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1 This study was designed to investigate the mechanism(s) of the negative inotropic effects of α1-adrenoceptor agonists observed in rat isolated left atria after exposure to free radicals. 2 Ouabain and calphostin C were used in contraction experiments to block the sodium pump and protein kinase C. Methoxamine-induced phospholipase C and Na+/K+ ATPase activities were measured. 3 Methoxamine (300 μM) increased contractile force by 1.6±0.2 mN in control atria but decreased contractile force in electrolysis-treated atria by 2.0±0.1 mN (P

1. The aim of this study was to investigate the time-dependency of the influence of dysthyroid states on the beta-adrenoceptor system in rat heart left ventricle. Therefore, the influence of acute and chronic hyper- and hypothyroidism on beta-adrenoceptor-induced left ventricular responses, beta-adrenoceptor density, cardiac noradrenaline tissue concentrations, Gs alpha-proteins, and basal and stimulated adenylate cyclase activities was determined. 2. Hyperthyroid rats were obtained by feeding with thyroxine (T4)-containing rat-chow for 1, 4 and 8 weeks. Hypothyroidism was induced by adding 0.05% propylthiouracil (PTU) to the drinking water. Rats of varying ages were used in order to compensate for the differences in the duration of the treatments. Rats were aged 3 and 5 months at the end of the experiments. 3. Thyroxine treatment for 4 and 8 weeks increased the cardiac sensitivity to isoprenaline, but maximal induced inotropic responses were decreased. Cardiac ventricular beta-adrenoceptor density was increased only in rats treated with T4 for 1 week. This transient effect of hyperthyroidism on cardiac beta-adrenoceptor density was not observed in older rats. The PTU treatment resulted in a stable decrease of cardiac beta-adrenoceptor density. 4. Left ventricular tissue noradrenaline concentrations were unaffected by hyperthyroidism, where a decrease was observed in hypothyroid rats. Density of Gs alpha proteins was increased in hearts from chronic hyperthyroid rats. 5. These results indicate that the increased sensitivity to beta-adrenoceptor-mediated stimulation in chronic hyperthyroidism cannot be attributed to changes in cardiac beta-adrenoceptor density, but is probably caused by an enhanced content of Gs alpha. Accordingly, in hyperthyroidism, the beta-adrenoceptor system is influenced time-dependently, whereas hypothyroidism affects the beta-adrenoceptor system independent of time.

1. The inhibitory effects of methylene blue (MB) on different types of cholinesterases and [3H]-N-methylscopolamine ([3H]-NMS) binding to muscarinic receptors were studied. 2. Human plasma from young healthy male volunteers, purified human pseudocholinesterase and purified bovine true acetylcholinesterase were incubated with acetylcholine and increasing concentrations of MB (0.1-100 mumol l-1) in the presence of the pH-indicator m-nitrophenol for 30 min at 25 degrees C. The amount of acetic acid produced by the enzymatic hydrolysis of acetylcholine was determined photometrically. 3. Rat cardiac left ventricle homogenate was incubated with [3H]-NMS and with increasing concentrations of MB (0.1 mmol l-1 mumol l-1) at 37 degrees C for 20 min. THe binding of [3H]-NMS to the homogenate was quantified by a standard liquid scintillation technique. 4. MB inhibited the esterase activity of human plasma, human pseudocholinesterase and bovine acetylcholinesterase concentration-dependently with IC50 values of 1.05 +/- 0.05 mumol l-1, 5.32 +/- 0.36 mumol l-1 and 0.42 +/- 0.09 mumol l-1, respectively. MB induced complete inhibition of the esterase activity of human plasma and human pseudocholinesterase, whereas bovine acetylcholinesterase was maximally inhibited by 73 +/- 3.3%. 5. MB was able to inhibit specific [3H]-NMS binding to rat cardiac left ventricle homogenate completely with an IC50 value of 0.77 +/- 0.03 mumol l-1, which resulted in a Ki value for MB of 0.58 +/- 0.02 mumol l-1. 6. In conclusion, MB may be considered as a cholinesterase inhibitor with additional, relevant affinity for muscarinic binding sites at concentrations at which MB is used for investigations into the endothelial system. In our opinion these interactions between MB and the cholinergic system invalidate the use of MB as a tool for the investigation of the L-arginine-NO-pathway, in particular when muscarinic receptor stimulation is involved.

Cardiac beta-adrenoceptors are assumed to play a key role in chronic heart failure. Although several radioligands labeled with 11C or 18F have been synthesized for imaging purposes with positron emission tomography (PET), so far no optimal ligands are available to image cardiac beta-adrenoceptors using single photon emission tomography (SPECT). In the present study, we characterized four new synthesized analogues of the nonselective beta-adrenoceptor antagonist 4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one (CGP12177) and one analogue of the nonselective beta-adrenoceptor antagonist penbutolol. Using classical in vitro displacement studies with left ventricular tissue of New Zealand White rabbits and [125I]iodocyanopindolol as a radioligand, binding affinity to the receptor was determined. From the four analogues, only (2'S,2"E)- [4-(3'-(1",1"-dimethyl-3"-Iodo-2" propenylamino)-2'-hydroxypropoxy)]-benzimidazol-2-one proved to have a high affinity, with Ki = 1.25 +/- 0.09 nM, n = 3. The other analogues showed relatively low affinity, with Ki-values > 1 nM. The analogue of penbutolol ((S)-(-)-[1-(2-Iodophenoxy)]-3'-(tert-butylamino)-2'-propanol) also showed a Ki value of 0.64 +/- 0.26 nM, n = 3. Subsequently, (2'S,2"E)-[4-(3'-(1",1"-dimethyl-3"-Iodo-2" propenylamino)-2'-hydroxypropoxy)]-benzimidazol-2-one and (S)-(-)-[1-(2-Iodophenoxy)]-3'-(tert-butylamino)-2'-propanol were radioactively labeled with 123I to study their biodistribution in New Zealand White rabbits and to determine specific binding. Significant uptake was observed in both lungs and left ventricles. However, both compounds showed high nonspecific binding in vivo because uptake of the radioligand could not be inhibited by preinjection of different (selective- and nonselective-adrenoceptor antagonists and hydrophilic and lipophilic antagonists) antagonists. In conclusion, although two analogues showed reasonable affinity in vitro for the receptor, their binding in vivo proved to be largely nonspecific, suggesting that these two compounds are unsuitable for imaging purposes. However, because marked differences in affinity for the receptor were observed with only little structural changes between compounds, the present results offer future perspectives for the synthesis of a more specific radioligand.

Background Potential new radioligands for the noninvasive imaging of cardiac β-adrenoceptors with single-photon emission computed tomography were investigated.

1. In the present study we investigated functional and binding characteristics of muscarinic receptors in experimental cardiac hypertrophy. 2. As models of cardiac hypertrophy we used hearts of spontaneously hypertensive rats (SHR) and Wistar rats with surgically induced abdominal aorta stenosis (ASR). Wistar Kyoto rats (WKY) and sham operated Wistar rats were used as respective controls. 3. The hypertrophy was more pronounced in hearts of ASR compared to SHR, although the mean arterial pressure was found to be lower. 4. Isolated, perfused Langendorff heart preparations (paced with 5 Hz) from both groups of hypertrophied hearts were less sensitive to the muscarinic agonists oxotremorin and methacholine (P < 0.05, all n = 6) when compared with control organs. The maximal reduction in contractile force induced by methacholine was 59.3 +/- 4.5% in SHR and 41.6 +/- 3.4% in ASR hearts versus 26.4 +/- 4.1% and 25.0 +/- 2.6% in control organs, respectively. 5. The density (fmol/mg protein-1) of muscarinic receptors in membrane homogenates of hearts from SHR (127.6 +/- 11.5) was unchanged, whereas in hearts from ASR (221.0 +/- 8.9) it was found to be reduced (P < 0.05) when compared to the respective controls (142.5 +/- 14.7 and 308.8 +/- 16.1, respectively, all n = 6). 6. From the present data we conclude that cardiac hypertrophy results in a loss of sensitivity towards muscarinic receptor stimulation. A corresponding reduction of left ventricular receptor density could only be demonstrated in massively hypertrophied hearts of ASR.

We investigated whether post-receptor alterations contribute to the diminished beta-adrenergic inotropic effects in the rat Langendorff heart following ischaemia (I) and reperfusion (R). We quantitated immunodetectable Gs and Gi protein alpha-subunit content, basal and stimulated adenylyl cyclase activity and cyclic AMP (cAMP) content in normoxic, ischaemic (30 min) and ischaemic reperfused (30 min) hearts. In addition, we measured the inotropic response of normoxic and reperfused Langendorff hearts to forskolin and dibutyryl cAMP (db-cAMP). Immunodetectable Gs and Gi alpha-subunits were unaltered by I or R. Basal adenylyl cyclase activity was decreased during I, but recovered during R. In membranes from normoxic hearts, isoprenaline, GTP, Gpp(NH)p, NaF, forskolin or Mn2+ enhanced adenylyl cyclase activity. This increase in activity was diminished in ischaemic hearts, but could be restored by R. cAMP content decreased time-dependently during I and did not recover by R, indicating ATP depletion. Forskolin and db-cAMP induced an inotropic response in normoxic hearts, which was virtually abolished after I and R. We conclude that adenylyl cyclase responsiveness is impaired during I. Since adenylyl cyclase responsiveness recovers during R, whereas inotropic responses to forskolin and db-cAMP are virtually absent in reperfused hearts, an additional mechanism downstream of cAMP formation appears to be defective during R, which prevents recovery of inotropic responses to hormonal stimulation.

Objectives Hypertension in humans and experimental animals is known to be associated with an increase in left ventricular myocardial mass. The development of cardiac hypertrophy is not caused by increased blood pressure alone; the autonomic nervous system may also play an important role. Design The functional responses to the beta-adrenoceptor agonists isoprenaline, dobutamine, salbutamol and terbutaline, and the alpha 1-adrenoceptor agonists methoxamine, cirazoline and phenylephrine were studied in isolated (Langendorff) hearts from spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) controls. The results were compared with data from radioligand binding experiments. Results There was no significant difference in the increase of left ventricular pressure induced by all beta-adrenoceptor agonists studied in SHR and WKY rat hearts. Although there was no significant difference in the response to phenylephrine, the inotropic responses to cirazoline and methoxamine proved to be significantly weaker in hearts from SHR than in those from WKY rats. Binding experiments with 3H-prazosin revealed no differences in density or affinity for cardiac tissues from SHR and WKY rats. Conclusions Long-standing hypertension leads to an impaired response of the isolated heart to alpha 1-adrenoceptor stimulation, without changes in alpha 1-receptor density or affinity. It seems likely that changes in postreceptor events are responsible for the impaired inotropic response to alpha 1-adrenoceptor agonists in hearts from SHR.

To gain more information about sources of activator Ca2+ involved in the contraction of rat and guinea-pig aorta evoked by angiotensin II and their sensitivity to Ca2+ entry blockers, measurement of slowly exchanging 45Ca2+ was established. A more physiological procedure was used, replacing La(3+)- and EGTA-containing solutions by a normal Ca(2+)-containing buffer. It was demonstrated that the angiotensin II-induced increase in slowly exchanging 45Ca2+ in rat aorta was incompletely (by approximately 60%-70%) inhibited by the organic Ca2+ entry blockers nifedipine, verapamil and diltiazem and by other Ca2+ entry blocking compounds like CoCl2 and chlorpromazine. 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) was able to inhibit the angiotensin II-induced increase in 45Ca2+ content completely, but this may be an intracellular storage effects. By contrast, the organic Ca2+ entry blockers completely inhibited that part of the angiotensin II-induced contraction of rat aorta which was dependent upon extracellular Ca2+. In guinea-pig aorta, the increase in 45Ca2+ content elicited by angiotensin II could be completely suppressed by all compounds under study. The results of these experiments correlated well with data from the functional experiments in guinea-pig aorta. In both preparations the release of Ca2+ from a rapidly as well as a slowly exchanging intracellular pool appears to contribute to the contractile response elicited by angiotensin II.

1. The aim of the present investigation was to analyse whether three prototype allosteric modulators of ligand binding to muscarinic receptors, i.e. alcuronium, gallamine, and the alkane-bis-ammonium compound W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropylammonium bromide]), may have allosteric effects on radioligand-binding characteristics at other G-protein-coupled receptors, such as cerebral A1 adenosine receptors (Gi-coupled), cardiac left ventricular alpha1-adrenoceptors (Gq), and beta-adrenoceptors (Gs). 2. The modulators were applied at concentrations known to be high with regard to the allosteric delay of the dissociation of the antagonist [3H]-N-methylscopolamine (NMS) from muscarinic M2-receptors: 30 micromol l(-1) W84, 30 micromol l(-1) alcuronium, 1000 micromol l(-1) gallamine. As radioligands, we used the adenosine A1-receptor ligand [3H]-cyclopentyl-dipropylxanthine (CPX), the alpha1-adrenoceptor ligand [3H]-prazosin (PRAZ), and the beta-adrenoceptor ligand (-)-[125I]-iodocyanopindolol (ICYP). Allosteric actions on ligand dissociation and the equilibrium binding were measured in the membrane fractions of rat whole forebrain (CPX) and of rat cardiac left ventricle (PRAZ, ICYP, NMS), respectively. 3. CPX and PRAZ showed a monophasic dissociation with half-lives of 5.88+/-0.15 and 12.27+/-0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influenced by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36+/-2% of the control value with alcuronium and to 42+/-2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately accelerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and after 3 h, more than 50% of the radioligand was still bound, so that a reliable half-life could not be calculated. ICYP dissociation was not affected by W84. In the presence of alcuronium and gallamine, the dissociation curve of ICYP revealed an initial drop from the starting level, followed by the major phase of dissociation being parallel to the control curve. 4. In summary, the allosteric action of the applied agents is not a common feature of G-protein-coupled receptors and appears to be specific for muscarinic receptors.

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 alpha 1-adrenoceptor agonist methoxamine were investigated. Since alterations in alpha-adrenoceptor density could explain the increased sensitivity to methoxamine in hearts from hypothyroid rats, alpha 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 alpha 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 alpha 1-adrenoceptors. Both T4 and PTU treatment resulted in a non-transient decrease of alpha 1-adrenoceptor density in left ventricular tissue. Furthermore, hypothyroidism increased the percentage of alpha 1A-binding sites, whereas in hyperthyroidism the distribution of the alpha 1-adrenoceptor subtypes was not affected. Myocardial tissue concentrations 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 alpha 1-adrenoceptor mediated myocardial functions.

Potential new radioligands for the noninvasive imaging of cardiac beta-adrenoceptors with single-photon emission computed tomography were investigated.Two iodinated derivatives of CGP12177 para (S-CYBL2B) and ortho (CYBL2A) substituted CGP12177 and an iodinated form of nadolol (CYBL1) were synthesized. Their affinity was tested in vitro (left ventricular homogenates). The biodistribution of [123I]S-CYBL2B was evaluated in rabbits. Specific binding was assessed by pretreatment of the animals with 0.1 mumol propranolol. The inhibition constant values (in nanomolars, means +/- SEM; n = 3 to 5) were determined at 1.17 +/- 0.42, 28800 +/- 9260, 11.1 +/- 2.1, 53.0 +/- 19.9, and 1790 +/- 700 for CGP12177, CYBL2A, S-CYBL2B, nadolol, and CYBL1. Myocardial uptake of [123I]S-CYBL2B was not inhibited by pretreatment of the animals with propranolol, but uptake by lung tissue could be blocked by propranolol (0.63% +/- 0.09% vs 0.33% +/- 0.02% % injected dose/g x kg; p0.05). In isolated right atria, preincubation with S-CYBL2B induced a parallel rightward shift of the concentration-response curve with isoprenaline.S-CYBL2B shows high affinity for cardiac beta-adrenoceptors, but binding proved nonspecific in vivo, whereas binding in lung tissue was specific. These results suggest that S-CYBL2B is probably not a suitable radioligand for receptor imaging.

A new (radio)iodinated, beta-adrenoceptor ligand, (S)-(-)-4-[3-[(1,1-dimethyl-3-iodo-(2E)-propenyl)-amino]-2- hydroxypropoxy]carbazole (CYBL8E, 1), was prepared. 1 is an iodinated analogue of the high-affinity beta-adrenoceptor antagonist carazolol (2). The asymmetric synthesis was achieved in four steps starting from 4-hydroxycarbazole. The iodine-123-labeled form was obtained by an iododestannylation reaction with [123I]NaI in the presence of H2O2. Using classical in vitro displacement experiments with membrane fractions of cardiac left ventricular muscle, 1 proved to have a high affinity for the receptor (Ki = 0.31 +/- 0.03). Biodistribution studies performed in New Zealand white rabbits demonstrated the specificity of the binding in vivo to the receptor. Uptake of [123I]1 was reduced significantly in both atrial muscle, left ventricular muscle, frontal cortex, cerebellum, and striatum, by the pretreatment of the animals with different beta-adrenoceptor antagonists. In conclusion, 1 is a potent nonselective beta-adrenoceptor antagonist, which binds specifically to the beta-adrenoceptor in vivo, and is therefore a promising radioligand for the imaging of beta-adrenoceptors using single photon emission computerized tomography.

In heart failure cardiac sympathetic neuronal function and activity appear to be altered. Although these changes are widely accepted, controversy exists concerning the neurohormonal changes occurring in pressure and volume overloaded hearts. The present study in rabbits was performed to assess the effects of mechanical overload on cardiac sympathetic neuronal function and beta-adrenoceptor density, in relation to left ventricular function.In nine rabbits the aortic valve was perforated to induce left ventricular volume overload. Pressure overload was induced by suprarenal banding of the aorta abdominalis (group 1). Five animals were sham operated (group 2). Subanalysis of group 1 was performed for non-failing (n = 5) and failing (n = 4) hearts. Heart failure was defined as any reduction in left ventricular fractional shortening 2 weeks after the second operation compared to baseline.In animals with cardiac overload, left ventricular weight was higher compared with the control animals, 7.99 +/- 1.13 vs. 6.16 +/- 0.86 g (P0.02). Left ventricular end diastolic diameter increased from 1.35 +/- 0.16 to 1.57 +/- 0.15 cm (P0.005) after surgically induced overload. Left ventricular end systolic diameter and fractional shortening did not change significantly. Myocardial noradrenaline (NA) concentration and beta-adrenoceptor density were significantly lower in group 1 than in group 2, 1005 +/- 393 vs. 1643 +/- 109 ng/g (P0.02) and 167 +/- 36 vs. 224 +/- 36 fmol/mg protein (P0.03), respectively. Myocardial [123I]-MIBG uptake did not significantly differ between group 1 and 2, 2.1 +/- 0.58 vs. 1.8 +/- 0.44 (%ID/g x kg). A significant positive correlation between myocardial NA concentration and beta-adrenoceptor density was found (r = 0.66, P0.02). Myocardial NA concentration was inversely related to left ventricular weight (r =-0.75, P0.003).The present data indicate that in a condition of cardiac volume and pressure overload, sympathetic activity is enhanced as shown by myocardial noradrenaline depletion and beta-adrenoceptor downregulation. In contrast, no cardiac neuronal dysfunction is observed, even in the stage of early heart failure.

Objectives: In heart failure cardiac sympathetic neuronal function and activity appear to be altered. Although these changes are widely accepted, controversy exists concerning the neurohormonal changes occurring in pressure and volume overloaded hearts. The present study in rabbits was performed to assess the effects of mechanical overload on cardiac sympathetic neuronal function and beta-adrenoceptor density, in relation to left ventricular function. Methods: In nine rabbits the aortic valve was perforated to induce left ventricular volume overload. Pressure overload was induced by suprarenal banding of the aorta abdominalis (group 1). Five animals were sham operated (group 2). Subanalysis of group 1 was performed for non-failing ( n = 5) and failing ( n = 4) hearts. Heart failure was defined as any reduction in left ventricular fractional shortening 2 weeks after the second operation compared to baseline. Results: In animals with cardiac overload, left ventricular weight was higher compared with the control animals, 7.99 ± 1.13 vs. 6.16 ± 0.86 g ( P < 0.02). Left ventricular end diastolic diameter increased from 1.35 ± 0.16 to 1.57 ± 0.15 cm ( P < 0.005) after surgically induced overload. Left ventricular end systolic diameter and fractional shortening did not change significantly. Myocardial noradrenaline (NA) concentration and beta-adrenoceptor density were significantly lower in group 1 than in group 2, 1005 ± 393 vs. 1643 ± 109 ng/g ( P < 0.02) and 167 ± 36 vs. 224 ± 36 fmol/mg protein ( P < 0.03), respectively. Myocardial [123I]-MIBG uptake did not significantly differ between group 1 and 2, 2.1 ± 0.58 vs. 1.8 ± 0.44 (%ID/g × kg). A significant positive correlation between myocardial NA concentration and beta-adrenoceptor density was found ( r = 0.66, P < 0.02). Myocardial NA concentration was inversely related to left ventricular weight ( r = −0.75, P < 0.003). Conclusion: The present data indicate that in a condition of cardiac volume and pressure overload, sympathetic activity is enhanced as shown by myocardial noradrenaline depletion and beta-adrenoceptor downregulation. In contrast, no cardiac neuronal dysfunction is observed, even in the stage of early heart failure.

The aim of the present study was to evaluate the use of the noradrenaline analogue iodine-123 metaiodobenzylguanidine ([123I]MIBG) for the assessment of cardiac sympathetic activity in the presence of diabetes mellitus and/or hypertension in animal models. One model used Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) rendered diabetic at 12 weeks of age by an intravenous injection of streptozotocin (STZ). The other model used lean and obese Zucker rats. In all groups basic haemodynamic values were established and animals received an intravenous injection of 50 microCi [123I]MIBG. Initial myocardial uptake and wash-out rates of [123I]MIBG were measured scintigraphically during 4 h. After sacrifice, plasma noradrenaline and left cardiac ventricular beta-adrenoceptor density was determined. The diabetic state, both in STZ-treated rats (direct induction) and in obese Zucker rats (genetic induction), appeared to induce a lower cardiac density of beta-adrenoceptors, indicative of increased sympathetic activity. Cardiac [123I]MIBG then showed increased wash-outs, thereby confirming enhanced noradrenergic activity. This parallism of results led to the conclusion that [123I]MIBG wash-out measurements could provide an excellent tool to assess cardiac sympathetic activity non-invasively. However, in hypertension (WKY vs SHR), both parameters failed to show parallelism: no changes in beta-adrenoceptor density were found, whereas [123I]MIBG wash-out rate was increased. Thus, either [123I]MIBG washout or beta-adrenoceptor density may not be a reliable parameter under all circumstances to detect changes in the release of noradrenaline. Changes in the initial uptake of [123I]MIBG were observed as well. This may be a good marker for the disappearance of cardiac innervation, but it seems not to be a good parameter for distinguishing between loss of sympathetic innervation and enhanced uptake of noradrenaline in pathological conditions.

The influence of global ischemia on cardiac beta-adrenoceptors was studied in rat and guinea pig Langendorff hearts (LH), both by functional and binding experiments using the specific beta-adrenoceptor ligand (-)-[125I]-iodocyanopindolol. Neither ischemia (30 or 60 min) nor postischemic reperfusion caused any change in beta-adrenoceptor density, affinity or in the beta 1/beta 2 ratio in LH of normal rats or in LH of rats pretreated with reserpine or 6-hydroxydopamine (6-OHDA), or in guinea pig LH, whereas perfusion of rat LH with 10(-5) M isoprenaline (15 min) caused the expected decrease in beta-adrenoceptor density. After ischemia, isoprenaline was no longer able to influence beta-adrenoceptor density, suggesting that the internalization mechanism is impaired. In functional studies, perfusion of the rat LH with 10(-5) M isoprenaline (15 min) shifted the concentration-response curve for isoprenaline to the right. Thirty-minute global ischemia virtually abolished the inotropic but not the chronotropic response to isoprenaline. Ischemia did not impair the inotropic response to ouabain or to calcium, indicating that the contractile apparatus itself was still largely intact. Our results suggest that the contractile failure after ischemia is not caused by a decrease in beta-adrenoceptor density or by a defect in the contractile apparatus but by an impaired second-messenger system.

For a series of 12 serotonin antagonists, largely varying in potency, the decrease in diastolic pressure was determined after intravenous injection into pentobarbitone-anaesthetized normotensive rats. The hypotensive activity of these antagonists was correlated with their affinity for α1 -adrenoceptors, established by (3H) prazosin radioligand displacement, and the 5-HT2 serotonergic receptor, determined by inhibition of specific (3H) mianserin binding. The radioligand binding assays were performed since they correspond to the in vivo antagonistic potencies of the antagonists at α1 - and 5-HT2 - receptors, respectively. A close correlation (r = 0.963) was found between the affinity for α1 -ad-renoceptors and hypotensive activity. On the other hand, a negative correlation of lower statistical quality (r = −0.808) existed between the affinity for 5-HT2 - receptors and the depressor potency. In this series of 12 compounds, the new antihypertensive drug ketanserin is included for which it has been speculated that it lowers blood pressure by virtue of its serotonin antagonistic activity. The results of the present study, however, point towards α1 -adrenolytic potency as an important mechanism in the hypotensive action of the drug.

A series of benzodioxane derivatives, structurally related to WB 4101 and piper-oxan as well as prazosin and its two analogues UK-18,596 and UK-33,274, was studied with respect to their affinity for α

The binding of 3[H]R 29814, the pharmacologically less active threoisomer of the potent hypotensive agent erythro-R 28935, was assayed in rat brain membranes and compared with published data on 3[H]R 28935 binding. The 3[H]R 29814 bound to homogenates of rat brain with one saturable component with high affinity (KD = 1.1 nM). The specific binding was rapid and reversible. It was not affected by sodium or magnesium ions, or by guanosine triphosphate. The maximum number of binding sites amounted to approximately 135 fmol/mg protein. The drug specificity of the 3[H]R 29814 binding site was not associated with that of any known drug recognition site, but corresponded to that of erythro 3[H]R 28935. A linear relationship was derived between the affinities of drugs for inhibiting 3[H]R 29814 and 3[H]R 28935 binding. The results suggest that identical sites were labeled by 3[H]R 29814 and 3[H]R 28935 in rat brain membranes with comparable affinity. Since the hypotensive activities of R 29814 and R 28935 greatly differ, it is concluded that the high affinity binding sites of 3[H]R 29814 as well as those of 3[H]R 28935 are not compatible with the sites responsible for the hypotensive effect.

The experimental drugs butanserin (R 53393), ritanserin (R 55667), R 56413, flufylline (Sgd 195/78) and fluprofylline (Sgd 144/80) were evaluated with respect to their antagonism at postjunctional α1- and α2-adrenoceptors and 5-HT2-receptors in pithed rats. Moreoever, affinity for [3H]mianserin, [3H]prazosin and [3H]yohimbine binding sites was assessed using rat brain preparations. In all experiments ketanserin was taken as a reference compound. It is concluded that of the compounds investigated butanserin is the most potent and selective α1-adrenoceptor antagonist, whereas ritanserin was found to be a potent and selective 5-HT2-antagonist. Of the other compounds, fluprofylline was a very selective though not very potent α1-adrenoceptor antagonist. The other compounds were less active and less selective in this respect.

A series of benzodioxane derivatives, structurally related to WB 4101 and piperoxan as well as prazosin and its two analogues UK-18,596 and UK-33,274, was studied with respect to their affinity for alpha 1-and alpha 2-adrenoceptors identified by 3H-prazosin (specific activity 33 Ci/mmol) and 3H-clonidine (specific activity 26.7 Ci/mmol), respectively, in isolated rat brain membranes. The structural variations made in these molecules gave rise to pronounced differences in affinity for alpha 1-adrenoceptors, whereas their binding affinity for alpha 2-adrenoceptors only slightly varied. Apart from piperoxan and its analogues, which showed some preference for alpha 2-adrenergic binding sites, all benzodioxane-like structures displayed a general selectivity for the alpha 1-adrenoreceptor sites labeled with 3H-prazosin. The drugs were 5-50 times more potent in inhibiting 3H-prazosin than 3H-clonidine from their specific binding sites in rat brain membranes. The highest alpha 1 selectivity was found for prazosin and UK-33,274. Within the present series of WB 4101-related benzodioxane compounds, the affinity for alpha 1-adrenoceptors is greatly reduced by alkyl substitution at the secondary amino nitrogen in the side chain. Ortho substitution of the phenyl moiety with methoxy increased affinity as did hydroxy at the para position. The side chain oxygen atom can be deleted or substituted by methylene without great loss in 3H-prazosin displacing effectiveness. The affinity for alpha 1-adrenoceptors was profoundly influenced by the configuration of the molecule. Upon introducing a second chiral center through a methyl group, the two resulting racemates differ 10-fold in activity and selectivity towards alpha 1-adrenoceptors. One of these racemates was even slightly more selective than WB 4101 itself. The selectivity of the drugs to bind to alpha 1-and alpha 2-adrenoceptors corresponded well with their in vivo selectivity to antagonize alpha 1- and alpha 2-adrenoceptor-mediated vasoconstriction in pithed normotensive rats. It is suggested that a systematic study of the structure-affinity relationship in benzodioxane antagonists may provide potent and selective blocking drugs of alpha 1-adrenoceptors.

The antagonistic properties of the cinchona alkaloids quinidine, dihydroquinidine, quinine and dihydroquinine were evaluated with respect to alpha 1- and alpha 2-adrenoceptor-induced vasoconstriction in pithed normotensive rats. Radioligand displacement studies were performed to determine the in vitro affinities of the alkaloids for alpha 1- and alpha 2-adrenoceptors. Quinidine and dihydroquinidine were more effective alpha 1-adrenoceptor antagonists than quinine and dihydroquinine. Their weak to moderate alpha 1-sympatholytic activities were compatible with their in vitro affinity for alpha 1-adrenoceptors. The potencies of (dihydro)quinidine and (dihydro)quinine in inhibiting vascular postsynaptic alpha 2-adrenoceptor-mediated pressor effects were also weak and comparable in the lower dose range (15-250 mumol kg-1). In a dose of 300 mumol kg-1, dihydroquinidine exceeded the other alkaloids in activity. The alpha 2-adrenoceptor antagonistic actions of the alkaloids did not correspond with their actual affinity for alpha 2-adrenoceptors observed in vitro. A calcium antagonistic action is proposed to contribute to the interference of these drugs with the vasoconstriction governed by alpha 2-adrenoceptors. The interaction of quinine, quinidine and their hydrogenated products with vascular alpha 1- and alpha 2-adrenoceptors may explain their hypotensive properties as well as their therapeutic effect in certain forms of vascular disorders.

Some alpha-adrenoceptor-mediated cardiovascular activities and alpha-adrenoceptor binding affinities for (+)- and (-)-lofexidine (Dexlofexidine and Levlofexidine) have been studied in comparison with racemic lofexidine. In pithed normotensive rats, i.v. (-)-lofexidine elicited pressor effects at doses (0.1-30 micrograms/kg), which were approximately 20 times lower than those of the (+)-isomer. Both yohimbine and prazosin in selective amounts of 1.0 and 0.1 mg/kg (i.v., -15 min), respectively, attenuated the increase in diastolic pressure induced by (+/-)-, (+)- and (-)-lofexidine, showing the involvement of alpha 1- as well as alpha 2-adrenoceptors in the vasopressor responses. No differences were observed in the sensitivity of the pressor effects of the (+)- and (-)-enantiomers to blockade by either yohimbine or prazosin. Following i.v. administration to pentobarbitone-anaesthetized normotensive rats, (-)-lofexidine (0.5-5.0 micrograms/kg) was found about 20 times more effective than the dextrorotatory isomer in decreasing mean arterial pressure and heart rate. The increase in heart rate evoked by electrical stimulation in pithed rats was dose-dependently reduced by (+/-)-, (-)- and (+)-lofexidine, the (-)-isomer being about 30 times more potent than the (+)-isomer. Similarly, the electrical stimulation-induced increase in diastolic pressure was also most effectively impaired by the laevorotatory enantiomer of lofexidine. (-)-Lofexidine showed an approximately 9-fold higher affinity than (+)-lofexidine for the alpha 2-adrenoceptor-like binding sites in rat brain membranes identified by [3H]-clonidine and was 4 times more potent at displacing [3H]-prazosin from alpha 1-adrenoceptors. It is concluded that the alpha-adrenoceptor activity of lofexidine resides predominantly in the (-)-isomer. The isomeric activity ratio of the enantiomers of lofexidine (about 20-fold) is higher than normally found for other imidazolines.

Dilazep, 1,4-bis-[3-(3,4,5-trimethoxybenzoyl-oxy)propyl]perhydro-1,4-diazep ine, is a novel antianginal agent with an unusual chemical structure. The drug is a weak calcium antagonist. In pithed rats dilazep (10-100 mg/kg i.v.) caused a transient hypotensive effect which was accompanied by a strong and persistent reduction in heart rate. Similarly as observed for other, more potent calcium antagonists dilazep (10-100 mg/kg) counteracted the vasoconstriction, evoked by the stimulation of postsynaptic alpha 2-adrenoceptors with the selective agonist B-HT 920. The antagonism proved noncompetitive. The vasoconstriction, induced upon selective stimulation of postsynaptic alpha 1-adrenoceptors with methoxamine, however, was hardly influenced by dilazep. These findings are in accordance with the calcium-antagonistic activity of dilazep, demonstrable at relatively high doses. From radioligand-binding studies it was concluded that dilazep is an extremely weak antagonist of alpha 1-adrenoceptors, whereas it does not possess any measurable affinity towards alpha 2-adrenoceptors. It seems unlikely that the antianginal activity of dilazep can be fully explained by its weak calcium-antagonistic potency. However, the bradycardic effect of dilazep is probably relevant to its antianginal activity.

Hypotensive activities were determined of some derivatives of erythro 1-(1-[2-(1,4-benzodioxane-2-yl)-2-hydroxyethyl]-4-piperidyl)-2-benzimidazolinone (R 28935) following intravenous administration to anaesthetized normotensive rats. Introduction of chlorine into the benzimidazolinone part of R 28935 negatively influenced the hypotensive potency as did opening of the dioxane ring. An appropriate substituent restored the hypotensive effectiveness of the "opened" structures. In general, the compounds were weak inhibitors of the specific binding of (3H)-prazosin (alpha 1-adrenoceptors), (3H)-clonidine (alpha 2-adrenoceptors) and (3H)-dihydromorphine (opiate-receptors) to rat brain membranes. The relative order of affinity for either receptor did not correspond with that of the hypotensive activity. Previous (-15 min) intravenous treatment with phentolamine (0.2 mg/kg) abolished the depressor effect of prazosin and diminished that of the threo form R 29814 as well as of all "opened" congeners, but did not significantly reduce the hypotensive response to R 28935 and the other erythro structures. It is concluded that neither alpha 1- and alpha 2- nor opiate-receptors are involved as the primary targets for R 28935 and its congeneric drugs to induce hypotension. The exact mechanism of action remains therefore unsolved. Erythro R 28935 and the other racemic erythro mixtures are centrally acting hypotensive agents. A peripheral alpha-sympatholytic component may contribute to the overall depressor effect of threo R 29814 and the "opened" derivatives.