Responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on aortae from normo-, pre- and hypertensive rats

The objective of this study was to determine the responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on the isolated aorta in the maturation of normotensive and hypertensive rats. The effects of a very slowly reversible antagonist, bromoacetylal-prenololmenthane (BAAM), on the relaxant responses of the aortae of 5- and 14-week-old Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs) to isoprenaline were determined. Five-week-old SHRs are pre-hypertensive and the aortic rings are less responsive to isoprenalinethan age-matched WKY (pD2 values: WKY, 8.40; SHRs, 8.03). Similar relaxant responses to forskol in were obtained on the aortae of 5- and 14-week-old WKY and SHRs. The K(A) value for isoprenaline at the aortic beta2-adrenoceptors of the 5-week-old WKY was 2.1 x 10(-7) M, and similar values were obtained on the aortae of 5-week-old SHR and 14-week-old WKY and SHRs. In the maturation of the WKY aortae from 5 to 14 weeks, there was a reduction in the maximum response, a major loss of sensitivity and a loss of beta2-adrenoceptor reserve for isoprenaline. On 5-week-old SHR aorta, the sensitivity to isoprenaline was 2.5-fold lower, and the beta2-adrenoceptor reserve was less than on age-matched WKY. In the development of hypertension on the SHR aorta from 5 to 14 weeks, there was a reduction in the maximum response to isoprenaline. At 14 weeks, the sensitivity and the beta-adrenoceptor reserve to isoprenaline were similar, but the maximum responses were lower on the SHR than WKY. As there are differences in pre-hypertensive SHR and age-matched WKY aortic responses to isoprenaline, it is no longer valid to consider that the loss of responsiveness to isoprenaline in hypertension is solely owing to the hypertension. There are no changes in affinity, but major changes in the sensitivity, maximum responses and aortic beta2-adrenoceptor reserves to isoprenaline in the maturation of normotensive and pre-hypertensive aortae.     

The alpha (1D)-adrenoceptor antagonist BMY 7378 is also an alpha (2C)-adrenoceptor antagonist

1 We have investigated the actions of the alpha(1D)-adrenoceptor selective antagonist BMY 7378 in comparison with yohimbine at alpha(1)- and alpha(2)-adrenoceptors. 2 In rat aorta (alpha(1D)-adrenoceptor), BMY 7378 (pA(2) of 8.67) was about 100 times more potent than yohimbine (pA(2) of 6.62) at antagonizing the contractile response to noradrenaline. 3 In human saphenous vein (alpha(2C)-adrenoceptor), BMY 7378 (pA(2) of 6.48) was approximately 10 times less potent than yohimbine (pA(2) of 7.56) at antagonizing the contractile response to noradrenaline. 4 In prostatic portions of rat vas deferens, BMY 7378 (10 mum) did not significantly affect the concentration-dependent inhibition of single pulse nerve stimulation-evoked contractions by xylazine (an action at prejunctional alpha(2D)-adrenoceptors). 5 In ligand-binding studies, BMY 7378 showed 10-fold selectivity for alpha(2C)-adrenoceptors (pK(i) of 6.54) over other alpha(2)-adrenoceptors. 6 It is concluded that BMY 7378, in addition to alpha(1D)-adrenoceptor selectivity in terms of alpha(1)-adrenoceptors, shows selectivity for alpha(2C)-adrenoceptors in terms of alpha(2)-adrenoceptors.     

Potency, affinity constants and receptor reserves for noradrenaline and adrenaline on aortae from aged normo- and hypertensive rats

Previously we have determined the potency, affinity constants (K(A) values), and alpha1-adrenoceptor reserves for noradrenaline and adrenaline on the thoracic aortae of 20-week-old Wistar Kyoto normotensive (WKY) and spontaneously hypertensive rats (SHRs). This study has investigated whether these parameters were altered on the thoracic aortae by ageing, and in hypertension/heart failure. The effects of phenoxybenzamine on the contractile responses of the aortae of 20-month-old WKYs and SHRs were determined. The pD2 values for noradrenaline and adrenaline were 7.1 and 7.0, respectively, on the aortae of 20-month-old WKYs, and similar values were obtained on age-matched SHRs. On the aortae of 20-month-old WKYs, the K(A) values for noradrenaline and adrenaline were 1.85 and 1.95 x 10(-6) M, and the receptor occupancies required for 50% maximum responses were 16 and 24%, respectively. There were lower affinities, by approximately twofold, but similar receptor reserves for noradrenaline and adrenaline on the aortae of age-matched SHRs. In comparison with the aortae of 20-week-old WKYs and SHRs, there was a 5-fold loss of sensitivity to noradrenaline and adrenaline between 20 weeks and 20 months. Between 20 weeks and 20 months there was a 50-fold loss of affinity with ageing and a further twofold loss with hypertension/heart failure, and an increase in alpha1-adrenoceptor reserves for noradrenaline and adrenaline between 20 weeks and 20 months. There were no differences in the sensitivity and affinity, and minor changes in the alpha1-adrenoceptor reserves for noradrenaline and adrenaline between the aortae of 20-month-old WKYs and SHRs. In contrast there were major changes in these parameters in the ageing of the WKY aorta from 20 weeks to 20 months. There were no additional changes in the sensitivity and alpha1-adrenoceptor reserves, but a small additional change in affinity for noradrenaline and adrenaline in hypertension/heart failure on the aortae of 20-month-old SHRs.     

Evidence showing that beta-adrenoceptor subtype responsible for the relaxation induced by isoprenaline is principally beta 2 but not beta 1 in guinea-pig tracheal smooth muscle

1 The present study was carried out to pharmacologically identify the β‐adrenoceptor subtype that mediates isoprenaline‐elicited relaxation in the isolated guinea‐pig tracheal smooth muscle, to answer the question whether it is β₁‐ or β₂‐subtype? 2 Isoprenaline as well as salbutamol, a well‐known β₂‐selective adrenoceptor agonist, produced a concentration‐dependent relaxation with a pD₂ value of 8.12 vs. 7.54 for salbutamol. 3 Isoprenaline‐elicited relaxation was not affected by β₁‐selective antagonists, atenolol and CGP‐20,712A, within the concentration ranges supposed to antagonize β₁‐subtype: atenolol, ≤10^?6 m ; CGP‐20,712A, ≤10^?8 m . 4 By contrast, the concentration–response curves for isoprenaline as well as salbutamol were shifted rightwards in a competitive fashion by atenolol at the concentrations ≥3 × 10^?6 m . However, pA₂ values of atenolol against isoprenaline (5.86) and salbutamol (5.71) were consistent with the value corresponding to β₂‐ but not to β₁‐subtype (around 7.00), and these values were not significantly different from each other. 5 Competitive antagonism of the relaxations to isoprenaline and salbutamol were also obtained with β₂‐selective antagonists, butoxamine and ICI‐118,551. Against isoprenaline and salbutamol, the pA₂ values of butoxamine (6.51 vs. 6.81) and ICI‐118,551 (8.83 vs. 8.90) were substantially identical. Thus the primary mediation of β₂‐receptor in the relaxations was strongly supported. 6 The present findings provide evidence that the β‐adrenoceptor which mediates isoprenaline‐elicited relaxation of guinea‐pig tracheal smooth muscle is essentially β₂‐ but not β₁‐subtype. The present study also indicates the importance of using multiple receptor antagonists with different pA₂ values to pharmacologically identify the responsible receptor subtype in smooth muscle mechanical responses.     

Responsiveness, affinity constants and receptor reserves for serotonin on aortae of aged normotensive and hypertensive rats.

We have previously shown that the potency and affinity constants (K(A) values) for serotonin (5-HT) are greater, and the 5-HT2A-receptor reserve is lesser, on the aorta of 6-month-old spontaneously hypertensive rats (SHRs) compared with age-matched Wistar Kyoto normotensive (WKY) rats. The present study was undertaken to investigate whether these parameters are altered on the aorta with ageing and as hypertension progresses to heart failure. The effects of phenoxybenzamine on the serotonergic responses of the aortae of 24-month-old WKY rats and SHRs were determined. On WKY rat aorta, ageing from 6 to 24 months was associated with an increase in sensitivity and affinity for serotonin, and a loss of 5-HT2A-receptor reserve. On SHR aorta, ageing from 6 to 24 months was also associated with an increase in sensitivity and affinity for serotonin, but a loss of 5-HT2A-receptor reserve. The sensitivity to serotonin was greater on the 24-month-old SHR aorta (pD2 6.53) than age-matched WKY rat aorta (pD2 5.89). On the aorta of the 24-month-old WKY rats, the K(A) value for serotonin was 4.5 x 10(-6) M, and the receptor occupancies required for 20 and 50 % maximum responses were 12 and 29%, respectively. There was a similar affinity, but greater receptor reserves, for serotonin on the aorta of age-matched SHRs. In summary, we have shown changes in sensitivity, affinity and 5-HT2A-receptor reserves for serotonin on the aorta with ageing and in hypertension/heart failure.     

Selective agonists reveal alpha(1A)- and alpha(1B)-adrenoceptor subtypes in caudal artery of the young rat

Multiple alpha(1)-adrenoceptors were evaluated in caudal artery of the young Wistar rat using selective agonists and antagonists. Arteries were exposed to the selective alpha(1A)-adrenoceptor agonist, A-61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulfonamide) or to phenylephrine and to prazosin (alpha(1)-adrenoceptor antagonist), or the selective alpha(1A)-adrenoceptor antagonists 5-methylurapidil, RS 100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione), RS 17053 (N-[2(2-cyclopropylmethoxy) ethyl]-5-chloro-alpha, alpha-dimethyl-1H-indole-3-ethanamide), and the selective alpha(1D)-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione). Results showed a 100-fold higher potency of A-61603 for the alpha(1)-adrenoceptor present in the artery, compared with phenylephrine. Prazosin displaced both agonists with high affinity, whereas 5-methylurapidil, RS 100329 and RS 17053 displaced A-61603 with high affinity, indicating the presence of alpha(1A)-adrenoceptors. The selective alpha(1A)-adrenoceptor antagonists blocked phenylephrine responses with low affinity, suggesting that phenylephrine activated a second receptor population in caudal artery. BMY 7378 antagonized with low affinity both A-61603 and phenylephrine-induced contractions, indicating absence of alpha(1D)-adrenoceptors in the vessel. The results suggest that functional alpha(1B)-adrenoceptors are present in caudal arteries of the young Wistar rat.     

Alpha 2-adrenoceptor and NO mediate the opioid subsensitivity in isolated tissues of cholestatic animals

1. Our previous report showed that in acute cholestasis, the subsensitivity to morphine inhibitory effect on electrical-stimulated contractions develops significantly faster in guinea-pig ileum (GPI) and in mouse vas deferens (MVD) (45.2 and 29.9 times, respectively) compared with non-cholestatic subjects. 2. The possible contribution of alpha2-adrenoceptor and nitric oxide (NO) pathways on the development of tolerance was assessed in GPI and MVD of cholestatic subjects. 3. Daily administration of naltrexone (20 mg kg(-1)), yohimbine (5 mg kg(-1)), and Nomega-nitro-l-arginine methyl ester (l-NAME) (3 mg kg(-1)) to cholestatic animals significantly (P-value < 0.05) inhibited the process of subsensitivity in all groups. 4. Consistent with the literature, it was concluded that both the alpha2-adrenergic system and NO have close interaction with the opioid system and may underlie some of the mechanisms involved in the subsensitivity development to opioids in acute cholestatic states.     

Captopril therapy decreases both expression and function of alpha-adrenoceptors in pre- hypertensive rat aorta

1.-- The effects of captopril on alpha(1)-adrenoceptor mRNA and protein and phenylephrine-induced contraction was assessed in aorta of pre-hypertensive spontaneously hypertensive rats. 2.-- Four-week-old SHR and WKY rats were treated with captopril [an angiotensin-converting enzyme (ACE) inhibitor] 3 mg kg(-1) day(-1) for 1 week. 3.-- pA(2) values for BMY 7378, an alpha(1D)-adrenoceptor antagonist, were 8.63-9.20 among the different groups. Schild slopes were close to unity suggesting that contraction was produced primarily by alpha(1D)-adrenoceptor stimulation and was not changed with therapy. 4.-- Alpha(1D)-adrenoceptor mRNA and protein values were higher in pre-hypertensive SHR than in WKY, whereas alpha(1A)-adrenoceptor mRNA was higher in WKY and alpha(1B)-adrenoceptors were similar in both strains, and protein was not significantly different for alpha(1A)- and alpha(1B)-subtypes. 5.-- Captopril decreased maximal contraction in SHR, without having effect in WKY rats, while alpha(1D)-adrenoceptor mRNA was decreased in both rat strains but alpha(1D)-adrenoceptor protein was significantly decreased only in SHR, and increased alpha(1A)-mRNA in SHR, no effect of captopril treatment was observed on alpha(1B)-adrenoceptor mRNA and protein nor on alpha(1A)-adrenoceptor protein. 6.-- These data suggest that ACE inhibition by captopril influences both expression and function of alpha(1D)-adrenoceptors in aorta of pre-hypertensive rats, probably avoiding alpha(1D)-subtype expression by blockade of angiotensin II synthesis.     

beta(3)-Adrenoceptor agonists: potential,pitfalls and progress

β₃-Adrenoceptor agonists are very effective thermogenic anti-obesity and insulin-sensitising agents in rodents. Their main sites of action are white and brown adipose tissue, and muscle. β₃-Adrenoceptor mRNA levels are lower in human than in rodent adipose tissue, and adult humans have little brown adipose tissue. Nevertheless, β₃-adrenoceptors are expressed in human white as well as brown adipose tissue and in skeletal muscle, and they play a role in the regulation of energy balance and glucose homeostasis. It is difficult to identify β₃-adrenoceptor agonist drugs because the pharmacology of both β₃- and β₁-adrenoceptors can vary; near absolute selectivity is needed to avoid β_1/2-adrenoceptor-mediated side effects and selective agonists tend to have poor oral bioavailability. All weight loss is lipid and lean may actually increase, so reducing weight loss relative to energy loss. β₃-adrenoceptor agonists have a more rapid insulin-sensitising than anti-obesity effect, possibly because stimulation of lipid oxidation rapidly lowers intracellular long-chain fatty acyl CoA and diacylglycerol levels. This may deactivate those protein kinase C isoenzymes that inhibit insulin signalling.     

Anatomical differences in responsiveness to vasoconstrictors in the mesenteric veins from normal and portal hypertensive rats

The present study evaluates the effects of pre-hepatic portal hypertension, induced in rats by partial portal vein ligation, on the responsiveness of rostral (proximal) and caudal (distal) rings from the mesenteric vein. The anatomical origin of the sample influenced the response to vasoconstrictors in sham-operated animals, and this pattern of reactivity was specifically modified in portal-ligated rats. In veins from sham-operated rats, contraction induced by a submaximal concentration of KCl (60 mM) was greater in proximal than in distal rings. Vasopressin and 5-hydroxytryptamine contracted mainly distal rings, methoxamine showed a greater effect on proximal rings, and endothelin-1 and angiotensin-II contracted vein rings independently of their anatomical origin. In veins from portal hypertensive rats, responses to KCl (60 mM) were increased in distal rings, and all rings exhibited enhanced reactivity to vasopressin and 5-hydroxyptyptamine as well as attenuation of the response to methoxamine. Responses to endothelin-1 were decreased in proximal vein rings from portal hypertensive rats whereas responses to angiotensin-II were not influenced by the anatomical origin. Incubation with atropine, propranolol or indomethacin, did not modify the responses to vasopressin and 5-hydroxytryptamine in tissues from either sham-operated or portal hypertensive animals. Likewise, the hyporeactivity to methoxamine and endothelin-1 in rings from portal hypertensive rats persisted in the presence of the nitric oxide inhibitor N ^G-nitro-l-arginine methyl ester. These results suggest the physiological existence of anatomical differences in the responsiveness to vasoconstrictors throughout the mesenteric vein and that changes in the responsiveness of the mesenteric vein induced by portal hypertension are specific for each agonist and possibly result from individual variations at a receptor or post-receptor level.     

Intrinsic sympathomimetic activity of beta-adrenoceptor antagonists: down-regulation of cardiac beta 1- and beta 2-adrenoceptors

Prolonged treatment of cultured rat heart muscle cells containing β₁- and non-muscle cells containing β₂-adrenoceptors with β-adrenoceptor antagonists devoid of intrinsic sympathomimetic activity had no effect on β-adrenoceptor density. In contrast, antagonists with intrinsic sympathomimetic activity decreased β-adrenoceptor density and response (adenylate cyclase stimulation) in both heart muscle (β₁) and non-muscle cells (β₂) by a maximum of about 50%. An even larger down-regulation of β-adrenoceptors and loss of receptor-stimulated adenylate cyclase activity was induced by the full endogenous agonist, noradrenaline, with the β-adrenoceptors of heart muscle cells (β₁) being much more sensitive to the β₁-selective noradrenaline than the heart non-muscle cell β₂-adrenoceptors. When combined with noradrenaline, the antagonists with intrinsic sympathomimetic activity prevented the action of noradrenaline at both β₁- and β₂-adrenoceptors, thereby leading to an apparent up-regulation of receptor density and response. This apparent reversal from an agonist to an antagonist action was observed at much lower concentrations of noradrenaline at β₁- than at β₂-adrenoceptors. The data presented indicate that the β-adrenoceptor antagonists with intrinsic sympathomimetic activity, but not those without, upon prolonged treatment decrease the density and responsiveness of both β₁- and β₂-adrenoceptors in cultured rat heart cells. This suggests that the intrinsic sympathomimetic activity of these agents is not a subtype-selective component. Furthermore, the agonist and antagonist activity of these agents apparently depends on the concomitant presence of an endogenous full agonist and an its own affinity and that of the partial agonist for the β-adrenoceptor subtype.     

The effects of bromoacetylalprenololmenthane on the responses to isoprenaline of the left ventricle from normo-, prehyper- and hypertensive rats

1 We have studied the effects of bromoacetylalprenololmenthane (BAAM), a very slowly reversible β-adrenoceptor antagonist, on the responses of the left ventricle of 5 and 22 week old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) to isoprenaline. At 5 weeks the SHRs were prehypertensive and at 22 weeks they had hypertension-induced hypertrophy of the left ventricle. 2 The mean potency of isoprenaline on the left ventricle from 5 week old WKY was similar to that obtained on left ventricles from 22 week old WKY, and 5 and 22 week old SHRs. 3 Following treatment of the left ventricle with BAAM at 10^?6m for 30 min, there was a parallel rightward shift of the isoprenaline concentration–response curves. Treatment with a higher concentration of BAAM (10^?5m) caused non-parallel rightward shifts of the concentration–response curves, with a depression of the isoprenaline maximum responses. These data were used to derive affinity (K_A) values for isoprenaline. The mean isoprenaline K_A value on the left ventricle from 5 week old WKY was 2.44 × 10^?6m , and similar K_A values were obtained on the left ventricles from 22 week old WKY and 5 and 22 week old SHRs. On all the left ventricles tested, isoprenaline produced a half maximal response by occupying less than 1%, and a near maximal response by occupying about 5% of the available β₁-adrenoceptors. 4 This study has shown that there are no differences in the cardiac responses to isoprenaline at β₁-adrenoceptors, isoprenaline K_A values or the β₁-adrenoceptor reserve for isoprenaline on the SHR left ventricle in prehypertension or in the early stages of hypertension-induced hypertrophy.     

Combining inhaled glucocorticoids and long acting beta(2)-adrenoceptor agonists in asthma and COPD

Inhaled long-acting beta(2)-adrenoceptor agonists and glucocorticoids form the mainstay of maintenance treatment of asthma and chronic obstructive pulmonary disease (COPD), usually given as a combination inhaler. Most patients will have good asthma control if they comply with this therapy, although it is generally less effective in COPD. The traditional dogma has been that these agents act on distinct components of disease pathophysiology with beta(2) agonists acting on the bronchospastic component and glucocorticoids acting on the inflammatory component. Considerable evidence has emerged recently, however, to suggest that these two classes of agents interact at a molecular level. Understanding the mechanisms of these interactions may enable the development of new therapies for asthma and COPD.     

WAY 405, a new silent 5-HT (1A) receptor antagonist with low affinity for vascular alpha (1)-adrenoceptors

1 The effect of WAY 405 ((R)-N-(2-methyl-(4-indolyl-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide), a putative 5-HT(1A) receptor antagonist, on cardiovascular function was studied. 2 In anaesthetized rats, the i.v. injection of WAY 405 did not significantly modify basal heart rate nor blood pressure at doses of 1, 3, 10 and 30 microg kg(-1); while the antagonist dose dependently antagonized the 5-HT(1A) receptor agonist, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin)-induced hypotension and bradycardia. 3 WAY 405 antagonized noradrenaline-induced contraction in isolated arteries, with pK(B) values of 6.6+/-0.1, 6.5+/-0.1 and 6.5+/-0.1, for rat tail artery (alpha(1A)-adrenoceptors), rabbit aorta (alpha(1B)-adrenoceptors), and rat aorta (alpha(1D)-adrenoceptors) respectively. 4 The results show that in the control of blood pressure the new compound, WAY 405, behaves as a silent 5-HT(1A) receptor antagonist in the anaesthetized rat, also having low affinity for vascular alpha(1)-adrenoceptors.     

Suppression of histamine-induced tachypnoea in the rhesus monkey by sibenadet: no role for dopamine D2 receptors

1. Sibenadet (Viozan), a dual dopamine D(2)/beta(2)-adrenoceptor agonist, suppresses histamine-induced tachypnoea in the dog by activating dopamine D(2) receptors. We here compare the effects of sibenadet and formoterol, a selective beta(2)-adrenoceptor agonist, on histamine-induced tachypnoea in the rhesus monkey. 2. Anaesthetized, spontaneously breathing, rhesus monkeys were set up for measuring airways resistance, respiratory rate, blood pressure and heart rate. 3. Both sibenadet and formoterol administered by aerosol, induced inhibition of the bronchoconstrictor response to aerosolized methacholine accompanied by tachycardia. Sibenadet, but not formoterol, also reduced blood pressure. 4. Administration of histamine by inhalation induced tachypnoea which was accompanied by bronchoconstriction. Tachypnoea to histamine was suppressed by both sibenadet and formoterol at doses which manifest anti-bronchoconstrictor activity. These effects and the accompanying tachycardia but not the hypotension induced by sibenadet were abolished by pretreatment with propranolol. 5. The dopamine D(2) receptor agonist, quinagolide, did not suppress tachypnoea to histamine despite inducing a fall in blood pressure indicating activation of dopamine D(2) receptors. 6. Thus, both sibenadet and formoterol suppress histamine-evoked tachypnoea in the rhesus monkey. The effect arises exclusively through activation of beta(2)-adrenoceptors and probably reflects the anti-bronchoconstrictor effects of these agents. The results reveal a fundamental difference in the role of dopamine receptors in the airways of dog and rhesus monkey.     

Toxicity of cholesterol oxidation products to Caco-2 and HepG2 cells: modulatory effects of alpha- and gamma-tocopherol

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g ml(-1)) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (ic(50) value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.     

Hydrophobicity of β-adrenoceptor blocking agents: Study of correlations between retention in reversed-phase HPLC systems and octanol-water partition constants

The capacity factors (k′) of seven β-adrenoceptor blocking agents in six different reversed-phase HPLC systems have been determined. Octanol-aqueous buffer (pH 7.4) partition constants (P) for these blocking agents were also obtained. By using target factor analysis (TFA), good empirical correlations between the log k′ and log P were derived. The resulting hydrophobicity order agrees well with the metabolic elimination pathways of these drugs.     

beta(1)- and beta(2)-adrenoceptor responses in cardiomyocytes derived from human embryonic stem cells: comparison with failing and non-failing adult human heart

BACKGROUND AND PURPOSE: Characterization of human embryonic stem cell-derived cardiomyocytes (hESC-CM) in relation to adult myocytes is essential for their future use in transplantation or as a model system. The beta-adrenoceptor pathways, which are known to be effective early in hESC-CM development, are of major importance because of their control of rate and force of beating, arrhythmia generation and apoptosis/necrosis. We have therefore performed detailed pharmacological analysis of the beta-adrenoceptor responses in developing hESC-CM.EXPERIMENTAL APPROACH: hESC-CMs were differentiated from H7 ESCs and studied up to 79 days of differentiation. Rate of beating and time course of contraction and relaxation were measured in superfused preparations.KEY RESULTS: Responses to the mixed beta(1)- and beta(2)-adrenoceptor agonist isoprenaline were evident from day 10 to day 79. Stability of the responses during an application, for repeated applications on the same experimental day and over the time of development, was determined. Concentrations for half-maximal response (12.9 nM) were similar to those from adult human heart, but closer to those obtained from failing rather than normal ventricle. Acceleration of both contraction and relaxation was quantitatively similar to that in adult ventricular myocytes, as was sensitivity to muscarinic inhibition. Use of specific antagonists showed that both beta(1)- and beta(2)-adrenoceptors contributed to contractile responses, as seen with adult myocytes.CONCLUSIONS AND IMPLICATIONS: These data show the compatibility of hESC-CM with adult human myocardium in terms of beta-adrenoceptor response. The experiments described here also confirm the utility of the hESC-CM preparation for detailed pharmacological analysis.     

Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and beta2-adrenoceptor mechanisms

Background and purpose:

Picomolar concentrations of the β₃-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via β₂-adrenoceptors. Effects of BRL37344 and β₂-adrenoceptor agonists are compared.

Experimental approach:

Mouse soleus muscles were incubated with 2-deoxy[1-¹⁴C]-glucose, [1-¹⁴C]-palmitate or [2-¹⁴C]-pyruvate, and BRL37344, β₂-adrenoceptor agonists and selective β-adrenoceptor antagonists. Formation of 2-deoxy[1-¹⁴C]-glucose-6-phosphate or ¹⁴CO₂ was measured. 2-Deoxy[1-¹⁴C]-glucose uptake and β-adrenoceptor mRNA were measured in C2C12 cells.

Key results:

10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33–54%. The effect of BRL37344 was prevented by 1 μM atenolol but not by 300 nM CGP20712A or IC3118551, or 1 μM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 st4mulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only β₂-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2-deoxyglucose uptake and the effect of clenbuterol was not significant.

Conclusions and implications:

Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via β₂-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake.     

Synthesis of new pyrrolo[2, 3-b]pyridines as a potent inhibitor of tumour necrosis factor alpha

The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and 1L-1beta. Accordingly, new pyrrolo[2, 3-]pyridine derivatives 5a-d were prepared from 2-amino-3-cyanopyrroles 3a-d via the intermediate propenylaminopyrroles 4a-d. Then the compounds 5a-d were tested for their ability to inhibit the production of TNF-alpha in vivo in rats. The most potent compounds 5a and 5b possess enhanced ability to inhibit the production of TNF-alpha stimulated with bacterial lipopolysaccharide.