaesthetized normotensive rats by establishing the following parameters: 1) the peripheral presynaptic activity (inhibition of tachycardia to stimulation of the spinal cord in pithed rats); 2) central hypotensive potency in intact rats (decrease in mean arterial pressure); 3) overall bradycardiac activity in intact rats (decrease in cardiac frequency); 4) sympathetic bradycardia in bilaterally vagotomized rat; and 5) vagal bradycardia in -blocked rats. The lipophilicity of the imidazolidines (log P) was measure in the octanol/buffer (pH=7.4) reference system at 37°C. Central hypotensive activity as well as vagal bradycardiac potency correlated with the ability to penetrate the central nervous system, as indicated by the overall lipophilic behavior (log P) of the compounds. In contrast, brain penetration was not a prerequisite for the provocation of bradycardia in bilaterally vagotomized rats. This sympathetic bradycardia resembled peripheral presynaptic inhibition. The hydrophilic — poor brain-penetrating — substances already induced maximal sympathetic bradycardia at doses which did not induce hypotension or vagal bradycardia. The lipophilic drugs, however, only showed a preferentially sympathetic bradycardiac effect over vagal bradycardia at low doses. In intact pentobarbitone-anaesthetized rats, the log dose-bradycardiac response curves of the hydrophilic imidazolidines displayed a bisigmoidal character in contrast to their lipophlic analogues, i.e. at the lower dose-range of the hydrophilic drugs, bradycardia in intact rats resembled peripheral presynaptic inhibition, whereas a contribution of increased vagal activity only was significant after maximal sympathoinhibition was achieved. These data provide evidence for the view that the bradycardiac action of clonidine in pentobarbitone-anaesthetized normotensive rats is due, at least in part, to the functional role of cardiac presynaptic ₂-adrenoceptors. The stimulation of these receptors will inhibit the sympathetic neurotransmission in the heart.     

Frequency-dependence of serotonin autoreceptor but not α2-adrenoceptor inhibition of [3H]-serotonin release in rat hypothalamic slices

Summary The overflow of tritium from stimulated rat hypothalamic slices preincubated with [³H]-serotonin (5-HT) was significantly enhanced by reducing the frequency of stimulation from 3 Hz to 1 Hz while keeping the number of impulses constant. The 5-HT receptor agonist 5-methoxytryptamine inhibited in a concentration-dependent manner the electrically-evoked release of [³H]-5-HT with IC₅₀ values of 560 nmol/l and of 34 nmol/l when the stimulations were delivered at 3 Hz and 1 Hz, respectively. The terminal 5-HT autoreceptor antagonist methiothepin enhanced in a concentration-dependent manner the electrically-evoked release of [³H]-5-HT and this effect was greater at a frequency of stimulation of 3 Hz than at 1 Hz. In the same paradigm, the 5-HT reuptake inhibitors citalopram and paroxetine did not alter the overflow of radioactivity elicited by stimulation at 3 Hz but significantly decreased it at 1 Hz. In the presence of 5-HT autoreceptor blockade achieved with methiothepin, citalopram increased the overflow of [³H]-5-HT to the same extent at 1 Hz and at 3 Hz. The IC₅₀ values for inhibition of [³H]-5-HT release by the selective ₂-adrenoceptor agonist UK 14.304 were 35 nmol/l at 3 Hz and 30 nmol/l at 1 Hz. It is concluded that modulation of 5-HT release by 5-HT autoreceptors, but not by ₂-adrenoceptors is dependent on the synaptic concentration of 5-HT as a function of the frequency of depolarization. Send offprint requests to S. Z. Langer at the above address     

α1A-Adrenoceptor subtype effectively increases Ca2+-sensitivity for contraction in rabbit thoracic aorta

• 1.1. Norepinephrine and phenylephrine increase cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and muscle tension, which shows positive correlation between [Ca²⁺]_i and tension development.
• 2.2. The slopes of regression lines between [Ca²⁺]_i and tension development for norepinephrine and phenylephrine in tissues treated with an irreversible α_1B-adrenoceptor selective blocking agent, 10⁻⁴M chloroethylclonidine, were significantly steeper than those with untreated tissues.
• 3.3. Myosin light chain kinase inhibitors, KT5926 (3 × 10⁻⁶M) and K252a (10⁻⁶M) more selectively reduced the contraction produced by norepinephrine (3 × 10⁻⁷M) than that produced by clonidine (3 × 10⁻⁶M).
• 4.4. In the chloroethylclonidine-treated tissues, KT5926 and K252a did not tend to affect the contraction induced by norepinephrine and clonidine.
• 5.5. These results suggest that the contractile response through the α_1B-adrenoceptor subtype causes a greater muscle tension than that through the α_1B-subtype at the same level of [Ca²⁺]_i and that the α_1A-adrenoceptor subtype mainly activates myosin light chain kinase independent pathways of contractile mechanisms in vascular smooth muscle of rabbit.

     

17β-Estradiol inhibits calcium-dependent,but not calcium-independent,contraction in isolated rat aorta

It is now well known that 17-estradiol has an endothelium-independent, non-genomic vasorelaxant effect. We hypothesized that 17-estradiol has its non-genomic effect on calcium-independent contraction in de-endothelialized rat aortic rings. Rat aortic ring preparations were mounted in organ baths and exposed to contractile agents. 17-Estradiol (8, 20 or 50 M), but not 17-estradiol, concentration-dependently decreased the tension induced by 1.0 M phenylephrine (PE) in the presence, but not in the absence, of calcium in the solution. Pretreatment with 17-estradiol concentration-dependently inhibited vascular contractions induced by cumulative addition of PE or calcium and almost completely abolished those induced by cumulative addition of Bay K8644, a calcium channel opener. Furthermore, 17-estradiol also concentration-dependently decreased the tension induced by 0.3 M phorbol 12,13-dibutyrate (PDBu), a protein kinase C activator, in the presence of calcium in the solution, but not in the absence of calcium in the solution. Pretreatment with 17-estradiol had little effect on vascular contractions induced by PDBu or PE or on PE-induced mitogen-activated protein kinase (MAPK) activation in calcium-free Krebs solution. These results suggest that 17-estradiol inhibits calcium-dependent, but not calcium-independent, vascular contraction.H.-A. Lee and Y. Seong contributed equally to this work.     

Enhancement of δ- but not μ-opiate agonist binding by calcium

Summary Present evidence for distinction of 2 types of opiate receptor sites in rat brain homogenates originates from different relative affinities of morphine-like alkaloids and enkephalins to -or enkephalin and - or morphine-receptor sites. We now report that Ca²⁺ in a physiological dose range (0.5–3 mM) enhances the binding of ³H-enkephalin in hypotonically treated rat brain membranes, whereas specific binding of ³H-morphine-like alkaloids is not affected. Furthermore, the potency of [d-Ala², d-Leu⁵]-enkephalin to inhibit [³H]-diprenorphine and [³H]-ethylketazocine binding increased in the presence of Ca²⁺, whereas an increase in potency of [d-Ala², d-Leu⁵]-enkephalin to inhibit binding of -receptor ligands was not observed. Kinetic analysis revealed that Ca²⁺ decreased the rate of dissociation of [d-Ala², d-Leu⁵]-enkephalin without affecting the rate of association, thereby increasing the affinity. However, in saturation binding studies, performed in diencephalic membranes, in which [d-Ala², d-Leu⁵]-enkephalin binds predominantly to -receptors, Ca²⁺ also increased the binding affinity of [³H]-[d-Ala², d-Leu⁵]-enkephalin. Double reciprocal analysis suggested a mixed competitive-noncompetitive type of inhibition of [d-Ala², d-Leu⁵]-enkephalin binding by dihydromorphine. Thus, the interactions of - and -opiate ligands with -receptors may involve topographically different, but closely related binding sites, located on a single receptor molecule.Abbreviations DADL [d-Ala², d-Leu⁵]-enkephalin - DHM dihydromorphine - met-enkephalin methionine-enkephalin - leu-enkephalin leucine-enkephaline - FK 33-824 [d-Ala², MePhe⁴, Met(O)-ol]-enkephalin - EGTA ethyleneglycol-bis-(-aminoethylether) N, N'-tetraacetic acid - TRIS Tris (hydroxymethyl)-aminomethan     

Only activated but not non-activated presynaptic α2-autoreceptors interfere with neighbouring presynaptic receptor mechanisms

Summary Experiments were carried out in rabbit cerebrocortical slices in order to find out whether the attenuation by presynaptic ₂-autoreceptors of effects mediated by presynaptic opioid - and adenosine A₁-receptors requires activation of the ₂-receptors. The slices were preincubated with ³H-noradrenaline and then superfused with medium containing desipramine 1 mol/l. They were stimulated electrically either with single pulses or with trains of 32 pulses at 1 Hz.The overflow of tritium elicited by a single pulse amounted to 0.21% of the tritium content of the tissue. It was Ca²⁺-dependent and tetrodotoxin-sensitive and not changed by rauwolscine 1 mol/l or yohimbine 0.3 mol/l. Ethylketocyclazocine (EK; 0.1–10 nmol/l) and R-(–)-N⁶-phenylisopropyladenosine (PIA; 1–1,000 nmol/1) potently inhibited the overflow evoked by a single pulse, and their effects were not changed by yohimbine. — The overflow of tritium elicited by trains of 32 pulses at 1 Hz amounted to 0.92% of the tritium content of the tissue and was increased approximately fourfold by yohimbine 0.3 mol/l. EK and PIA were less potent inhibitors than in the one pulse experiments. Yohimbine greatly enhanced the effects of EK and PIA. The enhancement was even more pronounced when the Ca²⁺ concentration in the medium was reduced in order to obtain a control tritium overflow similar to that evoked by 32 pulses in the absence of yohimbine.The results demonstrate that there is no ₂-adrenergic autoinhibition when noradrenaline release is elicited by a single pulse. Under these conditions, the non-activated presynaptic ₂-adrenoceptor does not interfere with presynaptic opioid - and adenosine A₁-receptor mechanisms. It is only when the autoreceptor is activated by released noradrenaline that it attenuates neighbouring presynaptic receptor mechanisms, and this attenuation is removed by ₂-adrenoceptor antagonists.Send offprint requests to N. Limberger at the above address     

Autoreceptors and α2-adrenoceptors at the serotonergic axons of rabbit brain cortex

Summary Slices of the rabbit occipito-parietal cortex were preincubated with ³H-serotonin and then superfused and stimulated electrically (2 min at 3 Hz). In the absence of drugs, the stimulation-evoked overflow of tritium was approximately 3% of the tritium content of the tissue. Unlabelled serotonin and 5-carboxamido-tryptamine, when administered in the presence of 6-nitroquipazine, reduced the evoked overflow of tritium. Their effects were antagonized by metitepin (apparent pA₂ value 8.1) and (±)-cyanopindolol (apparent pA₂ value 6.4). Metitepin, but not cyanopindolol, increased evoked tritium overflow; the effect of metitepin was greater in the presence than in the absence of nitroquipazine. The evoked overflow of tritium was also depressed by clonidine, an effect antagonized by idazoxan (apparent pA₂ value 7.0) but not by prazosin. Phenylephrine caused a decrease only at high concentrations that simultaneously accelerated basal tritium efflux. Prazosin and idazoxan did not change evoked tritium overflow, and phentolamine increased it significantly only when administered in the presence of (+)-oxaprotiline. Rauwolscine produced an inhibition that was prevented by metitepin. It is concluded that the serotonergic axons of the rabbit occipitoparietal cortex possess presynaptic, release-inhibiting serotonin autoreceptors and ₂-adrenoceptors. The receptors appear to receive an input of endogenous serotonin and, to a lesser extent, noradrenaline, under the conditions of these in vitro experiments.     

Investigation of α1-adrenoceptor subtypes mediating vasoconstriction in rabbit cutaneous resistance arteries

1. Cutaneous resistance arteries (c.r.a.) (internal diameter=240.94±5.42 μm, n=67/25 (number arteries/number animals)) from New Zealand white rabbits were mounted in wire myographs and a normalization procedure followed.
2. Cumulative concentration-response curves (CCRCs) were constructed for the α-adrenoceptor agonists noradrenaline (NA), (R)A61603 and phenylephrine (PE) in the presence of cocaine (3 μM), propranolol (1 μM) and corticosterone (10 μM). The effects of competitive α₁-adrenoceptor antagonists, prazosin, WB4101, 5-methyl-urapidil, HV723, BMY7378 and the irreversible α_1B selective compound chloroethylclonidine (CEC) were examined versus the potency and maximum response of the c.r.a.s to noradrenaline.
3. The high potency of A-61603 relative to PE has been shown to differentiate both functional and binding site α_1A- or α_1B-adrenoceptors from α_1D-adrenoceptors: A-61603 was 944 times more potent than phenylephrine (at EC₅₀) suggesting the presence of a functional α_1A or α_1B as opposed to an α_1D-subtype.
4. Exposure to chloroethylclonidine (CEC; 100 μM) decreased the maximum response to noradrenaline but did not significantly change noradrenaline sensitivity indicating that a substantial part of noradrenaline-induced vasoconstriction in rabbit cutaneous arteries is CEC-insensitive.
5. The potencies of prazosin (pA₂=9.14) and WB4101 (pA₂=9.30) indicate the involvement of prazosin-sensitive functional α₁-adrenoceptors. The slopes of corresponding Schild plots for prazosin and WB4101 did not include negative unity which implies the possible involvement of more than one functional α₁-adrenoceptor subtype in noradrenaline-induced vasoconstriction in rabbit cutaneous resistance arteries. In contrast to this, in the case of 5-methyl-urapidil and HV723, the Schild plot slope parameters were not significantly different from negative unity over the range of concentrations used; the low pA₂ value for 5-methylurapidil (7.27) suggests the non-involvement of an α_1A- or an α_1D-adrenoceptor; the low pA₂ value for HV723 (8.47) was similar to that against responses postulated as α_1L.
6. We conclude that rabbit cutaneous resistance arteries express a prazosin-sensitive functional α₁-adrenoceptor resembling the α_1B and another low affinity site for prazosin which on the basis of the functional antagonism produced by HV723 most closely resembles the α_1L-adrenoceptor; the low pA2 value for HV723 (8.47) is similar to that against responses postulated as α_1L.

     

Contractile responses and calcium movements induced by α1-adrenoceptor stimulant,norepinephrine, in rabbit iris dilator muscle

• 1.1. The mechanisms involved in contraction of rabbit iris dilator muscle induced by norepinephrine (NE) were studied.
• 2.2. The concentration-response curve of NE was not influenced by Ca²⁺ blockers in the normal physiological saline solution (PSS) and removal of Ca²⁺ from PSS.
• 3.3. In 0.01 mM EGTA containing Ca²⁺-free PSS, the NE-induced contraction was phasic, which was suppressed by TMB-8, cyclopiazonic acid, ionomycin and A23187 but still partly remained.
• 4.4. In 2 mM EGTA containing Ca²⁺-free PSS, NE increased the intracellular Ca²⁺ ([Ca²⁺]_i) and muscle tension. Ryanodine abolished the increase in [Ca²⁺]_i induced by NE but slightly inhibited the tension.
• 5.5. These results suggest that the NE-induced contraction of rabbit iris dilator in normal PSS is mainly due to the increase in the release of intracellularly sequestered Ca²⁺ and partly due to the Ca²⁺-independent processes.