Biomedical subjects
Publications and source records attributed to H S Margolius.
The chloride secretory response to bradykinin in T84 cells is regulated. The efflux rate of 125I from these cells can be used to measure this response, and to demonstrate that the sensitivity to bradykinin varies with time in culture. The response in NuT84 cells is greater than that in T84, and can be blocked by the B2 receptor antagonist HOE-140.
The production of tissue kallikrein and the short-circuit current (SCC) response in the human colonic epithelial cell line T84 to bradykinin have been studied. These cells produce true tissue kallikrein and secrete it into the growth medium, and addition of bradykinin results in a small increase in SCC. After growth of T84 cells as a xenograft in athymic (nude) mice (NuT84), however, the cellular concentration of the enzyme increases 38-fold, and the maximal change in SCC (delta SCC) induced by bradykinin increases 35-fold. The SCC response to prostaglandin E1 is not changed, and the response to forskolin increases eightfold. The bradykinin-induced delta SCC was inhibited by piretanide, suggesting that a secretory movement of chloride was responsible for the change, and the effect was attenuated by an antagonist to kinin receptors. We conclude that both the production of true tissue kallikrein and the chloride secretory response to bradykinin can be regulated in T84 cells by changes in the cell environment.
The effects of endogenous and exogenous glucocorticoids on renal active and prokallikrein levels (ng/mg protein) and in vivo kallikrein synthesis rate were studied in the conscious rat. Within two hours after low dose methylprednisolone (MP, 0.0125 to 0.05 mg/100 g body wt), active kallikrein and prokallikrein fell (29.1 +/- 2.3 and 35.1 +/- 2.7 ng/mg protein, respectively, compared to 38.4 +/- 3.7 and 42.7 +/- 3.4 in vehicle-treated rats, P less than 0.05 or less). These changes were accompanied by a significant fall in prokallikrein synthesis rate relative to total protein synthesis. The reductions in active and prokallikrein levels were transient, dissipating by six hours. With increasing MP doses, there was further dose-dependent reduction in active kallikrein. However, prokallikrein levels increased to normal as the MP dose was increased despite continued suppression of synthesis, suggesting that prokallikrein activation was inhibited. Renal kallikrein levels were also examined in relation to changes in endogenous glucocorticoid levels. In intact rats, three hours after plasma corticosterone peaked (10 p.m.), active and prokallikrein levels were 30.2 +/- 2.9 and 27.0 +/- 1.6 ng/mg protein, respectively, compared to 36.9 +/- 2.3 and 37.2 +/- 2.6 (P less than 0.005) three hours after the corticosterone nadir (11 a.m.). Furthermore, adrenalectomy increased active and prokallikrein (47.3 +/- 4.8 and 87.3 +/- 6.0 ng/mg protein, respectively), compared to levels in intact or shamoperated rats (intact: 32.9 +/- 2.9 and 54.9 +/- 5.3 ng/mg protein, P less than 0.01 or less). Adrenalectomy also eliminated the diurnal changes in kallikrein levels seen in intact rats. These data suggest that renal prokallikrein synthesis and activation are physiologically regulated by glucocorticoids.
The relationship between renal hemodynamic abnormalities and renal kallikrein activity was studied in streptozocin-induced diabetic rats. Diabetic rats were either not treated with insulin and had plasma glucose levels greater than 400 mg/dl (severely hyperglycemic diabetic [SD]) or were treated with 1.5-1.75 U/day protamine zinc insulin and had glucose levels of 200-300 mg/dl (moderately hyperglycemic diabetic [MD]). In SD rats, kidney tissue level and excretion of active kallikrein were reduced after 3 wk compared with age-matched nondiabetic control rats (tissue, 11.7 +/- 1.9 vs. 20.5 +/- 1.8 ng/mg protein, P less than 0.005; urine, 126 +/- 12 vs. 179 +/- 10 micrograms/24 h, P less than 0.005). Despite increased kidney size, renal plasma flow (RPF) was reduced in SD rats (5.38 +/- 0.23 vs. 6.37 +/- 0.20 ml/min, P less than 0.05). Glomerular filtration rate (GFR) was not significantly lower (2.77 +/- 0.60 vs. 3.02 +/- 0.56 ml/min). In MD rats, kidney tissue level and excretion of active kallikrein were increased after 5 wk compared with age-matched nondiabetic control rats (tissue, 28.4 +/- 1.3 vs. 23.3 +/- 1.7 ng/mg protein, P less than 0.05; urine, 289 +/- 16 vs. 196 +/- 13 micrograms/24 h, P less than 0.001). In MD rats, GFR and RPF were increased (3.80 +/- 0.11 and 8.04 +/- 0.17 ml/min, respectively) compared with control rats (3.22 +/- 0.05 and 7.28 +/- 0.09 ml/min, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
There is no convincing evidence presently for the notion that tissue kallikrein-regulated systems are the proximate cause of any form of human or animal disease. Nevertheless, the collection of facts very briefly mentioned here, suggest strongly that all of the conditions for such a possibility are being met and the right questions about such possibilities are being asked. Even without the answers, it is quite possible to anticipate what the abnormalities might be, and try to devise rational therapeutic strategies and new drugs. This is an area of endeavor which will be worthwhile because it will allow us to learn more about how kininogens, kallikreins, kinins and kininases function, even as new therapies are created.
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We studied GFR, RPF and renal kallikrein in rats fed 9%, 25%, or 50% protein (casein) diets for 8 to 13 days. CFR and RPF increased progressively with increasing dietary protein. Renal excretion of active kallikrein (microgram/day) was 128 +/- 9, 174 +/- 11 and 228 +/- 14 in 9%, 25%, and 50% protein-fed rats, respectively (P less than 0.02 or less between groups). Prokallikrein excretion in these groups was 23 +/- 7, 77 +/- 11 and 118 +/- 15 micrograms/day, respectively (P less than 0.005 or less between groups). The in vivo renal kallikrein synthesis rate, relative to total protein synthesis, was reduced in 9% protein-fed rats (2.74 +/- 0.24) compared to rats fed 25% (3.93 +/- 0.34, P less than 0.02) or 50% protein (4.41 +/- 0.30, P less than 0.001). These changes in synthesis and excretion rates were not accompanied by changes in renal tissue levels of active or prokallikrein. In all groups, GFR and RPF correlated directly with the renal excretion of active kallikrein, prokallikrein or total kallikrein (r = 0.41 to 0.66, P less than 0.01). Treatment of 50% protein-fed rats with aprotinin, a kallikrein inhibitor, markedly lowered renal and urinary kallikrein-like esterase activity. Left kidney GFR and RPF were significantly reduced in aprotinin-treated rats compared to vehicle-treated rats (1.54 +/- 0.15 and 4.86 +/- 0.38 ml/min vs. 1.89 +/- 0.10 and 5.93 +/- 0.22 ml/min, GFR and RPF, respectively, P less than 0.05 or less).(ABSTRACT TRUNCATED AT 250 WORDS)
The spectrum of cellular, or whole animal responses to kinins has expanded enormously in the last five years. The molecular basis for these consequences of kinin-kinin receptor interactions is being glimpsed as a series of cascading and parallel biochemical events occurring either in the same or adjacent, but communicating, cells of the same tissue (e.g. epithelial, endothelial, muscle, neural). Although it is likely that even more kinin-induced events remain to be discovered, like the interesting effects of the peptides on osteoclastic activity (128), a greater challenge in this field is the gathering of insights into precisely how the regulation of system component gene expression is coordinately carried out to allow component protein synthesis, transport and processing when necessary, and limitation of activities, when required. With these insights, we will be more able to understand the meaning of many observations of tissue kallikrein-kinin system abnormalities in common human diseases. This understanding will then make more obvious the drug design strategies to be used to stimulate or replace, and modulate or inhibit kallikrein-kinin system components in those diseases.
Little is known about the relationship between blood pressure and endogenous sex steroid hormones in patients with essential hypertension. Studies in hypertensive men have described decreased androgens. Men with cardiovascular disease may have estrogen levels which are increased or similar to healthy controls. We measured selected sex steroid hormones in 24 medication-free patients with uncomplicated essential hypertension (diastolic blood pressure less than or equal to 90 mmHg) and 24 normotensive subjects. The groups were equally divided by race, gender, age and weight. Hypertensive men had lower levels of both free and total testosterone and androstenedione than controls. The converse was true for hypertensive women. Androgen levels were similar in blacks and whites regardless of gender or blood pressure. Estradiol levels were higher in hypertensive men and women than controls and in blacks than whites. Levels of luteinizing hormone and sex hormone binding globulin were similar in all subjects. The clinical and pathophysiological significance of our findings merits further investigation.
We studied bradykinin effects upon electrogenic ion transport across voltage-clamped guinea pig gallbladder, and found stimulation after both mucosal (EC50 = 0.2 microM) and serosal addition (EC50 = 10 microM). The mucosal effect is dose-dependent, reproducible and sustained. Responses to bradykinin were unaffected by amiloride (1 mM) or piretanide (1 mM) but were attenuated by acetazolamide (1 mM), indicating the charge-carrying ion species to be bicarbonate. This was confirmed by abolishing the short circuit current response to bradykinin with bicarbonate-free solutions. Tetrodotoxin (1 microM) did not alter the response, indicating that neurons are not involved. This effect of bradykinin on gallbladder bicarbonate transport appears to be dependent upon arachidonate metabolism because the cyclooxygenase inhibitor piroxicam reversibly abolished the effect. In addition, prostaglandin E2 was more effective (EC50 = 0.01 microM) than bradykinin in stimulating short circuit current. This newly appreciated ion transporting effect of bradykinin occurs in a tissue where kinin effects upon afferent nerve endings and smooth muscle are also evident and raise several questions about kinin involvement in stone formation or local inflammatory events.
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The effects of dietary sodium upon serum and urinary calcium and selected vitamin D metabolites were studied in two groups (n = 10 each) of age and gender matched, white normotensive subjects and patients with normal-renin hypertension. Isocaloric diets were consumed on a metabolic ward with sequential daily sodium intake of 109 meq for 5 days and 9 meq and 259 meq for 6 days each. Values for serum and urinary calcium, phosphorus, magnesium and electrolytes, creatinine clearance, plasma immunoreactive parathyroid hormone, and serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were similar in both study groups on each diet. Measurements of plasma renin activity and serum aldosterone levels were higher in the hypertensive than in the normotensive group on each diet (p less than .05-.01). Serum 1,25-dihydroxyvitamin D and urinary calcium increased on the high sodium diet in the normotensive (p less than .05) and the hypertensive groups (p less than .01). When the data for normotensive subjects and hypertensive patients were pooled by gender, males had a 1 1/2 to 3 times the urinary calcium excretion than females, regardless of diet. The present study indicates that there are no differences in the selected components of calcium and vitamin D metabolism in response to sodium intake in patients with essential hypertension and normal plasma renin activity as compared to normal controls.
Anaphylactic reactions can be elicited by beta-lactoglobulin (beta LG) in the isolated colonic and ileal mucosae of guinea pigs fed cow milk. We have shown that these tissues respond to antigen challenge with a rapid rise in short-circuit current (SCC) due to net chloride secretion in the colon and less well-characterized ion movements in the ileum. Here, tissues were characterized morphologically at or near the peak of the SCC response 2 min after antigen addition. These sensitized preparations, or naive tissues passively sensitized with immune sera and challenged with beta LG, showed numerous structural alterations that correlated significantly with the change in SCC. In the ileum, these changes included compound exocytosis of mucous granules from goblet cells of crypts but not of villi and a decrease in the intercellular space of the crypts. In contrast, colonic goblet cells of both crypts and luminal surface showed no evidence of secretion, but crypt architecture was reproducibly and characteristically altered. Thus antigen challenge of intestinal mucosa results in temporally related but diverse changes in morphology and ion transport.
The level of tissue kallikrein in serum and urine, and of an erythrocyte kallikrein-like enzyme, were compared in 10 subjects without hypertension and in 10 patients with hypertension with normal renin levels. Each group consisted of five men and five women. All subjects were observed at a general clinical research center for consecutive 5- to 6-day periods of daily dietary sodium intake of 109, 9, and 259 mEq. Tissue kallikrein levels in serum and urine and levels of the erythrocyte kallikrein-like enzyme were measured with specific radioimmunoassays or with an activity assay, respectively. Mean active and total urinary kallikrein excretion rates were higher in women than in men (both with and without hypertension) when they were given all diets (p less than 0.05 to 0.025), and these rates varied inversely with sodium intake. The serum immunoreactive tissue kallikrein level was higher in men than in women when they were given all diets (p less than 0.05 to 0.001), but there was no difference between subjects with and without hypertension. There were no consistent changes in levels with altered sodium intake. Erythrocyte kallikrein-like esterase activity was greater in women without hypertension than in men without hypertension (p less than 0.05 to 0.001) when receiving the 9 and 109 mEq sodium diets, but values were similar in all groups receiving the 259 mEq sodium diet.(ABSTRACT TRUNCATED AT 250 WORDS)
A tissue kallikrein was purified from rat skeletal muscle. Characterization of the enzyme showed that it has alpha-N-tosyl-L-arginine methylesterase activity and releases kinin from purified bovine low-Mr kininogen substrate. The pH optimum (9.0) of its esterase activity and the profile of inhibition by serine-proteinase inhibitors are identical with those of purified RUK (rat urinary kallikrein). Skeletal-muscle kallikrein also behaved identically with urinary kallikrein in a radioimmunoassay using a polyclonal anti-RUK antiserum. On Western-blot analysis, rat muscle kallikrein was recognized by affinity-purified monoclonal anti-kallikrein antibody at a position similar to that of RUK (Mr 38,000). Immunoreactive-kallikrein levels were measured in skeletal muscles which have different fibre types. The soleus, a slow-contracting muscle with high mitochondrial oxidative-enzyme activity, had higher kallikrein content than did the extensor digitorum longus or gastrocnemius, both fast-contracting muscles with low oxidative-enzyme activity. Streptozotocin-induced diabetes reduced muscle weights, but did not alter the level of kallikrein (pg/mg of protein) in skeletal muscle, suggesting that insulin is not a regulator of kallikrein in this tissue. Although the role of kallikrein in skeletal muscle is unknown, its localization and activity in relation to muscle functions and disease can now be studied.
The effects of afferent vagal stimulation, cerebroventricular vasopressin, and intravenous nitroprusside on cerebrospinal fluid (CSF) kinin levels, mean arterial pressure (MAP), and heart rate (HR) were determined in anesthetized dogs in which a ventriculocisternal perfusion system (VP) was established. Following bilateral vagotomy, stimulation of the central ends of both vagi for 60 min significantly increased MAP and CSF perfusate levels of kinin and norepinephrine (NE). MAP was increased a maximum of 32 +/- 4 mmHg, and the rates of kinin and NE appearance into the CSF perfusate increased from 4.2 +/- 1.4 to 22.1 +/- 6.9 and from 28 +/- 5 to 256 +/- 39 pg/min, respectively. A significant correlation was found between CSF kinin and NE levels in these experiments. In other experiments the addition of arginine vasopressin to the VP system caused a significant increase in CSF perfusate kinin without affecting MAP or HR. Intravenous infusion of nitroprusside lowered MAP without affecting kinin levels in the CSF. However, on cessation of nitroprusside infusion, CSF kinin increased significantly in association with the return in MAP to predrug level. Collectively the data are consistent with the hypothesis that central nervous system kinins have some role in cardiovascular regulation, and furthermore that this role may involve an interaction between brain kinin and central noradrenergic neuronal pathways.
The effect of tryptase, a neutral protease released from human lung mast cell secretory granules, on the tissue prokallikrein present in human urine was examined. Tryptase has been shown previously to lack activity against plasma prokallikrein. Purified tryptase was incubated with a concentrated preparation of urinary prokallikrein. No increase in kallikrein-like enzymatic activity or immunoreactive tissue kallikrein was detected. Activation of urinary prokallikrein with trypsin served as a positive control. Furthermore, preincubation of urinary prokallikrein with tryptase did not diminish the subsequent activation of urinary prokallikrein by trypsin. Therefore, tryptase neither activates nor destroys human tissue or plasma prokallikreins.