Effect of covalent dimer conjugates of angiotensin II on receptor affinity and activity in vitro.

Angiotensin II [1-8 or 2-8] analogues and [4-8] fragments were dimerized through the amino- or carboxy-terminal groups in order to try to increase their potency as reported for other hormones. The binding affinity to the angiotensin II receptor subtypes A (A IIA) and B (A IIB) was tested and compared to the potency in rabbit aortic ring. The [2-8] dimers coupled through the N-terminus show no significant change in potency in aortic ring. The [4-8] fragments coupled through the N-terminus are inactive in the ring. They have however a significantly increased affinity for the A IIA receptor, the specific function of which has not yet been reported. When angiotensin II analogues or fragments are coupled through the C-terminus, there was a significant drop in affinity and potency, confirming the importance of the free carboxyl group in position 8 for binding and activity. It is concluded that binding to the A IIB receptor correlates well with the effectiveness in aortic ring. However, in contrast to the beneficial effect reported for a large number of other hormones, dimerization of angiotensin II or its fragments is not accompanied by an increased biological activity in aortic ring.

Reactivity and sensitivity of mesenteric vascular beds and aortic rings of spontaneously hypertensive rats to endothelin: effects of calcium entry blockers.

1. The vasoconstrictor effects of endothelin-1 were studied in perfused mesenteric vascular beds (MVB) and aortic rings of 14-16 week-old spontaneously hypertensive rats (SHR) and age-matched Wistar Kyoto rats (WKY). 2. Reactivity to endothelin-1 was increased in MVBs of SHR, as indicated by the maximum perfusion pressure obtained (264 +/- 8 and 141 +/- 9 mmHg respectively) (P less than 0.001), whereas sensitivity was not significantly different between the two strains (EC50 171 +/- 21 and 102 +/- 19, respectively). 3. In aortic rings, in contrast, reactivity to endothelin-1 was reduced in SHR as compared to WKY, whereas sensitivity was similar (EC50 0.78 +/- 0.08 and 0.87 +/- 0.09 nM). 4. As with endothelin-1, reactivity to noradrenaline and potassium chloride was increased in MVBs, but not in aortic rings of SHR. Endothelin-1 was 30 times more potent than noradrenaline in MVBs of SHR, and 15 times more potent than noradrenaline in aortic rings. 5. In both strains, nifedipine and nitrendipine almost completely blocked potassium-induced contractions in MVB and aortic rings, respectively, whereas contractions induced by endothelin-1 or noradrenaline were only partially inhibited. 6. It is concluded that calcium influx via the voltage-operated calcium channel is only partially responsible for the vasoconstrictor action of endothelin-1 in MVBs and aortic rings of SHR and WKY rats. The increased reactivity of the MVB of SHR to endothelin-1 at this stage of the hypertensive process is most likely to be the result of a change in vascular structure rather than due to a primary hypertensive mechanism.

Treatment of adjuvant arthritis in rats: vaccination potential of a synthetic nonapeptide from the 65 kDa heat shock protein of mycobacteria.

Adjuvant arthritis induced by mycobacteria in rats is a widely used model of chronic arthritis. A previously described nonapeptide (Thr-Phe-Gly-Leu-Gln-Leu-Glu-Leu-Thr, amino acid sequence 180-188) from the recombinant 65 kDa heat shock protein of Mycobacterium bovis BCG, which was found to contain a T-cell epitope recognized by both arthritogenic and protective T-cell clones in vitro, has been investigated for its vaccination and therapeutic potential in adjuvant arthritis in rats. The nonapeptide was found not to be arthritogenic, although the T cells from nonapeptide immunized rats cross-react in vitro with mycobacterial antigens. Intraperitoneal administration of 0.1 mg nonapeptide in oil at day -20 or days -2, -1 and 0, resulted in a marked reduction of incidence and severity of adjuvant arthritis. The disease process and severity were also influenced by therapeutic treatment with 0.1 mg nonapeptide injected intraperitoneally at days 7 to 10. Interestingly, subplantar or intravenous application of the nonapeptide had no influence on the disease process. Deletion of the N-terminal threonine led to complete loss of in vivo activity of the nonapeptide.

Synthesis and biological activity of seventeen analogues of human insulin.

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.

Analogues of human calcitonin. V. Influence of basic amino acids in positions 11, 17 and 24 on hypocalcaemic activity in the rat.

The influence of positions 11 and 24 on hypocalcaemic potency and duration of action was examined. These positions are respectively occupied by threonine and glutamine in HCT, but by the basic amino acids lysine and arginine in SCT. Replacement of threonine by lysine trebled the hypocalcaemic potency of HCT and slightly prolonged its duration of action. Substitution of arginine for glutamine reduced the activity to about one tenth. The simultaneous introduction of both basic amino acids yielded an analogue intermediate in activity between those obtained by the single substitutions. The analogue [Bmp1, Va18, Lys11, Arg24]-HCT displayed the same effects as [Lys11]-HCT.

[Structure-activity relationship of human calcitonin. III. Biological activity of synthetic analogues with shortened or terminally modified peptide chains (author's transl)]. / Struktur-Wirkungsbeziehungen beim menschlichen Calcitonin. III. Die biologische Aktivität verkürzter oder an den Kettenenden modifizierter, synthetischer Analoger

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.

Analogues of human calcitonin. IV. Influence of leucine substitutions 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) by 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-terminus yielded an analogue 10 times more potent 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.

Analogues of human calcitonin. II. Influence of modifications in amino acid positions 1, 8 and 22 on hypocalcemic activity in the rat.

The calcitonin analogues [Val8] -HCT and [Tyr22] -HCT, each with a single modification, and [Val8, Tyr22] -HCT and [Bmp1, Val8] -HCT, with two replaced amino acids were compared with synthetic human calcitonin (HCT) in respect of their hypocalcemic effects in the rat. The introduction of either valine in place of methionine in position 8 or of tyrosine for phenylalanine in position 22 of the HCT molecule yielded analogues 4 to 5 times as potent and nearly twice as long-acting as HCT. The doubly substituted peptide [Val8, Tyr22] -HCT displayed properties closely similar to those of [Val8] -HCT and [Tyr22] -HCT. The analgoue [Bmp1, Val8] -HCT, with a deaminated cysteine residue at the N-terminus, was about 6 times more potent than HCT and slightly longer-acting than [Val8] -HCT.