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P Molenaar

Publications and source records attributed to P Molenaar.

At least 19 recordsLinked to original sources
PubMed2001

Abolition of (-)-CGP 12177-evoked cardiostimulation in double beta1/beta2-adrenoceptor knockout mice. Obligatory role of beta1-adrenoceptors for putative beta4-adrenoceptor pharmacology.

Some beta1- and beta2-adrenoceptor-blocking agents, such as (-)-CGP 12177, cause cardiostimulant effects at concentrations considerably higher than those that antagonise the effects of catecholamines. The cardiostimulant effects of these non-conventional partial agonists are relatively resistant to blockade by (-)-propranolol and have been proposed to be mediated through putative beta4-adrenoceptors or through atypical states of either beta1- or beta2-adrenoceptors. We investigated the effects of (-)-CGP 12177 on sinoatrial rate and left atrial contractile force as well as the ventricular binding of (-)-[3H]CGP 12177 in tissues from wild-type, beta2-adrenoceptor knockout and beta1/beta2-adrenoceptor double knockout mice. The cardiostimulant effects of (-)-CGP 12177 were present in wild-type and beta2-adrenoceptor knockout mice but were absent in beta1/beta2-adrenoceptor double knockout mice. Thus, the presence of beta1-adrenoceptors is obligatory for the cardiostimulant effects of (-)-CGP 12177. It appears therefore that an atypical state of the beta1-adrenoceptor contributes to the mediation of the cardiostimulant effects induced by non-conventional partial agonists. Ventricular beta1- and beta2-adrenoceptors, labelled in wild-type with a K(D) approximately 0.5 nmol/l (approximately 16 fmol/mg protein), were absent in beta1/beta2-adrenoceptor double knockout mice. However, a high density binding site (approximately 154-391 fmol/mg protein) that did not saturate completely (K(D) approximately 80-200 nM) was labelled by (-)-[3H]CGP 12177 in the three groups of mice, being distinct from beta1- and beta2-adrenoceptors, as well as from the site mediating the agonist effects of (-)-CGP 12177.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Cardiostimulant effects of urotensin-II in human heart in vitro.

The effects of the recently identified human peptide urotensin-II (hU-II) were investigated on human cardiac muscle contractility and coronary artery tone. In right atrial trabeculae from non-failing hearts, hU-II caused a concentration-dependent increase in contractile force (pEC(50)=9.5+/-0.1; E(max)=31.3+/-4.8% compared to 9.25 mM Ca(2+); n=9) with no change in contraction duration. In right ventricular trabeculae from explanted hearts, 20 nM hU-II caused a small increase in contractile force (7.8+/-1.4% compared to 9.25 mM Ca(2+); n=3/6 tissues from 2 out of 4 patients). The peptide caused arrhythmic contractions in 3/26 right atrial trabeculae from 3/9 patients in an experimental model of arrhythmia and therefore has less potential to cause arrhythmias than ET-1. hU-II (20 nM) increased tone (17.9% of the response to 90 mM KCI) in 7/7 tissues from 1 patient, with no response detected in 8/8 tissues from 2 patients. hU-II is a potent cardiac stimulant with low efficacy.

Arrhythmias, Cardiac↗
PubMed2001

Murine ventricular L-type Ca(2+) current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor.

1. The functional coupling of beta(2)-adrenoceptors (beta(2)-ARs) to murine L-type Ca(2+) current (I(Ca(L))) was investigated with two different approaches. The beta(2)-AR signalling cascade was activated either with the beta(2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta(2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta(2)-ARs). Ca(2+) and Ba(2+) currents were recorded in the whole-cell and cell-attached configuration of the patch-clamp technique, respectively. 2. Zinterol (10 microM) significantly increased I(Ca(L)) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta(1)-AR subtype, since it was blocked by the beta(1)-AR selective antagonist CGP 20712A (300 nM). The beta(2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I(Ca(L)) to zinterol. 3. In myocytes with beta(2)-AR overexpression I(Ca(L)) was not stimulated by the activated signalling cascade. On the contrary, I(Ca(L)) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I(Ca(L)). The beta(2)-AR inverse agonist ICI 118,551 did not further decrease I(Ca(L)). PTX-treatment increased current amplitude to values found in control myocytes. 4. In conclusion, there is no evidence for beta(2)-AR mediated increases of I(Ca(L)) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta(2)-AR responses to zinterol, but augments beta(1)-AR mediated increases of I(Ca(L)). In the mouse model of beta(2)-AR overexpression I(Ca(L)) is reduced due to tonic activation of Gi-proteins.

Adrenergic beta-1 Receptor Agonists↗
PubMed2000

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of Fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein.

BACKGROUND: In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. METHODS AND RESULTS: Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 21/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC(50) [mol/L] NE 5.5+/-0.1, n=12; EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC(50) [mol/L] NE 5.8+/-0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC(50) [mol/L] NE 5.7+/-0.2; EPI 5.8+/-0.1). Ventricular membranes from Fallot infants, labeled with (-)-[(125)I]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. CONCLUSIONS: Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

Calcium-Binding Proteins↗
PubMed2000

The human heart endothelin system: ET-1 synthesis, storage, release and effect.

Endothelin-1 (ET-1) is synthesized, stored and released in the human heart. On release, ET-1 has the potential to activate specific receptors in a paracrine manner to modify heart function directly. Two binding sites corresponding to endothelin ETA and ETB receptors are localized to human heart. Contractility is modified by ETA receptors in the left ventricle and by ETA and ETB receptors, and a receptor that is possibly a different conformation of ETA and/or ETB receptors, in the right atrium. The endothelin system might directly support the failing heart but might also contribute to its pathology, providing a basis for the therapeutic use of endothelin receptor antagonists.

Animals↗
PubMed2000

Contribution of beta-adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wildtype and beta(3)-adrenoceptor knockout mice.

The smooth muscle relaxant responses to the mixed beta(3)-, putative beta(4)-adrenoceptor agonist, (-)-CGP 12177 in rat colon are partially resistant to blockade by the beta(3)-adrenoceptor antagonist SR59230A suggesting involvement of beta(3)- and putative beta(4)-adrenoceptors. We now investigated the function of the putative beta(4)-adrenoceptor and other beta-adrenoceptor subtypes in the colon, oesophagus and ureter of wildtype (WT) and beta(3)-adrenoceptor knockout (beta(3)KO) mice. (-)-Noradrenaline and (-)-adrenaline relaxed KCl (30 mM)-precontracted colon mostly through beta(1)-and beta(3)-adrenoceptors to a similar extent and to a minor extent through beta(2)-adrenoceptors. In colon from beta(3)KO mice, (-)-noradrenaline was as potent as in WT mice but the effects were mediated entirely through beta(1)-adrenoceptors. (-)-CGP 12177 relaxed colon from beta(3)KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (-)-noradrenaline and increase for (-)-CGP 12177 indicate compensatory increases in beta(1)- and putative beta(4)-adrenoceptor function in beta(3)KO mice. In oesophagi precontracted with 1 microM carbachol, (-)-noradrenaline caused relaxation mainly through beta(1)-and beta(3)-adrenoceptors. (-)-CGP 12177 (2 microM) relaxed oesophagi from WT by 61.4+/-5.1% and beta(3)KO by 67.3+/-10.1% of the (-)-isoprenaline-evoked relaxation, consistent with mediation through putative beta(4)-adrenoceptors. In ureter, (-)-CGP 12177 (2 microM) reduced pacemaker activity by 31.1+/-2.3% in WT and 31.3+/-7. 5% in beta(3)KO, consistent with mediation through putative beta(4)-adrenoceptors. Relaxation of mouse colon and oesophagus by catecholamines are mediated through beta(1)- and beta(3)-adrenoceptors in WT. The putative beta(4)-adrenoceptor, which presumably is an atypical state of the beta(1)-adrenoceptor, mediates the effects of (-)-CGP 12177 in colon, oesophagus and ureter.

Adrenergic beta-1 Receptor Antagonists↗
PubMed2000

Quantitative analysis of vascular to cardiac selectivity of L- and T-type voltage-operated calcium channel antagonists in human tissues.

1. Classical L-type voltage-operated calcium channel (VOCC) antagonists dilate blood vessels, depress myocardial contractility and slow cardiac conduction. 2. We compared four L-type VOCC antagonists and a novel tetralol derivative, mibefradil, reportedly 10-fold more selective for T- (transient) over L-type VOCC in two in vitro assays of human tissue, namely isolated small arteries from the aortic vasa vasorum in a myograph and right atrial trabeculae muscle under isometric force conditions. 3. In arteries contracted with K+ (62 mmol/L), the relaxation pIC50 values for the VOCC antagonists felodipine, nifedipine, amlodipine, verapamil and mibefradil were 8.30, 7.78, 6.64, 6.26 and 6.22, respectively. In atrial trabeculae, the pIC50 values to inhibit the inotropic response to a submaximal concentration of isoprenaline (6 nmol/L) for felodipine, nifedipine, verapamil, amlodipine and mibefradil were 7.21, 6.95, 6.91, 5.94 and 4.61, respectively. 4. Taking the anti-log (pIC50 vessel-pIC50 atrium) the vascular relaxation to cardiac depression potency ratios for mibefradil, felodipine, nifedipine, amlodipine and verapamil were 41, 12, 7, 5 and 0.22, respectively. 5. We conclude that, in human tissue assays, perhaps T- over L-type VOCC selectivity confers the most favourable vascular selectivity on mibefradil. Alternatively, splice variants of L-type VOCC in the vasculature (CaV1.2b) may be more sensitive to mibefradil than the splice variants in the heart (CaV1.2a).

Aorta↗
PubMed2000

Contractile and arrhythmic effects of endothelin receptor agonists in human heart in vitro: blockade with SB 209670.

It is known that binding sites with endothelin(A) (ET)(A) and ET(B) receptor characteristics coexist in human heart but little is known about the receptors that mediate cardiostimulant effects of ET receptor agonists or their consequences. Functional studies were performed on isolated human cardiac tissues. The maximal positive inotropic effects of ET-1 were right atrium > left atrium = right ventricle. The rank order of potencies of agonists in right atrium was sarafotoxin S6c > ET-1 = ET-2 > or = ET-3. The ET(A) receptor-selective compounds BQ123 (10 microM) and A-127722 (1 microM) only slightly blocked (<0.5 log-unit shift) the effects of lower concentrations of ET-1, and the ET(B) receptor antagonist Ro46-8443 (10 microM) did not cause blockade. SB 209670 caused concentration-dependent rightward shifts of ET-1 and sarafotoxin S6c concentration-effect curves with Schild slopes not different from one and affinities (-logM K(B)) of 7.0 and 7.9, respectively. ET-1 caused arrhythmic contractions in right atrial trabeculae that were prevented by 10 microM SB 209670 but not 10 microM BQ123 or 1 microM A-127722, precluding ET(A) receptors. ET-1 caused a higher incidence of arrhythmic contractions in tissues taken from patients treated with beta-blockers before surgery than in tissues from non-beta blocker-treated patients. Sarafotoxin S6c produced arrhythmias that were prevented by SB 209670. The positive inotropic effects of ET-1 in human right atrial myocardium are mediated mostly by a non-ET(A), non-ET(B) receptor. Ventricular inotropic ET receptors differ from atrial inotropic ET receptors. ET-1 induced arrhythmic contractions in human atria do not appear to be mediated by an ET(A) receptor.

Adult↗
PubMed1999

Putative beta 4-adrenoceptors in rat ventricle mediate increases in contractile force and cell Ca2+: comparison with atrial receptors and relationship to (-)-[3H]-CGP 12177 binding.

1. We identified putative beta4-adrenoceptors by radioligand binding, measured increases in ventricular contractile force by (-)-CGP 12177 and (+/-)-cyanopindolol and demonstrated increased Ca2+ transients by (-)-CGP 12177 in rat cardiomyocytes. 2. (-)-[3H]-CGP 12177 labelled 13 - 22 fmol mg-1 protein ventricular beta1, beta2-adrenoceptors (pKD approximately 9.0) and 50 - 90 fmol mg-1 protein putative beta4-adrenoceptors (pKD approximately 7.3). The affinity values (pKi) for (beta1,beta2-) and putative beta4-adrenoceptors, estimated from binding inhibition, were (-)-propranolol 8.4, 5.7; (-)-bupranolol 9.7, 5.8; (+/-)-cyanopindolol 10.0,7.4. 3. In left ventricular papillary muscle, in the presence of 30 microM 3-isobutyl-1-methylxanthine, (-)-CGP 12177 and (+/-)-cyanopindolol caused positive inotropic effects, (pEC50, (-)-CGP 12177, 7.6; (+/-)-cyanopindolol, 7.0) which were antagonized by (-)-bupranolol (pKB 6.7 - 7.0) and (-)-CGP 20712A (pKB 6.3 - 6.6). The cardiostimulant effects of (-)-CGP 12177 in papillary muscle, left and right atrium were antagonized by (+/-)-cyanopindolol (pKP 7.0 - 7.4). 4. (-)-CGP 12177 (1 microM) in the presence of 200 nM (-)-propranolol increased Ca2+ transient amplitude by 56% in atrial myocytes, but only caused a marginal increase in ventricular myocytes. In the presence of 1 microM 3-isobutyl-1-methylxanthine and 200 nM (-)-propranolol, 1 microM (-)-CGP 12177 caused a 73% increase in Ca2+ transient amplitude in ventricular myocytes. (-)-CGP 12177 elicited arrhythmic transients in some atrial and ventricular myocytes. 5. Probably by preventing cyclic AMP hydrolysis, 3-isobutyl-1-methylxanthine facilitates the inotropic function of ventricular putative beta4-adrenoceptors, suggesting coupling to Gs protein-adenylyl cyclase. The receptor-mediated increases in contractile force are related to increases of Ca2+ in atrial and ventricular myocytes. The agreement of binding affinities of agonists with cardiostimulant potencies is consistent with mediation through putative beta4-adrenoceptors labelled with (-)-[3H]-CGP 12177.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Validity of (-)-[3H]-CGP 12177A as a radioligand for the 'putative beta4-adrenoceptor' in rat atrium.

1. We have recently suggested the existence in the heart of a 'putative beta4-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta1- and beta2-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[3H]-CGP 12177A ([-]-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[3H]-CGP 12177A for this purpose for two reasons, because it is a non-conventional partial agonist and also because it is a hydrophilic radioligand. 2. Increasing concentrations of (-)-[3H]-CGP 12177A, in the absence or presence of 20 microM (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta1- and beta2-adrenoceptors (pKD 9.4+/-0.1, Bmax 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta4-adrenoceptor' (pKD 7.5+/-0.1, Bmax 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta1- and beta2-adrenoceptors, (-)-[3H]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pKD 7.6+/-0.1, Bmax 50.8+/-7.4 fmol mg(-1) protein). 3. The non-conventional partial agonists (-)-CGP 12177A (pKi 7.3+/-0.2), (+/-)-cyanopindolol (pKi 7.6+/-0.2), [-]-pindolol (pK1 6.6+/-0.1) and (+/-)-carazolol (pKi 7.2+/-0.2) and the antagonist (-)-bupranolol (pKi 6.6+/-0.2), all competed for (-)-[3H]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta4-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4. The catecholamines competed with (-)-[3H]-CGP 12177A at the 'putative beta 4-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pKiH 6.3+/-0.3, pKiL 3.5+/-0.1), (-)-adrenaline (pKiH 6.5+/-0.2, pKiL 2.9+/-0.1), (-)-isoprenaline (pKiH 6.2+/-0.5, pKiL 3.4+/-0.1), (+)-isoprenaline (pKi< 1.7), (-)-RO363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy++ +)-2-propranol)oxalate, pKi 5.5+/-0.1). 5. The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta4-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[3H]-CGP 12177A for one receptor state in the absence (pKi 7.3+/-0.2) or presence of GTP (pKi 7.3+/-0.2). (-)-Isoprenaline competed with (-)-[3H]-CGP 12177A for two states in the absence (pKiH 6.6+/-0.3, pKiL 3.5+/-0.1; % H 25+/-7) or presence of GTP (pKiH 6.2+/-0.5, pKiL 3.4+/-0.1; % H 37+/-6). In contrast, at beta1-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6. The specificity of binding to the 'putative beta 4-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta 3-adrenoceptor agonists, BRL 37344 ((RR+SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy-ethyl]amino]propyl]phenoxy]acetic acid, 6 microM), SR 58611A (ethyl{(7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl2-yloxy} acetate hydrochloride, 6 microM), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)-ethan-1-ol, 60 microM), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 microM) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 microM) caused less than 22% inhibition of (-)[3H]-CGP 12177A binding in the presence of 500 nM (-)propranolol. Histamine (1mM), atropine (1 microM), phentolamine (10 microM), 5-HT (100 microM) and the 5-HT4 receptor antagonist SB 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate, 10nM) caused less than 26% inhibition of binding. 7.Non-conventional partial agonists, the antagonist (-)bupranolol and catecholamines all competed for (-)[3H]-CGP 12177A binding in the absence of (-)propranolol at beta1-adrenoceptors, with affinities (pKi) ranging from 1.6-3.6 log orders greater than at the 'putative beta 4-adrenoceptor'. 8.We have established and validated a radioligand binding assay in rat atrium for the 'putative beta 4-adrenoceptor' which is distinct from beta1-, beta2- and beta 3-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta 4-adrenoceptor'.

Adrenergic beta-Agonists↗
PubMed1998

Human vascular to cardiac tissue selectivity of L- and T-type calcium channel antagonists.

1. Voltage-operated calcium channel (VOCC) antagonists are effective antihypertensive and antianginal agents but they also depress myocardial contractility. 2. We compared four L-type calcium channel antagonists, felodipine, nifedipine, amlodipine and verapamil and a relatively T-type selective calcium channel antagonist, mibefradil, on human and rat isolated tissue assays to determine their functional vascular to cardiac tissue selectivity (V/C) ratio. 3. The V/C ratio was calculated as the ratio of the IC50 value of the antagonist that reduced (by 50%) submaximally contracted (K+ 62 mM) human small arteries from the aortic vasa vasorum (vascular, V) mounted in a myograph and the IC50 value of the antagonist that reduced (-)-isoprenaline (6 nM) submaximally stimulated human right atrial trabeculae muscle (cardiac, C) mounted in organ chambers. 4. The average pIC50 values (-log IC50 M) for the human vascular preparations were felodipine 8.30, nifedipine 7.78, amlodipine 6.64, verapamil 6.26 and mibefradil 6.22. The average pIC50 values for the cardiac muscle were felodipine 7.21, nifedipine 6.95, verapamil 6.91, amlodipine 5.94, and mibefradil 4.61. 5. The V/C ratio calculated as antilog [pIC50V-pIC50C] is thus mibefradil 41, felodipine 12, nifedipine 7, amlodipine 5 and verapamil 0.2. 6. In rat small mesenteric arteries the pIC50 values for the five drugs were similar to the values for human vasa vasorum arteries contracted by K+ 62 mM. However for methoxamine (10 microM) contraction in the rat arteries the pIC50 values were lower for felodipine 7.24 and nifedipine 6.23, but similar for verapamil 6.13, amlodipine 6.28 and mibefradil 5.91. 7. In conclusion. in the human tissue assays, the putative T-channel antagonist mibefradil shows the highest vascular to cardiac selectivity ratio; some 3 fold higher than the dihydropyridine, felodipine, and some 200 fold more vascular selective than the phenylalkylamine, verapamil. This favourable vascular to cardiac selectivity for mibefradil, from a new chemical class of VOCC antagonist, may be explained by its putative T-channel selectivity.

Animals↗
PubMed1998

(-)-CGP 12177 causes cardiostimulation and binds to cardiac putative beta 4-adrenoceptors in both wild-type and beta 3-adrenoceptor knockout mice.

Some blockers of beta1- and beta2-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta4-adrenoceptor) that resembles the beta3-adrenoceptor. It is likely but not proven that the putative beta4-adrenoceptor is genetically distinct from the beta3-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta3-adrenoceptor gene (beta3 KO). (-)-CGP 12177, a beta1- and beta2-adrenoceptor blocker that causes agonist effects through both beta3-adrenoceptors and cardiac putative beta4-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta3 KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 microM) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta3 KO. (-)-[3H]CGP 12177 labeled a similar density of the putative beta4-adrenoceptor in ventricular membranes from the hearts of both WT (Bmax = 52 fmol/mg protein) and beta3 KO (Bmax = 53 fmol/mg protein) mice. The affinity of (-)-[3H]CGP 12177 for the cardiac putative beta4-adrenoceptor was not different between WT (Kd = 46 nM) and beta3 KO (Kd= 40 nM). These results provide definitive evidence that the cardiac putative beta4-adrenoceptor is distinct from the beta3-adrenoceptor.

Adrenergic beta-Agonists↗
PubMed1998

The contribution of classical (beta1/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities.

The role of beta3- and other putative atypical beta-adrenoceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta3-adrenoceptor (beta3AR) agonists with varying intrinsic activities and selectivities for human cloned betaAR subtypes. The ability to demonstrate beta1/2AR antagonist-insensitive (beta3 or other atypical betaAR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta3AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta1/2AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta3AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta1/2AR antagonism, despite it having very low efficacies at cloned beta1- and beta2ARs. A component of the response to another phenylethanolamine selective beta3AR agonist (SB-215691) was insensitive to beta1/2AR antagonism in some experiments. Because no [corrected] novel aryloxypropanolamine had a beta1/2AR antagonist-insensitive inotropic effect, these results establish more firmly that beta3ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta4ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned betaARs which betaARs will mediate responses to agonists in tissues that have a high number of beta1- and beta2ARs or a low number of beta3ARs.

Adipose Tissue↗
PubMed1997

Modulation of human cardiac function through 4 beta-adrenoceptor populations.

In human heart there is now evidence for the involvement of four beta-adrenoceptor populations, three identical to the recombinant beta 1-, beta 2- and beta 3-adrenoceptors, and a fourth as yet uncloned putative beta-adrenoceptor population, which we designate provisionally as the cardiac putative beta 4-adrenoceptor. This review described novel features of beta-adrenoceptors as modulators of cardiac systolic and diastolic function. We also discuss evidence for modulation by unoccupied beta 1- and beta 2-adrenoceptors. Human cardiac and recombinant beta 1- and beta 2-adrenoceptors are both mainly coupled to adenylyl cyclase through Gs protein, the latter more tightly than the former. Activation of both human beta 1- and beta 2-adrenoceptors not only increases cardiac force during systole but also hastens relaxation through cyclic AMP-dependent phosphorylation of phospholamban and troponin 1, thereby facilitating diastolic function. Furthermore, both beta 1 and beta 2-adrenoceptors can mediate experimental arrhythmias in human cardiac preparations elicited by noradrenaline and adrenaline. Human ventricular beta 3-adrenoceptors appear to be coupled to a pertussis toxin-sensitive protein (Gi?). beta 3-Adrenoceptor-selective agonists shorten the action potential and cause cardiodepression, suggesting direct coupling of a Gi protein to a K+ channel. In a variety of species, including man, cardiac putative beta 4-adrenoceptors mediate cardiostimulant effects of non-conventional partial agonists, i.e. high affinity beta 1- and beta 2-adrenoceptor blockers that cause agonist effects at concentrations considerably higher than those that block these receptors. Putative beta 4-adrenoceptors appear to be coupled positively to a cyclic AMP-dependent cascade and can undergo some desensitisation.

Atrial Function↗
PubMed1997

Effects of (-)-RO363 at human atrial beta-adrenoceptor subtypes, the human cloned beta 3-adrenoceptor and rodent intestinal beta 3-adrenoceptors.

1. Chronic treatment of patients with beta-blockers causes atrial inotropic hyperresponsiveness through beta 2-adrenoceptors, 5-HT4 receptors and H2-receptors but apparently not through beta 1-adrenoceptors despite data claiming an increased beta 1-adrenoceptor density from homogenate binding studies. We have addressed the question of beta 1-adrenoceptor sensitivity by determining the inotropic potency and intrinsic activity of the beta 1-adrenoceptor selective partial agonist (-)-RO363 and by carrying out both homogenate binding and quantitative beta-adrenoceptor autoradiography in atria obtained from patients treated or not treated with beta-blockers. In the course of the experiments it became apparent that (-)-RO363 also may cause agonistic effects through the third atrial beta-adrenoceptor. To assess whether (-)-RO363 also caused agonistic effects through beta 3-adrenoceptors we studied its relaxant effects in rat colon and guinea-pig ileum, as well as receptor binding and adenylyl cyclase stimulation of chinese hamster ovary (CHO) cells expressing human beta 3-adrenoceptors. 2. beta-Adrenoceptors were labelled with (-)-[125I]-cyanopindolol. The density of both beta 1- and beta 2-adrenoceptors was unchanged in the 2 groups, as assessed with both quantitative receptor autoradiography and homogenate binding. The affinities of (-)-RO363 for beta 1-adrenoceptors (pKi = 8.0-7.7) and beta 2-adrenoceptors (pKi = 6.1-5.8) were not significantly different in the two groups. 3. (-)-RO363 increased atrial force with a pEC50 of 8.2 (beta-blocker treated) and 8.0 (non-beta-blocker treated) and intrinsic activity with respect to (-)-isoprenaline of 0.80 (beta-blocker treated) and 0.54 (non-beta-blocker treated) (P < 0.001) and with respect to Ca2+ (7 mM) of 0.65 (beta-blocker treated) and 0.45 (non-beta-blocker treated) (P < 0.01). The effects of (-)-RO363 were resistant to antagonism by the beta 2-adrenoceptor antagonist, ICI 118,551 (50 nM). The effects of 0.3-10 nM (-)-RO363 were antagonized by 3-10 nM of the beta 1-adrenoceptor selective antagonist CGP 20712A. The effects of 20-1000 nM (-)-RO363 were partially resistant to antagonism by 30-300 nM CGP 20712A. 4. (-)-RO363 relaxed the rat colon, partially precontracted by 30 mM KCl, with an intrinsic activity of 0.97 compared to (-)-isoprenaline. The concentration-effect curve to (-)-RO363 revealed 2 components, one antagonized by (-)-propranolol (200 nM) with pEC50 = 8.5 and fraction 0.66, the other resistant to (-)-propranolol (200 nM) with pEC50 = 5.6 and fraction 0.34 of maximal relaxation. 5. (-)-RO363 relaxed the longitudinal muscle of guinea-pig ileum, precontracted by 0.5 microM histamine, with intrinsic activity of 1.0 compared to (-)-isoprenaline and through 2 components, one antagonized by (-)-propranolol (200 nM) with pEC50 = 8.7 and fraction 0.67, the other resistant to (-)-propranolol with pEC50 = 4.9 and fraction 0.33 of maximal relaxation. 6. (-)-RO363 stimulated the adenylyl cyclase of CHO cells expressing human beta 3-adrenoceptors with pEC50 = 5.5 and intrinsic activity 0.74 with respect to (-)-isoprenaline (pEC50 = 5.9). (-)-RO363 competed for binding with [125I]-cyanopindolol at human beta 3-adrenoceptors transfected into CHO cells with pKi = 4.5. (-)-Isoprenaline (pKi = 5.2) and (-)-CGP 12177A (pKi = 6.1) also competed for binding at human beta 3-adrenoceptors. 7. We conclude that under conditions used in this study, (-)-RO363 is a potent partial agonist for human beta 1- and beta 3-adrenoceptors and appears also to activate the third human atrial beta-adrenoceptor. (-)-RO363 relaxes mammalian gut through both beta 1- and beta 3-adrenoceptors. (-)-RO363, used as a beta 1-adrenoceptor selective tool, confirms previous findings with (-)-noradrenaline that beta 1-adrenoceptor-mediated atrial effects are only slightly enhanced by chronic treatment of patients with beta-blockers. Chronic treatment with

Adenylyl Cyclases↗
PubMed1996

Changes in sensitivity of 5-HT receptor mediated functional responses in the rat oesophagus, fundus and jejunum following chronic infusion with 5-hydroxytryptamine.

The effects of chronic infusion with 5-hydroxytryptamine (5-HT, 75 micrograms/kg per hour) for 5 or 10 days in vivo on the responses of rat oesophagus, fundus and jejunum to 5-HT and partial 5-HT receptor agonists in vitro were investigated. In the rat oesophagus, chronic treatment produced rightward shifts of the 5-HT4 receptor-mediated concentration-effect curves to 5-HT (dose-ratio = 3.8, day 5 and 2.8, day 10) and SC 53116 (1-S,8-S)-4-amino-5-chloro-N-[(hexahydro-1H-pyrrolizin-1-yl) methyl]-2-methoxy-benzamide hydrochloride, dose-ratio = 7.1, day 5 and 8.9, day 10) as compared to control tissues. The maximum effect of 5-HT and SC 53116 in rat oesophagus was reduced following the 10 day treatment. The 5-HT2B receptor-mediated contractile effect of 5-HT on rat fundus from treated animals responded with a significantly reduced potency (dose-ratio = 4.1, 5-day; 4.2, 10-day) compared to control tissues. The maximum response to 5-HT was reduced in tissues from animals treated for 10 days. The concentration-effect curve to the partial agonist 1-(3-chlorophenyl)-piperazine dihydrochloride (mCPP) was shifted to the right in fundic tissue from treated animals (dose-ratio = 2.5, 5-day; 2.8, 10-day) compared to control tissues. The maximum response to mCPP was also reduced in tissues from 5-HT treated animals. It has recently been shown in a preliminary characterisation that in rat jejunum, 5-HT produces a biphasic concentration-effect curve which is mediated by a putative 5-ht7 (first phase) and 5-HT3 (second phase) receptor mechanism. In the present study 5-HT produced a control biphasic concentration-effect curve in rat jejunum with a pEC50-1 value of 8.5 +/- 3.5 and a pEC50-2 value of 5.9 +/- 0.3 and maximum response values of 43.8 (32.5-55.0)% (Emax1) and 65.3 (41.7-88.9)% (Emax 2) of the response to acetylcholine. In jejunal tissues from treated animals, 5-HT produced its contractile effect in vitro with a 3.7 and 2.8 fold (5-day) and a 1.3 and 1.4 fold (10-day) rightward shift of the first and second phase respectively of the control concentration-effect curve to 5-HT. The maximum response produced by 5-HT in the first phase in jejunal tissues from animals treated for 10 days was significantly reduced by 49.1%. These findings represent the first report that chronic infusion of 5-HT produces a residual desensitisation of the 5-HT4, 5-HT2B, and putative 5-ht7/5-HT3 receptor-mediated responses in rat oesophagus, fundus and jejunum respectively when measured in vitro.

Animals↗
PubMed1996

Chronic (-)-isoprenaline infusion down-regulates beta 1- and beta 2-adrenoceptors but does not transregulate muscarinic cholinoceptors in rat heart.

Regulation of beta-adrenoceptor (beta-ar) subtypes and transregulation of muscarinic cholinoceptors (mAchr) was examined in regions of rat heart after chronic infusion of (-)-isoprenaline (450 micrograms/kg per hour) for 14 days. Following (-)-isoprenaline infusion systolic blood pressure was reduced for 10 days but then gradually returned to control levels, whereas heart rate was increased for 7 days before declining to a level significantly above control. Heart weight to body weight ratio was increased in (-)-isoprenaline treated rats. beta-ar subtype densities were measured by quantitative autoradiography with [125I]-cyanopindolol (CYP) in sinoatrial node (SA), atrioventricular node (AV), bundle of His (BH), left (LB) and right (RB) bundle branches, interventricular (IVS) and interatrial (IAS) septa, right atria (RA), apex (AX) and mitral valve (MV). beta 1-ars were reduced by 59.1-74.2% in the AV conducting regions, 53.4% in the SA node and 43.3-53.4% in myocardial areas, beta 2-ars were markedly reduced in myocardial regions (93.2-98.5%) and in pacemaker and conducting regions (87.7-97.8%). No changes in mAchr densities measured using [3H]-N-methyl scopolamine (NMS) occurred in the AV node, BH, LB, RB, IVS and IAS following (-)-isoprenaline infusion. Densities of beta 1- and beta 2-ars and mAchrs were also measured in ventricular homogenates from control and (-)-isoprenaline treated animals. beta-ar levels were significantly reduced (P < 0.05) in treated animals and the ratio of beta 1- to beta 2-ars increased after treatment. mAchr density in ventricular homogenates measured using either [3H]-NMS or [3H]-quinuclidinyl [phenyl-4-3H]benzilate (QNB) was unchanged. Homogenates of left and right ventricle also showed no change using [3H]-NMS. Organ bath studies were used to investigate the effect of (-)-isoprenaline infusion on negative inotropic and chronotropic effects of the non-selective muscarinic receptor agonist bethanechol in left and right atria, respectively. Lower concentrations of bethanechol (3 x 10(-10) to 10(-6) M) produced a negative inotropic response in isolated electrically driven left atria from (-)-isoprenaline treated rats, but not from control rats, with the slope of the curves being significantly different between groups (ANCOVA, P = 0.037). At concentrations of bethanechol from 10(-6) to 3 x 10(-4) M the negative inotropic response was not changed between (-)-isoprenaline treated and control animals. Bethanechol also produced a negative chronotropic response at lower concentrations (10(-10) to 10(-6) M) in (-)-isoprenaline treated rats, but not in controls. A second, steeper phase of the negative chronotropic response occurred at concentrations of bethanechol greater than 10(-6) M and was also seen in control rats. Expression of M2 (cardiac) mAchrs (m2Achr) in left and right ventricular tissues measured using a quantitative non-competitive polymerase chain reaction (PCR) assay showed a significant (P = 0.001) 28.5% increase in expression in left ventricle and a significant (P = 0.003) 21.5% decrease in expression in right ventricle after (-)-isoprenaline treatment, compared to controls. There was no significant difference in total ventricular m2Achr expression between the two groups of rats. The results suggest that chronic beta-ar stimulation down-regulates both beta 1- and beta 2-ars, and appears to differentially transregulate m2Achr expression, but not mAchr protein. Following (-)-isoprenaline infusion, muscarinic receptor mediated responses were sensitised, with no change in receptor densities, suggesting changes occur in the cell signalling system beyond the level of the receptor.

Animals↗