The authors have demonstrated previously that human plasma contains an inhibitor(s) of the enzymatic activity of renin. The purpose of this study is to identify the circulating renin inhibitor. Plasma initially was fractionated with preparative sephacryl S-300 chromatography. A fraction with low protein content inhibited the in vitro enzymatic activity of human renin by 76%. The inhibitory activity of this fraction was not altered by boiling and/or acidification. This fraction was applied to an affinity column, using purified mouse submaxillary renin as ligand. The inhibitor was recovered after elution with hypertonic NaCl. Linoleic acid, a previously identified renin inhibitor, was present in this fraction. The authors conclude that circulating linoleic acid inhibits renin. Conceivably, the overall activity of the renin-angiotensin-aldosterone cascade may be modified by alterations of plasma fatty acid concentrations.

The enzymatic activity of renin is increased in plasma of patients with renal failure, possibly because of the deficiency of a renin inhibitor. Our study was undertaken to identify renin inhibitors and to compare their activities in plasma of 10 normal humans and 10 patients with renal failure. In vitro, both human renal renin and purified mouse submaxillary renin were inhibited by intact plasma and by a high molecular weight (greater than 65,000 daltons) and a lower molecular weight (55,000 daltons) plasma fraction (HMF and LMF, respectively) obtained by Sephadex chromatography. Most plasma proteins were recovered in HMF, and LMF also contained a small amount of protein; however, renin inhibition by HMF and LMF was not dependent on the presence of intact protein. HMF extracted from normal and uremic plasma inhibited renin to a comparable extent. However, LMF from uremic plasma inhibited renin to a lesser extent (p less than 0.01) than LMF from normal plasma. HMF-free plasma of normal subjects contained a specific renin-inhibiting neutral lipid component that was absent in uremic plasma. Thus, circulating renin inhibitors may contribute to the overall activity of the renin-angiotensin system, and increased enzymatic activity of renin in plasma of patients with renal failure may be related to the deficiency of a normally occurring neutral lipid renin inhibitor.

We have previously reported that the in vitro enzymatic activity of exogenous renin, plasma renin reactivity (PRR), is increased in plasma of patients with chronic renal failure, possibly due to the deficiency of a renin inhibitor. To determine whether increases PRR is related to renal failure per se or to hyperlipidemia, PRR was measured in 10 control subjects, 10 patients with renal failure, and 10 hyperlipidemic patients with normal renal function. Compared to that in control subjects (52.6 ng angiotensin I generated per ml/h +/- 3.8 SE) PRR was increased (P < 0.05) in plasma of uremic patients (65.1 +/- 4.3) and hyperlipidemic patients (71.4 +/- 10.7). Renin substrate concentration did not differ among groups, and after denaturation of endogenous substrate by acidification of plasma, PRR was still increased. A "protein-free" extract of plasma from normal subjects inhibited renin, whereas little or no inhibition occurred with a comparable extract from uremic patients and hyperlipidemic patients. Thus, alterations in lipid metabolism may account for the increased enzymatic activity of renin in uremic plasma. Increased PRR may be related to the deficiency of a normally occurring renin inhibitor.

The in vitro enzymatic activity of renin is increased in plasma of patients with chronic renal failure and patients with glucocorticoid excess. To evaluate mechanisms for increased renin reactivity, measurements of plasma renin reactivity, renin substrate, and concentrations of previously identified inhibitors of renin were compared in normotensive controlsubjects, patients on chronic renal dialysis, and renal allograft recipients on maintenance prednisone therapy. After the addition of exogenous renin, the in vitro rate of angiotensin generation was significantly increased (P < 0.01) in the plasma of dialysis patients (mean ± SE, 60.6 ± 2.9 ng⁄ml-h) and steroidtreated subjects (63.5 ±5.0 ng⁄ml·h) compared to that in control subjects (46.3 ± 4.6 ng⁄ml-h). Renin substrate concentration was increased (P < 0.01) in steroid-treated patients but not in patients with chronic renal failure. In subjects on steroid therapy, there was a significant correlation between renin reactivity and renin substrate (r = 0.55; P < 0.02).Concentrations of polyunsaturated fatty acids, previously observed to inhibit renin, were decreasedin dialysis patients, and the ratio of saturated and monounsaturated fatty acids to polyunsaturated fatty acids was increased in these patients for each major lipid class. These results suggest that increased enzymatic activity of renin in chronic renal failure is related to the deficiency of a circulating renin inhibitor, whereas increased renin reactivity associated with steroid therapy is related to increased concentrations of renin substrate.

Plasma renin activity (PRA) increases after acidification of plasma or exposure of plasma to cold temperatures.The purpose of the present study is to evaluate the hypothesis that these increases of PRA reflect inactivation of circulating renin inhibiting factors.In each of eight normotensive subjects, mean PRA increased (p < 0.01) after acidification of plasma by acid dialysis (98%), after acidification of plasma by addition of HCI (47%), or after exposure of plasma to -4°C for 5 days (155%).Renin substrate-free plasma was obtained by passing plasma over Sephadex G-50-40.Both untreated plasma and the substrate-free extract of plasma inhibited the enzymatic activity of renin (p < 0.01); little or no inhibition occurred after addition of acidified plasma or the protein-free extract of acidified plasma to renin-renin substrate.After addition of exogenous renin to cold-exposed plasma, the rate of angiotensin I generation was greater than that in control plasma (p < 0.05), although renin substrate did not differ.These observations suggest that denaturation of a circulating renin inhibitor may contribute to increased PRA after acidification or cold exposure of plasma.In a separate experiment, the addition of polyunsaturated free fatty acids (linoleic and arachidonic) to renin-renin substrate inhibited angiotensin production, although no inhibition occurred after dialysis of these fatty acids against an acid buffer (pH 3.3).Prolonged exposure of linoleic acid to cold temperatures did not affect its capacity to inhibit renin.Thus, loss of long chain, unSaturated free fatty acids may contribute to increased PRA after acid dialysis of plasma but not to increased PRA after exposure of plasma to cold temperatures.(Hypertension 1: 190-196, 1979) KEY WORDS • acid activation renin • renin inhibitor • fatty acids plasma renin activity • cyroactivationR ENIN is a proteolytic enzyme that cleaves a circulating alpha-2-globulin substrate to form the decapeptide angiotensin I. Several reports suggest that plasma contains inhibitors of the reaction between renin and its substrate. 19Acetonesoluble neutral lipids extracted from plasma inhibit renin, 10 and we have recently demonstrated that physiologic concentrations of long chain, unsaturated fatty acids inhibit both the capacity of renin to.generate angiotensin in vitro and the pressor response to renin in vivo. 11Thus, in addition to renin and renin substrate concentrations, the measurement of plasma renin activity (PRA) may be affected by circulating renin inhibitors.We have used the term renin reactivity to refer to the capacity of added renin to generate angiotensin I either after its addition to crude

We have previously reported that the addition of plasma from normotensive human subjects to renin-renin substrate inhibits the in vitro rate of angiotensin generation. The purpose of the present study was to determine whether a renin-inhibiting factor could be identified in human plasma. Neutral lipids and phospholipids were extracted from plasma of seven normotensive control subjects. During 30-, 60-, and 180-minute incubations, neutral lipids inhibited the reaction between human renin and homologous renin substrate (P less than 0.01). Phospholipids did not affect the rate of angiotensin generation (P less than 0.05). The neutral lipid extract was further separated on silica gel chromatographic plates, and several fractions inhibited renin (P less than 0.01) Addition of each of six synthetic neutral lipids to renin-renin substrate did not affect the rate of angiotensin generation. In conclusion, several neutral lipid fractions extracted from human plasma inhibit the in vitro renin reaction.

After addition of exogenous human renin, the in vitro rate of angiotensin I generation is faster in plasma of patients with chronic renal insufficiency and, to a lesser extent, in plasma of patients with essential hypertension than in plasma from normotensive control subjects. The increased reactivity of renin in hypertensive and uremic plasma is not related to differences of endogenous renin activity, angiotensinase activity, renin substrate concentration, or substrate reactivity. Addition of normal, hypertensive, and uremic plasma to a human renin-sheep renin substrate system inhibited the rate of angiotensin generation, although significantly less inhibition was observed with uremic plasma. The reactivity of renin increased in normal plasma but not in uremic plasma after treatment with 95% acetone. After acetone extraction renin reactivity in normal and plasma inhibited the rate of angiotensin generation in a renin-renin substrate system. Less inhibition occurred with the acetone extract from a pool of uremic plasma. These results provide evidence for the existence of a naturally occurring acetone soluble renin inhibiting factor in normal and uremic plasma. The increased reactivity of renin in uremic plasma may be related to a deficiency of this factor.

The present study was undertaken to evaluate the effects of several prostaglandins (PG) on the in vitro velocity of the reaction between human renin and homologous renin substrate. In the presence of high renin concentrations, 1.4 × 10−6 moles PGA1 and PGA2 each significantly inhibited the rate of angiotensin I production (p < 0.005), whereas PGE1 and PGE2 did not (p > 0.05). At lower enzyme concentrations, PGA2 was a more potent inhibitor than PGA1. In the presence of physiologic renin concentrations, 1.4 × 10−9 moles PGA2 but not 1.4 × 10−10 moles PGA2 inhibited renin reactivity. Unrelated to differences of endogenous renin substrate concentration, renin reactivity in plasma of uremic patients was greater than that of normotensive control subjects, even after the addition of 1.4 × 10−9 moles PGA (p < 0.025). Renin inhibition was demonstrated by concentrations of PGA within the renal medulla, but not at the lower concentrations in peripheral venous plasma. Consequently, if PGA induced renin inhibition is of physiologic significance, it is probably within the kidney itself rather than in the peripheral circulation. Additionally, it is unlikely that the greater renin reactivity in plasma of uremic patients is related to a deficiency of circulating PGA.

The conversion of [32P] phosphatidylinositol to diphosphoinositide can be catalysed by the soluble fraction of a yeast cell homogenate; little activity could be demonstrated by the sedimentable fraction. A kinase appeared responsible for the reaction since the reaction required ATP and Mg2+ and since the γ-phosphate of [γ-32P] ATP appeared in the product. The activity could be precipitated with (NH4)2 SO4 and redissolved in a fully soluble form.

In cells of Saccharomyces cerevisiae uniformly labeled with 32P the levels of ATP, ADP, AMP, diphosphoinositide and triphosphoinositide were measured simultaneously after chromatographic separation. When a respiratory deficient mutant strain was removed from complete growth medium, there resulted an immediate and large drop in the levels of diphosphoinositide, triphosphoinositide, and ATP as well as the adenylate energy charge. Addition of glucose to starved cells in buffer resulted in a rapid increase in the concentrations of ATP, diphosphoinositide and triphosphoinositide. 2-Deoxyglucose caused a rapid decrease of the adenylate energy charge to intermediate values; again, the levels of diphosphoinositide and triphosphoinositide fell rapidly but not to the same extent as with simple starvation. The close association between the adenylate energy charge and the concentration of these polyphosphoinositides herein demonstrated is discussed in relation to the well known turnover of phosphomonoester groups of these lipids.