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Biomedical subjects

A G Scicli

Publications and source records attributed to A G Scicli.

At least 37 records · Page 2Linked to original sources
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

Uterine kallikrein in the early pregnant rat.

Uterine homogenates of cycling and early pregnant Sprague Dawley rats and purified rat urinary kallikrein showed similar curves of displacement of 125I-kallikrein binding to a polyclonal antibody. Uterine kallikrein concentration measured by RIA was 8.7 +/- 2 SEM ng/g wet weight during the cycle (n = 6 in diestrus and metestrus) and 20.8 +/- 2 SEM (n = 7) ng/g wet weight on Day 7 of pregnancy (P7) (p < 0.001). On P7, kallikrein concentration was increased 12.4-fold in the implantation nodes, as compared to the interimplantation segments. Uterine homogenates of rats on P7, submitted to DEAE-cellulose chromatography and Sephadex gel filtration, yielded two fractions containing kallikrein immunoreactivity and kininogenase activity, with molecular masses that ranged from 120-125 kDa and 39-43 kDa, respectively. In the RIA, both fractions displayed parallelism with purified kallikrein. Enzymatic activity was expressed after activation by trypsin. It was inhibited by aprotinin, PMSF, p-amino-benzamidine, and leupeptin, but not by soybean or ovomucoid trypsin inhibitors. Kallikrein mRNA was demonstrated by reverse transcriptase/polymerase chain reaction in uteri of nonpregnant and P7 rats. These results show that rat uterus synthesizes one or more serine proteases that are immunologically and enzymatically related to tissue kallikrein in the implantation node on P7--determined both by an increment of whole uterus kallikrein content and a depletion of the interimplantation segments--suggests that kallikrein may play a role in the vasoactive changes of implantation.

Animals↗
PubMed1993

Kallikrein release by vascular tissue.

Vascular tissue contains kallikrein and kallikrein mRNA, suggesting a vascular kallikrein-kinin system. We questioned whether 1) kallikrein concentration varies among large and small vessels; 2) kallikrein is released by vascular tissue; and 3) blocking protein synthesis inhibits release, suggesting de novo synthesis. Using rat vascular rings and isolated-perfused hindquarters, we examined kallikrein in the bath and perfusate. Active kallikrein was higher in tail arteries than the aorta (P < 0.001); tail veins had six times more kininogenase than the vena cava (P < 0.001). Total kallikrein showed a similar pattern, being highest in tail vessels. Arterial rings released active and total kallikrein. After 1, 2, and 3 h incubation, cumulative release was as follows: active, 90 +/- 13, 201 +/- 25, and 311 +/- 41 pg.h-1 x mg tissue-1; total, 170 +/- 14, 366 +/- 24, and 537 +/- 40 pg.h-1 x mg tissue-1, indicating constant release up to > or = 3 h. In contrast, lactic dehydrogenase fell from 6.7 +/- 2.5 to 2.5 +/- 0.4 U.h-1 x mg tissue-1. Total kallikrein in the rings was 302 +/- 51 pg bradykinin/mg wt tissue before 3 h and 298 +/- 68 afterward. Kallikrein released by the hindquarters after 3 h was as follows: active, 6.2 +/- 2.8 ng bradykinin.min-1 x kg.body wt-1; total, 85.2 +/- 17 ng bradykinin.min-1 x kg body wt-1. Puromycin pretreatment (10 mg ip) reduced total perfusate kallikrein from 105 +/- 19 to 8.5 +/- 3.6 (P < 0.005).(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↗
PubMed1993

Role of kinins and nitric oxide in the effects of angiotensin converting enzyme inhibitors on neointima formation.

Marked neointima formation occurs after balloon injury to the intima of rat arteries. Angiotensin II has been implicated as a growth factor in this process, since angiotensin converting enzyme (ACE) inhibitors block neointima formation after injury. However, ACE is an important kininase, and its inhibitors may act in part by a kinin-mediated mechanism. Kinins are also known to stimulate synthesis of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) and prostacyclin, both of which have antigrowth effects. To determine whether the effect of ACE inhibitors on neointima formation is due to blockade of angiotensin II synthesis alone and/or inhibition of kinin inactivation, we followed two approaches. First, we compared the inhibition of neointima formation induced by the AT1-type angiotensin II receptor antagonist losartan with that caused by the ACE inhibitor ramipril. We also studied whether a kinin receptor antagonist, Hoe 140, blocks the effect of two different ACE inhibitors, ramipril and enalapril, on neointima formation. In addition, we studied whether the effect of ramipril is blocked by an NO synthesis inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME). Although both ramipril and losartan significantly reduced neointima formation, ramipril had a more marked effect (p < 0.05 for ramipril versus losartan). The kinin antagonist Hoe 140 reduced the inhibitory effect of ramipril and enalapril by 73% and 62%, respectively. The remaining effect of the ACE inhibitors was now similar to that of losartan. Inhibition of neointima formation by ramipril was also blocked by the NO synthesis inhibitor L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1992

Kinins mediate the antiproliferative effect of ramipril in rat carotid artery.

Angiotensin-converting enzyme (ACE) inhibitors have been shown to inhibit neointimal proliferation in response to endothelial injury in the rat carotid artery. Since ACE inhibitors block degradation of kinins, our objective in this study was to determine whether kinins mediate the antiproliferative effect of the ACE inhibitor ramipril. Endothelial denudation was achieved in the left carotid artery of male Sprague-Dawley rats using a balloon catheter. The rats were divided into four groups: a) vehicle (saline); b) DuP 753 10 mg/kg/day; c) ramipril 5 mg/kg/day; and d) ramipril 5 mg/kg/day plus Hoe 140 70 micrograms/kg/day. Ramipril markedly reduced neointimal proliferation compared to control (vehicle) (p less than 0.05) and DuP 753-treated groups (p less than 0.05). When ramipril was given together with Hoe 140 its effect was significantly blunted (p less than 0.05). These results show that kinins are important mediators in the antiproliferative effect of ACE inhibitors.

Angiotensin II↗
PubMed1992

Submandibular enzymatic vasoconstrictor messenger RNA in rat kidney.

Recently, we reported the isolation and identification of a potent vasoconstrictor enzyme from the rat submandibular gland, a member of the rat kallikrein gene family, which we named submandibular enzymatic vasoconstrictor (SEV). We studied whether messenger RNA (mRNA) for SEV is present in the kidney and isolated glomeruli, using the polymerase chain reaction assay with primers specific to the entire rat kallikrein family that would amplify a 430-bp fragment from their mRNA. As a probe we used a phosphorus-32-labeled oligonucleotide specific for SEV mRNA. A fragment of the predicted size was obtained on Southern blot for amplified renal RNA; however, no signal was obtained with glomerular RNA. To further confirm the presence of SEV mRNA in the kidney, polymerase chain reaction was repeated using primers specific to SEV mRNA that would amplify a 372-bp fragment from SEV mRNA alone. Again, a fragment of the predicted size was obtained on Southern blot after amplification of renal RNA but not RNA from the glomeruli. Southern blot of polymerase chain reaction-amplified RNA with primers that amplified the entire kallikrein gene family, using kallikrein complementary DNA that recognizes all members of the kallikrein gene family as a probe, revealed a 430-bp fragment for both renal and glomerular RNA, indicating that glomeruli contain mRNA for a member or members of the kallikrein family other than SEV. When the Southern blots were hybridized with a 32P-labeled oligonucleotide probe specific for glandular kallikrein, a fragment of the predicted size was obtained from amplified renal RNA but not glomerular RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The kallikrein--kinin system in blood vessels.

We have previously reported that vascular tissue contains kallikrein and kallikrein mRNA. We can now show that kallikrein is present throughout the vascular tree and is released from arterial and venous rings incubated "in vitro". Using the isolated perfused rat hindquarters as a model, we found that kallikrein appeared in the perfusate in concentrations that increased linearly with time. Treatment with puromycin inhibited kallikrein release by 87% (p < 0.01), these data suggest that kallikrein is synthesized and released by the vascular wall. Local generation of kinins (autocrine/paracrine system) may contribute to the regulation of vascular homeostasis.

Animals↗
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

A novel serine protease with vasoconstrictor activity coded by the kallikrein gene S3.

A fraction separated from rat submandibular gland homogenates was found to contain a potent vasoconstrictor when tested on isolated rabbit aortic rings. The vasoconstrictor was purified by a series of chromatographic steps. The purified compound (2.77 x 10(-9) M) induced 40% of the maximum contractile response to 60 mM KCl. Constriction was slow in onset, long-lasting, rinse-resistant, and unchanged by de-endothelialization; in addition, it was dose-related and inhibited by both EGTA and verapamil, but it was not affected by DUP753, an angiotensin II receptor antagonist. The compound was found to be a protein having a pI of 7.36 and a molecular weight of approximately 29,000 and exhibiting partial immunologic identity to rat glandular kallikrein and rat tonin. After 2-mercaptoethanol treatment, it separated into heavy (approximately 19,900) and light (approximately 10,700) chains having amino-terminal sequences of AY(X)HNNDLMLL and VVGGYN(X)ETNSQ, respectively. We found that they correspond to the amino-terminal and internal sequence of a previously unidentified kallikrein-like serine protease whose mRNA, named S3, has been found in the rat submandibular gland and prostate. The vasoconstrictor is able to hydrolyze t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide (a thrombin substrate), although its Kcat/Km was only 0.02% that of rat thrombin. Both vasoconstrictor and enzymatic activity on t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide were completely suppressed by amidinophenylmethylsulfonyl fluoride and soybean trypsin inhibitor; however, they were unaffected by hirudin, a thrombin inhibitor. At pH 6.5, it released angiotensin II when incubated with sheep angiotensinogen, although it had approximately one-tenth the activity of tonin. The submandibular enzymatic vasoconstrictor is a kallikrein-like enzyme, having some properties of both tonin and thrombin. It directly contracts vascular smooth muscle, acting via a mechanism that requires intact enzymatic activity.

Amino Acid Sequence↗
PubMed1991

Tissue kallikrein processes small proenkephalin peptides.

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met5-enkephalin and Met5-Lys6-enkephalin. Met5-Lys6-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37 degrees C and pH 8.5, the KM values for formation of Met5-enkephalin and Met5-Lys6-enkephalin were 129 and 191 microM, respectively. Corresponding kcat values were 0.001 and 0.03 s-1 and kcat/KM ratios were 8 and 1.6.10(2) M-1.s-1, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met5-Arg6-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, KM was 64 microM, kcat was 4.5 s-1, and the kcat/KM ratio was 7.10(4) M-1.s-1. At 5.5, the pH of the secretory granules, KM, kcat and kcat/KM were 184 microM, 1.9 s-1 and 10(4) M-1.s-1, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.

Amino Acid Sequence↗
PubMed1991

Endothelin-1 and human platelet activity.

Endothelin-1, a peptide produced by endothelium, causes vascular smooth muscle contraction possibly by mobilizing intracellular calcium. Shifts in ionized calcium may also play a role in platelet activation. Accordingly, the effects of endothelin on platelet ionized calcium and aggregation were studied. The measurements were made in aequorin-loaded gel-filtered human platelets derived from healthy donors. Endothelin even in a final concentration of 10(-6) M did not cause a measurable change in platelet ionized calcium or aggregation. When tested in combination with collagen, thrombin and platelet activating factor, endothelin showed no synergistic effect. These observations raise the possibility that endothelin may not interact with platelets in a physiologically significant way.

Adult↗
PubMed1991

A potent vasoconstrictor in the rat submandibular gland.

We detected a novel vasoconstrictor in an arginine esterase fraction separated from fractions containing tonin and other esterases that were obtained from a rat submandibular gland extract. When tested on isolated rabbit aorta rings, the substance caused dose-related contractions that were slow in onset, long-lasting, and difficult to reverse by rinsing. The substance acts directly on vascular smooth muscle, since preincubation with plasma or intact endothelium is not required. The fact that the constrictor was destroyed by heat and incubation with pronase suggests that it is a protein. Molecular sieving indicates an estimated molecular weight of 24,000 Da. It has a neutral isoelectric point that is higher than the pI of tonin, from which it can be separated by anion exchange chromatography. A small amount of the vasoconstrictor was obtained by gel filtration and eluted from isoelectric focusing polyacrylamide gels. The purified substance showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a potent vasoconstrictor; an estimated concentration of 2.5 nM induced contraction of isolated rabbit aorta rings ranging from 15% to 40% of the maximum contraction obtained by 60 mM KCl. Contraction was completely blocked by 1 mM (p-amidinophenyl)methanesulfonyl fluoride, a serine protease inhibitor. Contractile activity was not affected by hirudin, a thrombin inhibitor, but was completely inhibited by soybean trypsin inhibitor and blunted by aprotinin; thus it may be a trypsin-like serine protease. Purified vasoconstrictor preparation showed hydrolyzing activity on Pro-Phe-Arg-methyl-coumarin amide, a kallikrein substrate. We conclude that a novel vasoconstrictor serine protease is present in the rat submandibular gland.

Animals↗