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M Bienert

Publications and source records attributed to M Bienert.

At least 73 records · Page 4Linked to original sources
PubMed1994

Functional studies with substance P analogues: effects of N-terminal, C-terminal, and C-terminus-extended analogues of substance P on nicotine-induced secretion and desensitization in cultured bovine adrenal chromaffin cells.

Substance P (SP) and SP analogues, including C-terminal, N-terminal, and C-terminus-extended analogues, have been investigated for their ability to modulate nicotine-induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line luciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10(-5) M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C-terminus-extended analogue SP-Tyr12-NH2, 65% by (Ala3)SP-NH2, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 x 10(-5) M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr12-NH2 > (Ala3)SP-NH2 > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala3)SP-NH2 protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (all at 3 x 10(-5) M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr12-NH2 at 3 x 10(-5) M did not protect against nicotinic desensitization. Cyclo-SP(3-9), Ac-SP(3-9)-NH2, SP(3-9), and SP(3-6) had neither inhibitory nor facilitatory effects on secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Spontaneous chemical degradation of substance P in the solid phase and in solution.

The instability of the undecapeptide substance P (SP), a neuropeptide implicated in several physiological processes, was occasionally observed when the peptide was stored in the solid state or in solution. The aim of the present study was to identify the decomposition products of SP stored as lyophilized peptide or in aqueous neutral solution. The main pathway of the decomposition of SP acetate consists of the subsequent release of N-terminal dipeptides via their diketopiperazines, cyclo(Arg-Pro) and cyclo(Lys-Pro). In contrast to the decomposition of the acetate of SP, the hydrochloride and trifluoroacetate salts were found to be considerably more stable. Under the studied conditions the release of N-terminal dipeptides dominates over other possible routes of spontaneous modifications, such as S-oxidation and deamidation.

Amino Acid Sequence↗
PubMed1993

Relaxing actions of corticotropin-releasing factor on rat resistance arteries.

1. Although it well established that corticotropin-releasing factor (CRF) injected i.v. can cause hypotension and vasodilatation, there is no in vitro evidence that CRF acts as a vasodilator. We have therefore tested the hypothesis that the hypotensive effect of i.v. CRF is due to a direct vasodilator action by carrying out experiments in vitro on rat resistance arteries (i.d. 150-300 microns). 2. Initial in vivo experiments confirmed that CRF (1.5 nmol.kg-1) injected i.v. caused hypotension in rats, this being partially antagonized by the CRF analogue CRF9-41. 3. For the in vitro experiments, vessels were taken from the mesenteric, cerebral and femoral vascular beds, and mounted as ring preparations in an isometric myograph. The vessels were pre-contracted with one of 3 agonists (prostaglandin F2 alpha, arginine vasopressin or noradrenaline) or with a high-potassium solution (K+). 4. With maximal concentrations of the agonists, CRF caused relaxation of mesenteric and cerebral vessels with 10 nM, and near complete relaxation with 100 nM. Femoral vessels pre-constricted with agonists and all vessels pre-constricted with K+ were less affected by CRF. In the mesenteric vessels, with sub-maximal levels of pre-constriction, CRF caused substantial relaxation at 1 nM and could cause complete relaxation at 10 nM. 5. The relaxant effect of CRF on contractions of mesenteric vessels was antagonized by 100 nM CRF9-41. Neither tetraethyl ammonium (30 mM) nor glibenclamide (3 microM) antagonized the relaxant effect of CRF. 6. The relaxant effect of CRF on mesenteric small arteries was found to be unaffected by removal of the endothelium. 7. The results indicate that CRF causes an endothelial-independent vasodilatation of rat resistance arteries under in vitro conditions at concentrations which are consistent with this being an important cause of the hypotension observed with i.v. injection of CRF.

Animals↗
PubMed1991

Gonadotropin-releasing hormone (GnRH) analogs: relationship between their structure, proteolytic inactivation and pharmacokinetics in rats.

There are two types of superactive agonists of gonadotropin-releasing hormone (GnRHa-I: (D-amino acid)6-GnRH and GnRHa-II: (D-amino acid)6-(desGly)10-GnRH- ethylamide) the high hormonal activity of which is understood to be due to their higher receptor affinity and their higher proteolytic stability as compared with the native GnRH sequence. Using the soluble fractions of various rat tissues in studies on the inactivation of GnRH peptides, we confirmed the higher proteolytic resistance of GnRHa-II, but not of D-Phe6-GnRH (GnRHa-I) and of another analog, D-Trp3-D-Phe6-GnRH, as compared with GnRH. The exact behaviour of the peptides during degradation was found to be dependent on the peptide concentrations used, showing the importance of using conditions as near to the physiological ones a possible. Towards the membrane fractions, however, the order of degradability was found to be GnRH much greater than D-Phe6-GnRH much greater than D-Trp3-D-Phe6-GnRH. The pharmacokinetic consequences of the different proteolytic degradabilities of the GnRH peptides, observed in rats, were a moderate increase in the biological half-life of D-Phe6-GnRH by 2.5-fold, as compared with GnRH, and a small increase in half-life of D-Trp3-D-Phe6-GnRH by 1.4-fold when compared with D-Phe6-GnRH. Whereas no intact GnRH was recovered in rat urine, small amounts of D-Phe6-GnRH (about 1% of dose) and high amounts of D-Trp3-D-Phe6-GnRH (25.5%) were excreted into urine. Combining the biochemical and pharmacokinetic data, it is concluded that proteolytic stability of GnRH analogs in pharmacological terms means stability towards membrane enzymes (pharmacologically-related stability) and that designing analogs with further increased proteolytic stability will be of only limited consequences with respect to their biological half-lives, the glomerular filtration rate of the kidney becoming the determining factor in the peptide clearance.

Amino Acid Sequence↗
PubMed1991

Effect of tertiary amine on the carbodiimide-mediated peptide synthesis.

The effect of tertiary amine (DIEA) on reaction rate and product purity of a carbodiimide/HOBt-mediated peptide synthesis was studied. It was found that very rapid activation can be achieved using carbodiimide/HOBt in non-polar solvents, such as DCM. Although the HOBt is poorly soluble in DCM, the activation proceeds within 2 min, probably forming the HOBt-ester. By such a preactivation followed by a coupling in the presence of DIEA the rate of coupling is comparable with other rapid methods using BOP or TBTU, and no racemization was found in a model coupling (less than 0.1%). For comparison, syntheses of neurotensin by means of different coupling reagents (BOP, TBTU, OPfp-esters) and the DIEA-catalyzed coupling after carbodiimide/HOBt-activation under comparable conditions have shown that these procedures are of the same value in view of coupling efficiency and product purity.

Amines↗
PubMed1991

Chymotrypsin-catalyzed fragment coupling synthesis of GnRH-analogs.

D-Xaa6-GnRH analogs (Xaa: Ala, Nal1), Phe, Ser(tBu), Trp) were prepared by chymotrypsin catalyzed 3 + 7 fragment coupling synthesis with conversion rates of the amino component in the range from 90.3 to 97.4%. For D-Phe6-GnRH the method was scaled up to production level.

Catalysis↗
PubMed1989

Influence of substance P and substance P--sequences on immunocompetent cells.

The influence of substance P (SP) and substance P-sequences on spleen cell cultures of mice and on rat lymph node mononuclear cells was studied. SP and the N-terminal fragments SP(1-4) and SP(1-7) were capable of inducing the secretion of lymphokines with chemotactic properties for polymorphonuclear leukocytes and lymphocytes. The peak of the dose-response curves appeared in a concentration range of 10(-11) to 10(-10) mol/l. The predominant migratory stimulatory activity was found at a molecular mass of 12,000 and 23,000 dalton and an inhibitory activity at 35,000 dalton. The C-terminal part of the peptide, SP(6-11), SP(7-11), SP(8-11), SP(9-11), was not able to induce such lymphokine secretion. The experimental results display the importance of the N-terminal fragment for the lymphokine secretion, and support the hypothesis that SP is also a peptide with modulatory functions in the immune response.

Animals↗
PubMed1988

Structural requirements for mast cell triggering by substance P-like peptides.

Experiments were based on the hypothesis that the histamine releasing action of substance P and some other neuropeptides is not due to the interaction of the neuropeptide with a specific membrane receptor. Rather, this may be a property of many amphiphilic compounds having a minimal number of positives charges and a hydrophobic chain which is not necessarily a peptide. According to this hypothesis, 18 substance P derivatives and fragments were synthesized and tested on rat and hamster peritoneal mast cells, among them five compounds which contain a non-peptide chain instead of the C-terminal substance P heptapeptide. The results are in accord with the above hypothesis.

Animals↗
PubMed1987

Mast cell activation--a receptor-independent mode of substance P action?

Substance P is a representative of a group of amphiphilic neuropeptides which act as mast cell secretagogues. Our experiments with some new substance P derivatives suggest that these effects are dependent on two structural elements: (i) a hydrophobic chain which is not essentially a peptide, and (ii) a hydrophilic part with two positively charged amino acids. The mast cell triggering effect is unlikely to be mediated by a selective substance P receptor, but has strong similarities to the mode of action of polycations.

Animals↗
PubMed1987

Histamine release induced by Arg-Pro-Lys-Pro(CH2)11CH3 from rat peritoneal mast cells.

The substance Arg-Pro-Lys-Pro-(CH2)11CH3 [SP1-4C12] was synthesized by forming a peptide bond between Arg-Pro-Lys-Pro, the N-terminal sequence of substance P and dodecylamine. The aim was to examine the roles of the N- and C-terminal sequences of substance P in stimulating histamine release from mast cells of the rat peritoneal cavity. SP1-4 C12 induces concentration-dependent histamine release in the range 8 to 200 nM. SP1-4C12 was 50 times more potent than substance P and 300 times more potent than dodecylamine. Unlike dodecylamine itself, SP1-4C12 induced noncytolytic histamine release which was inhibited by benzalkonium chloride and by the substance P antagonist [D-Pro4,D-Trp7,9,10]SP4-11. Histamine release induced by SP1-4C12 was inhibited at temperatures below 16 degrees C and did not require the presence of extracellular calcium ions. It is suggested that substance P and some other basic histamine liberators initiate histamine secretion by a mechanism that involves the insertion of a hydrophobic region into the membrane lipid which is necessary to present positively charged moieties to a receptor site involved in activating the secretory mechanism.

Animals↗
PubMed1986

The essential sequence of substance P for locomotion.

In rats the effect of substance P SP (1-11 and SP (5-11) heptapeptide on locomotion in open field was investigated after intrategmental application. SP (1-11) increase the locomotor activity significantly, SP (5-11) heptapeptide do not-do it. The effect of SP(1-11), SP(5-11) heptapeptide, SP(6-11) hexapeptide, and SP(1-4) tetrapeptide on the circadianly organized locomotor activity was researched after i.p. application at 11 a.m. (light phasis, low activity of rats) or 7 p.m. (dark phasis, high activity). An increased effect on locomotiou'slow activity of rats appears by SP(1-11) and SP(1-4) tetrapeptide application for several hours in light time. Both peptides display a decreasing effect on locomotion after application for several hours in dark time, too. SP(5-11) heptapeptide and SP(6-11) hexapeptide do not have any influence on locomotion. The effects of SP(1-11) are equal to results found after application into the ventral tegmental area. The experimental results display that SP acts as a regulatory peptide modulating the activity of rats by a levelling mechanism. The N-terminal SP-sequence, SP(1-4), acts in a similar manner. The effects are discussed in relation to the mediation by receptors which recognize the C- or N-terminal part of the SP molecule.

Amino Acid Sequence↗
PubMed1984

Substance P: binding to unilamellar and multilamellar liposomes made from mixtures of phosphatidylcholine and phosphatidic acid.

Binding of the undecapeptide substance P to unilamellar and multilamellar lipid vesicles, made from mixtures of phosphatidylcholine and phosphatidic acid, was investigated, because liposomes may be supposed to become a useful pharmaceutical tool for the delivery of low molecular mass peptide hormones. It is demonstrated that binding is largely electrostatic in nature, and that the amount of bound peptide may be varied within rather broad limits by changing the lipid matrix and/or the aqueous medium conditions. Concerning the distribution of the peptide between inner and outer surfaces of the liposomes, large differences may be realised too, as revealed by enzymatic hydrolysis of the outside bound portions of substance P. But additionally, electrostatic binding is demonstrated also to bring about peculiar, unexpected properties of the system. These imply special relevance to a potential pharmaceutical application of substance P liposomes as well as some general relevance to in vivo application of liposomes as drug carriers.

Drug Stability↗
PubMed1983

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown.

Analogs of luteinizing hormone-releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct 3H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe6,desGly10]-LHRH ethylamide and [D-Ser(But)6,desGly10]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al2O3 catalyst to high specific radioactivities. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various tissues of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe6,desGly10]- and [D-Ser(But)6,desGly10]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of the analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.

Animals↗