Biomedical subjects

M Freissmuth

Publications and source records attributed to M Freissmuth.

81 records · Page 5Linked to original sources

PubMed1987

The role of a low beta 1-adrenoceptor selectivity of [3H]CGP-12177 for resolving subtype-selectivity of competitive ligands.

On the basis of saturation binding studies on rat cardiac microsomes, which contained a mixed population of beta-adrenoceptor subtypes, [3H]CGP-12177 is presumed to be a non-selective beta-adrenergic radioligand. However, saturation binding studies carried out in the presence of subtype-saturating concentrations of the beta 2-selective antagonist ICI 118,551 and the beta 1-selective antagonist ICI 89,406, respectively, revealed a KD for beta 1-adrenoceptors of 0.33 +/- 0.02 nmol/l and a KD for beta 2-adrenoceptors of 0.90 +/- 0.14 nmol/l. Competition experiments with the highly selective antagonists revealed greatly different competition binding curves in the presence of either [3H]CGP-12177 or (-)[125I]iodocyanopindolol (ICYP), a beta-adrenergic radioligand considered to be as non-selective as [3H]CGP-12177. The following results are further suggestive for a selectivity of [3H]CGP-12177 for beta 1-adrenoceptors: (1) Using non-linear regression analysis, a significantly lower selectivity (expressed as the ratio of the IC50 for beta 2-adrenoceptors to the IC50 for beta 1-adrenoceptors) as well as a larger proportion of beta 1-adrenoceptors were calculated by competition of the beta 1-selective antagonist ICI 89,406 with [3H]CGP-12177 binding than by competition of ICI 89,406 with ICYP binding; (2) reducing the [3H]CGP-12177 concentration from 2 to 0.4 nmol/l, competition experiments with ICI 89,406 led to an increase in the estimated selectivity of the competitor and in the estimated proportion of beta 1-adrenoceptors; (3) reverse findings were obtained with ICI 118,551, a beta 2-selective antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗

PubMed1987

Effects of ischemia on the canine myocardial beta-adrenoceptor-linked adenylate cyclase system.

Several studies indicate that myocardial ischemia causes a redistribution of beta-adrenergic receptors from a presumably intracellular compartment to the cell surface. However, a decreased adenylate cyclase and contractile responsiveness to beta-adrenergic stimuli has also been reported. The aim of the present study was to investigate possible ischemia-induced changes in myocardial beta-adrenoceptor coupling to adenylate cyclase. Myocardial ischemia was induced by hydraulic occlusion of the LAD in mongrel dogs anesthetized with isoflurane. After 90 min of ischemia, tissue samples were removed from the ischemic and nonischemic regions for tissue catecholamine determinations and for the preparation of particulate fractions from tissue homogenates. Saturation experiments on microsomal fractions obtained from the ischemic and control areas did not reveal any significant changes in the calculated dissociation constant for (-)[125I]iodocyanopindolol binding nor in the calculated receptor density. Likewise, the relative numbers of beta 2-adrenergic receptors were comparable in both preparations (approximately 20%). On the other hand, the proportion of beta-adrenoceptors stabilized in the high-affinity state by (-)isoproterenol was significantly reduced in the ischemic region when compared with the control myocardium (17 +/- 5 vs. 41 +/- 4%). This change was accompanied by a significant decrease in the intrinsic activity of (-)isoproterenol in stimulating adenylate cyclase activity. We propose that the initial uncoupling of the beta-adrenoceptor from its effector is a physiologically important, protective mechanism which guards the ischemic myocardium against the deleterious effect of excessive sympathetic stimulation.

Adenylyl Cyclases↗

PubMed1986

Cardiac ventricular beta 2-adrenoceptors in guinea-pigs and rats are localized on the coronary endothelium.

In mammalian heart tissue beta 2-adrenoceptors are known to coexist with beta 1-adrenoceptors. In the present study, evidence that beta 2-adrenoceptors in guinea-pig and rat ventricles are primarily localized on the coronary endothelium is provided by competition binding studies with the subtype-selective beta-adrenoceptor antagonists ICI 89.406 (beta 1-selective) and ICI 118.551 (beta 2-selective) on four different plasma membrane preparations. (1) Following density gradient centrifugation of cardiac ventricular microsomes from rats or guinea-pigs, endothelial plasma membranes migrated at slightly higher density than the sarcolemmal membranes, as verified by endothelial (angiotensin converting enzyme) and sarcolemmal markers (adenylate cyclase, [3H]ouabain binding). At the activity peak of angiotensin converting enzyme, the relative amount of beta 2-adrenoceptors in guinea-pigs and rats was 25% and 65%, respectively. (2) On sarcolemmal membranes corresponding to the activity peak of adenylate cyclase, beta-adrenoceptors consisted of the beta 1-type exclusively (guinea-pig), or to at least 90% (rat). (3) Cultures of coronary endothelial cells derived from guinea-pigs revealed only beta 2-adrenoceptors. (4) Isolated guinea-pig cardiomyocytes contained only beta 1-adrenoceptors, a finding recently established in rat myocytes as well.

Animals↗

PubMed1986

Cardiac sarcolemmal purity is essential for the verification of adenylate cyclase inhibition via A1-adenosine receptors.

Inhibition of cardiac adenylate cyclase by adenosine receptor agonists was reinvestigated in a more homogeneous sarcolemmal vesicular preparation than used in a previous study. Microsomal particles obtained by differential centrifugation were further fractionated on a shallow density gradient of Percoll. Two populations of plasma membrane vesicles were partially resolved. Identical peaks were identified for adenylate cyclase activity and [3H]ouabain binding, whereas 5'-nucleotidase activity and beta-adrenoceptor binding displayed an additional peak at higher density, where angiotensin converting enzyme, a marker for endothelial plasma membranes, was at maximal activity. Significant inhibition by N6-cyclohexyladenosine (CHA), as measured in each fractionation step following homogenization, was observed only at the activity peak of adenylate cyclase. Moreover, analysis of the degree and rank order of potency of several adenosine analogs was indicative for interaction with A1-adenosine receptors. Accordingly, the peak in adenosine receptor binding, using (-)[125I]iodo-N6-hydroxyphenyl-isopropyladenosine as the radioligand, coincided with CHA-inhibitable adenylate cyclase activity. By contrast, adenylate cyclase was slightly stimulated by CHA in the higher density range, an action suggested to be mediated via A2-adenosine receptors, which recently have been demonstrated to exist on guinea-pig coronary endothelium. It is concluded that the full extent of adenosine receptor-mediated adenylate cyclase inhibition in the heart is only to be demonstrated if contamination of the sarcolemmal preparation with endothelial membrane components is kept to a minimum.

Adenosine↗

PubMed1986

Two saturable recognition sites for (-) [125I]iodo-N6-(4-hydroxyphenyl-isopropyl)-adenosine binding on purified cardiac sarcolemma.

Analysis of (-) [125]iodo-N6-(4-hydroxyphenylisopropyl)-adenosine [( 125I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 mumol/l) was used as non-specific binding marker. In the presence of 1 mmol/l theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [125I]HPIA binding, with a high affinity potency rank order typical of interaction with A1-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/l. In brain membranes, specific binding of [125I]HPIA as well as of [3H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [125I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.

Adenosine↗

PubMed1985

Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex.

Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.

Adenosine Triphosphate↗

PubMed1984

Interaction of the antihypertensive drug urapidil with cardiac beta-adrenoceptors in vitro.

A possible interaction of the antihypertensive drug, urapidil with beta-adrenoceptors was investigated in a guinea-pig ventricular membrane preparation. Urapidil at concentrations above 1 microM antagonized the isoproterenol-induced stimulation of adenylate cyclase activity. Urapidil 10 microM shifted the concentration-response curve for isoproterenol in a parallel manner to the right by a factor of 16. Urapidil competed with specific beta-adrenoceptor binding of [125I]iodocyanopindolol with an IC50 of 12 microM. Since the affinity of urapidil to beta-adrenoceptors appeared rather low, any beta-adrenoceptor blocking property may be of relevance in vivo at high dosages only.

Adenylyl Cyclases↗

PubMedDate not supplied

Binding of [3H]ouabain to endothelial cells derived from various vascular beds.

Binding experiments were performed with [3H]ouabain on plasma membranes derived from several types of isolated and cultivated endothelial cells. Identical saturation curves for [3H]ouabain binding to endothelial cells from pig aorta, caval vein, and pulmonary artery were obtained with a dissociation constant (KD) of 3.29 +/- 0.31 nmol/l and a binding capacity (Bmax) of 5.22 +/- 0.12 pmol/mg protein. On guinea-pig coronary endothelial cells, saturation of [3H]ouabain revealed much lower affinity (KD 95 +/- 15 nmol/l, Bmax 2.08 +/- 0.09 pmol/mg protein). All Scatchard plots were linear, indicating a homogeneous class of binding sites. In competition experiments, cardiac glycosides and their aglycons displaced the radioligand with a structure-activity relationship typical for interaction with Na+/K+-ATPase (proscillaridin A greater than ouabain greater than digoxin greater than g-strophanthidin greater than digoxigenin greater than dihydrodigoxin); in particular, removal of the sugar moiety results in considerable reduction of affinity. Furthermore, K+ displayed a steep inhibition curve with a half-maximal inhibitory constant of 2 mmol/l. All these findings suggest the presence of endothelial ouabain receptors linked to Na+/K+-ATPase. However, direct measurement of this enzyme was not possible due to an extremely high Mg2+-ATPase activity.

Animals↗

PubMedDate not supplied

Alterations of the glomerular beta-adrenoceptor-linked adenylate cyclase system in perinephritis hypertension.

The present study was undertaken to investigate beta-adrenoceptor-adenylate cyclase coupling in a myocardial ventricular membrane preparation and in isolated renal glomeruli of cellophane perinephritis hypertensive rats. Adenylate cyclase activity and [125I]iodocyanopindolol binding were differentially affected in these preparations. In isolated glomeruli of hypertensive rats a reduced intrinsic activity of isoproterenol was associated with an apparent loss of the guanine nucleotide-sensitive high-affinity state of the beta-adrenoceptors, whereas their absolute number was unchanged when compared with the normotensive control rats. On the other hand, in the sarcolemmal preparation of hypertensive rats adenylate cyclase activity, the relative amount of high-affinity states, and the density of beta-adrenoceptors were not different from the respective values in normotensive controls. Experiments performed on isolated glomeruli of rats with unilateral cellophane perinephritis that developed only moderate hypertension provide evidence that the apparent loss of the high-affinity state is a consequence of hypertension, since no difference was observed in glomeruli from the wrapped, as compared with the intact kidney.

Adenylyl Cyclases↗