Biomedical subjects

G Scicli

Publications and source records attributed to G Scicli.

At least 19 recordsLinked to original sources

PubMed1996

Short-term hemodynamic effects of endothelin receptor blockade in dogs with chronic heart failure.

BACKGROUND: Plasma endothelin levels are increased in heart failure and may contribute to the increased peripheral vasoconstriction that characterizes this disease state. In the present study, we examined the effects of intravenous bosentan, a nonpeptide, competitive endothelin-1 receptor antagonist, on hemodynamics in dogs with chronic heart failure. METHODS AND RESULTS: Chronic heart failure was produced in 11 dogs by multiple sequential intracoronary microembolization. At the time of study, left ventricular (LV) ejection fraction was 25 +/- 2%. Hemodynamic and echocardiographic measurements were made at baseline and at 15, 30, and 60 minutes after a bolus injection of bosentan (10 mg/kg). Bosentan had no significant effect on heart rate or mean aortic blood pressure. At 60 minutes, bosentan reduced LV end-diastolic pressure (17 +/- 2 versus 11 +/- 2 mm Hg; P < .05) and systemic vascular resistance (3891 +/- 379 versus 3071 +/- 346 dyne .s. cm-5; P < .05) compared with baseline and increased cardiac output (2.63 +/- 0.29 versus 3.33 +/- 0.46 L/min; P < .05), peak rate of change of LV pressure during isovolumic contraction and relaxation (1751 +/- 92 versus 2197 +/- 170 mm Hg/s; P < .05), and LV fractional shortening determined by echocardiography (30 +/- 2% versus 36 +/- 2%; P < .05). CONCLUSIONS: Short-term intravenous bosentan reduced systemic vascular resistance and improved overall LV performance in dogs with chronic heart failure. These results suggest that endothelin-1 receptor antagonists may be useful therapeutic agents in the treatment of heart failure.

Animals↗

PubMed1994

Effects of long-term monotherapy with enalapril, metoprolol, and digoxin on the progression of left ventricular dysfunction and dilation in dogs with reduced ejection fraction.

BACKGROUND: Recent clinical trials have suggested that therapy with angiotensin-converting enzyme inhibitors in asymptomatic patients with reduced left ventricular (LV) function can significantly reduce the incidence of congestive heart failure compared with patients receiving placebo. In the present study, we examined the effects of long-term monotherapy with enalapril, metoprolol, and digoxin on the progression of LV systolic dysfunction and LV chamber enlargement in dogs with reduced LV ejection fraction (EF). METHODS AND RESULTS: LV dysfunction was produced in 28 dogs by multiple sequential intracoronary microembolizations. Embolizations were discontinued when LVEF was 30% to 40%. Three weeks after the last embolization, dogs were randomized to 3 months of oral therapy with enalapril (10 mg twice daily, n = 7), metoprolol (25 mg twice daily, n = 7), digoxin (0.25 mg once daily, n = 7), or no treatment (control, n = 7). As expected, in untreated dogs, LVEF decreased (36 +/- 1% versus 26 +/- 1%, P < .001) and LV end-systolic volume (ESV) and end-diastolic volume (EDV) increased during the 3-month follow-up period (39 +/- 4 versus 57 +/- 6 mL, P < .001, and 61 +/- 6 versus 78 +/- 8 mL, P < .002, respectively). In dogs treated with enalapril or metoprolol, LVEF remained unchanged or increased after therapy compared with before therapy (35 +/- 1% versus 38 +/- 3% and 35 +/- 1% versus 40 +/- 3%, respectively, P < .05), whereas ESV and EDV remained essentially unchanged. In dogs treated with digoxin, EF remained unchanged but ESV and EDV increased significantly. CONCLUSIONS: In dogs with reduced LVEF, long-term therapy with enalapril or metoprolol prevents the progression of LV systolic dysfunction and LV chamber dilation. Therapy with digoxin maintains LV systolic function but does not prevent progressive LV enlargement.

Animals↗

PubMed1994

A local kallikrein-kinin system is present in rat hearts.

It has been reported that kinins mediate part of the beneficial cardiac effects induced by treatment with angiotensin-converting enzyme inhibitors in situations such as ischemia-reperfusion injury, myocardial infarction, and cardiac hypertrophy. However, it is not known whether the heart contains an independent kallikrein-kinin system. We measured kallikrein in tissue and in the incubation medium of heart slices. Heart slices released active and total (trypsin-activatable) kallikrein into the medium (46 +/- 5 and 380 +/- 18 pg bradykinin/mg, respectively, after 1 hour and 78 +/- 6 and 654 +/- 14 pg bradykinin/mg after 2 hours, n = 7). Release was not due to tissue damage because lactate dehydrogenase, a cytosolic marker, decreased from 8.9 +/- 2.9 to 2.9 +/- 1.0 U/mg per hour. Although kallikrein was released, total tissue kallikrein in the slices did not change (423 +/- 25 pg bradykinin/mg in nonincubated slices and 370 +/- 42 pg bradykinin/mg after 2 hours, P = NS), suggesting pool replenishment. Cardiac kallikrein activity was inhibited by incubation with anti-glandular kallikrein antibodies. Pretreatment with the protein synthesis inhibitor puromycin (10 mg IP) lowered release of active kallikrein from 78 +/- 6 to 22 +/- 4 pg bradykinin/mg and total kallikrein from 654 +/- 14 to 113 +/- 9 pg bradykinin/mg (P < .001). By using reverse transcription polymerase chain reaction with kallikrein family oligonucleotide primers and a specific kallikrein probe, we found that mRNA for tissue kallikrein is present in both atrial and ventricular RNA. Kallikrein activity was also detected in primary cultures of neonatal rat atrial and ventricular cardiocytes and their incubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Adrenal kallikrein.

Kallikrein was identified in the adrenal glands of the rat. The enzyme was present in active and inactive forms (n = 9), since preincubation with trypsin increased kininogenase activity from 54.8 +/- 11.8 to 230 +/- 23 pg bradykinin per milligram protein per minute. Adrenal kininogenase activity was inhibited by 91% by phenylmethylsulfonyl fluoride (2 mM), 81% by D-Phe-Phe-Arg-chloromethyl ketone (1 microM), 88% by aprotinin (1,000 KIU), and only 16% by soybean trypsin inhibitor (50 microM). Preincubation with antibodies against rat urinary kallikrein resulted in over 90% inhibition of kininogenase activity. Immunoreactive glandular kallikrein was 30.7 +/- 4.8 ng/mg protein (n = 11). The apparent molecular weight of the adrenal kininogenase on gel filtration chromatography was 33,000 +/- 500 D. Both the adrenal enzyme and the purified submandibular gland kallikrein used as a control had the same mobility on alkaline polyacrylamide gel electrophoresis. To determine whether messenger RNA (mRNA) for glandular kallikrein is present in adrenal gland RNA, we used the polymerase chain reaction employing oligonucleotide primers and glandular kallikrein 32P complementary DNA (cDNA) as a probe, which should give a cDNA fragment of 370 bp. Southern blots of the amplified products revealed a fragment of the predicted size. In conclusion, glandular kallikrein has been identified in the adrenal glands. The presence of mRNA for glandular kallikrein suggests that kallikrein is synthesized locally in this tissue. This provides an anatomic basis for possible participation of a local kallikrein-kinin pathway in the regulation of adrenal function.

Adrenal Glands↗

PubMed1992

Synthesis of renin by tubulocystic epithelium in autosomal-dominant polycystic kidney disease.

Evidence suggests an important role for the renin-angiotensin system in the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). Therefore, we studied the presence of immunoreactive renin in renal biopsies and measured the concentrations of renin in cyst fluids. Normal kidneys and kidneys with renal artery stenosis were used for comparison. In ADPKD, immunoreactive renin was present in juxtaglomerular apparatus, associated arterioles, and in some cells within the connective tissue surrounding the cysts. Vascular immunoreactive renin was less prominent than in renal artery stenosis. Increased amounts of tubular immunoreactive renin were noted in polycystic kidneys, as compared to normal kidneys and kidneys with renal artery stenosis. Cyst fluids contained renin detected by Western analysis and enzymatic activity; concentrations were greater in gradient cysts than in nongradient cysts. Seventy-four percent of the renin in gradient cysts was active as compared to 23% in nongradient cysts and 15% in plasma. To determine whether cyst epithelial cells are capable of synthesizing renin, these cells were isolated in tissue culture. Enzymatic assay of extracts from these cells revealed the presence of renin-like enzymatic activity (1.3 +/- 0.8 ng AI/mg protein/hr). The synthesis of renin by tubulocystic epithelium was confirmed by [35S]-methionine radiolabeling of cyst-derived cells, followed by immunoprecipitation and SDS-PAGE and by detection of renin mRNA by the polymerase chain reaction. These results indicate that the tubulocystic epithelium has the potential to synthesize renin. Elevated levels of active renin in renal cysts may be linked to the pathogenesis of hypertension in ADPKD. The occurrence of renin in the lining epithelium of cyst walls raises the possibility that abnormal expression of the renin-angiotensin system may, by a paracrine or autocrine mechanism, regulate epithelial hyperplasia in growing renal cysts.

Base Sequence↗

PubMed1992

Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells.

Stimulation of the release of endothelium-derived relaxing factor (EDRF) in the kidney has been shown to result in natriuresis without affecting glomerular filtration rate. This may be due to EDRF directly regulating solute transport in the cortical collecting duct (CCD). To test this hypothesis, we measured the effect of bradykinin (Bk) or acetylcholine (Ach) on short-circuit current (Isc; a measure of active transport) in a CCD cell line (M-1), in the presence or absence of cow pulmonary artery endothelial (CPAE) cells. 10(-9) M Bk or 10(-7) M Ach had no effect on M-1 Isc in which CPAE cells were absent. The addition of CPAE cells to M-1 cells also did not affect M-1 Isc. On the other hand, when 10(-9) M Bk or 10(-7) M Ach were added to M-1 cells in the presence of CPAE cells, Isc decreased from 43 +/- 4.5 to 26 +/- 4 and 64 +/- 9 to 33 +/- 4 microA/cm2, respectively (P less than 0.001). Nitroarginine (N-Arg, 10(-4) M), a competitive inhibitor of EDRF production, blocked the inhibition in M-1 Isc due to both agonists. Since cGMP is the second messenger of EDRF in vascular smooth muscle, we measured the effects of Bk on cGMP production in M-1 cells in the presence and absence of CPAE cells. Bk increased cGMP content in M-1 cells in the presence of CPAE cells from 33 +/- 3.4 to 132 +/- 11.7 fmol/10(6) M-1 cells (P less than 0.001). When cultures of M-1 and CPAE cells were treated with N-Arg and challenged with Bk, Bk's effect on cGMP was partially blocked (61.4 +/- 12 fmol/10(-6) M-1 cells; NS). These data suggest that EDRF inhibits transport and increases cGMP content in M-1 cells.

Acetylcholine↗

PubMed1992

The kallikrein-kinin system in cardiac tissue.

Kallikrein and minute amounts of kininogen have been found in rat cardiac tissue. The mRNA for kallikrein was also determined by the polymerase chain reaction using KK-specific probe. The existence of an intrinsic kallikrein-kinin system in the heart raises the possibility that the enzyme-peptide system is involved in local regulation of cardiac function and metabolism.

Animals↗

PubMed1991

T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen gamma.

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.

Amino Acid Sequence↗

PubMed1991

Effect of an angiotensin II and a kinin receptor antagonist on the renal hemodynamic response to captopril.

The role of angiotensin II and kinins on the renal cortical and papillary hemodynamic and on the sodium and water excretory responses to converting enzyme inhibition with captopril was examined in euvolemic Munich-Wistar rats. Cortical and papillary blood flows were measured using a laser Doppler flowmeter. Cortical blood flow increased 28% after blockade of angiotensin II receptors with DuP 753 (2 mg/kg i.v., n = 6). Captopril (2 mg/kg i.v., n = 6) had no effect on cortical blood flow in rats pretreated with the angiotensin II antagonist. DuP 753 had no effect on papillary blood flow, nor did it prevent the rise in papillary blood flow produced by captopril (2 mg/kg, n = 6). Infusion of a kinin receptor antagonist, D-Arg, [Hyp3,Thi5,8,D-Phe7]-bradykinin (2.5 micrograms/min i.v.), reduced basal papillary blood flow by 15% and blocked the rise in papillary blood flow produced by captopril. Renal blood flow rose by 11% after DuP 753 (2 mg/kg, n = 6), and subsequent administration of captopril and the kinin antagonist had no effect on renal blood flow. Urine flow and sodium excretion increased after DuP 753, but captopril produced additional increases in urine flow and sodium excretion of 68% and 46% respectively. Fractional sodium excretion rose from 0.85 +/- 0.15% to 1.56 +/- 0.14% after captopril. Infusion of the kinin antagonist returned sodium and water excretion to control levels, but fractional sodium excretion was not significantly altered. Glomerular filtration rate was not altered by DuP 753 or captopril; however, it fell from 1.6 +/- 0.1 to 1.2 +/- 0.1 ml/min/g kidney wt during infusion of the kinin antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗

PubMed1990

A kallikrein-like enzyme in blood vessels of one-kidney, one clip hypertensive rats.

Active and inactive kallikrein or a kallikrein-like enzyme are found in the aorta, vena cava, and tail artery and veins of the rat. We studied the concentration of vascular kininogenase in rats with one-kidney, one clip renovascular hypertension and in unilaterally nephrectomized normotensive rats. Six weeks after surgery, active and total vascular kininogenase activity (active plus trypsin-activated) was measured. Blood pressure was 212 +/- 4 mm Hg in the hypertensive rats (n = 33) and 120 +/- 1 mm Hg in the normotensive rats (n = 32) (p less than 0.001). Active kininogenase was lower in the hypertensive rats; although the difference was not significant in the thoracic aorta (56 +/- 8 versus 77 +/- 15), it was highly significant in the abdominal aorta (63 +/- 13 versus 167 +/- 17, p less than 0.001) and tail artery (48 +/- 8 versus 197 +/- 31, p less than 0.003). Total vascular kininogenase activity (active plus trypsin-activated) was lower in the hypertensive rats in all arteries examined: thoracic aorta (183 +/- 16 versus 380 +/- 38, p less than 0.003), abdominal aorta (565 +/- 61 versus 1,093 +/- 74, p less than 0.001), and tail artery (532 +/- 112 versus 1,243 +/- 135, p less than 0.003). Active kininogenase in the vena cava was higher in the hypertensive rats (213 +/- 56 versus 131 +/- 31); however, this difference was not statistically significant, whereas in the tail veins it was highly significant (1,803 +/- 221 versus 771 +/- 79, p less than 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Glandular kallikrein-like enzyme in adrenal glands.

A kallikrein-like kininogenase was identified in the rat adrenal gland. Most of the enzyme was present in an inactive form, since pre-incubation with trypsin markedly increased kininogenase activity from 54.8 +/- 11.8 to 230 +/- 23.0 pg bradykinin/mg protein/min. Adrenal kininogenase was inhibited 90% by phenyl methyl sulfonyl fluoride, 92% by D-Phe-Phe-Arg-chloromethylketone, 91% by aprotinin, and only 15% by soybean trypsin inhibitor. Pre-incubation with antibodies against rat urinary kallikrein resulted in 85% inhibition. The apparent molecular weight of adrenal kininogenase on gel filtration chromatography was 33 Kd. The enzyme was strongly adsorbed to immobilized rat urinary kallikrein antibodies and required drastic conditions for elution. In canine adrenal glands, we found that there was no difference in the cortical and medullary distribution of active and inactive SBTI resistant kininogenase activity. We conclude that an enzyme which closely resembles glandular kallikrein is present in adrenal glands.

Adrenal Cortex↗

PubMed1989

Kinins contribute to the contractile effects of rat glandular kallikrein on the isolated rat uterus.

Glandular kallikrein is known to promote contractions of the isolated, estrogenized rat uterus, perhaps independently of kinin formation. The recent availability of kinin receptor antagonists led us to study whether they might affect the oxytocic activity of kallikrein. DArg0-Hyp3-Thi5,8-DPhe7-bradykinin (8.5 x 10(-7) M) displaced the dose-response curves to both bradykinin (from 1.0 x 10(-9) to 4.0 x 10(-6) M) and kallikrein (from 4.7 x 10(-11) to 8.0 x 10(-9) M) approximately one order of magnitude to the right. This inhibition could not be due to a nonspecific effect on the uterine muscle, as the contractile response to oxytocin was not altered. In addition, carboxypeptidase B (a potent kininase) and kinin antibodies reduced the contractile response to kallikrein by 70 and 60%, respectively. Removal of the intervening agent restored the normal response. The effect of kallikrein depended on its enzymatic activity, inasmuch as kallikrein inactivated with D-Phe-Arg-Arg-CH2Cl was not oxytocic. Prolonged or multiple exposures to kallikrein completely abolished uterine response, whereas the effect of bradykinin was unaltered. Uterine horns rendered insensitive to kallikrein by prolonged exposure still contracted in response to trypsin. Kininogen was present in the uterine tissue in a concentration of 1.5 +/- 0.3 ng of bradykinin equivalents per mg wet wt. No more than 15.9 +/- 1.2% of this total was due to plasma contamination. Only 21.5 +/- 2.9% of total kininogen could be cleaved by kallikrein. We conclude that part of the oxytocic activity of kallikrein is related to generation of kinins from a kallikrein-sensitive kininogen present in the isolated rat uterus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Identification of a new kinin in human urine.

The types of kinins excreted in fresh urine of dogs, rats, and humans were compared. Urinary kinins were separated by reverse-phase (C18) high performance liquid chromatography and quantitated by radioimmunoassay using an antibody directed against the COOH-terminal region of the peptide. Kinins were found in the following proportions: 53 +/- 3% bradykinin, 23 +/- 4% Lys-bradykinin, and 13 +/- 7% des-Arg1-bradykinin in dog urine; 67 +/- 6% bradykinin, 6 +/- 3% Lys-bradykinin, and 10 +/- 3% des-Arg1-bradykinin in rat urine; and 12 +/- 4% bradykinin, 30 +/- 3% Lys-bradykinin, 2 +/- 1% des-Arg1-bradykinin, and 41 +/- 3% unknown kinin in human urine. The unknown kinin was purified from a pool of human urine. Amino acid sequencing revealed a structure similar to Lys-bradykinin except that proline in position 4 was replaced by alanine ([Ala3]Lys-bradykinin). Synthetic and endogenous [Ala3]Lys-bradykinins had similar high performance liquid chromotography elution volumes and both had vasodilator activity and contracted the rat uterus. Human urinary kallikrein incubated with semipurified human low molecular weight kininogen released 76% of the total kinins as Lys-bradykinin, 7% as bradykinin, and 17% as [Ala3]Lys-bradykinin. In contrast, rat urinary kallikrein released 86% bradykinin, 18% Lys-bradykinin, and negligible amounts of [Ala3]Lys-bradykinin. The study revealed the presence of a new kinin, [Ala3]Lys-bradykinin, in human urine and it also proves that the types of kinins generated intrarenally are species-dependent.

Amino Acid Sequence↗

PubMed1986

Purification and characterization of a serine protease (esterase B) from rat submandibular glands.

A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.

Amino Acids↗

PubMed1986

Kininogenase from rat vascular tissue.

A kininogenase resembling glandular kallikrein was partially purified from vascular tissue and characterized. Saline perfused rat tail arteries and veins were homogenized in 0.25 M sucrose containing 10 mM Tris-HCl (pH 7.4). The homogenate was centrifuged at 105,000 X g for 60 min and a vascular kininogenase was purified from the supernatant by chromatofocusing, affinity chromatography on immobilized antibodies against rat urinary kallikrein, and gel filtration on Sephadex G-100. The inhibitory effects of antibodies against rat urinary kallikrein were tested using equivalent kinin-forming concentrations of rat urinary kallikrein and vascular kininogenase. Kininogenase activities of both enzymes were similarly inhibited by urinary kallikrein antibodies. Aprotinin (1,000 KIU) completely inhibited vascular kininogenase activity while soybean trypsin inhibitor (100 micrograms) did not modify its kinin-forming activity. Vascular kininogenase and rat urinary kallikrein had the same elution volume when chromatographed on a Sephadex G-100 column and had similar mobilities in 10% polyacrylamide gel electrophoresis. Kinins released by vascular kininogenase were identified as bradykinin by reverse-phase high performance liquid chromatography. Rat vascular kininogenase appears to be similar to glandular kallikrein. Kinins released locally by vascular kininogenase may contribute to the regulation of vascular tone.

Animals↗

PubMed1985

Characterization of a kininogenase from rat vascular tissue resembling tissue kallikrein.

A kininogenase resembling glandular kallikrein was partially purified from vascular tissue and characterized. Saline-perfused rat tail arteries and veins were homogenized in 0.25 M sucrose containing 10 mM Tris-HCl (pH 7.4). The homogenate was centrifuged at 105,000 g for 60 minutes, and a vascular kininogenase was purified from the supernatant by chromatofocusing, affinity chromatography on immobilized antibodies against rat urinary kallikrein, and gel filtration on Sephadex G-100. The inhibitory effects of antibodies against rat urinary kallikrein were tested with equivalent kinin-forming concentrations of rat urinary kallikrein and vascular kininogenase. Kininogenase activities of both enzymes were similarly inhibited by both polyclonal and monoclonal antibodies. Aprotinin (1,000 KIU) completely inhibited vascular kininogenase activity, while soybean trypsin inhibitor (100 micrograms) did not modify its kinin-forming activity. Vascular kininogenase and rat urinary kallikrein had the same elution volume when chromatographed on a Sephadex G-100 column, and had similar mobilities in 10% polyacrylamide gel electrophoresis. Kinins released by vascular kininogenase were identified as bradykinin by reverse-phase high performance liquid chromatography. Rat vascular kininogenase appears to be similar to glandular kallikrein. Kinins released locally by vascular kininogenase may contribute to the regulation of vascular tone.

Animals↗

PubMed1982

Blood kinins, their concentration in normal subjects and in patients with congenital deficiency in plasma prekallikrein and kininogen.

Kinins are potent vasodilator peptides that may participate in the regulation of local blood flow and blood pressure. Here we report a new method to measure kinins in blood. For this, 6 ml of blood are collected in less than 10 sec directly into 25 ml of ethanol. Kinins are further purified by extracting lipids with ether and by removing kininogen and other interfering substances by chromatography on QAE Sephadex and BioRex 70; then they are measured by a sensitive RIA. In 22 normal subjects, after correction for recovery (50%), the kinin concentration in peripheral venous blood was 25.2 +/- 2.6 pg/ml (mean +/- S.E.M.). To determine whether the circulating kinins are formed by plasma kallikrein or other kininogenases, the concentration of blood kinins was measured in the venous blood of three patients with congenital deficiency in plasma prekallikrein (Fletcher trait) and in one patient with congenital deficiency in the substrate of plasma kallikrein, high-molecular-weight kininogen (Fitzgerald trait). In the three subjects with Fletcher trait, blood kinins were 16, 21, and 31 pg/ml, whereas in the subject with Fitzgerald trait they were 26 pg/ml. Normal subjects had concentrations in the same range (9 to 55 pg/ml), indicating that the concentration of blood kinins in normal subjects is much lower than previously reported (70 to 5000 pg/ml). These results also suggest that kininogenases other than plasma kallikrein may generate circulating kinins.

Animals↗

PubMed1979

Relation between structure and correcting activity of bovine high molecular weight kininogen upon the clotting time of Fitzgerald-trait plasma.

Bovine high molecular weight kininogen (bHMWK) partially corrects the activated plasma thromboplastin time (aPTT) of Fitzgerald trait plasma which is congenitally deficient in HMWK. The relationship between the structure and activity of HMWK was clarified by studying the effects of different fragments of bHMWK on the aPTT of Fitzgerald-trait plasma. The peptides studied were lys-bradykinin-free HMWK, bradykinin-fragment 1-2-free HMWK, heavy chain, fragment 1-2-light chain, and light chain. All fragments were tested in equimolar concentrations. Bradykinin-fragment 1-2-free HMWK, heavy chain, and light chain have little or no correcting activity upon Fitzgerald-trait plasma aPTr. Fragment 1-2 light chain has the same correcting activity as intact bHMWK, while that of lys-bradykinin-free HMWK appears to be higher. Both fragment 1-2 and fragment 2 inhibit the clotting time of normal human plasma. When compared on a molar basis, fragment 2 is a more active inhibitor than fragment 1-2. When the effects of bovine plasma kallikrein upon bHMWK and hHMWK were studied, it was found that it released kinins from both kininogens. However, while the correcting activity of bHMWK was completely destroyed after 60 min of incubation, that of hHMWK was fully retained. These data suggest that: (a) the active part of bHMWK is comprised of the fragment 1-2 light chain portion; (b) fragment 1-2 or fragment 2 is the binding site to negatively charged surfaces, while the light chain interacts with other components of the surface-mediated reactions; and (c) bovine plasma kallikrein releases kinins, but probably does not cause the release of fragment 1-2 from human HMWK.

Animals↗