Biomedical subjects

E Fink

Publications and source records attributed to E Fink.

At least 37 records · Page 2Linked to original sources

PubMed1999

Expression of plasma prekallikrein mRNA in human nonhepatic tissues and cell lineages suggests special local functions of the enzyme.

At present it is generally accepted that plasma prekallikrein (PPK) is synthesized in the liver and secreted into the bloodstream. Surprisingly, it has recently been shown that PPK mRNA is present also in RNA from the kidney, adrenal gland and placenta. In spite of its novelty and possible important physiological implications this finding has been neglected. Here we report that PPK mRNA is expressed also in the human brain, heart, lung, trachea, endothelial cells and leukocytes as well as in a variety of fibroblast and epithelial cell lines. Expression of PPK mRNA in fibroblasts, endothelial cells and leukocytes suggests that PPK mRNA detected in RNA preparations from whole tissue may originate solely from these ubiquitously occurring cells. However, PPK mRNA expression in various epithelial cell lines demonstrates that tissue-specific cells also transcribe the PPK gene. The presence of PPK mRNA in nonhepatic tissues and cells indicates that they have the capacity to synthesize the protein. The physiological role of PPK synthesized in extrahepatic tissue is unknown. It may participate in local actions within tissues as well as contributing to the PPK pool in blood plasma. Cultured cells will provide a valuable model for exploring the physiological significance of extrahepatic PPK expression.

Base Sequence↗

PubMed1998

Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech (Hirudo medicinalis)--primary structure, expression in yeast, and characterisation of native and recombinant inhibitor.

We have reported earlier the isolation and amino acid composition of bdellin A from medical leech, and characterised it as an inhibitor of trypsin, plasmin and acrosin [Fritz, H., Gebhardt, M., Meister, R. & Fink, E. (1971) in Proceedings of the international research conference on proteinase inhibitors (Fritz, H. & Tschesche, H., eds) pp. 271-280, Walter de Gruyter, Berlin]. In the present study, one of several chromatographic forms of this inhibitor was isolated from a semi-pure preparation. Elucidation of its amino acid sequence revealed that bdellin A is a member of the antistasin family. Therefore, it was renamed bdellastasin to avoid confusion with bdellin B, which is another trypsin-plasmin inhibitor from the medical leech, but of the Kazal type. Furthermore, a synthetic gene of bdellastasin was constructed, and the protein expressed in Saccharomyces cerevisiae with yields of 29 mg/l. The recombinant bdellastasin was purified by hydrophobic interaction and anion-exchange chromatography. Comparison by mass spectroscopy, far-ultraviolet circular dichroism studies, sequence determination, and inhibition characteristics demonstrated the identity of recombinant and native bdellastasin. The Ki values of bdellastasin for inhibition of bovine trypsin and human plasmin are in the nanomolar range; no inhibition was detected for factor Xa, thrombin, tissue kallikrein, plasma kallikrein and chymotrypsin. Circular dichroism analyses indicated that bdellastasin is devoid of secondary-structural elements.

Animals↗

PubMed1998

Purification and characterization of a kinin-releasing and fibrinogen-clotting serine proteinase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA.

Two forms of a proteinase, KN-BJ 1 and 2, were purified to homogeneity from the venom of Bothrops jararaca. In SDS/PAGE reduced KN-BJ 1 and 2 migrated as single bands with molecular masses of 38 kDa and 39 kDa. The two enzymes have similar N-terminal amino acid sequences and specific activities on synthetic chromogenic substrates, and both release bradykinin from bovine low-molecular-mass kininogen. KN-BJ 1 and KN-BJ 2 clot fibrinogen with specific activities of 245 NIH U/mg and 219 NIH U/mg, releasing only fibrinopeptide A. The amidolytic, kinin-releasing and coagulant activities are inhibited by phenylmethylsulfonyl fluoride, demonstrating that KN-BJ is a serine proteinase. Benzamidine derivatives, which are competitive inhibitors of trypsin-like proteinases, also inhibited the amidolytic activity of KN-BJ. A cDNA clone (HS104, 2.2 kb) has been isolated from a cDNA library of B. jararaca venom glands with an ORF of 771 bp. The deduced amino acid sequence contains segments that are identical to the sequences of the N-terminus and three tryptic peptides of KN-BJ 2. Therefore, the cDNA is believed to represent the gene of KN-BJ 2. The deduced amino acid sequence indicates that KN-BJ 2 is synthesized as a prezymogen of 257 amino acids with a putative signal peptide of 18 amino acids and an activating peptide of six amino acid residues. The sequence of 233 amino acids representing the mature enzyme exhibits high similarity to sequences of serine proteinases isolated from crotalid venoms.

Amino Acid Sequence↗

PubMed1998

Rat fetuin: distribution of protein and mRNA in embryonic and neonatal rat tissues.

Fetuin is a serum protein widely distributed in the animal kingdom and found in all mammalian species so far investigated. It is mainly a fetal protein, in the sense that the highest concentrations are found in serum and body fluids of embryos and fetuses. In order to elucidate possible biological functions of fetuin, we have studied its synthesis and distribution during the prenatal development of the rat with immunohistochemistry and in situ hybridization. We have isolated fetuin from rat serum and produced an antibody against this protein. In situ hybridization was performed using a 375-nucleotides-long digoxigenin-labeled riboprobe. Fetuin was unevenly distributed in all organ systems during development, with the most pronounced expression at E 10Fetuin is a serum protein widely distributed in the animal kingdom and found in all mammalian species so far investigated. It is mainly a fetal protein, in the sense that the highest concentrations are found in serum and body fluids of embryos and fetuses. In order to elucidate possible biological functions of fetuin, we have studied its synthesis and distribution during the prenatal development of the rat with immunohistochemistry and in situ hybridization. We have isolated fetuin from rat serum and produced an antibody against this protein. In situ hybridization was performed using a 375-nucleotides-long digoxigenin-labeled riboprobe. Fetuin was unevenly distributed in all organ systems during development, with the most pronounced expression at E16-E18. Fetuin expression was present in germinal cell populations, e.g., in the basal layer in the skin, in the germinal cell populations in the brain anlage and the gonads, and it was heavily expressed in the fetal hemopoietic liver. Furthermore, fetuin was expressed in the gastrointestinal epithelium prior to the development of glands and crypts. Fetuin was widely distributed in mesenchymal derived tissues, e.g., bone and muscle. In the developing kidney fetuin was heavily expressed is both mesenchymal condensations and glomerular anlages. Thus, fetuin was located in cells or structures undergoing differentiation and transformation. As fetuin has been shown previously to interfere with hormone signaling of transforming growth factor-beta, insulin and hepatocyte-growth factor, fetuin might be involved in cell differentiation and tissue transformation during the initial histogenesis, i.e., the time period in which cellular phenotypic characteristics are established.

Animals↗

PubMed1998

Anaphylactoid reactions during hemodialysis in sheep are ACE inhibitor dose-dependent and mediated by bradykinin.

Anaphylactoid reactions (AR) have been attributed to the generation of bradykinin (BK) when AN69 membranes are used together with angiotensin converting enzyme (ACE) inhibitors during hemodialysis. However, conclusive evidence for the involvement of the BK as the mediator of these AR is still lacking. This study examined the degree of contact activation in an animal model caused by three PAN membranes--AN69, PAN DX, and SPAN- and the effects of different doses of the ACE inhibitor enalapril (ENA) and the BK B2-receptor antagonist icatibant on AR during hemodialysis. Six sheep were dialyzed for one hour with or without ENA pre-treatment using the different membranes in random order. Severe AR were observed only during hemodialysis with AN69 dialyzers together with ENA pre-treatment; the severity of AR increased with the ENA dose. Mild hypotension was noted during hemodialysis with AN69 without ACE inhibition and with PAN DX and 20 mg ENA. Compared to pre-dialysis values, maximum generation of BK after blood passage through the dialyzer was found at five minutes: 73-fold (AN69 without ENA), 161-fold (AN69 with 10 mg ENA), 97-fold (AN69 with 20 mg ENA), 108-fold (AN69 with 30 mg ENA), 154-fold (AN69 with 30 mg ENA and 0.1 mg/kg icatibant), 18-fold (PAN DX without ENA), and 42-fold (PAN DX with 20 mg ENA). Elevated BK levels in arterial blood were detected during hemodialysis with AN69 membranes even without ACE inhibition (2.5-fold); pre-treatment with 20 mg ENA further increased arterial BK concentrations (4-fold). Furthermore, a marked decline of prekallikrein and high molecular weight kininogen concentrations was noted for both AN69 and PAN DX membranes. Anaphylactoid reactions during hemodialysis were completely prevented by icatibant even after pre-treatment with ENA and in the presence of high BK concentrations. Concentrations of prekallikrein, high molecular weight kininogen, and BK remained unchanged and no AR were observed during hemodialysis with SPAN and pre-treatment with 20 mg ENA. Our findings confirm that AR during hemodialysis with the negatively charged AN69 membrane are mediated by BK, since they can be prevented by the BK B2-receptor antagonist icatibant.

Acrylic Resins↗

PubMed1998

Enhanced blood pressure sensitivity to deoxycorticosterone in mice with disruption of bradykinin B2 receptor gene.

The renal kallikrein-kinin system is activated under conditions of mineralocorticoid excess. To evaluate whether endogenous kinins exert a protective role against the development of mineralocorticoid-induced hypertension, we studied the cardiovascular effects induced by long-term administration of deoxycorticosterone (DOC; 0.3 micromol/g body wt s.c. once per week for 6 weeks) or vehicle in transgenic mice (Bk2r-/-) lacking the bradykinin B2 receptor gene and in wild-type controls (Bk2r+/+). Under basal conditions, Bk2r-/- mice showed higher systolic blood pressure (tail-cuff plethysmography) than wild-type Bk2r+/+ and heterozygous Bk2r+/- mice (121+/-2 versus 114+/-2 and 115+/-2 mm Hg, respectively; P<0.05 for both comparisons). Heart rate was higher in Bk2r-/- and Bk2r+/- than in Bk2r+/+ (459+/-12 and 418+/-7 versus 390+/-7 bpm; P<0.05 for both comparisons). Systolic blood pressure was increased by DOC in transgenic as well as in wild-type mice, whereas no change was induced by the vehicle. The pressor response to DOC was more rapid and pronounced in Bk2r-/- than in Bk2r+/+ and Bk2r+/- (30+/-5 versus 15+/-4 and 6+/-3 mm Hg, respectively, at 3 weeks; P<0.01 for both comparisons). The difference in systolic blood pressure was consistent with that detected by direct intra-arterial measurements of mean blood pressure. Neither DOC nor its vehicle altered heart rate or gain in body weight over time. Under basal conditions, urinary sodium excretion did not differ between strains. During DOC administration, cumulative urinary sodium excretion was lower in Bk2r-/- than in Bk2r+/+ (2.59+/-0.15 versus 3.31+/-0.22 mmol, respectively, during the first week; P<0.05). Urinary kinin excretion was increased by DOC in both Bk2r-/- (from 0.65+/-0.17 to 4.27+/-0.80 pmol/24 h; P<0.01) and Bk2r+/+ (from 0.55+/-0.09 to 6.27+/-1.48 pmol/24 h; P<0.05). The increase in urinary kinin excretion was similar between strains. These results show that integrity of the bradykinin B2 receptor is essential for regulation of blood pressure and heart rate under basal conditions. In addition, they indicate that activation of the kallikrein-kinin system represents a compensatory response against the development of hypertension induced by mineralocorticoid excess.

Analysis of Variance↗

PubMed1997

Reducing milk production in ewes at weaning using restricted feeding and methscopolamine bromide.

Restricted feed (RF) and methscopolamine bromide (MB), an anticholinergic agent reported to block growth hormone secretion, were evaluated as potential means for reducing milk production in ewes at weaning. On d 58 +/- 1 postpartum (d 0), 40 fall-lambing ewes were allotted to treatments in a 2 (RF vs full feed [FF]) x 2 (MB vs saline [SAL]) factorial arrangement according to breed, age, and number of lambs born and suckled. On d 0 at 0800, ewes were separated from lambs, injected with 40 IU of oxytocin, and machine milked 30 s later. Ewes remained separated from lambs for 3 h, and at 1100 they were milked and yields were determined. From d 0 through 6, FF ewes received a daily ration of 1.8 kg of alfalfa hay and .9 kg of corn; RF ewes received only 1.8 kg of alfalfa hay. On d 7 at 0800 and 1400, ewes were separated from lambs, milked, treated with a s.c. injection of either 96 mg of MB dissolved in 2 mL of SA or SA alone, and after 3-h separations were milked at 1100 and 1700 and yields determined. Lambs were weaned at 1400. After milking at 1700, all ewes were placed in drylot without feed or water. At 1100 on d 9, residual milk yields were collected. Milk samples were retained for compositional analysis at each collection time. The RF ewes produced less milk than FF ewes (106 vs 137.4 g; P < .01) at 1100 on d 7, but yields of MB and SA ewes were similar. At 1700 on d 7, milk yield was only 15.9 g less (74.3 vs 90.2 g; P < .10) for RF than for FF ewes, but it was 26 g less (69.2 vs 95.2 g; P < .01) for MB than for SA ewes. On d 9, there was no significant difference in yields of RF and FF ewes; however, MB ewes continued to produce less milk than SA ewes (96.6 vs 125.4 g; P < .10). No significant interactions between feeding regimen and MB treatment were found for milk yield. These data provide evidence that MB treatment of ewes is effective in reducing milk production. Furthermore, even greater reduction can be achieved when MB is combined with RF.

Animal Feed↗

PubMed1996

Conditions for single strand conformation polymorphism (SSCP)analysis with broad applicability: a study on the effects of acrylamide, buffer and glycerol concentrations in SSCP analysis of exons of the p53 gene).

We examined the influence of electrophoretic conditions on the detectability of small sequence alterations in DNA fragments by single strand conformation polymorphism (SSCP) analysis. Three acrylamide concentrations, 7.5, 14 and 20%, were selected, and all three gel types were prepared with four different Tris/borate/EDTA buffer concentrations. In addition, all these twelve gels were prepared both with and without 10% glycerol. All electrophoretic runs were performed at ambient temperature of 20-24 degrees C. The resulting 24 different electrophoretic conditions were used for the SSCP analysis of DNA fragments of exons 5, 7 and 8 of the human p53 gene; the results were evaluated primarily in relation to detectability of mutants. Six out of the 24 conditions permitted the detection of all mutants. For practical reasons, 14% acrylamide, 44.5 mmol/1 Tris, 44.5 mmol/1 boric acid, 1 mmol/1 EDTA, pH 8.0, with and without addition of glycerol was chosen as the most suitable. These "selected conditions" were applied in the SSCP analysis of an arbitrarily chosen set of mutant DNA fragments with single base exchanges,and all but one of seven mutants were detected in the gel system containing glycerol. Our results indicate that the set of "selected conditions" is of broad applicability, permitting the detection of even small sequence differences like single base exchanges with high reliability. It should prove especially useful in screening for unknown mutations.

Acrylamides↗

PubMed1995

Complexes of tissue kallikrein with protein C inhibitor in human semen and urine.

An ELISA was developed for quantifying the complex between tissue kallikrein (tKK) and protein C inhibitor (PCI) (tKK:PCI) in seminal plasma and urine. The ELISA used purified tKK:PCI complex as a standard and was specific for this complex with a detection limit of about 1.1 pM. Purified tKK:PCI complex was obtained from human urine and was 95% homogeneous as judged by SDS/PAGE. The 90-kDa band corresponding to the purified tKK:PCI complex reacted with anti-tKK and anti-PCI antibodies as judged by immunoblotting. Seminal plasma collected in the absence of extrinsic inhibitors contained 1.8 +/- 0.6 nM tKK:PCI complex and 4.7 +/- 2.8 nM immunoreactive tKK (mean +/- SD, n = 10), which indicates that about 28% of the total tKK immunoreactivity is forming complexes with PCI. When semen was collected in the presence of tKK inhibitors it had only about 6% of the tKK complexed to PCI. In vitro studies showed that the tKK:PCI complex formation in semen was accomplished in about 1 h and that heparin stimulated both the rate and the extent of complexation of tKK with PCI. Native urine showed low levels of tKK:PCI complex, but after dialysis urine had 0.17 +/- 0.05 nM complex. Formation of tKK:PCI complex in urine and semen was also demonstrated by immunoblotting. These results suggest that PCI is a physiological inhibitor of tKK and provide additional evidence of the involvement of PCI in human reproduction.

Adult↗

PubMed1995

Purification, characterization, and amino acid sequence of a serine proteinase, PA-BJ, with platelet-aggregating activity from the venom of Bothrops jararaca.

A platelet-aggregating enzyme, PA-BJ, was isolated from the venom of the snake Bothrops jararaca. PA-BJ in a concentration of 3.2 x 10(-8) M promoted 95% platelet aggregation in platelet-rich plasma. SDS-polyacrylamide gel electrophoresis under reducing conditions showed a single protein band with an M(r) of 30,000. PA-BJ catalyzed the hydrolysis of several p-nitroanilide peptide substrates containing Arg or Lys at the scissile bond; among these the most sensitive were the thrombin substrates D-Phe-Pip-Arg-pNA and Tos-Gly-Pro-Arg-pNA. Both the platelet-aggregating and amidolytic activities of PA-BJ were abolished by reaction with phenylmethanesulfonyl fluoride. Several benzamidine derivatives, which are competitive inhibitors of trypsin-like serine proteinases, also inhibited the amidolytic activity of PA-BJ. Among the compounds tested, the thrombin inhibitor NAPAP [N alpha-[(2-naphthylsulfonyl)-glycyl]-4-amidinophenylalanine piperidide] showed the strongest inhibitor activity on PA-BJ. The complete amino acid sequence of PA-BJ, which, to the best of our knowledge, is the first of a platelet-aggregating enzyme from snake venom, was deduced from the N-terminal sequencing of overlapping fragments cleaved from the reduced and S-pyridylethylated protein by chemical and enzymatic methods. PA-BJ is composed of 232 amino acid residues and contains one N- and one O-glycosidically linked carbohydrate moiety at residues Asn20 and Ser23. Sequence comparison to other venom serine proteinases revealed significant homology, mainly in regions around the catalytic triad and conserved cysteine residues.

Amino Acid Sequence↗

PubMed1995

Anaphylactoid reactions during haemodialysis in sheep are associated with bradykinin release.

Anaphylactoid reactions have been observed in patients treated with AN69 dialysers and ACE inhibitors. Recently, it has been shown in vitro that AN69 membranes induce the release of high amounts of bradykinin in plasma. To verify the possible role of bradykinin in these shock-like reactions, six sheep were dialysed in a random fashion using AN69 or the new SPAN membrane with and without pretreatment with captopril. All animals were dialysed for 60 min via double-lumen Shaldon catheters. Blood samples were drawn at 0, 5, 10, 15, 30, and 60 min from the venous line. A total of 24 haemodialysis procedures was carried out: group A (n = 6), AN69 without captopril; group B (n = 6), SPAN without captopril; group C (n = 6), AN69 with captopril; group D (n = 6), SPAN with captopril. A significant bradykinin release was observed only in groups A and C, reaching peak values already after 5 min. Animals in group C showed the highest bradykinin values. In four of six animals in group C anaphylactoid reactions with severe hypotension were noted. From this animal model we conclude that dialysis with the AN69 membrane is associated with bradykinin release. Pretreatment with ACE inhibitors results in further increasing bradykinin levels, which lead to anaphylactoid reactions. In contrast, the new SPAN membrane was well tolerated without detectable changes in bradykinin concentrations.

Acrylic Resins↗

PubMed1995

No generation of bradykinin with a new polyacrylonitrile membrane (SPAN) in haemodialysis patients treated with ACE inhibitors.

BACKGROUND: Anaphylactoid reactions occurring in uraemic patients haemodialysed with polyacrylonitrile haemodialysis (HD) membranes and being treated with ACE inhibitors have been attributed to an excessive generation of bradykinin. METHODS: Here we tested in a prospective trial a new type of polyacrylonitrile membrane (SPAN) with respect to bradykinin generation in nine HD patients receiving either captopril or enalapril. Each patient had three consecutive HD sessions with each of the three tested membranes, high-flux SPAN, high-flux polysulphone (F60) and low-flux Hemophan (GFS Plus 16). RESULTS: No clinical signs of anaphylactoid reactions were observed in any of these patients but the number of patients was relatively small and the duration of exposure to different membranes relatively short. At 5 min after the start of HD session, plasma bradykinin levels were significantly higher in the venous than in the arterial line for all three HD membranes: SPAN, 18.5 +/- 11.9 versus 12.4 +/- 5.3 fmol/ml (P < 0.05); F60, 19.0 +/- 13.8 versus 11.5 +/- 6.5 fmol/ml (P < 0.01); and GFS Plus 16, 39.1 +/- 22.9 versus 15.8 +/- 12.4 fmol/ml (P < 0.005), mean +/- SD respectively. Higher venous line levels were still observed at the 15 and 60 min time points for F60 and GFS Plus 16, but not for SPAN. However, these levels were still insignificant compared to levels measured during episodes of anaphylactic shock from the literature. Plasma histamine and C5a anaphylatoxin levels did not show any increase during HD with SPAN. CONCLUSION: The SPAN membrane did not induce significant bradykinin release in dialysis patients on ACE-inhibitor therapy. It may therefore be used for high-flux dialysis in such patients.

Acrylic Resins↗

PubMed1994

Bradykinin is a mediator of anaphylactoid reactions during hemodialysis with AN69 membranes.

Anaphylactoid reactions (AR) are the most feared complications of hemodialysis. Recently, a high incidence of AR has been reported during dialysis with AN69 membranes in patients treated with ACE inhibitors. Plasma levels of C3a, histamine and bradykinin were measured in 12 patients at the onset of AR during dialysis with AN69. We also investigated bradykinin generation in 10 symptom-free patients dialyzed with four different membranes. None of the 12 patients studied during AR displayed excessive complement activation or histamine release. In contrast, high bradykinin plasma levels (2392 +/- 53 fmol/ml; mean +/- SEM) were observed in all nine patients of whom bradykinin was measured. One patient developed two consecutive episodes of hypersensitivity on AN69 membranes even without taking ACE inhibitors. Bradykinin levels were high in both episodes (5280 and 10467.7 fmol/ml). Furthermore, this patient showed no symptoms and normal bradykinin levels (123.4 fmol/ml) when dialyzed with other membranes. The role of the membrane type in the AR is further substantiated by the observation that AN69 also provoked a significantly higher bradykinin generation (327.6 +/- 18 fmol/ml; mean +/- SEM) during symptom-free sessions compared to other membranes like CuprophanR (5.1 +/- 7.3), HemophanR (17.2 +/- 6.3) and PolysulfoneR (39.7 +/- 6.6). Our findings strongly suggest that bradykinin is the principal mediator of AR during hemodialysis with AN69 membranes. To our knowledge it is the first time that data support the hypothesis of a more general role of bradykinin in shock-like symptoms. Furthermore, bradykinin generation must be regarded as a new marker of biocompatibility of extracorporeal treatments.

Acrylic Resins↗

PubMed1994

Inhibitor regulation of tissue kallikrein activity in the synovial fluid of patients with rheumatoid arthritis.

Tissue kallikrein (TK) and alpha 1-antitrypsin (AT)/TK complexes can be detected in SF from patients with RA if components of the fluids which interfere with the detection of TK are removed. alpha 2-Macroglobulin (alpha 2-M) in SF was demonstrated to contain trapped proteases which were still active in amidase assays. Removal of alpha 2-M from RA SF reduced their amidase activity. However, at least some of the remaining activity was due to TK because it was soya bean trypsin inhibitor resistant and trasylol sensitive and was partly removed by affinity chromatography on anti-TK sepharose. Removal of RF from the fluids reduced the values obtained for TK levels by ELISA. Addition of SF to human urinary kallikrein (HUK) considerably reduced the levels of TK detected suggesting the presence of a TK ELISA inhibitor in the fluids. Removal of components of > 300 kDa from SF markedly reduced the TK ELISA inhibitory activity and increased the values for both the TK and alpha 1-AT/TK levels in fluids as measured by ELISA. It is considered this novel inhibitor does not bind to the active site of TK but rather binds to the site reactive with anti-TK antibodies.

Adult↗

PubMed1994

Dextran sulfate activates contact system and mediates arterial hypotension via B2 kinin receptors.

To define some of the mechanisms underlying dextran sulfate (DXS)-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor des-Pro2-[Arg15] aprotinin (BAY x 4620) or the specific bradykinin B2-receptor antagonist Hoe-140 on the hypotensive response to DXS. In the first study, anesthetized miniature pigs were given DXS alone, DXS plus BAY x 4620 in various doses, or saline. As expected, DXS alone produced a profound but transient systemic arterial hypotension with a concomitant reduction in kininogen. Circulating kinin levels, complement fragment des-Arg-C3a, and fibrin monomer were all increased. Treatment with BAY x 4620 produced a dose-dependent attenuation of these effects with complete blockade of the hypotension as well as the observed biochemical changes at the highest dose (360 mg). In a second study, two groups of pigs were given either DXS alone or DXS plus Hoe-140. DXS-induced hypotension was completely blocked by Hoe-140 pretreatment; however, kininogen was again depleted. We conclude, therefore, that DXS-induced hypotension is produced by activation of plasma kallikrein that results in the production of bradykinin and that liberation of bradykinin and its action on B2 receptors in the vasculature are both necessary and sufficient to produce the observed effects on circulatory pressure.

Animals↗

PubMed1994

Kinin generation by hemodialysis membranes as a possible cause of anaphylactoid reactions.

1. Severe anaphylactoid reactions have been reported in some patients treated with angiotensin converting enzyme (ACE) inhibitors during hemodialysis with a polyacrylonitrile (PAN) membrane. Generation of bradykinin via contact activation at the negatively charged membrane surface and reduced bradykinin breakdown due to ACE inhibition have been suggested as possible causes. This hypothesis was evaluated in the present study. 2. PAN or cellulose dialyzer membranes were incubated with plasma at different concentrations of ACE inhibitor. The rate and extent of kinin accumulation was dependent on the ACE inhibitor concentration. 3. Bradykinin levels were determined in "historical" plasma samples drawn from patients treated with ACE inhibitor at the onset of anaphylactoid reactions during hemodialysis with PAN dialyzers. The kinin levels were significantly higher (2.4 +/- 0.05 pmol/ml) than in samples from a group of control patients (0.29 +/- 0.02 pmol/ml). 4. Plasma kinin levels were measured in patients who developed anaphylactoid reactions during dialysis with a PAN membrane though not being treated with ACE inhibitor. At the onset of the reaction, kinin levels increased to 2.1 pmol/ml in the line entering the dialyzer and to 10.5 pmol/ml in the line leaving the dialyzer compared to not more than 0.12 pmol/ml upon dialysis with other membranes. 5. These in vitro and in vivo results demonstrate that contact of blood with a polyacrylonitrile membrane leads to the generation of kinins which accumulate if ACE is inhibited. It is very likely that kinin accumulation in the circulation is the cause of anaphylactoid reactions during hemodialysis with PAN membranes in patients treated with ACE inhibitors and, in some cases, in patients not receiving ACE inhibitor medication.

Acrylic Resins↗

PubMed1994

Furosemide enhances the release of endothelial kinins, nitric oxide and prostacyclin.

Despite a wealth of data, the mechanism of the direct dilator effect of furosemide on the systemic arterial and venous systems is far from being satisfactorily understood. Therefore, we investigated whether furosemide is capable of stimulating the production of the endogenous vasodilators nitric oxide and prostacyclin in primary cultured bovine aortic endothelial cells by an enhanced synthesis and release of endothelium-derived kinins. Nitric oxide production was assessed in terms of intracellular guanosine cyclic-3',5' monophosphate accumulation; kinin and prostacyclin release were determined by specific radioimmunoassays. Furosemide concentration- and time-dependently increased the formation of nitric oxide and prostacyclin. Maximal increases of both autacoids were already obtained after a 5-min incubation with 3 x 10(-7) to 10(-6) mol/l of furosemide. In the same concentration range, furosemide led to an enhanced release of kinins into the supernatant of the cells. This observation was supported by the inhibitory effect of the specific B2 kinin receptor antagonist icatibant (Hoe 140) on the furosemide-induced increase of nitric oxide and prostacyclin. Thus the hemodynamic effects, and in particular the direct early dilator effect, of furosemide may be explained in part by an enhanced endothelial synthesis and release of bradykinin and related kinins, which in turn stimulates endothelial autacoid formation via B2 kinin receptor activation.

Animals↗

PubMed1993

Organization and sequence of the gene encoding the human acrosin-trypsin inhibitor (HUSI-II).

A complete cDNA encoding the acrosin-trypsin inhibitor, HUSI-II, was used as a probe to isolate genomic clones from a human placenta library. Three clones which cover the entire HUSI-II gene were isolated and characterized. The exon-intron organization of the gene was determined and found to be identical to other known Kazal-type inhibitor-encoding genes. The striking similarity in the amino acid sequences which was found previously in HUSI-II and glycoprotein hormone beta-subunits, is neither reflected in codon usage nor in the exon-intron arrangement of the genes. A 1.8-kb segment 5' of the gene was sequenced. The analysis of this sequence showed that HUSI-II contains a G + C-rich region upstream from the transcription start point (tsp) which fulfills the criteria for a CpG island. Furthermore, in the first intron, a potential glucocorticoid-responsive element was found as a half-palindrome flanked by two CACCC elements. Determination of the tsp by S1 mapping revealed that HUSI-II has multiple tsp. Genomic Southern hybridization was used to show that HUSI-II is a single-copy gene. The localization of the gene to chromosome 4 was determined by hybridization of a 5' genomic fragment to the DNA of a panel of somatic hybrids between human and rodent cells.

Acrosin↗