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Biomedical subjects

J W Ryan

Publications and source records attributed to J W Ryan.

At least 127 records · Page 7Linked to original sources
PubMed1981

Relationship of a hiatal hernia to the function of the body of the esophagus and the gastroesophageal junction.

One hundred two patients referred to our Esophageal Function Laboratory without endoscopic evidence of esophagitis were divided into two groups on the basis of the presence of a hiatal hernia on endoscopic examination. Fifty-three patients had a hiatal hernia and 49 did not. Both groups and 30 normal volunteer subjects had esophageal manometry and 24 hour esophageal pH monitoring. The incompetency of the cardia in patients with a hiatal hernia was dependent upon loss of components responsible for the antireflux mechanism, mainly a decrease in distal esophageal sphincter pressure and a decrease in the length of the sphincter exposed to the positive-pressure environment of the abdomen. These deficiencies were not related to the presence of a hiatal hernia and were similar to those of patients with an incompetent cardia without a hiatal hernia. Patients with a hiatal hernia and an incompetent cardia had significantly more esophageal exposure to refluxed acid than without a hiatal hernia. On the basis of the number of reflux episodes that lasted 5 minutes or longer and radioisotope transit studies, this increased acid exposure was due to both a loss of competency of the cardia and poor esophageal clearance secondary to the presence of a hiatal hernia. Reduction of the hernia and anchoring the distal esophagus into the abdomen not only may improve the antireflux mechanism, but corrects the clearance abnormality as well. The presence of a hiatal hernia has a detrimental effect on the clearance function of the body of the esophagus and may aggravate the effects of gastroesophageal reflux due to an incompetent cardia.

Adolescent↗
PubMed1980

Endothelial cells of bovine pulmonary artery lack receptors for C3b and for the Fc portion of immunoglobulin G.

Bovine pulmonary endothelial cells do not possess receptors for the 3b component of complement (C3b) or for the Fc portion of immunoglobulin G. The lack of these receptors may help explain the nonthrombogenic function of endothelial cells. Our findings rule out the possibility that endothelial cells participate in pulmonary immune complex disease through the binding of C3b or Fc fragments.

Animals↗
PubMed1980

Effects of aspirin and dipyridamole on the degradation of adenosine diphosphate by cultured cells derived from bovine pulmonary artery.

To improve understanding of the mechanisms by which ADP is degraded during passage through the pulmonary vascular bed, we examined cultured endothelial and smooth muscle cells of bovine pulmonmary artery for their abilities to metabolize [8-14C]ADP. ADP is rapidly converted to AMP and then to adenosine, hypoxanthine, and inosine. Inosine is the major metabolite produced by endothelial cells. Radioactivity (5-10%) is accumulated intracellularly primarily as ATP. Medium containing 50 micro M ADP incubated with endothelial cells rapidly loses its ability to aggregate platelets and becomes antiaggregatory under conditions in which prostacyclin is absent. The antiaggregatory activity is probably the result of accumulated adenosine. 10 micro M dipyridamole inhibits cellular uptake of radioactivity by greater than 90%, and inosine in the medium is largely replaced by adenosine. This is accompanied by increased anti-aggregatory activity of conditioned medium, which can be matched by authentic adenosine at the same concentration. 1 mM aspirin had no effect on the metabolism of ADP by endothelial cells. Our results suggest: (a) Metabolism of ADP during passage through the lung is mainly the result of endothelial ADPase. (b) ADP released from aggregating platelets can be converted to the antiaggregatory substance, adenosine. Dipyridamole may exert some of its antithrombotic actions by preventing the intracellular uptake of adenosine, thereby increasing its concentration near the site of thrombus formation. (c) The ability of the vessel wall to degrade ADP should not be compromised by the use of aspirin as an antithrombotic drug. (d) Endothelium may retain some of its antithrombogenicity when prostacyclin generation is impaired.

Adenosine Diphosphate↗
PubMed1980

Angiotensin-converting enzyme: I. New strategies for assay.

The disposition of converting enzyme (kininase II) on the luminal surface of pulmonary endothelial cells is well established. Further, it is known that there is a net conversion of angiotensin I into angiotensin II as blood passes through the lungs. However, little is known about modulations of converting enzyme activity that may arise through, e.g., changes in the quality of inhalants, blood flow, or blood oxygenation. There are few data on the effects of lung disease. A major barrier to studies to examine for pathophysiologic modulations of converting enzyme is that of assay. The enzyme can be measured in terms of the rate of formation of angiotensin II from a known quantity of angiotensin I. However, both peptides are biologically active, and lungs contain other enzymes capable of degrading them. We have developed a series of radiolabeled, acylated tripeptides to improve our ability to examine for changes in the net converting enzyme of intact lungs. The enzyme, a dipeptidyl carboxypeptidase, is capable of removing C-terminal dipeptides from a variety of oligopeptides. We have prepared benzoyl-Gly-Gly-Gly (I), benzoyl-Pro-Phe-Arg (II), benzoyl-Gly-His-Leu (III), benzoyl-Phe-Ala-Pro (IV), and benzoyl-Phe-His-Leu (V), each containing a (3)H-atom in the para position of the benzoyl moiety. Substrates I and III have been used previously in photometric assays of low sensitivity. II is the acylated C-terminal tripeptide of bradykinin, IV is an acylated tripeptide analog of BPP(5a) (<Glu-Lys-Trp-Ala-Pro) and V is the acylated C-terminal tripeptide of angiotensin I. These substrates can be used in vitro or in vivo to measure converting enzyme. The (3)H-labeled product is separable by partitioning between an organic solvent and acidified aqueous solution. The product is quantified by scintillation counting of the organic phase. The choice of substrate depends on the goals of the experiment: substrate I or III when wide variations in substrate concentrations are needed but high sensitivity is not; substrate IV when high sensitivity is needed.

Angiotensin I↗
PubMed1980

Angiotensin-converting enzyme: II. Pulmonary endothelial cells in culture.

Direct studies of the function of a given cell type often require that the cell type be obtained in pure culture. A number of different specific metabolic activities have been attributed to pulmonary endothelial cells, yet with few exceptions the conclusions were based on indirect evidence. Thus, to improve our ability to examine directly for specific metabolic activities, we began a program to obtain pulmonary endothelial cells in culture. Two methods have been developed: (1) cells can be obtained from pulmonary artery and vein of large animals (cow, pig), and (2) cells can be obtained from the microvasculature of small animals (rat, guinea pig, and rabbit). The latter technique can also be used to obtain cells from a lobe of lung from large animals and may be adaptable for use with human tissue. In the first technique, pulmonary arteries, free of blood, are filled with collagenase (0.25%, 500 units) in Puck's saline for 25 min. The collagenase mixture containing cells is removed and centrifuged. The pellet is resuspended and seeded into culture flasks. In the second method, lungs are perfused (artery to vein) with Krebs-Henseleit solution until the effluent is blood-free. Collagenase (0.25%, 500 units) is introduced, and the lungs are then perfused in the opposite direction (vein to artery) until the flow stops spontaneously (ca. 15 min). The detached cells are collected and seeded as before. The endothelial cells attach as small clumps (10-50 cells). Those flasks which contain more than 95% endothelial cells (by phase microscopy) are retained for culture and the lines are purified usng differential adherence procedures. The cells grow as monolayers with a cobblestone appearance. They contain Weibel-Palade bodies. They possess converting enzyme activity and are reactive with antibodies to converting enzyme, Factor VIII and alpha 2-macroglobulin. The cells synthesize prostaglandins and related substances. In addition, they possess ADPase and synthesize angiotensin-converting enzyme.

Adenosine Diphosphate↗
PubMed1979

Fate of bradykinin-potentiating peptide 9a after intravenous injection.

The fat of less than Glu1-3H-labelled bradykinin-potentiating peptide 9a [BPP9a; less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, an inhibitor of angiotensin-converting enzyme (peptidyl dipeptidase)] was studied in the rabbit. After intravenous injection, BPP9a was rapidly removed from blood and much of the associated radioactivity was excreted in urine. Approx. 8% of the radioactivity in urine collected 2h after drug administration occurred in the form of BPP9a itself, the remainder occurring in three lower homologues: less than Glu-Trp (60%), less Glu-Trp-Pro-Arg-Pro-Gln (20%) and less than Glu-Trp-Pro-Arg-Pro-Gln-Ile (12%). Hydrolysis was not accounted for by enzymes in blood or urine. Apparently hydrolysis occurred within the kidney, as less than Gl-Trp was obtained in 60% yield in urine of isolated rat kidney perfused with [less than Glu1-3H]BPP9a.

Animals↗
PubMed1979

Synthesis of renin substrate by rat liver.

The present study attempts to determine if the isolated rat liver is capable of synthesizing renin substrate from 14C-labelled amino acids added in the perfusate. The renin substrate is characterized via reaction with renin, forming a substance that is subsequently identified as proangiotensin. Extensive evaluation of the reaction product is carried out by using molecular-sieve chromatography, countercurrent distribution, reactivity with converting enzyme, radioimmunological technique and bioassay. The results demonstrate that isolated rat liver perfused with artificial salt solution is capable of synthesizing a protein that reacts with renin to form a radioactive substance indistinguishable from proangiotensin.

Angiotensinogen↗
PubMed1979

A direct radioimmunoassay for human urinary kallikrein.

A protein-binding radioimmunoassay (RIA) has been developed for human urinary kallikrein (HUK). HUK was purified to apparent homogeneity and rabbit anti-human urinary kallikrein serum was prepared. Radio-iodination of HUK was performed in 20 microgram batches with 1 mCi of 125I-Bolton-Hunter reagent in 0.1 M sodium borate buffer, pH 8.5, at 0 degree C. The 30 min reaction was terminated by adding 0.5 ml of 0.2 M glycine. Radiolabelled HUK was purified on a Sephacryl S-200 column (1x 50 cm) in 0.05 M Tris.HCl plus 0.2% gelatin, pH 7.4 (RIA buffer). 125I-HUK emerged as a single, uniform peak at Ve/Vo of 1.3. The RIA reaction mixture contained 15,000 cpm of 125I-HUK, 0.1 ml of diluted anti-kallikrein serum, 0.1 ml unlabelled kallikrein (1-100 ng) or the unknown sample and RIA buffer to a total of 0.5 ml. The reaction mixture was incubated at 22 degrees C for 18 hr, and 100 microliter to goat-anti-rabbit IgG serum was added to each tube. The tubes were incubated 18 hr at 4 degrees C, centrifuged 4,000 rpm for 30 min and, after decantation, the precipitate was counted for 125 I. The RIA reported here is sufficiently sensitive to detect 1 ng of kallikrein and should offer a useful technique to determine alterations of the kallikrein-kinin system in response to stimuli; both physiologic and pathologic.

Animals↗
PubMed1979

A simple radioassay for human urinary kallikrein.

We have developed a sensitive, highly selective assay for human urinary kallikrein (HUK) that uses Pro-Phe-Arg-[3H]benzyl-amide as substrate. The substrate was prepared from Pro-Phe-Arg-3-iodo-benzylamide by dehalogenation in 3H2 gas. HUK is measured by its ability to release [3H]benzylamine. The pH optimum is 9.5. Urokinase, plasmin and thrombin do not interfere. The assay can measure as little as 5 ng of HUK in a 15 min incubation at 37 degrees C. Typically, we use 50 microliter of dialyzed urine for HUK assays. Reactions are terminated by adding 0.1 M NaOH, and reaction product is separated from substrate by partitioning with an equal volume of toluene. A sample of the toluene phase is submitted for liquid scintillation counting. As judged by separations obtained on molecular sieve chromatography (Sephacryl), only one urinary enzyme possesses the ability to hydrolyze our substrate. The enzyme MW 45,000, is inhibited by Trasylol but not by soya bean trypsin inhibitor (SBTI). It is reactive with and is inhibited by antibodies prepared against pure HUK.

Acylation↗
PubMed1979

Components of the kallikrein-kinin system in urine.

The excretion of kallikrein in urine varies, but the pathophysiologic implications are not clear. To help clarify the role of the urinary kallikrein-kinin system, we have begun to define components of the system as they occur in urine. To minimize artifacts which may arise through extensive purification procedures, we studied urinary protein concentrates prepared by ultrafiltration. The concentrates were separated by chromatography on Sephacryl. Urine contains abundant kininase activity, but in strongly inhibited forms. Kininase II is separable into at least two forms. Another major kininase can hydrolyze benzoyl-Pro-Phe-Arg and is inhibited by arginine but not by BPP9a or SQ 14,225. Its molecular weight is approximately 63,000. A third kininase, not inhibited by BPP9a, is excluded from Sephacryl. Human urine appears to contain only one kallikrein-like enzyme (MW 45,000). In addition, urine contains a protein (MW approximately 80,000) which reacts with trypsin to release bradykinin and which inhibits the hydrolysis of Pro-Phe-Arg-[3H]anilide by urinary kallikrein. Thus, in addition to kallikrein and kinins, urine contains kininogen and at least three kininase enzymes. Urinary ultrafiltrate contains an inhibitory substance (approximately MW 400).

Animals↗
PubMed1979

How do kinins affect vascular tone?

Because kinins affect vascular tone, it is assumed that kinins act directly on smooth muscle. However, a direct interaction is difficult to conceive. Vessels containing smooth muscle are lined by a continuous endothelium with tight junctions. In addition, kinins act on endothelial cells to cause the release of prostaglandin-related substances; possibly through receptors. Furthermore, endothelial cells have a great capacity for hydrolyzing kinins to inactive products. Hence, even invoking active transport, less than 1% of kinins might be expected to reach the first layer of smooth muscle cells. However, kinins may not act directly on smooth muscle as endothelial cells and smooth muscle cells form specialized cell contacts. Myoendothelial junctions occur, and we have shown, in pulmonary arterioles, that smooth muscle cells send large numbers of projections into the cytoplasm of the endothelial cells. In addition, smooth muscle cells attach directly to the abluminal surface of endothelial cells, as do pericytes. Thus, there is a morphologic basis by which kinins can affect tone of smooth muscle without acting directly on smooth muscle cells.

Animals↗
PubMed1979

Pulmonary anaphylaxis and the kallikrein-kinin system.

The kallikrein-kinin system has been thought to participate in the pathogenesis of anaphylaxis. Kallikrein, released from lungs, has been postulated to contribute to cardiovascular collapse. Further to test the hypothesis, we examined for the occurrence of a kallikrein-like enzyme in guinea pig lungs and examined for release of such an enzyme by isolated, perfused lungs of guinea pig sensitized to and challenged with egg albumin. In addition, we treated guinea pigs with the bradykinin potentiating agents, BPP9a and SQ 14,225. In parallel experiments, we examined for effects of non-steroidal anti-inflammatory agents on the supposition that prostaglandin-related substances may mediate or modulate actions of kinins during anaphylaxis. A plasma kallikrein-like enzyme was found in lung homogenates and occurred in concentrations greater than that of plasma itself. Similarly, a store of kininogen occurs in lungs. However, using a sensitive radioassay for kallikrein-like enzymes, we were unable to confirm that antigenic challenge of sensitized lungs causes the release of enzyme into pulmonary venous effluent. Further, we were unable to modify the acute course of anaphylaxis by pretreatment of guinea pigs with bradykinin potentiating agents. However, indomethacin and aspirin at 20-40 mg/kg were found to greatly increase the severity of pulmonary anaphylaxis in terms of increased resistance to ventilation and increased numbers of lung hemorrhages. Paradoxically, aspirin or sodium salicylate at 80-100 mg/kg prevents the characteristic rise of insufflation pressure and the formation of lung hemorrhages.

Anaphylaxis↗