Biomedical subjects

L C Katwa

Publications and source records attributed to L C Katwa.

28 records · Page 2Linked to original sources

PubMed1995

Local regulation of extracellular matrix structure.

The extracellular matrix (ECM) is composed of various collagens, glycosaminoglycans, and elastin bathed by a tissue fluid found throughout the interstitial space. It is this substratum in which fibroblasts and macrophages normally reside, where fibroblast phenotypic transformation occurs, and into which inflammatory cells migrate when called upon during tissue repair. Many diseases, expressed in an organ-specific manner, require organ-specific ECM remodeling. Regulation of fibrillary type I collagen synthesis, whose disproportionate (relative to degradation) accumulation is characteristic of the tissue fibrosis that adversely alters organ function, is therefore of considerable importance. Emerging evidence implicates angiotensin converting enzyme (ACE), found in fibroblast-like cells, and ACE-related peptides, angiotensin II and bradykinin, in serving important regulatory functions that influence wound healing and thereby ECM structure in health and disease. The heart and its collagen matrix have been targeted for discussion in this brief review.

Animals↗

PubMed1995

Angiotensin converting enzyme and kininase-II-like activities in cultured valvular interstitial cells of the rat heart.

OBJECTIVE: The function of angiotensin converting enzyme (ACE) at cell sites of high collagen turnover, such as heart valves, is uncertain. The aim of this study was to assess ACE and kininase-II-like activities and collagen turnover in cultured valvular interstitial cells of the adult rat heart. METHODS: The valvular interstitial cell phenotype was determined by immunolabelling (rhodamine phalloidin, desmin, and Griffonia simplicifolia lectin), and the presence of ACE mRNA and protein was confirmed by reverse transcriptase-polymerase chain reaction analysis, ACE monoclonal antibody and in vitro autoradiography, respectively. ACE and kininase-II-like activities in valvular interstitial cells were analysed by high performance liquid chromatography. Angiotensin II (AT1) and bradykinin receptors in valvular interstitial cell membranes were examined by western immunoblotting and binding assay. Type I collagen and collagenase in valvular interstitial cell culture media were determined by ELISA and zymography, respectively. Type I collagen mRNA expression in cultured valvular interstitial cells was determined by northern blot analysis and in situ hybridisation. RESULTS: In intact valvular interstitial cells or their cell membrane we found: (1) actin microfilaments, but not desmin or lectin labelling; (2) ACE mRNA expression and binding activity; (3) conversion of angiotensin I to angiotensin II, which was completely inhibited by 50 microM lisinopril, while kinase-II-like activity exceeded ACE activity and was not inhibited by lisinopril; (4) AT1 and bradykinin receptors in valvular interstitial cell membrane preparations; (5) type I collagen mRNA expression and collagenase activity; and (6) angiotensin II induced increase in type I collagen synthesis and mRNA expression. CONCLUSIONS: Cultured valvular interstitial cells represent a nonendothelial, non-smooth-muscle cell type that expresses mRNA for ACE and type I collagen. ACE and kininase-II-like activities in valvular interstitial cells may be involved in the regulation of peptides that influence collagen turnover. Angiotensin II stimulates type I collagen synthesis and mRNA expression in these cells.

Angiotensin II↗

PubMed1993

Cryogenic solvents inhibit the binding of the Escherichia coli heat-stable enterotoxin to intestinal brush border membranes.

The cryogenic solvents ethylene glycol, glycerol, dimethyl sulfoxide (Me2SO) and dimethylformamide (Me2FM), with increasing potency, produced concentration-dependent inhibition of the binding of the Escherichia coli heat-stable enterotoxin (STa) to pig intestinal brush border membranes. Inhibition increased with time, and both Me2SO and Me2FM appeared to decrease both the affinity of the STa receptor and the effective receptor number. Both solvents stimulated the release of previously bound 125I-STa (Me2FM > Me2SO), 3 M Me2FM inducing 93% release by 120 min. These effects were reversible, and preincubation of membranes with up to 3 M Me2SO or Me2FM at 37 degrees C for 30 min, followed by washing, did not alter subsequent 125I-STa binding. Also, 125I-STa released from membranes by 3 M Me2FM was shown to rebind to the membranes after 10-fold dilution of Me2FM. Since pretreating membranes with the thiol reagent p-chloromercuribenzenesulfonate had no effect on the release of bound 125I-STa by Me2SO or Me2FM, and since neither of these can reduce disulfide bonds, the formation of mixed disulfides between STa and receptor is unlikely. Me2SO inhibition of 125I-STa binding was greater with membranes than with a partially purified receptor preparation, which may result from the substitution of detergent for the phospholipid normally associated with the receptor(s).

4-Chloromercuribenzenesulfonate↗

PubMed1993

Endothelin receptors in cultured adult rat cardiac fibroblasts.

OBJECTIVES: Endothelins, released by vascular endothelial cells, are known growth promoters of various mesenchymal cells that contribute to stromal protein accumulation. Whether endothelins could contribute to myocardial fibrosis depends, in part on whether cardiac fibroblasts have endothelin receptors. The identification and binding characteristics of endothelin-1 and endothelin-3 and their ETA and ETB receptor subtypes in cultured adult rat cardiac fibroblasts represented study objectives. METHODS: Cultured rat cardiac fibroblasts (passages 5-10) grown until confluence were used to study radioligand binding assays, receptor subtypes, association and dissociation, effects of agonist and antagonist on binding kinetics, and affinity cross linking. RESULTS: Binding association of 125I-endothelin-1 and 125I-endothelin-3 was rapid, specific, and saturable within 60 minutes. The dissociation of receptor bound 125I-endothelin-1 was slow and partially reversible (30%-40%), suggesting more than one class of endothelin receptors, whereas the dissociation of 125I-endothelin-3 was time dependent and reversible. Competitive displacement with unlabeled endothelin-1, endothelin-3, endothelin-receptor nonselective sarafotoxin (S6b), and ETA receptor selective antagonist PED-3512-PI were used to identify receptor subtypes. Displacement of 125I-endothelin-1 by cold endothelin-1, resulted in a low affinity, high binding site (IC50 5.4 x 10(-9) M; 3.6 x 10(4) binding sites.cell-1) and a high affinity, low binding site (IC50 4.2 x 10(-4) M; 11,830 binding sites.cell-1). With 125I-endothelin-1 the IC50 s for sarafotoxin, endothelin-3, and PED-3512-PI were 1.8 x 10(-10), 1.7 x 10(-9), and 3.7 x 10(-9) M, respectively; for 125I-endothelin-3 these IC50s were 2.28 x 10(-11), 1.9 x 10(-10), and 1.7 x 10(-9) M, respectively. Endothelin receptor subunits of 53, 37, 34, and 24 kDa were identified by affinity cross linking. CONCLUSION: Endothelin-1 and endothelin-3 binding and ETA and ETB receptor subtypes are present in cardiac fibroblasts with ETB predominant. The presence of these receptors support the hypothesis that endothelins may regulate cardiac fibroblast function.

Animals↗

PubMed1993

Effects of endothelins on collagen turnover in cardiac fibroblasts.

OBJECTIVES: Endothelins (ET-1 and ET-3) may be promoters of tissue remodeling, including fibrillar collagen accumulation. The hypothesis that ET-1 and ET-3 each modify cardiac fibroblast collagen turnover was tested. METHODS: Confluent adult rat cardiac fibroblasts were maintained for 24 hours in medium with 0.4% fetal calf serum, and varying concentrations of ET-1 or ET-3. Collagen synthesis was measured by 3H-proline incorporation. The synthesis of type I and III collagens was measured by enzyme linked immunosorbent assay (ELISA). The effects of endothelins on collagenase activity were estimated by zymography. RESULTS: ET-1 and ET-3 increased collagen synthesis in a concentration-dependent manner with a maximal 1.7-fold increase (p < 0.001 v control cells). The effects of ET-1 or ET-3 on collagen synthesis were not blocked by PED-3512-PI (10(-6) M), an ETA receptor antagonist. ET-1 and ET-3 each significantly (p < 0.01) increased the synthesis of both type I and III collagens. ET-1 caused a 5.8-fold decrease in collagenase activity (p < 0.01 v control), and this effect was blocked by PED-3512-PI (10(-6) M). ET-3 did not alter collagenase activity. CONCLUSION: ET-1 and ET-3 increased the synthesis of type I and III collagens, whereas ET-1, but not ET-3, reduced collagenase activity. The effects of endothelins on collagen synthesis in cardiac fibroblasts seem to be mediated through ETA and ETB receptors, whereas their effects on collagenase seem to be mediated by ETA receptors.

Animals↗

PubMed1992

Nucleotide regulation of heat-stable enterotoxin receptor binding and of guanylate cyclase activation.

Certain nucleotides were found to regulate the binding of the Escherichia coli heat-stable enterotoxin (STa) to its receptor in pig intestinal brush border membranes. ATP and adenine nucleotide analogues inhibited 125I-STa binding, while guanine nucleotide analogues stimulated binding, with maximal effects at 0.5-1.0 mM. The strongest inhibitors were adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) (36%) and adenosine 5'-[beta-thio]diphosphate (ADP[S]) (41%). Inhibition did not require Mg2+, and was blocked by p-chloromercuribenzenesulphonate (PCMBS). Stimulation of binding required Mg2+, was not prevented by PCMBS and was maximal with GDP[S] (41%). While App[NH]p and MgGDP[S] appeared to be acting at different sites, they also interfered with each other. These nucleotides exerted only inhibitory effects on STa-stimulated guanylate cyclase activity, in contrast with the stimulatory effects of adenine nucleotides on atrial natriuretic peptide (ANP)-stimulated guanylate cyclase. Inhibition by low concentrations of MgApp[NH]p and MgATP was weaker above 0.1 mM, while MgGDP[S] and magnesium guanosine 5'-[gamma-thio]triphosphate (MgGTP[S]) inhibited in a single phase. Inhibition by MgApp[NH]p, at all concentrations, was competitive with the substrate (MgGTP), as was that by MgGDP[S] and MgGTP[S]. Whereas membrane guanylate cyclases usually show positively co-operative kinetics with respect to the substrate, STa-stimulated activity exhibited Michaelis-Menten kinetics with respect to MgGTP. This changed to positive co-operativity when Lubrol PX was the activator, or when the substrate was MnGTP. These results suggest the presence of both a regulatory and a catalytic nucleotide-binding site, which do not interact co-operatively with STa activation.

4-Chloromercuribenzenesulfonate↗

PubMed1992

Presence of functional receptors for the Escherichia coli heat-stable enterotoxin in the gastrointestinal tract of the chicken.

Receptors for the Escherichia coli heat-stable enterotoxin (STa) were shown to be present throughout the digestive tract of the chicken, with binding activity present not only in the intestinal epithelium but also in the intestinal smooth muscle. Brush border membrane vesicles (BBMV) purified from chicken enterocyte homogenates and plasma membranes (SMPM) purified from intestinal smooth muscle homogenates were compared with pig enterocyte BBMV. All had similar 125I-STa binding affinities, but the 50% effective concentration for STa activation of guanylate cyclase was higher in SMPM than in BBMV. Maximal STa-stimulated guanylate cyclase activities were similar in chicken and pig BBMV and were seven- to eightfold higher than in SMPM, and the STa receptor density was five- to sixfold higher. Patterns unique to each membrane were demonstrated after affinity labelling of STa receptors with 125I-STa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The results demonstrated STa-stimulated guanylate cyclase activity in birds as well as mammals and suggested that there are different functional STa receptors in chicken BBMV and SMPM.

Animals↗

PubMed1991

Solubilization and reprecipitation from intestinal brush border membranes of a complex containing guanylate cyclase activatable by the heat-stable enterotoxin.

Extraction of pig intestinal brush border membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (Chaps) in the presence of 0.5 M KCl yielded a solution which contained 60-70% of the receptor for the Escherichia coli heat-stable enterotoxin (STa) and of the Lubrol PX-activated guanylate cyclase activity present in the membrane. When the supernatant solution was diluted fivefold with 10 mM Hepes buffer (pH 7.4) and kept at 4 degrees C overnight, a precipitate formed. Centrifugation yielded a pellet (P2) which contained 25-30% of both the cyclase and the receptor in the original membranes, with a 2.5- to 3-fold enrichment of both. The process could be repeated for further enrichment (P4). The addition of MgCl2 to the diluted extract affected both basal and STa-stimulated activity of P2; 1 mM was optimal. P2 resembled membranes with respect to competitive inhibition of 125I-STa binding by STa, and the concentration-dependent activation of cyclase by STa. Guanylate cyclase in resolubilized P2 was also activated by STa. Most of the enzymes interfering with guanylate cyclase determinations were removed, as were the brush border marker enzymes sucrase and gamma-glutamyltransferase, and a GTP-binding protein that is a pertussis toxin substrate. Specific cross-linking of 125I-STa to receptors in the membrane was preserved in P2 and P4, the three proteins showing the strongest radioactivity having relative molecular masses of 55,000-60,000, 70,000-80,000, and 135,000-140,000. P2 and P4 appear to contain a complex of membrane proteins with certain functional properties intact.

Adenosine Diphosphate Ribose↗

PubMed1991

Age-dependent changes in affinity-labeled receptors for Escherichia coli heat-stable enterotoxin in the swine intestine.

Intestinal brush border membranes from 1-day-old and 4-week-old (day of weaning) pigs were affinity labeled with an Escherichia coli heat-stable enterotoxin (STa) by cross-linking 125I-STa to receptor proteins with disuccinimidyl suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that a radioactive protein with a relative molecular weight of 137,000 to 145,000 was present in both age groups. A strongly radioactive protein with an apparent Mr of 90,000 was present in the 1-day-old animals but not in those that were 4 weeks old. The major radioactive protein present in the older pigs had an Mr of 64,000 to 67,000, but this protein was missing or very weakly radioactive in the younger pigs. There was no significant difference between the groups in receptor affinity for STa, although the receptor density in the older animals was marginally significantly greater. STa-stimulated guanylate cyclase activity in membranes from 1-day-old pigs was only one-sixth that in 4-week-old pigs, although the basal and Lubrol PX-stimulated activities were similar.

Affinity Labels↗

PubMedDate not supplied

Effects of angiotensin II on canine and porcine coronary epicardial and resistance arteries.

Coronary resistance arteriolar diameter importantly regulates myocardial blood flow, and is influenced by circulating neurohumoral agents. Angiotensin II (A-II) is a circulating polypeptide that is chronically elevated in heart failure and serves as a potent peripheral vasoconstrictor agent. However, its effects on isolated coronary resistance arterioles is relatively unknown. We compared the vasomotor effects of A-II on coronary epicardial and resistance arterioles in vitro from both the canine and porcine heart in order to determine the effects of A-II in different vascular beds and species. Epicardial rings were studied under isometric recording conditions, while resistance arterioles (50-150 microns) were studied in vitro using a video imaging system to record diameter. A-II, whether applied to passively distended or preconstricted porcine resistance arterioles, did not cause vasoconstriction when applied as a bolus or as cumulative doses. In preconstricted canine resistance arterioles, A-II elicited dose-dependent vasodilation (EC50 = 0.2 nM). In passively distended canine arterioles, high concentrations of A-II (0.1 microM) applied as a bolus elicited transient vasoconstriction in 28% of the vessels studied. In large epicardial rings, A-II was a weak vasoconstrictor, with greater potency in canine arteries compared to porcine arteries. In canine arteries, vasoconstriction to A-II was augmented after incubation with indomethacin. In contrast to the findings in canine arteries, the A-II vasoconstrictor response in porcine coronary arteries was decreased after incubation with indomethacin or removal of the endothelium. Thus, A-II elicits the release of a vasodilator prostanoid in epicardial canine coronary arteries and a vasoconstrictor prostanoid in porcine vessels which modulate the vasomotor action of A-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗