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R J Vavrek

Publications and source records attributed to R J Vavrek.

At least 37 records · Page 2Linked to original sources
PubMed1989

Inhibition of human and rat tissue kallikreins by peptide analog antagonists of bradykinin.

Three representative bradykinin receptor antagonists have been examined for their capacity to inhibit purified human and rat urinary kallikreins. In an amidolytic assay, the Ki values for the inhibition of human urinary kallikrein were 0.5, 0.3, and 2.5 microM for B4307 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg), B4308 (Lys-Lys-Arg-Hyp-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg) and B3852 (Arg-Pro-Hyp-Gly-Phe-Ser-DPhe-Phe-Arg), respectively. B4308 and B3852 inhibited rat urinary kallikrein with Ki's of 1.0 and 5.5 microM, respectively. B4308 (0.4 to 1.6 microM) also inhibited the kininogenase activity of human urinary kallikrein on human low molecular weight kininogen in a dose-dependent fashion. These bradykinin receptor antagonists may inhibit the kallikrein-kinin system both by blocking the binding of kinins to B2 receptors and by inhibiting the cleavage of kinins from kininogens.

Amino Acid Sequence↗
PubMed1989

Characterization of [3H]bradykinin binding sites in guinea-pig central nervous system: possible existence of B2 subtypes.

Specific [3H]bradykinin(BK) binding was investigated in membranes from guinea-pig brain. In kinetic experiments, specific [3H]BK binding (100 pM) reached equilibrium within 15 min at 25 degrees C (k + 1 = 1.40 nM-1min-1) and the binding was reversed by the addition of 1 microM BK (k-1 = 0.069 min-1). The presence of a high affinity BK binding site was also revealed in the guinea-pig brain by equilibrium saturation studies with a Kd value of 75 pM and a Bmax value of 4.9 +/- 0.9 fmol/mg protein. In inhibition experiments, the B2 antagonists (D-Phe7-BK and Thi5,8,D-Phe7-BK) inhibited [3H]BK binding, but not the B1 antagonist (des-Arg9[Leu8]-BK). D-Arg[Hyp3, D-Phe7]BK (B4801) showed a pseudo Hill coefficient of less than one. The KH and KL values are 1.8 and 94 nM. The regional distribution study shows the highest density of BK binding sites in the pons + medulla oblongata and the spinal cord, a moderate density in the cerebral cortex and hippocampus, and a low density in other brain regions. These data support the presence of B2 BK receptors in the guinea-pig brain and spinal cord and suggest the existence of B2 subtypes in the brain. The presence of these receptors suggests that BK acts as a neurotransmitter or a neuromodulator in these tissues.

Animals↗
PubMed1989

Guanine nucleotide-dependent, pertussis toxin-insensitive regulation of phosphoinositide turnover by bradykinin in bovine pulmonary artery endothelial cells.

In this paper we examine the effect of the vasodilator peptide bradykinin on endothelial cell regulation of phosphoinositide (PI) turnover. The data show that the activation of PI turnover by bradykinin in bovine pulmonary artery endothelial cells is insensitive to pertussis toxin, which ADP ribosylates a membrane protein of mol wt 40,000. However, this effect of bradykinin can be potentiated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S), an activator of G proteins, and depressed by guanosine 5'-O-(2-thio)diphosphate (GDP beta S), an inhibitor of G proteins. After endothelial cells were preincubated for 1 h with GTP gamma S, there was a three- to fourfold increase in PI turnover. Preincubation of cells with GDP beta S did not affect the basal level of PI turnover, but completely prevented activation of PI turnover by bradykinin. 4 beta-Phorbol-12 beta-myristate-13 alpha-acetate can block the bradykinin-stimulated inositol monophosphate formation in cultured endothelial cells. The effects of bradykinin on PI turnover were blocked by B2 antagonists but not by B1 antagonists. Taken together, these results indicate that in endothelial cells the bradykinin B2 receptor is coupled to phospholipase C via a G protein (or proteins) that is not a substrate for pertussis toxin (neither Gi nor Go).

Adenosine Diphosphate Ribose↗
PubMed1988

Bradykinin as a pain mediator: receptors are localized to sensory neurons, and antagonists have analgesic actions.

Autoradiographic studies localize [3H]bradykinin receptor binding sites to the substantia gelatinosa, dorsal root, and a subset of small cells in both the dorsal root and trigeminal ganglia of the guinea pig. [3H]Bradykinin labeling is also observed over myocardial/coronary visceral afferent fibers. The localization of [3H]bradykinin receptors to nociceptive pathways supports a role for bradykinin in pain mediation. Several bradykinin antagonists block bradykinin-induced acute vascular pain in the rat. The bradykinin antagonists also relieve bradykinin- and urate-induced hyperalgesia in the rat paw. These results indicate that bradykinin is a physiologic mediator of pain and that bradykinin antagonists have analgesic activity in both acute and chronic pain models.

Analgesia↗
PubMed1988

High-field NMR and circular dichroism solvent-dependent conformational studies of the bradykinin C-terminal tetrapeptide Ser-Pro-Phe-Arg.

The conformational properties of the tetrapeptide Ser1-Pro2-Phe3-Arg4, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H2O and CD3OH/D2O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD3OH/D2O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe3 and Arg4 exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg4 and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH3+(Ser1)-COO-(Arg4) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.

Bradykinin↗
PubMed1987

Bradykinin receptor antagonists used to characterize the heterogeneity of bradykinin-induced responses in rat vas deferens.

The application of bradykinin to the isolated, transmurally stimulated rat vas deferens caused two effects: increase of the basal tension of the tissue and potentiation of the magnitude of electrically driven twitches. These bradykinin responses were not evenly distributed along the ductus. The direct contractile action of bradykinin was found to be stronger in the epididymal half of the tissue while the potentiation of the muscle twitches was more pronounced in the prostatic half of the rat ductus. Bradykinin is more potent to potentiate the electrically driven twitches than to act as a postjunctional agonist. Tyr-bradykinin, [Tyr5]bradykinin and [Tyr8]bradykinin exhibited significant differences in the potency ratio to produce each of these responses. [Thi5,8,D-Phe7]bradykinin is a weak postjunctional agonist but was a full agonist to potentiate the electrically induced twitches. Furthermore, this compound antagonized the bradykinin-induced contractions. [Hyp3,Thi5,8,D-Phe7]bradykinin was devoid of agonist activity at either pre- or postjunctional sites; it behaved as a pure antagonist and was more than potent its non-hydroxylated analog. The addition of a D-Arg residue at the amino terminal increased the antagonist potency significantly. The pA2 of D-Arg-[Hyp3,Thi5,8,D-Phe7]bradykinin to antagonize the postjunctional effect of bradykinin was 6.35, a value that differed significantly from the value of 6.93 required to block the prejunctional effect of the peptide. The bradykinin receptor antagonists did not modify significantly the magnitude of the contractile responses caused by angiotensin II, norepinephrine or 5-hydroxy-tryptamine.

Animals↗
PubMed1987

Analysis of the receptors mediating vascular actions of bradykinin.

A range of bradykinin (BK) analogues was assessed for their ability to antagonise the action of BK on rabbit jugular vein, a B2-receptor containing tissue, and compared with their action against BK-induced increases in skin vascular permeability in the rabbit and rat. The results demonstrate that modification of the BK (nonapeptide) structure by the insertion of certain D-amino acids in positions 5, 7 and 8 in addition to elongation of the amino terminal resulted in compounds with potent antagonistic action against BK on rabbit jugular vein and rabbit skin vascular permeability. The same BK analogues did not antagonise the action of BK on rat skin vascular permeability. It is concluded that the kinin receptor mediating an increase in vascular permeability in the rabbit is the same as that mediating contraction of the rabbit jugular vein in vitro, that is the B2-type. The kinin receptor mediating an increase in skin vascular permeability in the rat is difficult to classify but does not appear to be of the B2 type.

Amino Acid Sequence↗
PubMed1987

The effect of kinin agonists and antagonists on the pain response of the human blister base.

1. The effect of bradykinin (BK) and some analogues of BK on the human blister base was studied. 2. BK produced reproducible dose-related increases in pain responses. A characteristic delay, which was not dose-related occurred between application of BK and the resultant response. 3. The rank order of potency of several kinin analogues on the pain response was BK much much greater than sigma-cyclo-(Lys1-Gly6)-BK = sigma-cyclo-kallidin greater than des-Arg9-BK. 4. No increase in pain response was seen with repeated application of the selective B1 receptor agonist des-Arg9-BK to the same blister base at 4 h intervals. The B1 receptor antagonist des-Arg9-Leu8-BK was without effect against BK-induced responses. 5. The B2 receptor antagonists, D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Phe-Thi-Arg-TEA and D-Pro-Phe-Arg-heptylamide produced significant antagonism of the bradykinin-induced pain responses at doses which had no effect against 5-hydroxytryptamine or potassium chloride. 6. It is concluded that the kinin receptor mediating pain on the human blister base is of the B2 type.

Blister↗
PubMed1987

CD-n.m.r. study of the solution conformation of bradykinin analogs containing alpha-aminoisobutyric acid.

The conformation in aqueous solution of several alpha-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and 1H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro2 and/or Pro3 in BK stabilizes a degree of beta-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB7]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a beta-turn involving their common Arg1-Pro2-Pro3-Gly4 moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis' Pro and the propensity for a beta-turn at the N-terminus of the peptide.

Amino Acid Sequence↗
PubMed1987

New synthetic antagonists of bradykinin.

1 A new synthetic bradykinin analogue was found to be an antagonist of bradykinin-induced vascular permeability in rabbit skin. It was effective in equimolar concentrations. 2 These analogues also antagonized the action of bradykinin in contracting the guinea-pig isolated ileum. The mean pA2 values of five different antagonists ranged from 5.3-6.4 respectively, on this preparation. 3 Our observations, together with those of others suggest that these antagonists act on the same receptor types, viz., B2, in rabbit blood vessels and in smooth muscle of guinea-pig ileum. 4 Our results support the view that the way is now promising for the synthesis of potent specific antagonists of bradykinin for experimental and therapeutic use.

Animals↗
PubMed1987

Effects of bradykinin and angiotensin II on intracellular Ca2+ dynamics in endothelial cells.

The purpose of this study was to investigate the effects of angiotensin II and bradykinin on intracellular Ca2+ dynamics in cultured endothelial cells. We used the "second-generation" fluorescent Ca2+ indicator fura-2, in conjunction with dual-wavelength fluorescence spectroscopy, in cultured adherent pulmonary arterial endothelial cells. Angiotensin II (up to 2 microM) had no consistent effect on intracellular Ca2+ levels. In contrast, bradykinin (10 nM) elicited a transient increase of cytosolic free Ca2+, from the resting value of 37 +/- 5 to 647 +/- 123 nM, followed by a decline to a steady-state value of 113 +/- 14 nM, which was significantly higher than the resting Ca2+ levels. Bradykinin's Ca-stimulatory effect was dose dependent, having a half-maximally effective concentration of approximately 1 nM and a maximally effective concentration of 10 nM. A B1-receptor agonist, Des-Arg9-bradykinin, was much less effective than bradykinin as modulator of cytosolic Ca2+. Moreover, a B1-receptor antagonist, Des-Arg9, [Leu8]-bradykinin, did not significantly affect the increase of cytosolic Ca2+ elicited by bradykinin. On the other hand, the bradykinin-elicited increase of Ca2+ was almost completely inhibited by a novel B2-receptor antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin. Bradykinin increased cytosolic free Ca2+ levels in cells maintained in Ca2+-deficient extracellular medium, suggesting that the peptide mobilized Ca2+ from intracellular stores. However, the absence of extra-cellular Ca2+ resulted in an 80-90% attenuation of the transient Ca2+ response, whereas the posttransient steady-state response was completely absent. These findings are consistent with the notion that the bradykinin-elicited transient Ca2+ response is dependent on both extra- and intracellular Ca2+ and that the posttransient steady-state response is entirely dependent on extracellular Ca2+. Endothelial cells were responsive to a second dose of bradykinin after a 10-min interim period of incubation in the absence of the peptide hormone. The absence of extracellular Ca2+ during the interim period, or the pretreatment of cells with ionomycin in the absence of extracellular Ca2+, prevented the response of the cells to a second dose of bradykinin. Bradykinin- or ionomycin-desensitized cells could be resensitized by a brief incubation period in Ca2+-replete medium. The results are consistent with the notions that cellular resensitization requires the replenishment of intracellular Ca2+ and that bradykinin, but not angiotensin II, modulates intracellular Ca2+ dynamics in endothelial cells by interacting with a B2-type receptor.

Angiotensin II↗
PubMed1987

Competitive analog antagonists of bradykinin in the canine hindlimb.

Six structural analogs of bradykinin were tested to determine whether they antagonize the vasodilator response to bradykinin. The dog hindlimb preparation was used as a bioassay. Mongrel dogs were anesthetized and the femoral arteries were isolated and fitted with a noncanulating electromagnetic flow probe. An indwelling catheter was also placed for administration of saline, bradykinin, or the various analogs. The vasodilatory responses of the hindlimb circulation to bolus doses of bradykinin from 1 of 20 ng were tested during vehicle or analog administration at 1 and 10 micrograms/min. Bradykinin analogs which were characterized by amino acid replacement by beta-(2-thienyl)-L-alanine (Thi) at positions 5 and 8, D-phenylalanine (D-Phe) at position 7, and an additional replacement of hydroxyproline at position 2 or 3 were effective antagonists of bradykinin. The decapeptide bradykinin analog (BKA06) D-Arg-(Hyp3-Thi5-D-Phe7-Thi)-BK was the most potent analog tested, producing a full log dose shift in the dose-response curve to bradykinin at the 10 micrograms/min (4 nmole/min) infusion rate. None of the analogs we tested produced vasodilation or had any effect upon systemic blood pressure at the concentrations tested. Our results suggest that these structural analogs of bradykinin may be effective pharmacologic tools to study the role of endogenous kinins in the control of vascular resistance and circulatory homeostasis.

Amino Acid Sequence↗
PubMed1986

Bradykinin competitive antagonists for classical kinin systems.

The substitution of D-phenylalanine for proline at position 7 of bradykinin (BK) converts BK into a specific antagonist. Additional modifications of the nonapeptide structure, especially the inclusion of beta-2-thienylalanine residues (Thi) for phenylalanine at positions 5 and 8, increase antagonist potency in the classic smooth muscle (isolated rat uterus and guinea pig ileum) and rat blood pressure kinin assays. [Thi5,8,DPhe7]-BK had a pA2 value of 6.5 for inhibition of the BK response on rat uterus, and 6.3 on guinea pig ileum. Addition of Lys-Lys- or a D-Arg- residue to the N-terminal of D-Phe7-substituted antagonists decreases uterine agonist activity, but does not affect inhibitory potency on the ileum. Addition of a D-proline residue in place of proline in position 3 of D-Arg- extended antagonists produces specific uterine inhibitors which show no antagonism on the ileum. The D-Phe7-analogs did not inhibit smooth muscle responses to substance-P or angiotensin-II, and the antagonism of kinin responses was competitive.

Amino Acids↗
PubMed1986

Smooth muscle selectivity in bradykinin analogs with multiple D-amino acid substitutions.

Two novel analogs of bradykinin (BK), [DAla7]-BK and DArg-BK, exhibited a dissociation of smooth muscle activities toward higher potency on the uterus than on the ileum, in spite of increased metabolic stability to pulmonary enzymatic breakdown in the rat blood pressure assay. Analogs having a combination of the substitution of D-alanine in position 7 with a D-aromatic amino acid residue in position 6 show even greater uterus specificity. The addition of a D-arginine residue to the N-terminal greatly enhances this selectivity. Relative to BK, these poly-substituted analogs are up to 17 times as potent on the uterus as on the ileum. Still greater uterus selectivity is found among analogs in which glycine replaces the serine residue at position 6 and a D-hydrophobic amino acid residue (such as D-phenylalanine or D-p-chloro-phenylalanine) is present in position 7. These [Gly6, D-Hydrophobic7]-BK analogs are 20-40 times as potent on uterus as on ileum. [Gly6,DPhe7]-BK is the most uterus-selective analog described. In contrast, [Aib7]-BK is quite ileum-selective, having a uterus/ileum potency ratio of 0.1.

Amino Acids↗