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L A Carbini

Publications and source records attributed to L A Carbini.

16 recordsLinked to original sources
PubMed2002

Angiotensin II stimulates migration of retinal microvascular pericytes: involvement of TGF-beta and PDGF-BB.

We studied the promigratory effect of angiotensin II (ANG II) on cultured bovine retinal microvascular pericytes. ANG II stimulated migration of pericytes by 86% at 10(-8) M, but this effect was lost at 10(-4) M. Migratory responses were inhibited by the ANG II type 1 (AT(1)) receptor antagonist losartan but not by PD-123319, an AT(2) antagonist. Addition of PD-123319 to the 10(-4) M ANG II dose restored migratory responses. The promigratory effect of ANG II (10(-7) M) was reduced by 59% in absence of gradient. Although ANG II augmented the latent matrix metalloproteinase-2 (MMP-2) activity of the pericyte by 35%, it also doubled tissue inhibitors of MMPs. ANG II-induced migration was not altered by a broad-spectrum MMP inhibitor (GM6001); it was inhibited by ~50% by antibodies against transforming growth factor (TGF)-beta(1/2/3) and was abolished by antibodies against platelet-derived growth factor (PDGF)-BB. We conclude that ANG II induces chemotactic responses on retinal microvascular pericytes acting through the AT(1) receptor. This effect is opposed by the AT(2) receptor. ANG II-induced chemotaxis is mediated by PDGF-BB and involves TGF-beta, but it is independent of MMP activity. It is also independent of vascular endothelial growth factor (VEGF) because VEGF did not stimulate pericyte migration. ANG II can contribute to the regulation of retinal neovascularization by stimulating pericyte migration.

Angiotensin II↗
PubMed1999

Angiotensin II and retinal pericytes migration.

Angiotensin II (Ang II) appears to participate in the regulation of neovascularization processes in the retina. Migration of perimural cells such as pericytes plays a key role in regulation of angiogenesis. We hypothesize that Ang II stimulates migration of retina pericytes. For this we studied the effects of Ang II on migration of bovine retinal pericytes using modified Boyden chambers and collagen IV-covered polyester membranes. Ang II stimulated migration of pericytes by 54.8 +/- 9.7% (n = 10, p < 0.001). This effect was blocked by an AT(1) receptor antagonist (Losartan) but not by an AT(2) receptor antagonist (PD123319). We determined using checkerboard assays (n = 3) that Ang II induces migration of pericytes by chemotaxis (gradient-dependent), in opposition to chemokinesis (nondirected). Thus, Ang II via its AT(1) receptor acts as a chemotactic factor and stimulates migration of retina microvascular pericytes. This effect may contribute to Ang II-induced regulation of neovascularization processes in the retina.

Angiotensin II↗
PubMed1999

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-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.

Acetylcholine↗
PubMed1998

Activation of kininogen expression during distal nephron differentiation.

Previous studies have shown that the epithelial precursors of the connecting tubule and collecting duct express tissue kallikrein and bradykinin B2 receptors, respectively, suggesting the presence of a local kinin-producing/responsive system in the maturing distal nephron. However, evidence for the existence of kininogen in the developing nephron is still lacking. This study examined the spatiotemporal relationships between segmental nephron differentiation and the ontogeny of kininogen and kinins in the rat. Kininogen immunoreactivity is detectable in the metanephros as early as embryonic day 15. In the nephrogenic zone, the terminal ureteric bud branches are the main kinin-expressing segments. Kininogen is also observed in the stromal mesenchyme. In contrast, proximal ureteric bud branches, metanephrogenic mesenchyme, and pretubular aggregates express little or no kininogen. After completion of nephrogenesis, kininogen distribution assumes its classic "adult" pattern in the collecting ducts. Peak kininogen mRNA and protein expression occur perinatally, corresponding to the period of active nephrogenesis in the rat, and declines gradually thereafter. Estimations made by RT-PCR, Western blotting, and radioimmunoassays indicate that renal kininogen mRNA and protein levels are at least 20-fold higher in newborn than adult rats. Likewise, immunoreactive tissue kinin levels are 2.3-fold higher in newborn than adult kidneys (P < 0.05). In summary, the present study demonstrates the activation of kininogen gene expression and kinin production in the developing kidney. The terminal ureteric bud branches and their epithelial derivatives are the principal kinin-producing segments in the maturing nephron. The results suggest an autocrine/paracrine role for the kallikrein-kinin system in distal nephron maturation.

Animals↗
PubMed1998

Angiotensin 1-7 induces bradykinin-mediated relaxation in porcine coronary artery.

Angiotensin 1-7 (Ang 1-7) has been reported to induce relaxation which is partially blocked by a kinin receptor antagonist. We investigated the relationship between kinins and angiotensin peptides with use of preconstricted isolated pig coronary arteries. Ang 1-7 alone (up to 10(-5) M) had no relaxant effect. Bradykinin (BK) (10(-10)-10(-7) M) induced transient relaxation, returning to basal tone, although BK remained in the bath. In these BK-stimulated rings, Ang 1-7 but not BK (both 5 x 10(-6) M) again relaxed the rings by approximately 50%. This relaxation was blocked by a BK B2 antagonist, a kininase, and a nitric oxide synthase inhibitor. Ang 1-7 inhibited purified angiotensin-converting enzyme (ACE) by 30 +/- 3.5% (n = 4) at 10(-6) M. However, in BK-pretreated rings, the ACE inhibitor ramiprilat did not induce relaxation, nor did it affect the relaxant response to Ang 1-7, which suggests that the effect of Ang 1-7 was not caused by ACE inhibition. Ang 1-7-induced vasodilation was reduced by 69.9 +/- 6.2% by an AT2 receptor blocker, PD-123319, and 29.3 +/- 7.3% by an AT1 antagonist, losartan. Neither the nonselective AT1/AT2 receptor antagonist sarthran nor saralasin inhibited the response to Ang 1-7. Ang II did not elicit relaxation either alone or in the presence of losartan, which suggests that activation of AT2 receptors does not cause relaxation. Thus, in the presence of bradykinin, Ang 1-7 relaxes pig coronary arteries via a PD-123319-sensitive mechanism involving nitric oxide, kinins and the BK B2 receptor. The kallikrein-kinin and renin-angiotensin systems may be linked through the interaction of Ang 1-7 and BK.

Angiotensin I↗
PubMed1997

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.

Animals↗
PubMed1997

Submandibular enzymatic vasoconstrictor increases DNA and phosphoinositol synthesis by mesenchymal cells.

Submandibular enzymatic vasoconstrictor (SEV, rK9) induces vascular contraction and platelet aggregation by a mechanism requiring intact enzymatic activity. On the basis of a published report demonstrating growth-promoting enzymatic activity in an extract of the rat submandibular gland, we hypothesized that SEV would affect DNA synthesis. Recombinant SEV (rSEV), expressed in a baculovirus system, increased DNA synthesis 3- to 25-fold in Chinese hamster lung (CCL39) fibroblasts and in rabbit and rat vascular smooth muscle cells in a dose-dependent manner dose eliciting 50% of maximal response: 0.1-1 nM); this effect was inhibited by pertussis toxin (PTX). Inactive rSEV failed to enhance DNA synthesis. In CCL39 fibroblasts, rSEV increased total phosphoinositol (PI) formation (6- to 10-fold at 10 nM), which was inhibited 49% by PTX; it was also partially inhibited by the tyrosine kinase inhibitor genistein (33%) but was not affected by the protein kinase C inhibitor bisindolylmaleimide. These results show that rSEV increases DNA synthesis and PI formation in mesenchymal cells in a dose- and enzymatic activity-dependent manner through a pathway partially mediated by a PTX-sensitive G protein. Thus SEV can induce growth-associated responses, perhaps through a protease-activated receptor mechanism.

Animals↗
PubMed1997

Angiotensin-(1-7) induces bradykinin-mediated hypotensive responses in anesthetized rats.

Angiotensin-(1-7) [Ang-(1-7)] reportedly potentiates hypotensive responses to bradykinin. We studied whether increases in circulating bradykinin would alter responses to Ang-(1-7). In rats anesthetized with thiobutabarbital, bradykinin infusion (5 microg/kg per minute I.A.) resulted in a rapid decrease in mean arterial pressure (MAP) of about 20 mm Hg (P<.01, n=9), although MAP slowly increased by 10 mm Hg after 15 minutes. When Ang-(1-7) (20, 80, and 380 nmol per rat I.A.) was given during bradykinin infusion, it elicited hypotension at 80 and 380 nmol (deltaMAP: -15+/-2.7 and -21+/-3.3 mmHg, respectively; P<.001); this hypotension was not affected by the angiotensin type 1 antagonist L-158,809 (200 microg/kg I.A.), the angiotensin type 2 antagonist PD 123319 (10 mg/kg I.A.), saralasin, or sarthran (10 microg/kg per minute). The bradykinin type 2 receptor antagonist icatibant (30 microg per rat) eliminated the hypotensive responses to Ang-(1-7), which now increased MAP at all doses tested (P<.005). Thus in the presence of bradykinin, Ang-(1-7) induces hypotensive responses that are blocked by icatibant and unaffected by angiotensin receptor antagonists. Ang-(1-7) given to saline-infused rats elicited hypertensive responses at all doses (deltaMAP: 6.4+/-1.5, 12+/-1.6, and 16.3+/-2.7 mmHg, respectively; P<.01); these responses were abolished by L-158,809 and sarthran. In rats pretreated with saralasin, Ang-(1-7) induced hypotension at 80 and 380 nmol (deltaMAP: -7.7+/-2.3 and -9.5+/-2.7, respectively; P<.05), whereas icatibant abolished this response. Thus in the rat, Ang-(1-7) can decrease blood pressure by a mechanism involving the bradykinin type 2 receptor and participates with bradykinin in a vasodepressor pathway that may serve a counterregulatory role, modulating the vasoconstrictor effects of Ang II.

Angiotensin I↗
PubMed1995

Potentiation by aminopeptidase P of blood pressure response to bradykinin.

We examined whether a specific aminopeptidase P (APP) inhibitor, apstatin, increases vasodepressor responses to bradykinin in anaesthetized rats, and whether it would augment blood pressure responses further after treatment with the angiotensin-converting enzyme inhibitor (ACEi), lisinopril. Apstatin doubled the maximum blood pressure response to bradykinin. The area under the curve (AUC), which incorporates both peak blood pressure changes and duration of response, was doubled in apstatin-treated rats vs controls and in the apstatin+lisinopril group vs lisinopril alone. These data demonstrate that APP is an important kinase in vivo.

Aminopeptidases↗
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

Functional analysis of conserved histidines in choline acetyltransferase by site-directed mutagenesis.

The choline acetyltransferase (ChAT) reaction involves the transfer of the acetyl group of acetyl-CoA to choline, in which an active site histidine is believed to act as a general acid/base catalyst. A comparison of the deduced amino acid sequences of the enzyme from Drosophila, pig, rat, and Caernohabditis elegans revealed three conserved histidines: Drosophila His268, His393, and His426. Each of these histidines was replaced by a leucine and a glutamine, and the kinetic properties of each of the recombinant mutant enzymes were determined. The mutations yielded active His268Leu-ChAT, His268Gln-ChAT, and His393Gln-ChAT and inactive His393Leu-ChAT, His426Leu-ChAT, and His426Gln-ChAT. The kinetic constants Km(CoA), Km(acetylcholine), and Vmax were essentially the same for all of the active mutants. When the integrity of the CoASAc binding site was investigated in the inactive mutants, the data suggested that the binding site in His393Leu-ChAT is disrupted but conserved in His426Leu-ChAT and His426Gln-ChAT. These results suggest that His426 is an essential catalytic residue and could serve as an acid/base catalyst.

Amino Acid Sequence↗
PubMed1990

Developmental expression of choline acetyltransferase mRNA in Drosophila.

We have measured the steady state levels of choline acetyltransferase (ChAT, EC 2.3.1.6) mRNA during different developmental stages of Drosophila melanogaster using a ChAT specific cRNA probe. ChAT mRNA was first detected approximately 6-7 h after oviposition, increased until the 1st-2nd larval instar, decreased into early pupal stages and increased again during late pupation, reaching a maximum in adults. Northern analysis showed a major RNA band with a Mr of 4.7 kilobases and Western analysis also showed a single major 75 kD protein band at all developmental stages. Our results support the hypothesis that a major point of regulation of ChAT expression may be at the transcriptional level.

Animals↗