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J W Ryan

Publications and source records attributed to J W Ryan.

At least 145 records · Page 8Linked to original sources
PubMed1979

Preparation of intrinsically-labelled kinins.

As part of a program to prepare bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) labelled at high specific radioactivities, we have synthesized three analogs for dehalogenation in tritium gas: [4-Br-Phe5]-bradykinin (BK), [4-Br-Phe8]-BK and [4-Br-Phe5,8]-BK. The analogs were synthesized by the Merrifield solid-phase method and were purified by molecular sieve and partition chromatography. The analogs themselves possess biological activity (as assayed for effects on mean arterial blood pressure and isolated rat uterus). [4-Br-Phe8]-BK was 1.5 to 3 times as active as bradykinin. [4-Br-Phe5,8]-BK was approx. 22% as active as BK and [4-Br-Phe5]-BK was approx. 18% as active. [4-Br-Phe5]-BK was submitted to catalytic dehalogenation with 10% Pd/C and 5% Rh/CaCO3 in H2O and DMF (1:1) plus 10 Ci of 3H2. [4-3H-Phe5]-BK was obtained at 6.7 Ci/mmole in an overall yield of 15%. [4-3H-Phe8]-BK was prepared similarly to yield an intrinsically-labelled peptide with a specific radioactivity of 21 Ci/mmole.

Animals↗
PubMed1979

Isolation and culture of endothelial cells from the lungs of small animals.

Techniques are descirbed for the isolation and culture of endothelial cells from the lungs of small animals. The cells are collected by retrograde perfusion of blood-free lungs with buffered saline containing collagenase. The cells are characterized by light microscopy, electron microscopy of thin sections and surface replicas, and by the presence of angiotensin-converting enzyme (ACE). ACE was assayed using 3H-benzoyl-Phe-Ala-Pro as substrate and was localized by indirect immunofluorescence using guinea pig endothelial cells incubated with rabbit antibodies to guinea pig lung ACE followed by goat anti-rabbit globulins conjugated to fluorescein. Thus, endothelial cultures can be established using small animals commonly employed in studies of pulmonary processing of vasoactive substances.

Animals↗
PubMed1979

Endothelial cells and components of the kallikrein-kinin system.

Endothelial cells are a major source of kininase enzymes including kininase II. Kininase II is situated along the plasma membrane, not as an ecto-enzyme but as an enzyme synthesized by the endothelial cells themselves. However, it is likely that endothelial cells do more than degrade kinins. These cells are contractile and may possess kinin receptors; a possibility supported by the fact that kinins stimulate endothelial cells to form and release prostaglandin-related substances. In addition, we have found that endothelial cells in culture are reactive with antibodies to alpha 2-macroglobulin. Endothelial cells can hydrolyze [3H]Pro-Phe-Arg-anilide, a kallikrein substrate, but the reaction is not inhibited by soya bean trypsin inhibitor (SBTI) or Trasylol. Possibly kallikrein or a related trypsin-like enzyme is bound to alpha 2-macroglobulin and is not free to react with the inhibitors. Thus, endothelial cells can bind and inhibit kallikrein-like enzymes, degrade kinins and respond to kinin stimulation.

Animals↗
PubMed1979

Mammalian inhibitors of angiotensin converting enzyme (kininase II).

Urines and sera (human, guinea pig and rat) contain low molecular weight inhibitors of angiotensin converting enzyme (ACE). The urines contain ACE, but the enzyme is scarcely measurable without prior ultrafiltration or dialysis. The activity increases strikingly through three ultrafiltration steps using a membrane with a 10,000 MW retention limit. As implied, the ultrafiltrates contain inhibitory activity and can prevent the hydrolysis of [3H]benzoyl-Gly-His-Leu by ACE from any source, including lung and serum. Human urinary ultrafiltrate contains at least three inhibitors separable on Bio-Gel P-2. The inhibitors are acidic and can be partially purified on Bio-Rex 70 developed with an acetic acid gradient. The smallest of the inhibitors can be purified to apparent homogeneity by partition chromatography (sephadex G-25; butanol, acetic acid, H2O; 4:1:5). The excretion of inhibitory activity varies in response to dietary salt: Activity is low when rats are maintained on a high NaCl diet and is high (3 x's control) on a low NaCl diet. Thus, the activity of ACE may be modulated in vivo by naturally-occurring enzyme inhibitors. Whether some hypertensive patients are deficient in ACE inhibitory activity remains to be determined.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1978

Studies on the hydrolysis of bradykinin by angiotensin-converting enzyme (kininase II).

Arg-Pro-Pro-Gly-Phe, the N-terminal pentapeptide of bradykinin, is not an inhibitor of angiotensin-converting enzyme and is not hydrolyzed by the enzyme. Arg-Pro-Pro, the N-terminal tripeptide is a relatively potent (IC50 = 2.3 X 10(6) M) inhibitor but its higher homolog. Gly-Arg-Met-Lys-Arg-Pro-Pro is not an inhibitor of angiotensin-converting enzyme.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1978

A simple high-yield procedure for isolation of human urinary kallikreins.

Two human urinary kallikreins (fractions A-1 and A-2) were purified to apparently homogeneous forms. The two kallikreins were separated by affinity chromatography using Trasylol (aprotinin) covalently bound to Sepharose. The kallikreins were eluted with a pH gradient (pH 9.5-3.0). Fraction A-1 was eluted between pH 6.2 and 4.2 and fraction A-2 was eluted between pH 4.2 and 3.1. Final purification was obtained by chromatography on Sephacryl SS-200. Antibodies prepared against fraction A-2 were also reactive with fraction A-1; thus it may be possible to measure both by radioimmunoassay using the same antibody preparation.

Antibodies↗
PubMed1978

Isolation and culture of pulmonary artery endothelial cells.

It has become increasingly evident that endothelial cells function as far more than a mechanical barrier between blood and parenchyma. Endothelial cells from one vesicular bed are known to differ structurally from those of another, and it has been suggested that they may differ functionally. Further to test the hypothesis that endothelial cells from one site may differ in terms of function from those of another site, it is necessary to test endothelium from various source after having obtained these cells in pure, well-characterized cultures. To facilitate such studies, we herein describe in detail means for the isolation, culture and characterization of endothelial cells from calf pulmonary artery. These cells may be of major interest in terms of specific metabolic activities as it has become evident that the lungs play a prominent role in determining the hormonal composition of systemic arterial blood.

Cell Adhesion↗
PubMed1978

New substrates for the radioassay of angiotensin converting enzyme of endothelial cells in culture.

To develop means of measuring angiotensin converting enzyme of endothelial cells in culture, we have synthesized benzoyl-Phe-Ala-Pro-OH (I), benzoyl-Pro-Phe-Arg-OH (II) and benzoyl-Gly-His-Leu-OH (III), each bearing a 3H-atom on the para-position of its benzoyl moiety. All three of the acylated tripeptides are substrates for the enzyme. Substrate I exhibits the lowest Km (12.5 micrometer) and yields the most sensitive assay: the enzyme of 10(6) cells can be measured in a 30 min incubation at 37 degrees C. Radiolabelled reaction product is separated from substrate by extraction of acidified reaction mixture with an organic solvent, and the rate of formation of product can be quantified by liquid scintillation counting of the organic phase. Substrate III can also be used to measure angiotensin converting enzyme of cells but requires longer incubations (180--240 min) and high salt concentrations (0.75 M Na2SO4). Substrate II is not specific: it is hydrolyzed by more than one enzyme of endothelial cells.

Cells, Cultured↗
PubMed1977

A simple radioassay for angiotensin-converting enzyme.

Angiotensin-converting enzyme can be measured by the rate of release of 3H-labelled hippurate from p-[3H]benzoylglycylglycylglycine. The product is separable from the substrate by extraction of acidified reaction mixtures with ethyl acetate. Assay results for human serum angiotensin-converting enzyme can be obtained within 1.5 h of receipt of serum samples. Within the limits tested, the assay appears to be specific. However, interference by hitherto unrecognized enzymes of abnormal sera must be ruled out.

Hippurates↗
PubMed1976

Formation of angiotensin III by angiotensin-converting enzyme.

Angiotensin III is formed from des-Asp1 -angiotensin I by angiotensin-converting enzyme. The Km (11 muM) of the reaction is one-third of that for the conversion of angiotensin I into angiotensin II. As suggested by the Km values, bradykinin, peptide BPP9a and angiotensins II and III are better inhibitors of the formation of angiotensin II than of the formation of angiotensin III.

Angiotensin II↗