Bruno Kamber

Preliminary biochemical characterization of two angiotensin II receptor subtypes

Two angiotensin II receptor subtypes (A and B) are described from rat and human tissues. They have been characterised using specific peptidic and non-peptidic ligands with affinities differing by 1000 fold or more. These subtypes are present in adrenal glomerulosa of both species. Human uterus contains only subtype A, whereas both subtypes are found in rat uterus. Vascular smooth muscle cells in culture express only subtype B. Dithio-threitol totally inhibits binding to subtype B, but enhances the affinity to subtype A. There is a good correlation between the affinities of the selected agonists and antagonists for the two subtypes in the various tissues tested which is a usual requirement for receptor classification.

Biochemical Characterization of Two Angiotensin II Receptor Subtypes in the Rat

Two angiotensin II receptor subtypes, A and B, have been described by means of specific and selective ligands (Biochem Biophys Res Commun 1989; 163:284-91). The present report describes the binding characteristics and the distribution of these subtypes in various rat tissues. Adrenal and uterus expressed both subtypes but in different proportions. Subtype B predominated in the adrenal glomerulosa (60%), whereas there was a greater proportion of subtype A in the medulla (70%). In uterus, subtype B was preferentially expressed (70%), and there was no difference in receptor distribution between muscle layers and serosa. Liver, kidney, and cultured aortic smooth muscle cells expressed only subtype B. In all of the tissues tested, the Ki values of various competing ligands were similar for each subtype expressed. It is proposed that subtype B is the vascular receptor. The function of subtype A, however, is still to be determined.

Radioiodinated CGP 42111A: A novel high affinity and highly selective ligand for the characterization of angiotensin AT2 receptors

CGP 42112A, a potent angiotensin AT2 receptor selective ligand, was radio-iodinated and its binding characteristics compared with those of [125I]angiotensin II. In human myometrium (only AT2 expressed), binding was saturable (Kd 1.03 x 10(-10) M; Bmax 807 fmol/mg) and reversible (K+1 1.89 x 10(8) M-1.min-1; K-1 3.77 x 10(-3) min-1). The order of potency of a number of peptides and non-peptides was the same as when [125I] angiotensin II was used as tracer. No specific binding could be detected on membranes from vascular smooth muscle cells (only AT1 expressed). In rat adrenal glomerulosa membranes (mixed AT1/AT2), [125I]CGP 42112A bound only to AT2. [125I]CGP 42112A can therefore be used as a specific probe for AT2 receptors and will be especially useful in tissues where other subtypes are also present.

A General Strategy for the Synthesis of Large Peptides: r1~he Combined Solid-Phase and Solution Approach.

Abstract In the synthesis of large peptides. the yield and purity of the end-products will be greatly improved when smaller segments are purified prior to their use for fragment coupling either on a solid-phase resin or in solution. The Naα-Fmoc(9-fluorenylmethoxycarbonyl) method allows the selective acidolytic cleavage of fully protected peptides with a free α-carboxyl group from the solid-phase resin. For this cleavage, the highly acid-labile HMPB linker, 4-(4-hydroxymethyl-3- methoxyphenoxy)-butyric acid, has been developed. The lipophilic protecting groups, in particular Trt on asparagine, glutamine, and histidine, as well as Pmc (2,2,5,7,8-pentamethylchroman-6- sulfonyl) on arginine, confer a good solubility on most protected peptide segments in organic solution and enable their purification by silicagel chromatography. Whereas the addition of segments on solid-phase resins is often difficult, they can as a rule be coupled easily in solution to give products in high yield and purity. The combined solid-phase and solution strategy is illustrated by the syntheses of human calcitonin-(l-33), human neuropeptide Y, and the sequence 230-249 of mitogen-activated 70K S6 kinase. Large peptides are built up in solution from smaller segments with lipophilic protecting groups, which can be prepared by solid-phase synthesis. The scheme below depicts the synthesis of human NPY using this method.

Binding characteristics and vascular effects of various angiotensin II antagonists.

Subtypes of angiotensin II (Ang II) receptors have been recently identified using specific ligands (see Whitebread et al. Biochem Biophys Res Comm 1989; 163:284-291). The present study compares the binding characteristics of different structural classes of Ang II receptor ligands in rat aortic smooth muscle cells (Ang IIB subtype) and in human uterus (Ang IIA subtype) and their effects on the constrictor response to Ang II in isolated rabbit aortic rings. Saralasin and [Sar1 Ile8]-Ang II displayed similar affinity for the two subtypes. In contrast, CGP 42112A bound with high affinity to the uterus (Ki 0.24 nM), but showed a low affinity for the aortic receptor (Ki 1,760 nM). Compound 89 displayed affinity for the aortic receptor only (Ki 26 nM) whereas Ex 169 recognized specifically the uterus receptor (Ki 310 nM). In rabbit aortic rings, saralasin, [Sar1 Ile8]Ang II, CGP42112A and compound 89 inhibited Ang II-induced contractions at concentrations similar to those required to bind to the Ang IIB receptor subtype. IC50s were 3, 0.7, 1,850, and 23 nM respectively. Ex 169 was ineffective in concentrations up to 100 microM. There was a highly significant correlation between inhibition of Ang II-induced contraction in aortic rings and binding to smooth muscle cells. This correlation does not exist with human uterus. Our results indicate that the Ang IIB receptor subtype is responsible for vascular contractions. Antagonists of this vascular receptor subtype are potential antihypertensive agents.

[Structure-activity relationship of human calcitonin. III. Biological activity of synthetic analogues with shortened or terminally modified peptide chains (author's transl)].

Assays of 8 synthetic analogues of human calcitonin in rats showed that their hypocalcaemic activity was drastically reduced by deletion of the C-terminal amide group, chain-shortening or opening of the disulphide ring, but unaffected or enhanced by modification of the N-terminal amino group.Assays of 8 synthetic analogues of human calcitonin in rats showed that their hypocalcaemic activity was drastically reduced by deletion of the C-terminal amide group, chain-shortening or opening of the disulphide ring, but unaffected or enhanced by modification of the N-terminal amino group.

The Tasp Concept - Mimetics of Peptide Ligands, Protein Surfaces and Folding Units

Abstract The TASP (Template Assembled Synthetic Protein) concept in protein de novo design has been introduced for the construction of protein-like molecules exhibiting tailor-made functional properties. After establishing some synthetic and conformational foundations in TASP strategy, we describe a 4α - helix bundle TASP molecule mimicking immunological properties of human MHC-I. Furthermore, we focus on structural motifs (e.g. topological templates) disposing functional groups in defined spatial positions as candidates to mimic structural and conformational features of peptides and proteins for molecular recognition studies.

Human cardiac plasma concentrations of atrial natriuretic peptide quantified by radioreceptor assay

The presence of high affinity receptors for atrial natriuretic peptide in bovine adrenal cortex has enabled the development of a sensitive, specific and rapid radioreceptor assay for this peptide in human plasma. In 18 normal subjects, venous plasma atrial natriuretic peptide concentration ranged from 6 to 65 pM. This plasma concentration was two-fold higher in right atrium as compared to venous blood in 12 patients investigated by cardiac catheterisation, confirming that the right atrium is the site of release of atrial natriuretic peptide into circulation. There was a further step up in plasma atrial natriuretic peptide concentration between pulmonary arterial and aortic plasma. This finding indicates that released hormone in man may undergo further activation in the lungs, or that there may be direct release from the left atrium.

ANALOGUES OF HUMAN CALCITONIN: IV. INFLUENCE OF LEUCINE SUBSTITUTIOS IN POSITIONS 12, 16 AND 19 ON HYPOCALCAEMIC ACTIVITY IN THE RAT

The replacement of the three aromatic amino acids in positions 12, 16 and 19 of human calcitonin (HCT) leucine residues, which occupy the corresponding positions in ultimobranchial, e.g. salmon and eel, calcitonins, increased the hypocalcaemic potency of the peptide, as determined by bioassay on the rat, about 10‐fold. The individual substitutions were not all equally augmentative: leucine (12) enhanced the activity of HCT about 4‐5 times, but leucine (16) and (19) afforded no increase at all. Combination of leucine (12) with a deamino cysteine at the N‐terminmus yielded an analogue 10 times more potnet than HCT. Another analogue containing valine in position 8 in place of methionine as well as the three leucine substituents in position 12, 16 and 19 proved more active than the tri‐leucine analogue, but the additional introduction of tyrosine (22) nearly doubled the hypocalcaemic potency of the latter. The duration of the hypocalcaemic effects of the substituted peptides closely followed the changes in potency.

The Angiotensin II AT2 Receptor Subtype

During the past 30 years, evidence has accumulated from pharmacological studies indicating that there are multiple subtypes of the angiotensin II (ANG II) receptor. A great many peptide analogues of ANG II have been synthesized to obtain final proof of the existence of such subtypes.1