Biomedical subjects

A J Kaumann

Publications and source records attributed to A J Kaumann.

At least 19 recordsLinked to original sources

PubMed2001

5HT4(a) and 5-HT4(b) receptors have nearly identical pharmacology and are both expressed in human atrium and ventricle.

5-Hydroxytryptamine (5-HT) increases human heart rate and atrial contractile force and hastens atrial relaxation through 5-HT4 receptors. Moreover, 5-HT may be arrhythmogenic and give rise to atrial fibrillation. It is not clear which splice variant(s) of the 5-HT4 receptor is expressed and mediates these effects of 5-HT in the human heart. Previous studies have indicated different pharmacological properties of 5-HT4 receptors in human heart and mouse colliculi neurones, possibly due to expression of different splice variants. We therefore cloned the human 5-HT4(b) receptor and compared its pharmacological properties with those of the cloned human 5-HT4(a) receptor and searched for the corresponding mRNA in human tissues. The primary structures of the two human 5-HT4 receptor splice variants are identical except for divergent C-terminal tails of 28 and 29 amino acids in the 5-HT4(a) and 5-HT4(b) receptors, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that both variants were coexpressed in various tissues, including cardiac atrium and ventricle. Additional bands suggested the presence of more than two human 5-HT4 receptor splice variants. With cloned receptors stably expressed in HEK293 cells or transiently expressed in COS-7 cells, [3H]GR 113808 consistently showed slightly higher binding affinity to h5-HT4(b) than to h5-HT4(a) receptor (pKd 0.1-0.2 log units higher). Competition of agonists, partial agonists and antagonists for [3H]GR113808 binding revealed no significant differences between the two receptors. For 5-HT4 receptor agonists and antagonists, their potencies in stimulating or inhibiting, respectively, 5-HT-stimulated adenylyl cyclase activity correlated well with their binding affinities. Tropisetron and SB207710 showed partial agonist activity at high receptor expression levels for both isoforms. Cisapride and renzapride were both partial agonists at moderate receptor levels and full agonists at high receptor levels. Cisapride was more potent than renzapride while the converse was the case in human atrium, for which cisapride had lower affinity and agonist potency than at the recombinant receptors. The binding affinities and agonist potencies of ligands for both 5-HT4(a) and 5-HT4(b) receptors correlated with the corresponding affinities and potencies in human atrium. The agonist potency of SB207710 was around 10 times lower than its binding and blocking affinity for both splice variants, suggesting that activation of adenylyl cyclase and blockade of 5-HT responses are mediated through different conformational states. The similar pharmacological properties of the two human 5-HT4 receptor splice variants together with their expression in human atrium would be consistent with mediation of the cardiostimulant effects of 5-HT through both variants. However, the effects of cisapride appear either mediated through non-a and non-b splice variants of the 5-HT4 receptor or 5-HT4(a) and 5-HT4(b) receptor expression in human atrial cells alters somewhat their pharmacological profile through still unknown mechanisms.

Adenylyl Cyclases↗

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↗

PubMed2000

Pertussis toxin suppresses carbachol-evoked cardiodepression but does not modify cardiostimulation mediated through beta1- and putative beta4-adrenoceptors in mouse left atria: no evidence for beta2- and beta3-adrenoreceptor function.

Activation of beta1-, beta2-, beta 3- and putative beta4-adrenoceptors modifies cardiac function. These receptors are usually coupled to Gs protein, but beta2- and beta3-adrenoceptors could also couple to Gi/o proteins. The mouse heart is used increasingly for studies of genetically disrupted or overexpressed proteins, including beta-adrenoceptor subtypes. We therefore investigated in contracting mouse left atria (2 Hz, 37 degrees C) if inactivation of Gi/o proteins with pertussis toxin modifies or uncovers effects mediated through beta-adrenoceptor subtypes. The negative inotropic effects of carbachol in atria exposed to catecholamine or high calcium (6.8 mmol/l) were assumed to be mediated through activation of muscarinic receptors coupled to Gi/o. We report conditions under which incubation of left atria with 200 ng/ml pertussis toxin for 24 h nearly abolished the carbachol responses. Although it has been reported that muscarinic receptor-mediated cardiodepression has an obligatory contribution of nitric oxide, the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (0.1-1 mmol/l) did not modify the negative inotropic effects of carbachol, inconsistent with an involvement of nitric oxide. The positive inotropic effects of (-)-noradrenaline and (-)-adrenaline, mediated through beta1-adrenoceptors, were not affected by pertussis toxin. (-)-Adrenaline did not cause positive inotropic effects attributable to beta2-adrenoceptor-mediation, in the presence of CGP 20712A (300 nmol/l) to block beta1-adrenoceptors, in control atria or atria pretreated with pertussis toxin. The positive inotropic effects of (-)-CGP 12177 (1 micromol/l), a compound with agonist activity at the putative beta4-adrenoceptor, were unaffected by pertussis toxin. The beta3-adrenoceptor-selective agonist BRL 37344 (1 micromol/l), in the presence of (-)-propranolol (200 nmol/l), did not cause positive or negative inotropic effects in control and pertussis toxin-treated atria. In left atria obtained from mice injected with 150 microg/kg i.p. pertussis toxin which abolished carbachol-evoked cardiode-pression, the positive inotropic effects of (-)-adrenaline were antagonised by CGP 20712A. The beta2-adrenoceptor-selective antagonist ICI 118551 (50 nmol/l) did not cause additional blockade of the effects of high (-)-adrenaline concentrations in the presence of CGP 20712A, ruling out the involvement of beta2-adrenoceptors. The results with intraparenteral PTX validate our in vitro PTX method. We conclude that inhibition of murine Gi/o proteins does not alter atrial positive inotropic effects mediated through beta1- and putative beta4-adrenoceptors and does not reveal functional beta2- and beta3-adrenoceptors.

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

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

Blockade of putative beta4- and beta1-adrenoceptors by carvedilol in ferret myocardium.

The putative beta4-adrenoceptor mediates positive inotropic effects, action potential shortening and arrhythmias in ferret ventricle. Here we compared the affinity of carvedilol at the putative beta4-adrenoceptor and beta1-adrenoceptor, activated by (+/-)-CGP 12177 and (-)-isoprenaline, respectively. In paced right ventricular preparations, carvedilol (0.01-10 micromol/l) was a simple competitive antagonist of the positive inotropic effects of (+/-)-CGP 12177 (slope of Schild-plot = 1.02, pK(B) = 6.8) and (-)-isoprenaline (slope of Schild-plot = 0.98, pK(B) = 8.1). Carvedilol also blocked putative beta4- and beta1-adrenoceptors of left ventricle, left atrium and sino-atrial pacemaker. Carvedilol therefore interacts with the putative beta4-adrenoceptor according to the law of mass action and may provide a lead in the development of putative beta4-adrenoceptor-selective antagonists.

Adrenergic beta-Agonists↗

PubMed1999

Beta4-adrenoceptors are more effective than beta1-adrenoceptors in mediating arrhythmic Ca2+ transients in mouse ventricular myocytes.

Putative beta4-adrenoceptors mediate cardiostimulation and arrhythmias in mammalian heart. Both beta1- and putative beta4-adrenoceptors mediate arrhythmias but through different mechanisms. To elucidate further the mechanisms of cardiostimulation and arrhythmias we measured Ca2+ transients and L-type Ca2+ currents in mouse ventricular myocytes. We used (-)-CGP 12177, an antagonist of beta1- and beta2-adrenoceptors with agonist properties at the putative beta4-adrenoceptor, and (-)-isoprenaline as an agonist for beta1- and beta2-adrenoceptors. (-)-CGP 12177 increased Ca2+ transients in electrically stimulated cells loaded with Indo-1. The maximum increase of Ca2+ transients caused by (-)-CGP 12177 amounted to approximately one-third of that caused by maximally effective (-)-isoprenaline concentrations. Both (-)-CGP 12177 and (-)-isoprenaline caused concentration-dependent arrhythmic Ca2+ transients. The arrhythmias appeared at paced Ca2+ transients and between paced Ca2+ transients. The arrhythmic potency of (-)-CGP 12177 (-logEC50=9.4) was approximately 40 times greater than that of (-)-isoprenaline (-logEC50=7.8). L-type Ca2+ current was measured in the whole cell configuration of the patch clamp technique. In the presence of both 3-isobutyl 1-methylxanthine (6 micromol/l) and (-)-propranolol (500 nmol/l), (-)-CGP 12177 (100 nmol/l) increased significantly L-type Ca2+ current by 19% of the effect of (-)-isoprenaline. The (-)-CGP 12177-evoked increase of Ca2+ transients contrasts with the smaller effects on L-type Ca2+ current, suggesting that activation of the putative beta4-adrenoceptor causes a more efficient Ca2+-induced Ca2+ release than activation of the beta1-adrenoceptor. Beta4-Adrenoceptors mediate arrhythmias with smaller Ca2+ transients and smaller increases of L-type Ca2+ current than beta1-adrenoceptors, in line with different but still unknown mechanisms as previously suggested for the intact heart.

1-Methyl-3-isobutylxanthine↗

PubMed1999

L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac beta 2-adrenoceptor.

OBJECTIVES: The reported increase in basal activity of hearts from transgenic mice (TG4) overexpressing the human beta 2-adrenoceptor (beta 2-AR) was explained by spontaneously active beta 2-ARs that stimulate the beta-adrenergic cascade in the absence of an agonist. In order to examine altered myocardial function on a cellular level, we have investigated L-type calcium current (ICa,L) and cell shortening in ventricular myocytes from TG4 hearts. Myocytes from littermates (LM) and wild type animals (WT) served as controls. METHODS: Cardiac beta-AR density was measured by [125I]-iodocyanopindolol binding to ventricular membranes. ICa,L was assessed by standard whole-cell voltage clamp technique. Contractility was measured as cell shortening in ventricular myocytes and as force of contraction in electrically stimulated left atria. RESULTS: Overexpression of beta 2-ARs was confirmed by an almost 400-fold increase in beta-AR density. The beta 1:beta 2-AR ratio in WT mice was 71:29. Myocytes from TG4 and LM mice were similar in size as judged by membrane capacitance and two dimensional cell area. ICa,L amplitude was significantly lower in TG4 than in LM myocytes (with 2 mM [Ca2+]o -4.82 +/- 0.48 vs. -6.56 +/- 0.38 pA/pF, respectively). In TG4 myocytes, the ICa,L response to isoproterenol (1 microM) was almost abolished. Cell shortening was not different in physiological [Ca2+]o, but smaller in maximum [Ca2+]o when comparing TG4 to control myocytes. Basal force of contraction in left atria did not differ between TG4 and LM at any age investigated. In TG4 left atria the inotropic response to isoproterenol was also absent, whereas responses to high [Ca2+]o or dibutyryl-cAMP (1 mM) were present but reduced. The rate of spontaneous beating of right atria was elevated in TG4 mice. CONCLUSIONS: Since only spontaneous beating rate but neither basal ICa,L amplitude nor basal contractile activity were elevated, our data fail to reveal evidence for spontaneously active, stimulating beta 2-ARs in left atrium and ventricle. A contractile deficit unrelated to the beta-adrenoceptor pathway is evident in TG4 myocytes and left atria.

Adrenergic beta-Agonists↗

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↗

PubMed1999

Simple O-acylated derivatives of lysergol and dihydrolysergol-I: synthesis and interaction with 5-HT2A, 5-HT2C and 5-HT1B receptors, and alpha1 adrenergic receptors.

A series of simple O-acylated derivatives of the naturally occurring clavine alkaloids lysergol and dihydrolysergol-I were synthesized and tested in-vitro for their ability to interact with 5-HT2A receptors in rat tail artery, 5-HT2C receptors in piglet choroid plexus, 5-HT1B receptors in guinea-pig iliac artery and alpha1-adrenergic receptors in rat aorta. In contrast to the classical ergoline 5-HT2A receptor antagonists methysergide and LY53857, the compounds produced competitive antagonism of the 5-HT response in rat tail artery. Affinities of ergolines 3-14 were higher (pA2 values of 7.33-8.40) than those of the parent alcohols lysergol (1) and dihydrolysergol-I (2), respectively. The introduction of an isopropyl substituent at the N(1) position of the compounds failed to enhance 5-HT2A receptor affinity. Compounds 3-14 exhibited lower affinities for alpha1-adrenergic receptors than for 5-HT2A receptors. In particular, those lysergol derivatives that had an isopropyl substituent at the N(1) position were highly specific 5-HT2A receptor antagonists (ratio 5-HT2A/alpha1 = 302-3548). Selected derivatives of lysergol (3-5, 9-11) which were assayed for radioligand binding at 5-HT2C receptors in piglet choroid plexus had affinities that were similar to those found in rat tail artery. Additionally, lysergol and its N(1)-unsubstituted derivatives were found to be partial agonists (alpha of 0.2-0.4) for 5-HT2C receptor-mediated inositol phosphate accumulation in piglet choroid plexus. On the other hand, analogues with an isopropyl substituent at N(1) showed no measurable agonist activity. The observation that N(1)-unsubstituted derivatives of lysergol possessed agonist properties at 5-HT2C receptors whereas their agonist activity at 5-HT2A receptors was marginal (alpha of 0.05 for compound 3 at 1 microM) or not measurable, suggests that these compounds have different abilities to cause conformational change at the two receptor types. Selected derivatives of lysergol (3-5, 9-11) which were examined as ligands for 5-HT1B receptors in guinea-pig iliac artery caused insurmountable blockade of the contractile effect of 5-HT. N(1)-isopropyl derivatives had 30-50-fold lower affinities for 5-HT1B receptors of this tissue than their N(1)-unsubstituted analogues. It is concluded that O-acylated derivatives of the clavine alkaloids lysergol and dihydrolysergol-I mimic therapeutically relevant ergolines due to the complexity of their pharmacological profile as partial agonists and antagonists at 5-HT2A, 5-HT2C and 5-HT1B receptors, and at alpha1-adrenergic receptors.

Animals↗

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

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression.

1. In the human temporal artery both 5-HT1-like and 5-HT2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT1-like receptors resemble more closely recombinant 5-HT1B than 5-HT1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1'-methyl-5-[2-methyl-4'[(5-methyl-1,2,4-oxadiazole- 3-yl) biphenyl-4-yl] carbonyl] furo[2,3-f]indole-3-spiro-4'-piperidine oxalate) and the 5-HT1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT1B, 5-HT1D and other 5-HT receptors. 2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent KB = 1 nM) and ketanserin (1 microM) but not by BRL-15572 (500 nM). 3. Sumatriptan evoked contractions (EC50, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM). 4. The potency of 5-HT (EC50) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20-0.67, the remainder being mediated through ketanserin-sensitive receptors. 5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT1B (8/8), 5-HT1D (2/8), 5-HT1F (0/4), 5-HT2A (0/8) 5-HT2B (0/8), 5-HT2C (0/8), 5-HT4 (4/8) and 5-HT7 (4/8). 6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.

Adolescent↗

PubMed1998

Action potential shortening through the putative beta4-adrenoceptor in ferret ventricle: comparison with beta1- and beta2-adrenoceptor-mediated effects.

The electrophysiological responses to (-)-CGP 12177 ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one), an agonist for the putative beta4-adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min(-1) and compared to those of (-)-noradrenaline and (-)-adrenaline, mediated through beta1- and beta2-adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; beta3-adrenoceptor-selective agonists had no effect. (-)-CGP 12177 was the most potent, but (-)-noradrenaline the most efficacious; both agonists caused ventricular extra-systoles. Because only (-)-noradrenaline but not (-)-CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through beta1- and putative beta4-adrenoceptors may be different.

Action Potentials↗

PubMed1998

Effects of the novel high-affinity 5-HT(1B/1D)-receptor ligand frovatriptan in human isolated basilar and coronary arteries.

The contractile actions of the novel high-affinity 5-hydroxytryptamine (5-HT(1B/1D)) ligand, frovatriptan (formerly VML 251/SB-209509) were investigated in human isolated basilar and coronary arteries in which the endothelium had been removed. Basilar arteries were obtained post mortem, and coronary arteries were obtained from patients undergoing heart transplant (recipient) or from donor hearts that were not suitable for transplant. Frovatriptan was a potent contractile agent in isolated basilar artery with a -log mean effective concentration (EC50) value of 7.86 +/- 0.07 and intrinsic activity of 1.25 +/- 0.10 relative to 5-HT (n = 4). Frovatriptan was 8.5-fold more potent than sumatriptan, which produced a -log EC50 value of 6.93 +/- 0.09 and intrinsic activity 11.1 +/- 0.08 relative to 5-HT (n = 4). In coronary arteries, frovatriptan produced contraction with -log EC50 values of 7.38 +/- 0.12 and 7.81 +/- 0.2 in recipient (n = 7) and donor (n = 3) arteries, respectively. The relative degree of contraction of frovatriptan was lower than that of 5-HT, with relative intrinsic activities of 0.42 +/- 0.06 and 0.40 +/- 0.09, respectively. Sumatriptan produced contraction of human recipient and donor arteries with -log EC50 values (intrinsic activity) of 6.57 +/- 0.13 (0.79 +/- 0.27; n = 6) and 7.35 (1.41; n = 2), respectively. Furthermore, marked bell-shaped responses were apparent for frovatriptan in coronary arteries, with relaxation occurring at concentrations >6 microM in some tissues. In contrast, no bell-shaped concentration-response curves were apparent for sumatriptan or 5-HT. Threshold concentrations for frovatriptan-induced contractions were also different between basilar (>2 nM) and coronary arteries (>20 nM). No separation of threshold activity was observed with sumatriptan or 5-HT. These data show that frovatriptan produces constriction of human isolated basilar and coronary arteries. However, frovatriptan produces a complex pharmacologic response in the coronary artery, with threshold contractile activity requiring approximately 10-fold greater concentrations of agonist than in the basilar artery. Frovatriptan also shows a differential pharmacologic profile compared with sumatriptan in coronary arteries, with reversal of tone predominating at high concentration.

Basilar Artery↗

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

Simultaneous measurement of [3H]noradrenaline release and neurogenic contraction under identical conditions, to determine the prejunctional inhibitory effects of SKF 99101H and BRL 56905 in dog saphenous vein.

Using a tissue bath system which allowed the simultaneous measurement of electrically-induced [3H]noradrenaline release and neurogenic contraction under identical conditions, we investigated the prejunctional inhibitory activity of the selective 5-HT(1D/1B) receptor agonists BRL 56905 ((+/-)-3-amino-6-carboxamido-1,2,3,4-tetrahydrocarbazole) and SKF 99101H (3-(2-dimethylaminoethyl)-4-chloro-5-propoxyindole hemifumarate), compared to sumatriptan and 5-HT. Transmural electrical stimulation (2 Hz) of dog saphenous vein induced consistent increases in [3H]noradrenaline release as well as reproducible contractile responses (<10% decrease over four stimulation periods). BRL 56905, SKF 99101H, sumatriptan and 5-HT (60 nM-6 microM) inhibited electrically-evoked [3H]noradrenaline release and neurogenic contractile responses in dog saphenous vein. However, despite being measured under identical conditions, the inhibition of [3H]noradrenaline release was consistently greater than the inhibition of neurogenic contraction induced by a particular concentration of agonist, suggesting that neurogenic contractile responses in dog saphenous vein result from the combined release of noradrenaline and other non-noradrenergic neurotransmitters. Under the present assay conditions, since the agonists produced only small (BRL 56905, sumatriptan and 5-HT) or marginal (SKF 99101H) contractile responses, it is unlikely that this is the cause of the discrepancy observed between inhibition of release and inhibition of contraction. The inhibitory effects of BRL 56905, sumatriptan and 5-HT were blocked by the 5-HT(1D/1B) receptor antagonist methiothepin, consistent with the involvement of canine ca-5-HT(1D/1B) receptors in inhibiting neurotransmitter release and subsequent smooth muscle contraction in dog saphenous vein. The present results show that the novel 5-HT(1D/1B) receptor agonists BRL 56905 and SKF 99101H are at least as potent as sumatriptan and 5-HT, at activating prejunctional inhibitory ca-5-HT(1D/1B) heteroreceptors on sympathetic axon terminals in dog saphenous vein. In addition, when measured simultaneously in the same tissue preparation, [3H]noradrenaline release was inhibited to a much greater extent than neurogenic contraction by any particular agonist.

Animals↗

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↗