Interaction of a neutral endopeptidase inhibitor with an ANP-C receptor ligand in anesthetized dogs.

Inhibition of important degradative pathways of atrial natriuretic peptide (ANP) in vivo could be a valuable therapeutic tool for regulating endogenous levels of ANP. The aim was to investigate the in vivo effects of both blockade of atrial natriuretic peptide clearance receptor and inhibition of neutral endopeptidase 24.11, an enzyme shown to be involved in ANP breakdown. Therefore, we infused a specific neutral endopeptidase inhibitor ((S)-thiorphan) and an ANP-C receptor ligand (AP 811) alone or in combination into anaesthetized beagle dogs. Compared with vehicle controls, coadministration of (S)-thiorphan and AP 811 (100 micrograms/kg/min and 10 micrograms/kg/min, resp.) had greater effects on endocrine and renal parameters than administration of either substance alone. Coadministration of both compounds increased urinary excretion of volume and sodium, cGMP and ANP. We found also increased plasma cGMP, plasma ANP and decreased plasma renin activity. No effects were observed with respect to blood pressure, left ventricular pressure or heart rate during the infusion period of 2 h. We conclude from these investigations, that blocking both degrading pathways of ANP with the ANP-C receptor ligand AP 811 and the neutral endopeptidase inhibitor (S)-thiorphan is more effective than inhibition of either system alone. Such a combination might therefore be a useful therapeutic tool in cardiovascular diseases.

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.

Structural fluctuations of a helical polypeptide traversing a lipid bilayer.

Time-resolved fluorescence anisotropy (FA) measurements are reported for five helical bilayer-spanning henicosapeptides, each containing one tryptophan at sequence position 1, 6, 11, 16, or 21. The FA decay reflects two molecular processes in all cases: local internal fluctuations of the tryptophan side chain with a relaxation time of 200-500 ps, and motions of the whole polypeptide molecule with a relaxation time of 9-10 ns. The amplitudes of the fast fluctuation are largest at the helix ends and decrease toward the center of the helix. A similar observation was made for the helical polypeptides dissolved in organic solvents showing that the mobility gradient along the polypeptide sequence is an inherent property of the polypeptide helix and not due to the lipid bilayer. However, the amplitudes of the fast fluctuations can be modulated by the physical state of the lipid bilayer. With decreasing temperature, the internal mobility of the tryptophan in all positions decreases with an abrupt change at the lipid-phase transition. Concomitant molecular dynamics calculations indicate a correlation between the fast FA decay and conformational fluctuations within the helix. According to the simulation, the conformation of the polypeptide is on average predominantly helical, but actually the molecule can fluctuate between a variety of different substructures. The conformational fluctuations are largest at the helix ends and provide the free space required for rotation of the indole ring around the tryptophan side chain bonds.

Depth-dependent fluorescent quenching of a tryptophan residue located at defined positions on a rigid 21-peptide helix in liposomes.

Five lipophilic 21-peptide analogs of the potential-dependent pore-former, alamethicin, were synthesized bearing tryptophan residues at the position 1, 6, 11, 16 and 21 on a long, conformationally rigid, alpha-helix. The alpha-helical conformation was induced and stabilized using the sequential oligomers (Ala-Aib-Ala-Aib-Ala)n as analyzed by CD and NMR. The partitioning of the N-t-butoxycarbonyl 21-peptide methyl esters and the N-terminally deprotected alpha-helices was followed by fluorescence enhancement in phospholipid bilayer vesicles. Quenching experiments were performed by titrating with n-doxyl stearic acids bearing the nitroxide label at positions 5, 7, 10, 12 and 16. This well-defined system revealed that the N- and C-terminal tryptophan residues become situated in the hydrophilic region. Tryptophan at position 11 was found in the lipophilic core, whereas the tryptophan at positions 6 and 16 were localized at intermediate depths of the lipid membrane. Therefore, the helices span the lipid bilayer with their long axis normal to the membrane surface.

Voltage-dependent channel formation by rods of helical polypeptides.

The voltage-dependence of channel formation by alamethicin and it natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts alpha-helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length. In order to test this hypothesis we synthesized a family of lipophilic polypeptides--Boc-(Ala-Aib-Ala-Aib-Ala)n-OMe, n = 1-4--which adopt alpha-helical conformation for n = 2-4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed quantitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase. The steady-state voltage-dependent conductance depends on the 8th-9th power of polypeptide concentration and involves the transfer of 4-5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 A is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 alpha-helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular shape and dipole moment of alamethicin-like synthetic peptides.

The peptides Boc-(L-Ala-Aib-L-Ala-Aib-L-Ala)n-OMe, with n = 2 (P10) and n = 4 (P20), have been synthesized as purely hydrophobic models of the antibiotic alamethicin, which is known to be a voltage-dependent pore former in membranes and is apparently alpha-helical in lipophilic media. These peptides were investigated in 1-octanol, a solvent which resembles the membrane environment. From dielectric dispersion studies quantitative information on the molecular shape and dipole moments could be derived. Further independent data concerning conformation and extent of aggregation of the peptides were obtained by circular dichroism and ultracentrifuge measurements. The results suggest that the peptides assume the form of elongated particles having a significant amount of ordered secondary structure and carrying a dipole parallel to the long axis. Apparently the monomeric peptide molecules undergo, to some extent, a head-to-tail aggregation which is slightly enhanced at lower temperatures. Based on the high-frequency parts of the dielectric dispersion curves the lengths, diameters, and dipole moments of the monomer particles have been determined as 22.5A, 10A, 36 D (P10) and 28.5A, 12A, 64D (P20).