R. Horton

Altered Regulation of Renin Secretion by Insulinlike Growth Factors and Angiotensin II in Diabetic Rats

The etiology of the low renin state in DM is not clear. To assess the role of certain growth and regulatory factors in this process, we studied the effects of insulin, IGF-I, and IGF-II on the renin-angiotensin system in normal and 8-wk STZ-induced diabetic rats. Renin secretion was studied both in static incubations and by perifusion of rat renal cortical slices. In diabetic rats, both plasma renin activity (0.65 ±1.6 vs. 4.0 ±1.2 ng ANG I · ml−1 · h−1) and tissue renin concentrations (27 ± 5 vs. 51 ± 8 ng ANG I · mg tissue−1 · h−1) were reduced. Insulin (0.1–1.0 mu/ml) and IGF-I (10−9 to 4 × 10−9 M) stimulated renin secretion in normal tissue (control, 95 ± 3%; insulin [0.5 mu/ml], 134 ± 7%; IGF-I «4 × 10−9 M], 149 ± 7%). IGF-I stimulated renin secretion in perifusions as early as 30 min, whereas IGF-II had no effect. However, in diabetic renal tissue, neither insulin (0.1–1.0 mu/ml) nor IGF-I (10−9 to 4 × 10−9 M) had an effect on renin. This lack of effect was overcome by adding up to 100-fold higher concentrations of these growth factors. ANG II (10−10M–10−8 M) had an exaggerated inhibitory effect on renin secretion in diabetic tissue. This study suggests that the low renin state in DM may be explained by the enhanced inhibitory effect of ANG II and the resistance to the secretogogue actions of insulin and IGF-I.

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Renal prostaglandins and sodium balance in normal man

We investigated the relationship between urinary prostaglandin E (PGE) excretion and sodium and water balance. PGE excretion was measured in thirteen healthy male volunteers on the metabolic ward during conditions of high sodium (200 mmols/day) and low sodium diets (40 mmols/day) and during intravenous administration of saline and of dextrose and water, using each subject as his own control. PGE excretion was higher on the high sodium than on the low sodium diet (191 +/- 37 SE versus 98 +/- 41 ng/6h, p less than 0.01). Saline and dextrose and water infusions significantly increased PGE excretion while subjects were on low sodium diets (to 314 +/- 74 and 443 +/- 152 ng/6h, respectively, p less than 0.01). While on high sodium diets the increase in PGE excretion during infusions was not significant. To further evaluate the role of prostaglandin in sodium excretion the study was repeated with simultaneous administration of indomethacin or ibuprofen to inhibit prostaglandin synthesis. Sodium excretion from saline and dextrose and water infusions were unaltered. The data suggest that dietary content of sodium may alter PGE excretion, but that acute changes in PGE excretion during saline administration reflect water balance rather than sodium load.

Effects of transforming growth factor β and epidermal growth factor on steroid 5α-reductase activity in genital skin fibroblasts ☆

Regulation of steroid 5α-reductase (5αR) activity and dihydrotestosterone (DHT) formation is central to prostate and sexual skin (hair) growth and cell function. Transforming growth factor-β1 (TGF-β1) is a ubiquitous peptide present in skin and scrotal tissue and its receptor is universally expressed. We have explored the role of TGF-β1 and -β2 on androgen formation in skin. Rat or human sexual skin fibroblasts were grown in primary cultures (passage 3–7). 5α-Reductase activity was measured by the %-conversion of tracer 3H-testosterone to dihydrotestosterone over a 4 h period. Incubation of scrotal fibroblasts (2 × 105 cells) in serum and growth factor free media with androgen, such as DHT for two days significantly stimulates 5αR in these cells (1.6-fold, p < 0.05 vs control). TGF-β1 alone at picomolar concentrations (2 × 10−11 M to 2 × 10−10 M) was a potent inducer of 5αR activity in both rat (1.8-fold and 2.8-fold, respectively, p < 0.001 vs control at both doses) and human cells (TGF-β1 2 × 10−10 M 3.3-fold, p< 0.001 vs control). Combined exposure of these fibroblasts to TGF-β1 (2 × 10−10 M) and androgen (10−10 M) further potentiated 5αR activity (rat cells 6.5-fold, human cells 6.4-fold, p< 0.001 vs DHT or TGF-β1 alone). TGF-β2 (2 × 10−10 M), a closely related peptide produced similar effects on 5αR activity in human cells (DHT 1.5-fold, TGF-β2 2-fold, TGF-β2 + DHT 5.5-fold), again synergism was noted with DHT. These effects were not associated with changes in cell number or increased 3H-thymidine incorporation. In contrast, epidermal growth factor (EGF, 5 × 10−9 M) which has a sequence homology with transforming growth factor-α (TGF-α) and interacts with the TGF-α type receptor did not significantly induce basal 5αR or in combination with an androgen. These studies indicate that TGF-β is much more potent than an androgen and is synergistic with androgen in inducing 5αR activity and DHT formation. EGF (TGF-α) has no significant effects on these events. TGF-β may play a role in androgen formation in sexual tissue.

Antihypertensive Effect of Enalapril in Essential Hypertension: Role of Prostacyclin

The effects of enalapril alone and in combination with the cyclooxygenase inhibitors sulindac and indomethacin on blood pressure (BP), plasma aldosterone, renin activity and converting enzyme activity were evaluated in 29 patients with mild to moderate essential hypertension, 26 of whom had low plasma renin activity. Patients were randomly assigned to one of three treatment groups. All patients underwent a 4-week placebo phase (phase I), then received enalapril (20 mg BID) for 4 weeks (phase II). In phase III, group I (n = 10) continued on enalapril alone; group II (n = 9) received sulindac 200 mg BID plus enalapril, and group III (n = 10) received indomethacin 50 mg BID plus enalapril, all for 4 weeks. Enalapril lowered BP significantly (mean supine BP 149/100 in phase I vs. 134/90 in phase II, p < 0.05) without inhibiting aldosterone production. The BP effect was not blunted by concomitant administration of sulindac or indomethacin. Enalapril lowered converting enzyme activity to 25% to 30% of baseline and tended to increase renin activity. In the 10 patients who received indomethacin (group III), the effects of enalapril alone and enalapril plus indomethacin on urinary excretion of 6-keto prostaglandin F1α (PGF1α, a stable metabolite of prostacyclin (PGI2), were examined. Enalapril increased urinary 6-keto PGF1α in group III from 118 ± 23 to 194 ± 38 ng/g creatinine (p < 0.05), while addition of indomethacin reduced 6-keto PGF1α to basal levels (138 ± 26 ng/g creatinine). There was no significant correlation between the fall in blood pressure and the increase in 6-keto PGF1α excretion in enalapril-treated patients. These data indicate that the antihypertensive effect of enalapril in essential hypertension occurs in the absence of an active renin-angiotension system and is associated with increased excretion of 6-keto PGF1α but is not blunted by cyclooxygenase inhibitors. These findings indicate (1) enalapril administration is associated with enhanced prostacyclin production in this patient group but (2) the antihypertensive action of enalapril is not critically dependent on prostacyclin, since blockade of prostacyclin production with a cyclooxygenase inhibitor does not blunt the antihypertensive effect of the drug.

Isolation and identification of androstanediol glucuronide from human plasma

[3H]Dihydrotestosterone (50 microCi) was infused into normal men and women for 8 h. It was previously shown that this was sufficient time for this material to reach a steady state. Venous plasma was obtained at 6 and 8 h, pooled, and the unconjugated steroids removed by ether extraction. The remaining plasma was adjusted to pH 4.9 and the steroid conjugate was extracted first with ethyl acetate and then with an ether-ethanol mixture. The extracts were combined and taken to dryness. Steroid sulfates were solvolyzed using dioxane, and the mixture partitioned between ether and 1% NaOH. The aqueous phase was acidified and added to an XAD-2 column, washed with water, and the glucuronide fraction eluted with methanol. The solvent was concentrated and the methanol extract was passed through a C18 Sep-Pak, filtered through an Acrodisc CR and then subjected to gradient high performance liquid chromatography [HPLC] (Nova-Pak C18, KH2PO4, pH 3, and methanol). The fractions containing steroid glucuronides were collected and esterified with diazomethane and then acetylated with acetic anhydride in pyridine. The glucuronide triacetyl methyl ester (GAME) derivatives were then run in a second HPLC system (3 Lichrosorb 5 mu columns, 4 mm x 25 cm) using a gradient of ethanol-heptane and heptane. We clearly established that this system separates 3 alpha-diol GAME conjugated at the 17 and 3 positions (44 vs 50 min) with authentic samples previously synthesized in our laboratory. We concluded that the pooled plasma contained only the 17-GAME conjugate. No significant activity of the 3-glucuronide was detected. The natural compound in circulation, therefore, is 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.

The effect of adrenergic stimulation on urinary prostaglandin E2 and 6 keto PGF1α in man

Abstract To evaluate the details of the adrenergic stimulation of urinary prostaglandins in man, ten normal volunteers were given various agonists and antagonists. The effect of 4 hour IV infusions of norepinephrine (NE), NE + phentolamine (PHT), NE + phenoxybenzamine (PHB), NE + prazosin (PZ), isoproterenol (ISO), and PHT alone on urinary PGE2 and PGI2 (6 keto PGF1α) were determined. PGE2 and 6 keto PGF1α were measured by radioimmunoassay from 4 hour urine samples. NE stimulated both PGE2 (196±40 to 370±84 ng/4 hrs/g creatinine and 6 keto PGF1α(184±30 to 326±36), both p 0.2). PHT alone stimulated only 6 keto PGF1α. PHB and the specific α1 antagonist PZ similarly eliminated the NE induced prostaglandin release. These results suggest that adrenergically mediated urinary prostaglandin release in man is via an alpha receptor with α1 characteristics.

Is urinary flow rate a major regulator of prostaglandin E excretion in man

Urinary PGE2 excretion is enhanced in several polyuric states in man suggesting that PGE2 synthesis could be a mediator of diuresis. To explore the alternate hypothesis that polyuria is the cause of the increased PGE2 excretion, we increased urine flow rate by intravenous administration of dextrose and water with different magnesium, calcium and potassium solutions in four normal males. Urinary PGE excretion rose in parallel with urine volume (r = 0.65 p < 0.01) independently of the electrolyte solution. To determine the effects of chronic alterations in water balance in 5 female subjects, we sequentially regulated oral water intake to induce 1, 2, 4 and 8 liters of urine volume/day. During low (40 mEq) sodium diets, PGE increased from 540 +/- 50 to 4880 +/- 1240 ng/d with increasing urinary volume (r = 0.81, p < 0.01). Similarly, for 200 mEq sodium intake PGE paralleled urinary volume (from 630 +/- 100 to 4740 +/- 800 ng/d, r = 0.61, p < 0.01). In vitro sample dilution studies demonstrated no interference from method blank, and the addition of thin layer chromatography prior to Sephadex chromatography failed to alter assay measurements. We conclude that extreme increases in urinary flow rate may directly enhance PGE excretion in man.

Induction of renin release by exogenous prostaglandins in hyporeninemic hypoaldosteronism.

A deficiency in renal prostaglandin synthesis has been proposed as the cause of the syndrome of hyporeninemic hypoaldosteronism. To determine if renin release could be stimulated by pharmacologic infusions of PGA1, we infused PGA1 0.075 to 0.60 microgram/kg/min to nine patients with the syndrome. Total renal PGE production as measured by urinary PGE excretion was normal (650 +/- 169 vs 400 +/- 55 ng/24hr in normal subjects). Renin (PRA) was markedly depressed in all patients despite stimulation with upright posture and furosemide (1.0 +/- 0.4 vs 9.3 +/- 0.7 ng/ml/hr, p < 0.001). But in two patients PGA1, induced an increase in renin similar to that of normal subjects. PRA increased to a lesser degree in two other patients and plasma aldosterone slightly increased. Five showed no response. Infusions of nitroprusside in doses and duration that mimicked the hypotensive effects of PGA1 failed to increase PRA or aldosterone. The data suggest that total renal PGE production is normal in patients with the syndrome of hyporeninemic hypoaldosteronism. Although orthostasis, furosemide and nitroprusside do not increase renin, prostaglandin A1 infusion appears to be a potent stimulus to renin release in some of the patients.

Activin and inhibin have opposite effects on steroid 5α-reductase activity in genital skin fibroblasts☆

The transforming growth factor beta (TGF-beta) superfamily includes several closely related peptides including the activins and inhibins. Since we recently reported that TGF-beta 1 and beta 2 are potent inducers of steroid 5 alpha-reductase (5 alpha R), we have now studied the effects of these other peptides using primary cultures of human scrotal skin fibroblasts. Recombinant human activin A or inhibin A were added to cultured cells (2 x 10(5) cells) for 2 days in a serum free media and 5 alpha R activity was measured by the %-conversion of tracer [3H]-testosterone to dihydrotestosterone (DHT) over a 4-h period. Activin significantly stimulated 5 alpha R activity in a dose related manner (control 3.0 +/- 0.4%, activin (1.2 x 10(-9) M) 6 +/- 0.7%, P < 0.01, (2.4 x 10(-9) M) 8.5 +/- 0.6%, P < 0.001). In comparison, androgen (DHT 10(-7) M) induction of 5 alpha R was 4.7 +/- 0.2%, P < 0.05. Combined exposure of fibroblasts to activin (1.2 x 10(-9) M) and androgen (10(-7) M) did not result in additive or synergistic effect on 5 alpha R activity. In contrast, exposure of cells to an androgen (10(-7) M) and TGF-beta (2 x 10(-10) M) led to synergistic effects on 5 alpha R activity (control 1.5 +/- 0.1%, DHT 2.6 +/- 0.2% TGF-beta 1 4.8 +/- 0.5, TGF-beta 1 + DHT 9.2 +/- 1.2%).(ABSTRACT TRUNCATED AT 250 WORDS)