α1-adrenoceptor subtype involved in the positive and negative inotropic responses to phenylephrine in rat papillary muscle

• 1.1. In rat papillary muscle, stimulation of α₁-adrenoceptors results in a biphasic inotropic response: a transient negative inotropic phase and a subsequent sustained positive inotropic phase. This study was designed to determine whether the positive and negative inotropic effects in this tissue are mediated by different α₁-adrenoceptor subtypes.
• 2.2. After treatment with the tumor-promoting compound, phorbol 12,13-dibutyrate, phenylephrine (in the presence of propranolol) produced only a positive inotropic effect. The selective α_1A-adrenoceptor antagonist, WB4101, significantly inhibited the positive inotropic effect. In contrast, inactivation of α_1B-adrenoceptors with chloroethylclonidine (CEC) did not alter the positive effect.
• 3.3. In the presence of the Ca²⁺ channel antagonist, nifedipine, phenylephrine induced only a sustained negative inotropic effect. The negative inotropic effect was significantly attenuated by WB4101, but was not affected by CEC.
• 4.4. We conclude that both the positive and negative inotropic responses of rat papillary muscle to phenylephrine are mediated exclusively by the WB4101-sensitive but CEC-resistant α₁-adrenoceptor subtype. The α₁-adrenoceptor subtype with such a property may correspond to the α_1A-subtype.

     

Characterization of subtype of α_1-adrenoceptor mediating vasoconstriction in perfused rat mesenteric vascular bed

目的：研究去甲肾上腺素（ＮＥ）介导大鼠肠系膜血管床（ＭＶＢ）收缩的α１肾上腺素受体（α１ＡＲ）亚型．方法：用灌流大鼠ＭＶＢ标本收缩功能实验和克隆细胞放射配体结合实验测定α１ＡＲ亚型选择性拮抗剂ｐＡ２和ｐＫｉ,并作相关分析．结果：α１ＡＡＲ选择性拮抗剂ＲＳ１７０５３、ＷＢ４１０１、５ＭＵ及α１ＤＡＲ选择性拮抗剂ＢＭＹ７３７８的ｐＡ２分别为８９８±０２８,９１６±０２０,８．６９±００２和６０３±０２６,Ｓｃｈｉｌｄ作图斜率值与１０差别无显著性．其ｐＡ２值与α１ＡＡＲ的ｐＫｉ相关系数为０９７,与α１Ｂ和α１ＤＡＲ的相关系数分别为０５２和００４．结论：介导外源性ＮＥ收缩大鼠ＭＶＢ的功能性受体为α１ＡＡＲ     

Quantification and distribution of α1-adrenoceptor subtype mRNAs in human proximal urethra

1. We performed RNase protection assays and in situ hybridization to investigate the ratio of the three α₁-adrenoceptor subtype mRNAs, α1a, α1b and α1d, in human proximal urethra, and their localization in urethral cross-sections. As revealed by the RNase protection assays, α1a was the predominant subtype mRNA in both male and female urethral samples. α1d mRNA was detected only in the female sample, and α1b mRNA was not detected in any of the samples tested. The ratio of the abundance of the subtype mRNAs, α1a : α1b : α1d, was 100 : 0 : 0 in the male urethra and 90 : 0 : 10 in the female urethra.
2. In situ hybridization studies showed no significant differences in the cross-sectional distribution of α₁-adrenoceptor subtype mRNAs between male and female urethras. Intense α1a staining was observed in the smooth muscle of the urethra, but α1b and α1d staining was much less intense.
3. Of the three cloned α1 subtypes, α1a is the most likely to be responsible for the contraction of the human urethra. Owing to the side effects of nonselective α₁ drugs, α₁-selective drugs may be clinically superior to nonselective drugs for the treatment of urethral disorders.

     

Enhancement of contractile responses to partial α-adrenoceptor agonists during warming in rat aorta

Summary We examined the effects of warming on the contractile responses to full and partial -adrenoceptor agonists in rat aorta. The contractions elicited by norepinephrine and methoxamine were not affected during warming (40°C, 42°C), whereas those induced by clonidine and St 587 were significantly enhanced. KCl-induced contractions of rat aorta were not affected by warming. The dissociation constants of clonidine and St 587 at 40°C were not different from those at 37°C. At 40°C, the receptor occupancy-contractile response curve of clonidine was a hyperbolic curve similar to that of methoxamine at 37°C, although at 37°C the curve was almost linear. The responses of St 587 at both 37°C and 40°C were related inversely hyperbolic to the receptor occupancy, but the receptor occupancy-contractile response curve was shifted to the left and upward during warming. Clonidine and St 587 elicited equal responses at lower fractional occupancies at 40°C than at 37°C. The relative efficacies of clonidine and St 587 to methoxamine were significantly augmented during warming. It is suggested that the contractile responses to partial -adrenoceptor agonists in rat aorta are enhanced during warming, and that this effect is related to the intrinsic efficacy of the agonists rather than to any function of their relative selectivity for ₁- or ₂-adrenoceptors. Such enhancement is due to augmentation of the efficacy rather than to augmentation of the affinity of the agonists. Send offprint requests to H. Kitagawa     

Characterization of α1-adrenoceptor subtypes mediating contractions to phenylephrine in rat thoracic aorta,mesenteric artery and pulmonary artery

1. The subtype of α₁-adrenoceptor mediating contractions to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery were investigated by use of antagonists which show selectivity between the cloned α₁-adrenoceptor subtypes in binding studies.
2. Cumulative concentration-contraction curves for phenylephrine were competitively antagonized in the rat thoracic aorta by prazosin (pA₂ 9.9), WB4101 (pA₂ 9.6), 5-methylurapidil (pA₂ 8.1), benoxathian (pA₂ 9.2) and indoramin (pA₂ 7.4). These compounds were also competitive antagonists in the mesenteric and pulmonary arteries (except for 5-methylurapidil in the pulmonary artery), (prazosin pA₂ 9.9 and 9.7; WB4101 pA₂ 9.8 and 9.6; 5-methylurapidil pA₂ 7.9 and pK_B estimate 8.0; benoxathian pA₂ 8.8 and 9.3; indoramin pA₂ 7.2 and 7.5, respectively).
3. RS 17053 was not a competitive antagonist in any blood vessel as Schild plot slopes were greater than unity. The pK_B estimates for RS 17053 were 7.1 in aorta, 7.0 in the mesenteric artery and 7.7 in the pulmonary artery.
4. The α_1D-subtype selective antagonist BMY 7378 appeared to be non-competitive with shallow Schild plot slopes. The data were better fitted with two lines in all tissues, with Schild plot slopes that were no longer different from unity, except in the pulmonary artery. The higher affinity site for BMY 7378 in the aorta had a pA₂ of 9.0, while it was 8.8 and 8.9 in the mesenteric and pulmonary arteries, respectively.
5. MDL73005EF acted in a non-competitive manner in all three blood vessels, with shallow Schild plot slopes. The pK_B estimates for MDL73005EF were 8.4 in aorta, 7.5 in the mesenteric artery and 8.0 in the pulmonary artery.
6. In all three blood vessels the functionally determined antagonist affinity estimates correlated best with published pK_i values for their displacement of [³H]-prazosin binding on membranes expressing cloned α_1d-adrenoceptors compared with α_1a- or α_1b-adrenoceptors. The antagonist affinity estimates in the aorta, mesenteric and pulmonary arteries correlated highly with their previously published pA₂ values in rat aorta (α_1D) and less well with those for α_1A- and α_1B-adrenoceptors mediating contraction of the rat epididymal vas deferens and rat spleen, respectively.
7. The results of this study suggest that the contraction to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery are mediated in part via the α_1D-subtype of adrenoceptor. The data for both BMY 7378 and MDL73005EF in all three blood vessels are consistent with receptor heterogeneity. However, the identity of the second site is unclear.

     

Age and region-dependent contraction to α-adrenoceptor agonists in rat and guinea-pig isolated trachea

1. The influence of age and of region on α-adrenoceptor-mediated contraction to (−)-adrenaline and (−)-noradrenaline was examined in rat (4–136 weeks) and guinea-pig (2–156 weeks) isolated tracheal ring preparations with particular emphasis on the early (up to 12 weeks) maturation phase.
2. In rat tracheal rings, significant regional variation was observed with respect to maximal (−)-adrenaline-induced contraction, such that the greatest activity was seen in ring preparations from the laryngeal end of the trachea. Tracheal rings from the carinal end responded very poorly or were unresponsive to (−)-adrenaline, depending on animal age. These regional differences were seen across the age range. The potencies of (−)-adrenaline and (−)-noradrenaline remained unchanged with respect to animal age, but the maximum contractile tension that developed in response to these agonists increased with increasing animal age in all regions of the trachea.
3. In guinea-pig isolated tracheal tissue, maximum contractile responses (E_max) to (−)-adrenaline and (−)-noradrenaline remained unchanged with increasing animal age. In addition, there was no evidence for a region-dependence in the responsiveness of tracheal tissue to α-adrenoceptor-mediated contraction in this species.
4. In both guinea-pig and rat isolated tracheal tissue, α-adrenoceptor-mediated contraction appeared to involve the activation of α₁-adrenoceptors.

     

Properties of β-adrenoceptor sites in metabolizing and nonmetabolizing rat reticulocytes and in resealed reticulocyte ghosts

Summary Intact cells and resealed ghosts of a homogeneous reticulocyte population isolated from the blood of phenylhydrazine-treated rats bound -adrenergic ligands reversibly, stereospecifically and with high affinity. Maximal specific binding capacity under control conditions (37° C), corresponded to 9.9 ± 0.8 fmol/l cells ( 600 sites/cell) and was similar in intact cells and ghosts. Pretreatment with metabolic inhibitors decreased the density of binding sites in intact cells to 6.48 ± 1.1 fmol/l cells, but had no effect in ghosts. Incubation at 1°C reduced specific binding in paired experiments by 68 and 44% in intact cells and ghosts, respectively. Rewarming to 37°C increased specific binding in cells and ghosts by 270 and 190%, respectively. A temperature shift from 1 to 37°C reduced the K _D value for the antagonist (-)timolol from 8.6 ± 1.5 to 1.1 ± 0.3 nmol/l in intact cells, while no significant reduction in K _D was observed with ghosts. Under all conditions the receptor population was homogeneous with respect to antagonist affinity but inhomogenous with respect to agonist affinity. Low affinity agonist binding sites predominated in native cells and in GTP- loaded resealed ghosts (apparent K _D values 447 and 680 nmol/l, respectively). High affinity binding sites predominated in both preparations at 1°C (K _D 29 and 14 nmol/l, respectively). -Adrenoceptor sites in starved cells and ghosts at 37°C showed intermediate apparent K _D values. GTP had no effect on antagonist affinity or on the density of -adrenoceptors. The results suggest that intact metabolizing cells can regulate -adrenoceptor density by mechanisms which are not shared by ghost cells. The fractional contribution of high and low affinity states of the receptor to the overall binding of agonists seemed to be determined largely by the intracellular GTP concentration.     

β 1- andβ 2-adrenoceptor binding and functional response in right and left atria of rat heart

Summary The properties of ₁- and ₂-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses to-adrenoceptor agonists were examined. [¹²⁵I](-)-pindolol (¹²⁵IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. Thek ₁'s for association in right and left atria were 6.5×10⁹ l/mol-min and 2.3×10⁹ l/mol-min respectively, while thek _–1's for dissociation were 0.20 min^–1 and 0.17 min^–1. The kinetically determinedK _D's were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibriumK _D's determined from Scatchard analysis of saturation isotherms of specific¹²⁵IPIN binding. Inhibition of¹²⁵IPIN binding by-adrenoceptor antagonists was stereoselective and the order of potency was timolol > 1-propranolol > d-propranolol > sotalol. Inhibition by ₁- and ₂-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement by ₁-selective (practolol, atenolol and metoprolol) and ₂-selective (ICI 118,551) antagonists gave estimates of the proportion of ₁- and ₂-adrenoceptors present in rat atria. Right atria contained 67±4.2% ₂-adrenoceptors and 33±4.2% ₂-adrenoceptor, while left atria contained 67±2.8% ₁- and 33±2.8% ₂-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused by-adrenoceptor agonists were also measured. pA₂ values for non-subtype selective-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated withK _D values determined for specific¹²⁵IPIN binding. pA₂ values for ₁- and ₂-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pK _D values of these drugs in binding to ₁-adrenoceptors, but not with the pK _D values in binding to ₂-adrenoceptors. Dose-response curves for stimulation of both rate and force by the ₂-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade of ₁-adrenoceptors with metoprolol than by selective blockade of ₂-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activating ₁-adrenoceptors. These results suggest that ₁- and ₂-adrenoceptors coexist in both left and right atria of rat heart in approximately a 21 ratio, however only ₁-adrenoceptors mediate the chronotropic and inotropic effects of-adrenoceptor agonists.Supported by a grant from the American Heart Association — Georgia Affiliate     

Comparison of cloned and pharmacologically defined rat tissue α1-adrenoceptor subtypes

Multiple ₁-adrenoceptor subtypes have been defined by pharmacological and receptor cloning techniques, but the precise alignment of cloned and pharmacologically-defined subtypes is still unclear. We have compared the affinities of 8 subtype-selective compounds at three cloned ₁-adrenoceptor subtypes (rat _1B, bovine _1C rat _1A/D) with those previously determined by the same methods in rat spleen, cerebral cortex, and kidney (Naunyn-Schmiedeberg's Arch. Pharmacol. 348: 385–395, 1993). Among all compounds tested to date at cloned ₁-adrenoceptor subtypes (+)-tamsulosin appears to be the most selective with a rank order of potency _1C > _{1A/D 1B}. Affinities for the _1A-selective 5-methyl-urapidil, methoxamine, oxymetazoline, phentolamine and (–)- and (+)-tamsulosin and for noradrenaline and SDZ NVI-085 at the splenic _1B-adrenoceptors and at their low affinity sites in cerebral cortex and kidney correlated best with those at the cloned _1B-adrenoceptor. Affinities of these drugs at their high affinity sites in cerebral cortex (pharmacologically-defined _1A-adrenoceptor) were matched best by those at the cloned _1C-adrenoceptor. Rat kidney appears to contain two chloroethylclonidine-resistant ₁-adrenoceptor subtypes one of which is similar to the cloned at _1C- and one to the cloned _1A/D-adrenoceptor. We conclude that the cloned _1B-adrenoceptor is the genetic correlate of the pharmacologically-defined _1B-adrenoceptor. An ₁-adrenoceptor subtype corresponding to the cloned _1A/D-adrenoceptor appears to exist in rat kidney. Among cloned ₁-adrenoceptor subtypes, the bovine _1C-adrenoceptor bears the closest resemblance to the pharmacologically-defined _1A-adrenoceptor in rat cortex and to one of the chloroethylclonidine-insensitive subtypes in rat kidney.     

A difference in the affinity of some selective α2-adrenoceptor antagonists when compared on isolated vasa deferentia of rat and rabbit

Summary The ₂-adrenoceptor antagonist potencies of the benzoquinolizines (Wy 26 703, Wy 25 309, Wy 26 392), the benzodioxans (RX 781 094, RS 21 361), yohimbine and rauwolscine have been compared at presynaptic ₂-adrenoceptors in the isolated vasa deferentia of the rat and rabbit. Yohimbine and rauwolscine are of equal potency as antagonists in both the rat and rabbit against the agonists clonidine or UK 14 304. The benzoquinolizines and benzodioxans are very weak antagonists of clonidine or UK 14 304 at the presynaptic ₂-adrenoceptors of the rabbit vas deferens when compared to their potency at the presynaptic ₂-adrenoceptors of the rat vas deferens. This suggests that the presynaptic ₂-adrenoceptors present in the rat vasa deferentia may be different from those present in the rabbit vasa deferentia.     

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.

     

Reduction with age in the relaxation to β-adrenoceptor agonists and other vasodilators in rat aorta

Summary We have examined the relaxation produced by the -adrenoceptor agonists isoprenaline and procaterol, and by sodium nitroprusside, forskolin and isobutyl-methylxanthine in KCl (40 mmol/1)-contracted aortae from 6 week, 3 month and 6 month old Sprague-Dawley rats. The maximum relaxation to all agents was greatest in aortae from 6 week old animals. The relaxation to the -adrenoceptor agonists was virtually abolished, and the relaxation to the other agents significantly reduced, in aortae from 3 and 6 month old animals. There were no significant differences in the response to any agent between aortae from 3 and 6 month old animals, so that the changes seen occurred in maturation from 6 week to 3 month. The reduced vasodilator response does not occur at the level of the -adrenoceptor or adenylate cyclase, since the response to sodium nitroprusside was also affected, but is a general reduction in the ability of the aorta to relax. Send offprint requests to J. R. Docherty at the above address     

Effect of endothelium on basal and α-adrenoceptor stimulated calcium fluxes in rat aorta

Summary The rate of unstimulated influx of Ca²⁺ into rat aorta smooth muscle, measured as uptake of ⁴⁵Ca, was inhibited in the presence of endothelium as compared to influx in the absence of endothelium. Efflux of ⁴⁵Ca from unstimulated prelabelled tissues was also reduced in the presence of endothelium. In normal physiological solution the rate of influx and efflux of Ca²⁺ stimulated by B-HT 920 (1 and 10 M), but not that stimulated by phenylephrine (30 nM and 1 M), was also reduced in the presence of endothelium. In the presence of the calcium entry blocker flunarizine (3 M), phenylephrine (1 M) stimulated efflux of Ca²⁺ was inhibited by the presence of endothelium. A correlation between inhibition of Ca²⁺ influx and modulation of -adrenoceptor agonist-induced contractions by endothelium could not be demonstrated, and methylene blue, an antagonist of endothelium mediated inhibition of B-HT 920 contractions, did not affect Ca²⁺ influx stimulated by the agonist. The effects of endothelium on Ca²⁺ influx and efflux are unlikely to be due to alterations by endothelium of diffusion of ⁴⁵Ca or the agonists in the vessel. The results demonstrate that an endothelial derived factor or factors can reduce calcium influx into smooth muscle cells and also modulate the release of calcium from cells, perhaps by affecting intracellular calcium pumping mechanisms. A reduction of calcium influx cannot be the sole explanation for the modulatory effect of endothelium on -adrenoceptor agonist-induced contractions but an effect on intracellular calcium metabolism may be important.     

Differential region-specific regulation of central α1-adrenoceptor binding following chronic haloperidol and clozapine administration in the rat

Rationale Many antipsychotics exhibit potent anti-₁-adrenergic receptor activity, which has been suggested to contribute to typical and atypical antipsychotic effects and to the production of centrally mediated side effects.Objectives To assess the relative contribution of ₁-adrenoceptors to the mechanism of action of haloperidol and clozapine and to identify possible sites of action.Methods We examined the effect of chronic haloperidol and clozapine treatment on ₁-adrenoceptor characteristics in several rat brain regions. For comparison, D₂-like dopamine receptor density in the striatum was also determined.Results Clozapine administration (25 mg/kg/day i.p., 21 days) significantly increased ₁-adrenoceptor density in the frontal cortex (44%), remaining cortex (49%) and thalamus (93%) but binding levels in the hippocampus and spinal cord were unchanged relative to vehicle. Haloperidol treatment (1.5 mg/kg/day i.p., 21 days) also significantly increased the density of ₁-adrenoceptor binding in the thalamus (73%), but had no effect on ₁-adrenoceptor levels in any other region examined. ₁-Adrenoceptor affinity in the cortex was not significantly altered by either antipsychotic treatment. Haloperidol, in contrast to clozapine, significantly upregulated dopamine D₂-like binding in the striatum.Conclusions Central ₁-adrenoceptors are differentially regulated after chronic haloperidol and clozapine treatment. It is suggested that thalamic ₁-adrenoceptors may represent a common anatomical locus contributing to the antipsychotic activity and/or ₁-adrenoceptor centrally mediated side effects of both drugs, whereas the selective upregulation of cortical ₁-adrenoceptor density by clozapine may contribute, in part, to its superior atypical properties.     

Do β-adrenoceptor agonists induce homologous or heterologous desensitization in rat urinary bladder?

β₃-Adrenoceptor agonists have recently been introduced for the symptomatic treatment of the overactive bladder syndrome. As such treatment is not curative, long-term treatment is anticipated to be required. As the susceptibility of β₃-adrenoceptors to undergo agonist-induced desensitization is cell type- and tissue-dependent, we have explored whether pre-treatment with a β-adrenoceptor agonist will attenuate subsequent relaxation responses to freshly added agonist using rat urinary bladder as a model. We have used the prototypical β-adrenoceptor agonist isoprenaline, the β₂-selective fenoterol and the β₃-selective CL 316,243 and mirabegron as well as the receptor-independent bladder relaxant forskolin. We show that a 6-h pre-treatment with agonist can significantly reduce subsequent relaxation against KCl-induced smooth muscle tone, but agonist-induced desensitization was also observed with longer pre-treatments or against passive tension. The agonist-induced desensitization was prominent for the β₂ component of rat bladder relaxation but much weaker or even absent for the β₃ component. Moreover, β-adrenoceptor agonist pre-treatment reduced contractile responses to the muscarinic agonist carbachol and the receptor-independent stimulus KCl. Taken together these data do not support the hypothesis that the long-term clinical efficacy of β₃-adrenoceptor agonists in the treatment of the overactive bladder syndrome will be limited by receptor desensitization. Rather they raise the possibility that such treatment may not only cause smooth muscle relaxation but also may attenuate hyper-contractility of the bladder.     

Noradrenergic modulation of serotonin release in rat dorsal and median raphé nuclei via alpha(1) and alpha(2A) adrenoceptors.

The rat rostral raphé nuclei receive catecholaminergic innervation from the locus coeruleus and other areas. In the present study, we investigated noradrenergic modulation of 5-HT release in rat dorsal and median raphé nuclei (DRN and MRN) slices (350 microm thick) superfused with artificial cerebrospinal fluid (aCSF). The raphé was locally stimulated (0.1 ms pulses, 10 mA) and 5-HT release was monitored at carbon fibre microelectrodes using fast cyclic voltammetry. The selective noradrenaline reuptake inhibitor desipramine (50 nM) did not increase stimulated (20 pulses, 100 Hz) 5-HT release but significantly slowed 5-HT reuptake in both DRN and MRN. On short stimulus trains (10 pulses, 200 Hz), the alpha(2)-selective agonist dexmedetomidine (10nM) decreased evoked 5-HT release in DRN and MRN (to 44+/-3 and 43+/-7% of pre-drug values, respectively, at minimum). In both nuclei, this response was antagonised by the selective alpha(2A)-antagonist BRL 44408 (1 microM: P<0.001 vs. dexmedetomidine) but not by the selective alpha(2B/C)-adrenoceptor antagonist ARC 239 (500 nM), the selective 5-HT(1A) antagonist WAY 100635 (100 nM) or the alpha(1)-selective antagonist prazosin (1 microM), suggesting that the effect of dexmedetomidine is wholly attributable to alpha(2A)-receptor activation. The alpha(1)-adrenoceptor agonist phenylephrine (5 microM) significantly decreased 5-HT release (to 49+/-7 and 41+/-4% of pre-drug values in DRN and MRN, respectively). The response was blocked by prazosin (P<0.001) and BRL 44408 (P<0.01) in DRN and by prazosin, BRL 44408 and WAY 100635 (all P<0.05) in MRN, suggesting that the effect of phenylephrine is, under these conditions, only partly mediated via alpha(1)-adrenoceptors. On long stimuli (30 pulses, 10 Hz), BRL 44408 (1 microM) increased evoked 5-HT efflux to 187+/-17 and 178+/-2% of pre-drug values in DRN and MRN, respectively (both P<0.001 vs. vehicle). Collectively, these data show that activation of both alpha(1) and alpha(2A)-adrenoceptors can decrease stimulated 5-HT release in the rostral raphé nuclei. Since the effect of dexmedetomidine was not antagonised by prazosin, we suggest that its effect was mediated directly, possibly through alpha(2A) receptors located on 5-HT cell elements, and not transduced indirectly through alpha(1)-adrenoceptor activation, as previously suggested by others.     

Effect of pretreatment with the selective β1-adrenoceptor antagonist bisoprolol on the subsequent cardiovascular actions and β-adrenoceptor subtype specific occupancy of celiprolol in healthy man

Summary The cardiovascular effects at rest and during exercise and ₁- and ₂-adrenoceptor occupancy following a single dose of 1200 mg celiprolol p. o. were investigated in 8 healthy subjects with or without pretreatment with a single dose of 20 mg bisoprolol p. o., using a place-bo-controlled, 2-way cross-over design.The ergometric responses of heart rate (HR) and systolic blood pressure (SBP) after celiprolol were reduced to a similar extent as after bisoprolol, but the cardiovascular function at rest was affected in a different way: there was a rise in HR, clear enhancement of cardiac systolic performance, and a considerable drop in the estimated total peripheral vascular resistance, associated with median ₁-RRA and ₂-RRA occupancies of 88 and 34%, respectively. The cardiovascular effects of celiprolol were not affected by pretreatment with bisoprolol. Celiprolol thus binds extensively to ₁-adrenoceptors, moderately to ₂-adrenoceptors, acts as ₁-adrenergic antagonist (exemplified by the ergometric effects) but has vasodilator, positive chronotropic and cardiac systolic performance enhancing properties, which do not involve either direct or indirect ₁-adrenergic agonism, but which might reflect ₂-adrenergic agonism.     

Irreversible binding of a carbostyril-based agonist and antagonist to the β-adrenoceptor in DDT1 MF-2 cells and rat aorta

1. The chemoreactive ligands 5(2-(((1′-(4′-isothiocyanatophenylamino)thiocarbonyl)-amino)-2-methyl-propyl)amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC) and 8-hydroxy-5(2-(((1′-(4′-isothiocya-natophenylamino)thiocarbonyl)amino)-2-methylprop-2-yl)amino-1-hydroxyethyl)-carbostyril (HCITC)were synthesized and shown to be potent irreversible antagonist and agonist ligands, respectively, for the β-adrenoceptor in DDT₁ MF-2 (DDT) cells and the rat isolated aorta.
2. In DDT cell membranes DCITC and HCITC inhibited (−)[¹²⁵I]-iodocyanopindolol (CYP) binding to the β-adrenoceptor with IC₅₀ values of 1.1 and 18 nM, respectively. (−)-Isoprenaline inhibited [¹²⁵I]-CYP binding with an IC₅₀ of 355 nM. Pretreatment of membranes with either chemoreactive ligand produced a time- and concentration-dependent decrease in the β-adrenoceptor content, indicating irreversible receptor binding. DCITC at concentrations up to 10 μM did not stimulate cyclic AMP accumulation in DDT cells nor did it amplify forskolin-stimulated cyclic AMP accumulation.
3. In the rat isolated aorta, DCITC (0.1 μM) did not affect either the phenylephrine-mediated tissue contraction or the acetylcholine-mediated relaxation. DCITC attenuated the maximal (−)-isoprenaline-mediated relaxation of a phenylephrine contracted aorta in a concentration-dependent manner and shifted the dose-response curves for (−)-isoprenaline to the right. The DCITC-induced decrease in maximal response was not reversed by extensive tissue washing. By use of the operational model of agonism, the calculated dissociation constant for (−)-isoprenaline ws 286 nM and the estimated receptor reserve for this agonist was 23% at the maximal response.
4. HCITC and (−)-isoprenaline stimulated cyclic AMP accumulation in DDT cells with pD₂ values (negative logarithm to base 10 of EC₅₀) of 7.95 and 7.97, respectively, and both mediated the same maximal stimulation. In the rat isolated aorta, HCITC produced a concentration-dependent relaxation of the tissue with a pD₂ value of 6.62, whereas the pD₂ for (−)-isoprenaline was 7.03. However, HCITC produced a greater maximal relaxation of the tissue than (−)-isoprenaline. The HCITC-mediated stimulation of cyclic AMP accumulation and relaxation of the isolated tissue were blocked when the β-antagonist propranolol was added concurrently. In contrast, once the HCITC-mediated responses were established, the addition of propranolol did not result in any attenuation indicating that HCITC is an irreversible β-agonist.

     

Does 4-methylimidazole have tumor preventive activity in the rat?

4-Methylimidazole (4-MEI) is found in a wide array of food products. The National Toxicology Program (NTP) recently conducted a two-year feeding cancer bioassay of 4-MEI in B6C3F₁ mice and F344/N rats. In rats, NTP found “equivocal evidence of carcinogenic activity” in females based on increased incidences of mononuclear cell leukemia and “no evidence of carcinogenic activity” in males. However, dose-related, statistically significant decreases in multiple tumors were observed in both male and female rats exposed to 4-MEI in the NTP bioassay. For example, 4-MEI was associated with a 25-fold decrease in the incidence of mammary tumors among high dose females. NTP noted briefly that the decreases in certain tumors, including mammary tumors, were greater than could be attributed to body weight alone. The present paper provides a more detailed evaluation of the evidence that 4-MEI exhibits tumor preventive activity in the rat based upon the results of the NTP bioassay. Reduced body weight offers a partial explanation for the reduction in tumors, but does not appear to be the primary cause of the decreased tumor incidences, indicating that 4-MEI itself may possess an ability to prevent tumor formation.     

Rat β3-adrenoceptor protein expression: antibody validation and distribution in rat gastrointestinal and urogenital tissues

β₃-Adrenoceptors play important roles in the regulation of urogenital and probably gastrointestinal function. However, despite recent progress, their detection at the protein level has remained difficult due to a lack of sufficiently validated selective antibodies. Therefore, we have explored the selectivity of two antibodies for the detection of rodent β₃-adrenoceptors in immunoblots and immunohistochemistry. Of two reportedly promising candidates, antibody AB15688 did not exhibit subtype selectivity in immunoblots. In contrast, the antibody Sc1473 exhibited at least some selectivity in immunoblots and more promising results in immunocytochemical and immunohistochemical stains in cells transfected with cloned β-adrenoceptor subtypes and in rat and mouse tissues. In a systematic screening of rat gastrointestinal and urogenital tissues, Sc1473 produced selective staining in the epithelial cell lining of the stomach and the urothelium of ureter and bladder. We conclude that the two tested antibodies are inappropriate or at least insufficient for immunoblotting applications, but Sc1473 appears to be useful for immunohistochemical detection of β₃-adrenoceptor protein in rodent tissues. The β₃-adrenoceptor protein exhibits a distinct expression pattern in the rat gastrointestinal and urogenital tract, which is at least partly in line with previously reported functional data.