Harald Rogg

Identification and characterization of angiotensin II receptor subtypes in rabbit ventricular myocardium

CGP 42 112 A and DuP 753 block [125I]-angiotensin II binding in rabbit ventricular myocardial membranes in a clearly biphasic manner, indicating the existence of high- and low-affinity sites for these highly selective agents. Assays using concentrations of either agent large enough to prevent high-affinity binding show that their respective high-affinity sites are distinct, and each corresponds to the low-affinity site of the other. The two receptor subsets, present in nearly equal proportions, are also distinguishable by their different sensitivities to dithiothreitol. These findings afford strong evidence for the existence of two distinct angiotensin II receptors in rabbit myocardium, corresponding to the A and B subtypes recently described in adrenals.

Enhancement of calcium influx in human platelets by CGP 28392, a novel dihydropyridine.

CGP 28392, a novel compound structurally related to the dihydropyridine Ca2+-entry blockers, causes a dose-dependent increase in intracellular free Ca2+ in human platelets, as measured with the Quin-2 Ca2+ indicator, with a semimaximal effective concentration of 2.2 X 10(-7) M. This effect occurs in a concentration range in which CGP 28392 competes for specific [3H]nitrendipine binding in guinea pig heart membranes. It can be inhibited by nitrendipine. The data presented furnish direct evidence of the Ca2+-entry-stimulating properties of CGP 28392 and indicate the presence of dihydropyridine-susceptible structures in human platelets.

An improved method for the separation and quantitation of the modified nucleosides of transfer RNA.

A method is described which allows a very efficient determination of the modified nucleosides of tRNA. The technique involves enzymatic degradation of the tRNA to nucleosides at pH 7.6 and their separation by two-dimensional thin-layer chromatography on cellulose-coated aluminum foils. Based on the analysis of two mammalian tRNAs it is shown that the technique is suitable for the determination of chemically unstable nucleosides as well as the ribose-methylated compounds. At least 36 of the 45 known modified nucleosides can be separated and quantitatively determined by the method described. This procedure is especially suitable for the estimation of the nucleoside composition of unlabeled tRNAs as well as for studying the post-transcriptional modifications of tRNA.

Stereospecific action of diltiazem on the mitochondrial NaCa exchange system and on sarcolemmal Ca-channels

The benzothiazepine diltiazem is a potent Ca-channel blocker, which also inhibits the Na-dependent Ca-efflux from heart mitochondria. In this study, the action of the 4 stereoisomers of diltiazem has been investigated using guinea-pig heart and liver mitochondria. The rate of the Na-dependent Ca-efflux from liver mitochondria has been found to be 10 times smaller than in heart mitochondria. Otherwise, the exchange systems from the two tissues have been found to be pharmacologically indistinguishable. Both the (+)-optical isomers of the cis- and trans-forms of diltiazem inhibit Na-Ca exchange activity with comparable potency (IC50 of 10-20 microM), while the (-)-optical isomers are ineffective (IC50 greater than 200 microM). Radioligand binding experiments have revealed that only one stereoisomer of diltiazem, the (+)-cis form, interacts with high affinity with the Ca-channel receptors of guinea-pig heart sarcolemma preparations (KD = 120 nM). The results have shown that the Ca-channel of plasma membranes and the mitochondrial Na-Ca exchanger have different stereospecific requirements for the binding of diltiazem.

LOCALIZATION OF ANGIOTENSIN II RECEPTOR SUBTYPES IN THE RABBIT HEART

Angiotensin II (Ang II) is an essential component of the renin-angiotensin system and is partially responsible for the maintenance of hypertension. Two major receptor subtypes have been defined for Ang II and have been detected in the heart of various species. Most of the known functions of Ang II are mediated via the AT1 subtype, whereas the function of the AT2 receptor remains ill defined. In this study we aimed to localize both receptor subtypes in the rabbit heart using film and light microscope autoradiography as well as radioligand binding assays on membranes. Total receptor densities in the atrium and nervous tissue were respectively four and nine times greater than in the ventricle. Conductive tissue shows a density between that of atrial and nervous tissue. In the ventricle, approximately 20% of the Ang II receptors were AT2. This receptor subtype was almost totally absent from nervous, conductive and atrial tissue. The limited resolution of the microscope autoradiography method did not allow us to specify the exact cell-type at this stage.

CGP 28392, a Dihydropyridine Ca2+ Entry Stimulator

Agents termed Ca2+ antagonists or Ca2+ channel blockers (e. g., nifedipine, verapamil, diltiazem) reduce Ca2+ influx through specific channels located in the membranes of vascular smooth muscle and myocardial cells. This action results in vasodilation and decreased myocardial contractility [1-5]. Consequently, this group of agents has clinical value in the therapy of angina pectoris, arrhythmias, and hypertension [6]. Compounds that act to increase Ca2+ influx through specific membrane channels might be expected to possess pharmacological activities opposite to those of the Ca2+ antagonists (e. g., vasoconstriction and increased myocardial contractility) [7]. The first Ca2+ entry stimulator (or Ca2+ agonistic compound) structurally related to nifedipine was YC-170 [8]. Like nifedipine, YC-170 is a 1,4-dihydropyridine derivative, but produced vasopressor effects in anesthetized rats and dogs as well as vasoconstriction in the isolated rabbit aorta. Based on indirect evidence, the authors suggested that these effects were due to an enhanced Ca2+ influx into vascular smooth muscle cells.

On the specificity of ribonuclease U2

Abstract The breakdown products of 5 dinucleotides, several trinucleotides and the pentanucleotide hU-hU-A-A-Gp after digestion with varying amounts of ribonuclease U2 have been analyzed. The data show the following specificities of the enzymatic attack. A-X > G-X, Pu-C > Pu-U, terminal bonds are cleaved more readily than internal bonds, and substrates carrying 2′,3′ cyclic phosphate end groups are much more resistant than those carrying an open phosphate group. Certain pyrimidine bonds such as in hU-hU-A are also attacked.

[12] Thin-layer chromatography of oligonucleotides

Publisher Summary This chapter explores the application of thin layer chromatography (TLC) of oligonucleotides for analysis of nucleic acid. Thin-layer chromatography of oligonucleotides is useful in nucleic acid analysis for two purposes: the separation of mixed oligonucleotides from column chromatograms and the analysis of partial digest of large oligonucleotides. The main advantage of thin-layer over paper chromatography lies in the sensitivity of the method, the near quantitative recovery, and the fact that upon elution, contaminants are extracted in smaller quantity. The disadvantage lies in the more limited capacity of the thin-layer plates and a somewhat greater sensitivity to salt effects. Therefore, it is important to remove salts carefully before application or to use volatile buffers when desalting seems impractical-for instance, after enzymatic digestion. The oligonucleotides are located under an ultraviolet lamp. Adenosine derivatives, by their longer phosphorescence can be distinguished from guanosine derivatives, which show a relatively shorter phosphorescence. On the other hand, some pyrimidine derivatives can also be determined by this method, because of their strong, but very short and characteristic phosphorescence.