β-Adrenoceptors potentiate α1-adrenoceptor-mediated inotropic response in rat left atria

AIM: To investigate whether stimulation of β-adrenoceptor (AR) and its subtypes augment α1-AR-evoked positive inotropic response and inositol phosphate (InsP) accumulation in isolated rat left atria. METHODS: Inotropic response was determined by contractile function experiment in isolated electrically driven rat left atria. ^3H-InsP accumulations were measured by ^3H-inositol incorporation and column chromatography. RESULTS: (1) Stimula-tion of α1-AR by phenylephrine (PE) or norepinephrine (NE) in the presence of propranolol (Prop) evoked positive inotropic response and ^3H-InsP accumulations, while stimulation of β-AR by isoprenaline (ISO) or NE in the presence of phentolamine (Phen) only evoked positive inotropic response, but not ^3H-InsP accumulations. (2) Simultaneous stimulation of α1- and β-AR by NE or ISO plus PE significantly shifted the concentration-dependent inotropic response curves and ^3H-InsP accumulation curves to the left and upward compared with individual α1-AR stimulation by PE or NE in the presence of Prop. (3) In the presence of ICI118551 (selective β2-AR antagonist) or CGP12177 (selective β1-AR antagonist), stimulation of either β1- or β2-AR did not change α1-AR-evoked inotropic response and ^3H-InsP accumulations. CONCLUSION: Stimulation of β1-AR and β2-AR potentiates α1-AR-mediated positive inotropic response and InsP accumulation in isolated rat left atria.     

β-Adrenoceptors potentiate α_1-adrenoceptor-mediated inotropic response in rat left atria

AIM: To investigate whether stimulation of β-adrenoceptor (AR) and its subtypes augment α1-AR-evoked positive inotropic response and inositol phosphate (InsP) accumulation in isolated rat left atria. METHODS: Inotropic response was determined by contractile function experiment in isolated electrically driven rat left atria. 3H-InsP accumulations were measured by 3H-inositol incorporation and column chromatography. RESULTS: (1)     

Effect of prednisolone and ketotifen onβ 2-adrenoceptors in asthmatic patients receivingβ 2-bronchodilators

Summary In 13 patients with bronchial asthma, who were on ₂-adrenergic bronchodilator therapy, the effects of prednisolone and ketotifen on lymphocyte ₂-adrenoceptor density and -responsiveness were investigated. The mean lymphocyte ₂-adrenoceptor density and -responsiveness was significantly lower than in healthy controls, presumably due to the long-term ₂-adrenergic bronchodilator treatment. Both prednisolone 100 mg i.v. and ketotifen 1 mg b.d.p.o. for 6 days rapidly improved lymphocyte ₂-adrenoceptor function. 16 h after prednisolone and about 6 days after the first dose of ketotifen lymphocyte ₂-adrenoceptor density and-responsiveness had risen to values within the range in normal volunteers.The improvement of lymphocyte ₂-adrenoceptor function was accompanied by a significant increase in peak expiratory flow rate before and after inhalation of salbutamol.It is concluded that prednisolone and ketotifen may act beneficially on the recovery of ₂-adrenoceptor responsiveness to ₂-adrenergic bronchodilators in tolerant asthmatic patients.     

Modelling of α1-adrenoceptor-mediated temporal dynamics of inotropic response in rat heart to assess ligand binding and signal transduction parameters

Background and purpose:

In order to use the transient response to an antagonist (prazosin) to evaluate properties of agonist interactions with the α₁-adrenoceptor system, an integrative mechanistic model of cardiac uptake of prazosin and its competitive interaction with phenylephrine at the receptor site was developed. Based on the operational model of agonism, the aim was to evaluate both the receptor binding and signal transduction process as determinants of the inotropic effect of phenylephrine.

Experimental approach:

In Langendorff-perfused rat hearts, prazosin outflow concentration and left ventricular developed pressure were measured, first in the presence of 12.3 µmol·L⁻¹ phenylephrine following a 1 min infusion of 1.27 nmol [³H]-prazosin, and second, when after 30 min the phenylephrine concentration in perfusate was reduced to 6.1 µmol·L⁻¹, the 1 min infusion of 1.27 nmol [³H]-prazosin was repeated.

Key results:

The kinetic model accounted for cardiac uptake and receptor binding kinetics of prazosin (dissociation constant, mean ± SD: 0.057 ± 0.012 nmol·L⁻¹), assuming that the competitive displacement of phenylephrine (dissociation constant: 101 ± 13 nmol·L⁻¹) reduced the receptor occupation by the agonist and, consequently, contractility. This competitive binding process appeared to be the rate-determining step in response generation. The relationship between receptor occupancy and inotropic response was described by an efficacy parameter (τ, ratio of receptor density and coupling efficiency) of 4.9.

Conclusions and implications:

Mechanistic pharmacodynamic modelling of the kinetics of antagonism by prazosin allows quantitative assessment of the α₁-adrenoceptor system both at the receptor and post-receptor levels.     

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.

     

Relationship between α1-adrenoreceptor density and functional response of rat vas deferens

Summary The relationship between the density of ₁-adrenoreceptors and the longitudinal contractile response of rat vas deferens was examined by using phenoxybenzamine to irreversibly decrease ₁-adrenoreceptor density. Receptor density was measured by Scatchard plots of saturation analysis of specific ¹²⁵I-BE 2254 (¹²⁵IBE) binding and compared to the potency of -adrenoceptor agonists in causing contraction and the maximum contraction elicited by these agonists. Treatment of isolated vasa deferentia in organ baths with phenoxybenzamine caused a dose-dependent decrease in the density of ¹²⁵IBE binding sites, the potency of the full agonist phenylephrine in activating contraction, and the maximum contractile response. The percentage of functional receptors remaining after phenoxybenzamine treatment (q value) was calculated from the contraction data and compared to the percentage of ¹²⁵IBE binding sites remaining. The reduction in the number of functional receptors was much greater than the reduction in the number of ¹²⁵IBE binding sites at all doses of phenoxybenzamine. Since the magnitude of the contractile response caused by a weak partial agonist should be approximately proportional to the density of functional receptors in the tissue, partial agonists were used as an independent measure of the degree of functional receptor inactivation. Bath application of two different doses of phenoxybenzamine caused a decrease in the maximal contraction caused by the partial agonists clonidine and ephedrine which was similar to the calculated decrease in the q value, but not to the observed decrease in the density of ¹²⁵IBE binding sites. Phenylephrine also caused contraction of spiral strips prepared from vas deferens, suggesting that ₁-adrenoreceptors exist on the inner circular muscle as well as the outer longitudinal muscle of this tissue. To determine whether access barriers in the layered muscle structure of the vas deferens could result in a preferential inactivation of a particular subpopulation of receptors, phenoxybenzamine was applied more uniformly by intravenous administration to anesthetized rats. Intravenous administration of phenoxybenzamine also resulted in a dose-dependent decrease in the density of ¹²⁵IBE binding sites, the potency of phenylephrine in activating contraction, and the maximum contractile response. The degree of functional receptor inactivation after intravenous phenoxybenzamine administration (q values) agreed reasonably well with the observed reduction in ¹²⁵IBE binding sites, particularly at lower doses of phenoxybenzamine. The equilibrium constant for activation of the receptor by phenylephrine (K _act) was the same whether calculated using data from bath application of phenoxybenzamine or intravenous application of phenoxybenzamine. These data suggest that only a portion of the ₁-adrenoreceptors in rat vas deferens affect longitudinal contraction. There is, however, a large pool of spare receptors for longitudinal contraction and these receptors are preferentially inactivated by application of phenoxybenzamine to the isolated tissue in an organ bath. When phenoxybenzamine is applied intravenously, the receptors affecting longitudinal contraction are inactivated to a degree similar to that of the total pool of receptors in this tissue.Supported by Grant HL 29871 from the National Institutes of Health     

Phentolamine and hypoxia: modulation of contractility and α1-adrenoceptors in isolated rat atria

The hypoxia-induced effects on the binding sites and affinity constant of adrenoceptors, in the presence and absence of phentolamine, were determined for atrial membranes of hearts from normal and genetically hyperlipidaemic Yoshida (YOS) rats. Atrial function was also measured during normoxia and hypoxia, in the presence and absence of phentolamine.Hypoxia increased a1-adrenoceptor density in atrial membranes of normal rats (B_max 10.6 to 26.7 fmoles/mg protein). Phentolamine prevented the increase in the B_max of ₁-adrenoceptors and increased the equilibrium dissociation constant of these receptors (K _D 0.17 to 0.53 nmol/l). Beta-adrenoceptors did not change during hypoxia, but the B_max was slightly increased (26%) in the presence of phentolamine. Thus, the ₁/\ ratio increased from 0.40 in normoxia to 1.06 in hypoxia. In normoxic atria from YOS rats, the ₁/\ ratio was already elevated (0.86) in comparison to control rats (mainly due to a higher density of at-adrenoceptors in atrial membranes from YOS rats). This ratio was not modified by hypoxia (0.84), but decreased when phentolamine was present (0.30).Hypoxia reduced the force of contraction and increased diastolic tension of atria of normal rats, while the sinus rate was not significantly modified. Phentolamine abolished the increase in diastolic tension and reduced the negative effect of hypoxia on contractile force. In YOS rat atria, functional parameters were modified by hypoxia in a qualitatively similar way to that of normal rat atria.The observed increase in ₁-adrenoceptor density during hypoxia is in accordance with the results of experiments with animal models of the ischaemic heart and with findings in human heart failure. The possible therapeutic significance of these data is considered. Correspondence to: G. Fassina at the above address     

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.     

The pharmacokinetics of theβ 2-adrenoceptor agonist fenoterol in healthy women

Summary We have studied the pharmacokinetics of fenoterol in healthy women during and after a 3 h intravenous infusion of different doses within the therapeutic range for tocolysis (0.5 g·min^–1, 1.0 g·min^–1, and 2.0 g·min^–1). A specific and sensitive radioimmuno-assay was used for the determination of fenoterol. For compartmental analysis the plasma concentration time data were fitted with the TOPFIT program, assuming two exponentials.The total clearance of fenoterol increased with dose (1299 ml·min^–1 at 0.5 g·min^–1, 1483 ml·min^–1 at 1.0 g·min^–1, and 1924 ml·min^–1 at 2.0 g·min^–1), as did the apparent volume of distribution (from 491 at the lowest to 851 at the highest dose).In contrast, the apparent half-lives were not dose-dependent, with t_{1/2· 1} 4.8 min and t_{1/2· 2} 52 min.This paper is dedicated to Prof. Dr. Ellen Weber, Heidelberg, FRG     

Desensitization of ETA endothelin receptor-mediated negative chronotropic response in right atria–species difference and intracellular mechanisms

1. Desensitization of ET_A endothelin receptor (ET_AR) was compared between the rat and guinea-pig with regard to negative chronotropic response (NC) in the right atria (RA).
2. ET-1 (100 nM) produced distinct NC in the presence of BQ788 (300 nM), and positive chronotropic response (PC) in the presence of BQ123 (1 μM) in both species, showing that ET_AR and ET_B endothelin receptor (ET_BR) mediate NC and PC, respectively.
3. Repetitive applications of ET-1 (50 nM) desensitized PC, and the second application only induced a strong NC in both species. Later applications of ET-1 produced virtually no response in the rat RA, whereas they produced BQ123-sensitive NCs repetitively in guinea-pig RA, exhibiting marked species difference in desensitization of ET_AR-mediated NC.
4. Pretreatment with staurosporine (100 nM) prevented desensitization of ET_AR in the rat RA altogether. However, phorbol 12-myristate 13-acetate (PMA, 300 nM) failed to induce, but rather hampered, desensitization of ET_AR.
5. Partial amino acid sequencing of ET_ARs, spanning from the 2nd through the 4th intracellular loops, revealed that all the potential Ser/Thr phosphorylation sites, including a protein kinase C (PKC) site, are conserved among guinea-pigs, rats, rabbits, bovines and humans.
6. In guinea pig RA, pretreatment with okadaic acid (1 μg ml⁻¹) and PMA did not facilitate desensitization of ET_AR whereas these agents successfully desensitized ET_AR during combined stimulation of β-adrenoceptor and ET_AR by isoproterenol (300 nM) and ET-1 (100 nM).
7. These results suggest that species differences in desensitization of ET_AR are not caused by differences in the site(s) of, but caused by differences in the environment for phosphorylation of the receptor. Desensitization of ET_AR appears to require phosphorylation of the receptor by PKC as well as a kinase stimulated by β-adrenoceptor activation.

     

Effect of left ventricular pressure and volume overload on α-adrenoceptor activity in patients with rheumatic heart valvular disease

We have investigated the possibility that the various left ventricular load conditions may exert different effects on the sympathetic function by comparing the influence of volume (VOL) and pressure (POL) overload on platelet α-adrenoceptor activity, plasma catecholamines and cAMP in 44 patients with rheumatic heart valvular disease. Receptor activity was determined by radioligand binding methods, catecholamines by HPLC using an electrochemical detector, and cAMP by radioimmunoassay. The mean a-adrenoceptor density (B_max) of the control group (n = 29) was 4.71±0.41 fmol per 10⁷ platelets and the corresponding dissociation constant (K_d) was 2.47±0.15 nM. In VOL patients, the density was elevated by 70% (P<0.001), but it remained unchanged in the POL patients. In contrast to the B_max the K_d of the VOL group was not changed, and it increased by 34% (P<0.01) in the POL group. Norepinephrine was elevated by 91% (P<0.05) in POL, and epinephrine increased by 65% (P<0.05) in POL and 71% (P<0.05) in VOL. These results suggest that the sympathetic nervous system responds to left ventricular volume overload by increasing α-adrenoceptor density with no apparent change in receptor affinity toward [³H]-yohimbine binding, and to left ventricular pressure overload by decreasing their binding affinity without a parallel decrease in receptor density. The increase in receptor density in VOL is accompanied by an increase in plasma epinephrine, and the decrease in binding affinity in POL is associated with increased plasma norepinephrine and epinephrine levels     

Autoradiographic localization of “peripheral-type” benzodiazepine binding sites in the rat brain,heart and kidney

Summary The localization of peripheral-type benzodiazepine binding sites has been accomplished using quantitative receptor autoradiography after labeling slidemounted tissue sections with [³H]-RO 5-4864. Specific [³H]-RO 5-4864 binding sites were observed in the choroid plexus and ependyma cells within the brain. Lower levels of specific binding were seen in areas corresponding to the glomerular layer of the olfactory bulb. Stereotaxic administration of the excitotoxin, ibotenic acid, into the piriform cortex produced a dramatic increase in binding to the peripheral-type site in this brain region. Binding in the kidney was associated with the ascending limb of the loop of Henle and the distal convoluted tubule, while in the heart a more diffuse binding was found throughout the ventricle wall. The localization of peripheral-type benzodiazepine binding sites to areas involved in ion transport gives additional support to previously suggested physiological roles for these sites.     

Effect of bunazosin, α1-adrenoceptor blocker,on multidirectional contractile response and localization of bunazosin binding sites in human hypertrophied prostate

• 1.1. Effects of bunazosin, an α₁-adrenoceptor blocker, on the contraction induced by norepinephrine in human hypertrophied prostate were examined in vitro.
• 2.2. Prostatic specimens showed maximum contraction at 10⁻⁴ M norepinephrine in longitudinal and circumferential directions to the urethra.
• 3.3. Bunazosin (10⁻⁷ M) blocked norepinephrine-induced contraction with a parallel shift of the dose-response curve in both directions (pA₂: 8.76 ± 0.15; pA₂: 8.90 ± 0.08, respectively).
• 4.4. Serial sections of prostates were also evaluated by autoradiography. The binding sites were diffusely distributed in the interstitium.
• 5.5. We concluded that bunazosin affects multidirectional contraction in prostates.

     

Binding of agonists and antagonists to β-adrenoceptors in rat vas deferens: relationship to functional response

Summary The properties of -adrenoceptors in rat vas deferens were examined using radioligand binding assays of ¹²⁵I-pindolol (¹²⁵IPIN) and inhibition of electrically-evoked contractions of vas deferens in vitro. ¹²⁵IPIN labelled a single class of high affinity binding sites with apparently mass action kinetics in membrane preparations of vas deferens with properties consistent with an essentially homogeneous population of ₂-adrenoceptors. Isoprenaline inhibited electrically evoked (60 V, 1.0 ms, 0.1 Hz) contractions of vas deferens with an EC₅₀ of 18.0±2.1 nM. K _B values for antagonists in competitively antagonizing this response correlated well (r ²=0.99) with the K _D values for inhibition of ¹²⁵IPIN binding. Inhibition of ¹²⁵IPIN binding by isoprenaline, adrenaline, noradrenaline and salbutamol was determined under conditions designed to produce high and low affinity agonist binding. In the presence of 10 mM MgCl₂, agonists inhibited specific ¹²⁵IPIN binding with a relatively high potency and low Hill slope, while in the presence of 154 mM NaCl and 300 M guanosine-5-triphosphate, agonists inhibited specific ¹²⁵IPIN binding with a lower potency and an apparent Hill slope closer to 1. To determine which affinity state was relevant to functional receptor stimulation, receptor density was decreased with bromoacetylalprenololmenthane (BAAM). Treatment of membrane preparations with 0.3 M BAAM produced a 45% decrease in the B _max for ¹²⁵IPIN with no change in the apparent K _D. Treatment of intact vasa deferentia with increasing concentrations of BAAM resulted in a progressive rightward shift in the dose-response curve to isoprenaline or salbutamol folowed by a decreased maximum response. K _A values for isoprenaline and salbutamol in activating the functional -adrenoceptors were compared with K _I values for agonist inhibition of specific ¹²⁵IPIN binding. The K _A values for both agonists were not significantly different from the low affinity K _I values, but were significantly different from the high affinity K _I values. These data suggest that 1) a homogeneous population of ₂-adrenoceptors inhibiting contraction of rat vas deferens can be labelled with ¹²⁵IPIN, 2) there is a substantial -adrenoceptor reserve in rat vas deferens; and 3) the initial event in signal transduction by -adrenoceptors in rat vas deferens is the binding of agonists to the low affinity form of the receptor which is not complexed with the guanine nucleotide binding protein.Supported by HL29871 and an Advanced Predoctoral Fellowship from the Pharmaceutical Manufacturers Association Foundation to J.M.M.Portions of this work were presented at the FASEB meeting April 4, 1984, St. Louis, MO, USA, at the ASPET meeting August 18–22, 1985, Boston, MA, USA, and appeared in Fed Proc 43:744     

Thyroid hormone modulates inotropic responses, α-adrenoceptor density and catecholamine concentrations in the rat heart

We investigated the influence of hyper- and hypothyroidism on basal parameters of isolated perfused hearts of rats. In addition the effects of different extracellular calcium concentrations ([Ca²⁺]_o), the calcium entry promoter Bay K8644 and the ₁-adrenoceptor agonist methoxamine were investigated. Since alterations in -adrenoceptor density could explain the increased sensitivity to methoxamine in hearts from hypothyroid rats, ₁-adrenoceptor density in the left ventricle was also established. Different time-schedules of exposure to hyper- and hypothyroidism were used to investigate whether the influence of chronic dysthyroid states on ₁-adrenoceptor density is transient and time-dependent. Simultaneously myocardial noradrenaline and adrenaline tissue concentrations were determined, since they might correlate with the observed changes. Hyperthyroidism was induced by feeding rats for 1, 4 and 8 weeks with 5 mg/kg L-thyroxine (T4)-containing rat chow. Hypothyroid rats were obtained by adding 0.05% propylthiouracil (PTU) to the drinking water during 1, 4 and 8 weeks. For the functional experiments animals were treated during 4 weeks, to mimic the clinical situation of a chronic endocrine disease. Langendorff hearts from hyperthyroid hearts showed an increased maximally developed relaxation velocity, whereas Langendorff hearts from hypothyroid rats showed an increased left ventricular pressure (LVP). We observed an increased maximal inotropic response to [Ca²⁺]_o in hearts from both hyperthyroid and hypothyroid rats, indicating that both dysthyroid states interfere with the handling of calcium ions by the contractile apparatus. Unchanged responses to Bay K8644 in hearts from hyperthyroid and depressed responses in hearts from hypothyroid rats suggest that the involvement of L-type calcium channels is rather unlikely. Furthermore, the reflex increase in coronary flow in response to enhanced contractile force appeared to fail in hearts from hypothyroid rats. Sensitivity of the response to methoxamine was increased in hearts from hypothyroid rats, which was accompanied by a decrease in the number of myocardial ₁-adrenoceptors. Both T4 and PTU treatment resulted in a non-transient decrease of ₁-adrenoceptor density in left ventricular tissue. Furthermore, hypothyroidism increased the percentage of _1A-binding sites, whereas in hyperthyroidism the distribution of the ₁-adrenoceptor subtypes was not affected. Myocardial tissue concencentrations of noradrenaline and adrenaline were unchanged in hyperthyroid rats and decreased in hypothyroid rats. The present study indicates that thyroid hormones have a direct rather than a sympathetically mediated effect on ₁-adrenoceptor mediated myocardial functions.     

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.     

Pertussis toxin does not attenuate α2-adrenoceptor mediated inhibition of noradrenaline release in mouse atria

Summary 1. The ₂-adrenoceptor agonist clonidine (0.03 and 0.1 ol/l) significantly inhibited stimulation-induced overflow of radioactivity from mouse isolated atria pre-incubated with [³H]-noradrenaline. This effect of clonidine was blocked by idazoxan (0.3 gmol/l) but not prazosin (0.3 ol/l), indicating that an ₂-adrenoceptor was involved. 2. In some experiments mice were injected with pertussis toxin (1.5 g/mouse) 4 days before their atria were removed and subsequently incubated with [³H]-noradrenaline. Alternatively, isolated atria from untreated mice were suspended in Krebs-Henseleit solution, incubated for 16 h with pertussis toxin (1.0 and 4.0 g/ml) or vehicle and subsequently incubated with [³H]-noradrenaline. The effectiveness of pertussis toxin pretreatment was assessed indirectly using carbachol. Carbachol caused a dose dependent fall in both the rate and force of contraction of isolated, spontaneously beating atria from mice pretreated with vehicle in vivo or in vitro. This effect of carbachol was not seen in atria from mice pretreated with pertussis toxin in vivo or in vitro, suggesting that active toxin penetrated the myocardium. 3. Pertussis toxin pretreatment, either in vivo or in vitro did not alter the inhibitory effect of clonidine (0.03 and 0.1 gmol/l), or the facilitatory effect of the -adrenoceptor antagonist phentolamine (1.0 mol/l), on the stimulation-induced overflow of radioactivity. These results suggest that ₂-adrenoceptor modulation of noradrenaline release from sympathetic nerve terminals is not dependent on an inhibitory guanine-nucleotide-binding protein. Send offprint requests to I. Musgrave     

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.     

Comparative effects ofβ 2-adrenoceptor agonists on intracranial self-stimulation,Sidman avoidance,and motor activity in rats

The effects of -adrenoceptor agonists were compared in various operant behavioral tasks, particularly intracranial self-stimulation (ICSS). Clenbuterol, salbutamol, and terbutaline all reduced responding by rats that lever-pressed for low stimulation intensities. The effects of clenbuterol in this test were completely reversed by propranolol, and those of salbutamol were partly reversed. Intermediate doses of clenbuterol and salbutamol slowed the initiation of rewarding brain stimulation in a shuttlebox but had little or no effect on the termination latencies. However, higher doses of both drugs lengthened the termination latencies. Motor activity was reduced at doses that attenuated ICSS responding. Complete tolerance occurred within 4 days to the effects of clenbuterol and salbutamol on leverpressing ICSS and to the effects of clenbuterol on motor activity. The apparent performance deficits induced by these drugs were overcome by more intense motivation. For example, even at high doses, clenbuterol reduced ICSS leverpressing only partially when animals bar-pressed for high rather than low stimulation intensities. Furthermore, all three drugs failed to alter Sidman avoidance responding at doses up to 100 times those that attenuated ICSS responding. It is concluded that although -adrenoceptor agonists cause apparent sedation in rats, this sedation is limited and shows rapid tolerance.