Tomislav Barth

Kinetic and pharmacological characterization of vasopressin membrane receptors from human kidney medulla: Relation to adenylate cyclase activation

Abstract Membranes from the medullopapillary portions of three human kidneys unsuitable for transplantation were prepared and stored in liquid nitrogen. Specific vasopressin binding sites were detected on these membranes and characterized using tritiated lysine vasopressin. The biological activities of vasopressin and of structurally related peptides were estimated on the same membrane preparations by measuring dose-dependent adenylate cyclase activation. The population of vasopressin binding sites was homogeneous and displayed the following characteristics: equilibrium dissociation constant K D 27 ± 7 nM (for lysine vasopressin); maximal specific binding capacity 0.7±0.1 pmol [ 3 H]LVP bound/mg protein. Maximal adenylate cyclase activation by lysine vasopressin was 5.4 times the basal value of 50 ± 4 pmol cAMP formed/5 min per mg protein. Half-maximal activation (K act ) was obtained at a concentration of 34 ± 5 nM. Both lysine vasopressin binding and hormone-induced adenylate cyclase activation were sensitive to guanyl nucleotides. 5′-Guanylyl imidodiphosphate reduced K act by a factor of 3 and increased K D by a factor of about 8. K D and K act were determined for a series of peptides including natural vasopressins, oxytocin and their structural analogues which were found to have enhanced or reduced antidiuretic activities in the rat. The concentration range of the K D and K act values covered a concentration range of more than three orders of magnitude. The most active peptide was the natural arginine vasopressin (K D 4.4 ± 0.7 nM; K act 3.1 ± 1.6 nM). There was a highly significant correlation between paired K D and K act values. The two vasopressin structural analogues [1-(β- mercapto-β, β-cyclopentamethylene propionic acid), 4-valine, 8-D-arginine]vasopressin and [1-β-mercapto-β, β-cyclopenthamethylene propionic acid), 2-O-ethyltyrosine, 4-valine]arginine vasopressin, behaved like competitive inhibitors of vasopressin-induced adenylate cyclase activation (K i = 65 and 59 nM respectively). Freezing the membranes in liquid nitrogen reduced the affinity of vasopressin receptors but fully preserved their selectivity towards vasopressin structural analogues. It is concluded that the vasopressin binding and/or adenylate cyclase assays might be useful for estimating several aspects of the pharmacological activities of vasopressin structural analogues in man.

Theory of the correlation between capillary and free-flow zone electrophoresis and its use for the conversion of analytical capillary separations to continuous free-flow preparative processes. Application to analysis and preparation of fragments of insulin.

A basic theoretical description of the correlation between capillary zone electrophoresis (CZE) and free-flow zone electrophoresis (FFZE) is presented. The theory of the correlation between CZE and FFZE results from the fact that both methods are based on the same separation principle, zone electrophoresis, and both are performed in the carrierless separation medium with the same composition of the background electrolyte. The equations describing the movement of the charged and noncharged particles in the d.c. electric field applied in the capillary and in the flow-through electrophoretic chamber are presented and used for the quantitative description of the correlation between CZE and FFZE. Based on the theory of the correlation between CZE and FFZE a procedure has been developed for conversion of analytical, microscale CZE separations into continuous preparative separation processes realized by FFZE. Practical application of the developed procedure is demonstrated by CZE analysis and FFZE preparation of an octapeptide fragment of human insulin.

Oxytocin receptor binding and uterotonic activity of carbetocin and its metabolites following enzymatic degradation

Metabolites of the analogue 1-deamino-1-carba-2-tyrosine(O-methyl)-oxytocin (carbetocin) following incubation with a rat kidney homogenate were isolated and their pharmacodynamic properties investigated. Apart from the parent compound two metabolites were identified namely desGlyNH2-carbetocin (carbetocin metabolite I) and desLeuGlyNH2-carbetocin (carbetocin metabolite II). Both carbetocin, carbetocin metabolite I and carbetocin metabolite II displayed binding affinities to the myometrial oxytocin receptor of a similar magnitude as oxytocin. Carbetocin was found to have agonistic properties on isolated myometrial strips and it was found to exert this effect through generation of inositol phosphates, as is the case for oxytocin. However, maximal contractile effect of carbetocin was approximately 50% lower than that of oxytocin (2.70 +/- 0.12 g compared to 5.22 +/- 0.26 g) and EC50 was approximately ten times higher (48.0 +/- 8.20 nM compared to 5.62 +/- 1.22 nM). Neither carbetocin metabolite I nor carbetocin metabolite II were able to contract isolated myometrial tissue. All three compounds displayed antagonistic properties against oxytocin in vitro, with carbetocin being the strongest inhibitor (pA2 = 8.21) and carbetocin metabolite II (pA2 = 8.01) being stronger than carbetocin metabolite I (pA2 = 7.81). These results indicate that carbetocin is a partial agonist/antagonist to the oxytocin receptor while the two metabolites carbetocin metabolite I and carbetocin metabolite II are pure antagonists. All three analogues bound to the myometrial vasopressin V1 receptor, albeit with much lower affinities than to the oxytocin receptor. Carbetocin metabolite II showed the weakest binding affinity of 33.7 +/- 7.34 nM compared to 7.24 +/- 0.29 nM for carbetocin and 9.89 + 2.80 nM for carbetocin metabolite I. Only carbetocin bound to the renal vasopressin V2 receptor though the binding affinity was very low (61.3 +/- 14.6 nM).

Induced ovulation in tench (Tinca tinca L.) by various LH-RH synthetic analogues: Effect of site of administration and temperature

Abstract No significant effect was found of the site of administration (intramuscular or into the pericardial cavity) of [D-Ala 6 ] LH-RH ProNH-Et (at 2 or 20 μg kg −1 ) on the spawning of tench. When [D-Ala 6 ] LH-RH ProNH-Et (10 μg kg −1 ) was administered to female tench kept at different water temperatures (within the physiological range) a decrease of relative working fecundity was found with increasing water temperature. Chicken LH-RH ([Gln 8 ] LH-RH) at 0.16–20 μg kg −1 was inactive, while [D-Tle 6 ] LH-RH ProNH-Et at 20 μg kg −1 was found to be as effective as [D-Ala 6 ] LH-RH ProNH-Et at the same dose.

Vasopressin analogs: Sedative properties and passive avoidance behavior in rats

The effects of several types of vasopressin analogs that are considered to be resistant to some of the physiologically significant enzymatic systems were investigated utilizing rats trained in a passive avoidance task. Enhancement of avoidance latencies was observed 2, 7 and 13 days after the single learning trial when deamino-carbavasopressins, triglycyl-8-lysine-vasopressin or its des-glycinamide derivative, and deamino-D-arginine-vasopressin were given shortly after the learning trial in the dose of 1 microgram s.c. (8-L-Arginine)deamino-6-carba-vasopressin and (8-L-ornithine)deamino-6-carba-vasopressin were also active in the dose of 0.1 microgram. Lysine vasopressin and its des-glycinamide derivative failed to enhance avoidance latencies in part of the experiments if doses of 0.3--3 micrograms were administered and 7 or 13 day intervals were used between the learning and the test trials. Enhancement of avoidance latencies was also observed, if some of the peptides were injected 20 min but not 120 or 180 min before the test trial. Marked depression of exploratory behavior of rats in an open field was found after s.c. injections of low doses (1--3 micrograms kg-1) of deamino-carba-vasopressins. Higher doses (10--30 micrograms kg-1) induced sleep-like immobility not accompanied by ataxia or catalepsy.

Application of capillary and free-flow zone electrophoresis and isotachophoresis to the analysis and preparation of the synthetic tetrapeptide fragment of growth hormone-releasing peptide

The use of high-performance electromigration separation methods, capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) and continuous free-flow arrangements of these two separation principles, free-flow zone electrophoresis (FFZE) and free-flow isotachophoresis (FFITP), was investigated in the analysis and purification of the synthetic C-terminal tetrapeptide fragment (H2N-Ala-Trp-D-Phe-Lys.NH2) of the growth hormone-releasing peptide. CZE and CITP were used for microanalysis of peptide preparations after different steps of their purification. The homogeneity of the peptide preparations, including fractions of preparative separations, was quantified by relative zone length (CITP) and/or relative peak height (CZE). In addition, the data obtained by CZE and CITP (electrophoretic and electroosmotic flow migration velocities) were utilized for conversion of micro-scale capillary separations (nano- to picomole level) into the preparative separations realized by FFZE and FFITP with a capacity from tens to hundreds of milligrams per hour.

Pharmacology of cyclic analogues of deamino-oxytocin not containing a disulphide bond (carba analogues)

Abstract A study was made of the uterotonic, antidiuretic and pressor activities and the effect on the isolated mammary gland of 6 deamino-oxytocin analogues not containing a disulphide bond: [1,6-α-deaminocystathionine] -oxytocin (deamino-carba 1 -oxytocin, Car-1 -OT), [6,1-β-deaminocystathionine] -oxytocin (deamino-carba 6 -oxytocin; Car-6-OT), [1,6-α-aminosuberic acid] -oxytocin (deamino-dicarba-oxytocin; Asub-OT), [1,6-deaminolanthionine] -oxytocin (Lan-OT), [1,6-α-aminopimelic acid] -oxytocin (Apim-OT), and [1,6-deaminohomolanthionine] -oxytocin (Hlan-OT). The values of biological activity obtained in the individual tests show that the presence of the disulphide bridge in the hormone molecule is not a prerequisite for the interaction of oxytocin with the receptors. The carba analogues of deamino-oxytocin with a modified ring size have all in all decreased activity with the exception of the effect of Hlan-OT on the mammary gland where this analogue has the highest activity of all the compounds tested.

Separation of structurally related peptides by open-tubular capillary electrochromatography using (metallo)porphyrins as the adsorbed stationary phase.

Several (metallo)porphyrins, particularly the porphyrin derivative tetraphenylporphyrin, and complexes of porphyrin derivatives with metal ions (Zn2+, Cu2+, Ni2+, Co2+, Co3+) have been employed as the stationary phase physically adsorbed onto the inner fused-silica capillary surface for open-tubular capillary electrochromatography, and applied for the separation of structurally related peptides. Four octapeptides, derivatives of the B23-B30 fragment of the B-chain of human insulin with minor changes in their sequences (presence of lysine or ornithine in position B-29, presence or absence of phenylacetyl protecting group on the amino group of lysine/ornithine or N-terminal amino group of glycine), were studied as model analytes. Separations were performed both in alkaline (pH 9.0) and in acidic (pH 2.25) background electrolytes, and the changes in the migration/retention behaviour of the model set of peptides were investigated with respect to the porphyrin periphery/central metal atom and the charge of the octapeptides modified. The key moment of successful separation of these peptides seems to be the accessibility of functional groups of the peptides to the interaction with the modifiers tested herein.

Use of capillary electrophoresis and high-performance liquid chromatography for monitoring of glycosylation of the peptides dalargin and desmopressin

Capillary electrophoresis (CE) and HPLC procedures have been developed and tested for monitoring of glycosylation of the peptide hormones dalargin and desmopressin, permitting identification and determination of the reaction products and establishing the reaction rate. Good separations have been obtained that can also be used for preparative purposes. Reversed-phase HPLC employs a gradient of methanol in a mobile phase of 0.1% trifluoroacetic acid, pH 2.2, with a diode-array UV detection. The CE systems involve 150 mM phosphoric acid, pH 1.9, or a micellar system containing 50 mM dodecyl sulfate and 20 mM sodium tetraborate, pH 9.2, both with UV photometric detection at 190 nm; the latter system is less suitable for glycoconjugate isolation. Whereas CE is superior to HPLC in analytical application, HPLC is preferable for preparative purposes.

Capillary zone electrophoresis with external radial electric field control of electroosmotic flow and its application to the separation of synthetic oligopeptides

Abstract A new way of applying external radial electric field for the control of electroosmotic flow in capillary zone electrophoresis (CZE) has been developed. It is based on a new type of low-conductivity coating of the outer capillary surface by a polyaniline dispersion in hydroxypropylcellulose and on the application of a set of three high-voltage power supplies to form a constant radial electric field across the capillary wall as far as possible along the capillary length. Two power supplies are connected to the ends of the outer low-conductivity coating and the third one is applied to the ends of the inner capillary compartment filled with background electrolyte. The difference of electric potentials at the inner and outer capillary surface determines the voltage of radial electric field across the capillary wall and affects the electrokinetic potential at the solid–liquid interface inside the capillary. The effect of magnitude and polarity of external radial electric field on the flow-rate of electroosmotic flow, on the migration times of charged analytes (speed of analysis) and on the separation efficiency and resolution of CZE separations of synthetic oligopeptides, diglycine, triglycine, dalargin and dalargin–ethylamide has been evaluated. Application of external radial electric field has proved to be an efficient tool for regulation of electroosmotic flow in CZE and for optimization of migration times and resolution of oligopeptides in their CZE separations and analyses.