A. Guillermo Scicli

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.

Effects of aminopeptidase P inhibition on kinin-mediated vasodepressor responses

We studied in anesthetized rats whether aminopeptidase P (AMP) may be involved in bradykinin (BK) metabolism and responses. For this we inhibited AMP with the specific inhibitor apstatin (Aps). Studies were done with Aps alone or together with the angiotensin-converting enzyme inhibitor lisinopril (Lis). Aps increased the vasodepressor response to an intravenous bolus of BK (400 ng/kg): vehicle, -3.0 ± 0.7 mmHg; Aps, -7.8 ± 0.7 mmHg ( P < 0.01 vs. vehicle); Lis, -23.8 ± 1.8 mmHg; Aps + Lis, -37.5 ± 1.9 mmHg ( P < 0.01 vs. Lis). Aps did not affect the vasodepressor response to BK given into the descending aorta. Plasma BK increased only in Aps + Lis-treated rats (in pg/ml): control, 48.0 ± 1.4; Lis, 57.5 ± 7.6; Aps + Lis, 121.8 ± 30.6 ( P < 0.05 vs. control or Lis), whereas in rats infused with BK (400 ng ⋅ kg-1 ⋅ min-1for 5 min), Aps increased plasma BK (in pg/ml): control, 51.9 ± 2.5; Aps, 83.5 ± 20.5; Lis, 725 ± 225; Aps + Lis, 1,668 ± 318 ( P < 0.05, Aps vs. control and Lis vs. Aps + Lis). In rats with aortic coarctation hypertension, the acute antihypertensive effects of Aps plus Lis were greater than Lis alone ( P < 0.01). Hoe-140, a BK B2-receptor antagonist, abolished the difference. We concluded that in the rat AMP contributes to regulation of BK metabolism and responses.We studied in anesthetized rats whether aminopeptidase P (AMP) may be involved in bradykinin (BK) metabolism and responses. For this we inhibited AMP with the specific inhibitor apstatin (Aps). Studies were done with Aps alone or together with the angiotensin-converting enzyme inhibitor lisinopril (Lis). Aps increased the vasodepressor response to an intravenous bolus of BK (400 ng/kg): vehicle, -3.0 +/- 0.7 mmHg; Aps, -7.8 +/- 0.7 mmHg (P < 0.01 vs. vehicle); Lis, -23.8 +/- 1.8 mmHg; Aps + Lis, -37.5 +/- 1.9 mmHg (P < 0.01 vs. Lis). Aps did not affect the vasodepressor response to BK given into the descending aorta. Plasma BK increased only in Aps + Lis-treated rats (in pg/ml): control, 48.0 +/- 1.4; Lis, 57.5 +/- 7.6; Aps + Lis, 121. 8 +/- 30.6 (P < 0.05 vs. control or Lis), whereas in rats infused with BK (400 ng. kg-1. min-1 for 5 min), Aps increased plasma BK (in pg/ml): control, 51.9 +/- 2.5; Aps, 83.5 +/- 20.5; Lis, 725 +/- 225; Aps + Lis, 1,668 +/- 318 (P < 0.05, Aps vs. control and Lis vs. Aps + Lis). In rats with aortic coarctation hypertension, the acute antihypertensive effects of Aps plus Lis were greater than Lis alone (P < 0.01). Hoe-140, a BK B2-receptor antagonist, abolished the difference. We concluded that in the rat AMP contributes to regulation of BK metabolism and responses.

Role of nitric oxide in the effect of blood flow on neointima formation

PURPOSEnNeointima formation after arterial injury is inhibited by increased blood flow. The object of this study was to determine whether nitric oxide mediates the effect of increased blood flow on neointima formation.nnnMETHODnBalloon catheter-denuded rat carotid arteries were exposed to increased blood flow or control blood flow by ligation of the contralateral carotid artery. Beginning 2 days before balloon denudation, rats were given either saline vehicle alone or the nitric oxide synthase inhibitor N-nitro-L-arginine-methyl ester (L-NAME) at a dose of 10 mg/kg/day or 2 mg/kg/day intraperitoneally. The normalized neointima area was measured 14 days after denudation.nnnRESULTSnBlood flow was significantly increased by ligation of the contralateral carotid artery for all drug treatments (p<0.008). In rats given saline vehicle only, normalized neointima area was significantly reduced after increased blood flow compared with control blood flow (0.33+/-0.04 compared with 0.48+/-0.03; p=0.006). Systolic blood pressure was significantly elevated by treatment with high-dose L-NAME (p=0.002 compared with vehicle), but was not altered by low-dose L-NAME (p=NS compared with vehicle). Normalized neointima area was not significantly reduced after increased carotid blood flow for rats treated with either dose of L-NAME (p=NS).nnnCONCLUSIONnThe inhibition of neointima formation by increased blood flow was abolished with hypertensive and nonhypertensive doses of the nitric oxide synthase inhibitor L-NAME, which suggests that the L-NAME effects are independent of systemic hemodynamic alterations. It is concluded that flow-induced inhibition of neointima formation is mediated in part by nitric oxide.

Purification and characterization of canine urinary kallikrein.

The renal kallikrein-kinin system may play a role in the regulation of sodium and water balance. Although the dog is a frequently used experimental animal in the study of the renal kallikrein-kinin system, dog urinary kallikrein (DUKK) has been poorly studied. We have purified DUKK by a series of chromatographic and electrophoretic procedures including anion-exchange chromatography, filtration through p-aminobenzamidine-Sepharose (to remove contaminating nonkallikrein esterases), gel filtration, isoelectric focusing, and molecular sieve HPLC. This DUKK preparation gave three protein bands on polyacrylamide gel electrophoresis, each having similar esterolytic and kininogenase activities and immunological identity. Preparative isoelectric focusing indicated the presence of multiple forms of kallikrein with pIs of 3.93, 4.05, 4.24, and 4.44, the species with a pI of 4.24 constituting the major component. Neuraminidase treatment converted all of the forms into the component with a pI of 4.44, suggesting the charge heterogeneity was due mainly to differences in sialic acid content. DUKK has a specific activity of 3 mg bradykinin eq/min/mg protein when partially purified dog kininogen is used as a substrate. It is a glycoprotein with a molecular weight of 40,500 (amino acid analysis best fit method) and an alkaline pH optimum (9.0-9.5). DUKK is resistant to soybean trypsin inhibitor and lima bean trypsin inhibitor but is inhibited by several serine protease inhibitors such as antipain, leupeptin, and p-aminobenzamidine. Phe-Phe-Arg-chloromethyl ketone is a very potent inhibitor of DUKK. Contrary to previous reports, DUKK is also inhibited by N-alpha-p-tosyl-L-lysine chloromethyl ketone and aprotinin, the inhibition by the latter being inversely related to the concentration of NaCl in the medium. The esterolytic and amidolytic activities of DUKK are inhibited by an increase in NaCl concentration of the medium. This inhibition may be related to a NaCl-induced conformational change in the enzyme moiety.

The molecular biology of the kallikrein-kinin system

There are numerous indications that the kallikrein-kinin system is underexpressed in various forms of hypertension: for example, urinary kallikrein excretion is decreased in human essential hypertension and in various models of genetic hypertension [1–4]. Low urinary kallikrein excretion in children is one of the major genetic markers associated with a family history of essential hypertension, and children with high urinary kallikrein excretion have less probability of a genetic background of hypertension [5]. We found that a restriction fragment length polymorphism (RFLP) for the kallikrein gene family in spontaneously hypertensive rats (SHR) is linked to high blood pressure [6]. Hypertension itself may alter expression of the kallikrein-kinin system; for example, in renovascular hypertension, urinary and arterial kallikrein are decreased [1].

Excess antibody immunoassay for the measurement of tonin in rat tissues and plasma

Tonin, a proteolytic enzyme isolated from the rat submandibular gland, can generate angiotensin II directly from angiotensinogen. To date a method for the measurement of tonin in plasma has not been available and the present paper describes a sensitive and specific excess antibody immunoassay for determination of tonin in tissue homogenates and plasma. Interference from immunologically cross-reacting proteins was evaluated and the assay was found to be specific for tonin. Tonin measured in various tissue homogenates was directly proportional to the amount of sample added, giving a linear dose-response curve. The slope of this curve was determined by the recovery of tonin, which was better than 55% for urine and all tissues tested. The highest concentration of tonin was seen in the submandibular and sublingual gland (69 and 0.7 microgram/mg protein, respectively). The parotid gland, the exorbital lacrimal gland, liver, kidney, pancreas, and lung contained only negligible amounts (less than 4 ng/mg protein). Tonin in plasma was bound to one major inhibitor with a molecular weight of about 650,000-750,000. A partial splitting of the tonin-inhibitor complex was obtained by preincubating plasma with guanidine, allowing tonin to be measured with a recovery of 38 +/- 13% (n = 16) and with a linear dose-response curve. The concentration of immunoreactive tonin in normal arterial plasma from adult male rats was 0.90 +/- 0.53 ng/ml (n = 16). The concentration decreased after removal of the submandibular glands and increased after sympathetic stimulation.

PROTEOLYTIC ACTIVATION OF A PUTATIVE RECEPTOR LEADING TO VASOCONSTRICTION AND PLATELET AGGREGATION

Submandibular enzymatic vasoconstrictor (SEV), a member of the kallikrein family of enzymes, elicits biological effects by a proteolytically mediated mechanism. We studied 1) whether SEV is able to aggregate platelets and 2) whether SEV may activate a receptor other than the cloned thrombin receptor. SEV (10(-8)M) aggregated platelets, released ATP and increased intracellular Ca2+. Elastase treatment rendered human platelets unresponsive to SEV and thrombin (TH), but not to cathepsin G. In desensitization experiments performed with gamma-TH, after two successive additions of approximately 50 nM gamma-TH, a third dose elicited 15.8 +/- 3.4% of the initial response (n = 4), but platelets responded to approximately 20 nM SEV by 33.8 +/- 7.2% of control (p < 0.03 vs last response to gamma-TH). After desensitization to SEV (n = 4), the response to a third dose was 4 +/- 1.3% of control, but gamma-TH still induced 37.7 +/- 12.4% aggregation (p < 0.02 vs last response to SEV). Incubation of washed rabbit platelets with alpha-TH digested with elastase (10(-10) M TH added to 7 micrograms/ml elastase for 1 min) rendered them unresponsive to additional challenges with TH, but they still responded to an equipotent dose of SEV (2.7 x 10(-9) M) by 86 +/- 48% of control. In isolated rabbit aortic rings contracted with 10(-6) M norepinephrine (NE) to 42 +/- 3% of maximum. SEV (2.8 x 10(-8) M) caused further contraction to 87 +/- 4%. In contrast, alpha-TH (1.6 x 10(-7) M) tended to relax both NE- and SEV-contracted rings by 14 +/- 2 and 16.2 +/- 2%, respectively (n = 3 each). We concluded that part of the platelet-aggregating effect of SEV may be mediated by activation of a receptor(s) different from that of TH.

Purification and characterization of a non-kallikrein arginine esterase from dog urine.

A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34,000 and 33,300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a pI of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and DPhe-Phe-Arg-chloromethyl ketone (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 values in the 10(-5)-10(-7) M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.