Anti-mitochondrial effects of bisethyl polyamines in mammalian cells.

The effects of three bisethyl polyamine analogs on mitochondrial structure and function were examined in human HeLa and L1210 murine leukemia cells. N, N' Bis-[3(ethylamino)-propyl]1-7- heptane diamine (BEPH), and its octane (BEPO), and butane (BESPM) derivative, were shown by electron microscopy and/or Rhodamine 123 uptake studies to alter the structural integrity of mitochondria when both cell lines were treated at the approximate IC50 dose of each drug. At this dose, BEPH had no marked effects on levels of the naturally occurring polyamines, putrescine, spermidine or spermine, in either cell line whereas BEPO and BESPM treatment did result in pool depletion. Southern blot analysis demonstrated a time and dose-dependent loss of mitochondrial DNA from BEPH-treated L1210 cultures suggesting that loss of mitochondrial integrity extended to the DNA level. Treatment of L1210 cells with all three analogs revealed marked reductions in the activity of two mitochondrial enzymes citrate synthase and cytochrome C oxidase. HeLa cells treated with all three analogs exhibited markedly reduced levels of ATP, complete loss of cytidine triphosphate (CTP) and near total depletion of uridine triphosphate (CTP) and near total depletion of uridine triphosphate (UTP). There was also a loss of colony forming ability in HeLa cells which could be nearly completely reversed by the addition of either uridine or cytidine suggesting that NTP reduction may be the primary antiproliferative determinant in these cells. Growth inhibition by BEPH in L1210 cells was markedly potentiated by the glycolysis inhibitor, 2-deoxyglucose, which had no such effect in otherwise untreated cells. This suggests that BEPH treatment of L1210 cells results in impairment of mitochondrial ATP synthesis and activation of the glycolytic pathway for energy production. 2-deoxyglucose treatment also completely prevented the increase of ATP by BEPH treatment of L1210 cells. It is concluded that all three bisethyl polyamines alter HeLa and L1210 mitochondria both structurally and functionally and that these alterations may play a primary role in the antiproliferative activity of these agents in HeLa cells. In L1210, the different spectra of cellular biochemical changes following bisethyl polyamine treatment suggests that additional mechanisms may be in effect.

Characterization of a dual inhibitor of angiotensin I-converting enzyme and neutral endopeptidase.

In the present study we characterize key activities of an agent designed to simultaneously inhibit angiotensin I-converting enzyme (ACE) and neutral endopeptidase (NEP). MDL 100,240 is a thioester prodrug of MDL 100,173, which is a potent competitive inhibitor of both ACE and NEP in vitro. MDL 100,240 was shown in an ex vivo study to inhibit both of these enzymes in rat kidney. When administered to anesthetized rats, MDL 100,240 enhanced the effect of infused ANP on blood pressure, diuresis and natriuresis and of infused bradykinin on blood pressure. Moreover, MDL 100,173 and MDL 100,240 inhibited the pressor response to angiotensin I. These results indicate that MDL 100,173 and its prodrug, MDL 100,240, produced effects, in vivo, consistent with inhibition of both ACE and NEP.

Inhibition of lipid peroxidation promoted by iron(III) and ascorbate.

Peroxidation of rat liver microsomes and of phospholipid isolated from them was studied using iron(III) and ascorbate initiation. One-half equivalent of citrate per iron equivalent maintained solubility of the metal ion at neutral pH. Several metal chelators, including additional citrate, blocked peroxidation, but catalase did not. These characteristics are consistent with those reported by others (D. M. Miller and S. D. Aust (1989) Arch. Biochem. Biophys. 271, 113-119). Several antioxidants, principally tocopherol analogues and nitroxides, and, as well, a nonenzymatic component of "thymol-free" catalase, potently blocked lipid peroxidation, or, equivalently, dioxygen depletion from suspensions of peroxidizing microsomes. Chromanols were the most active antioxidants. No thiol studied had significant antioxidant activity in the test system.

A diet containing n-3 and n-6 fatty acids favorably alters the renal phospholipids, eicosanoid synthesis and plasma lipids in nephrotic rats.

The nephrotic syndrome was induced in rats by intravenous adriamycin (3 mg/kg). The rats were then divided into four groups which, for six weeks, were pair-fed diets containing beef tallow (BT), fish oil (FO), a source of n-3 fatty acids, evening primrose oil (EPO), a source of n-6 fatty acids, or a combination of evening primrose oil and fish oil, 75:25 (EPO:FO). The fat content of the diets was 15%. Significant incorporation of the fatty acids into kidney phospholipids was demonstrated. Diets containing FO, EPO and EPO:FO lowered plasma triglycerides and total cholesterol levels as compared with diets containing BT. Only EPO:FO raised high density lipoprotein (HDL) cholesterol levels, as compared with BT. The combination EPO:FO prevented the tenfold suppression of aortic 6-keto-PGF1 alpha caused by FO. These changes in plasma lipids and eicosanoid production are potentially antiatherogenic and may prevent glomerular sclerosis. The combination of EPO and FO, containing n-6 and n-3 fatty acids may offer advantages over either family of fatty acids in this model of nephrotic syndrome.

The influence of n-6 and n-3 fatty acids on kidney phospholipid composition and on eicosanoid production in aging rats.

Changes in eicosanoid production may contribute to some of the complications of the aging process such as atherosclerosis and glomerular sclerosis. Polyunsaturated fatty acids of the n-6 and n-3 series are precursors of eicosanoids. We fed diets containing safflower oil as a source of n-6 fatty acids, fish oil as a source of n-3 fatty acids or beef tallow as a source of saturated fats to three groups of normal rats from 2-18 months of age. We demonstrated incorporation of the n-3 fatty acids, 20:5n-3 and 22:6n-3 into kidney phospholipids. Feeding of the diet containing n-3 fatty acids was associated with a markedly decreased glomerular production of PGE, 6-keto-PGF1 alpha and TXB2. It also decreased the aortic production of 6-keto-PGF1 alpha and platelet production of TXB2. No significant effect of n-6 fatty acids on dienoic eicosanoid production was observed. There were no adverse effects on kidney function as measured by urinary protein excretion and serum creatinine levels or on renal morphology by any diet. A diet enriched in n-3 fatty acids for 18 months remains effective in decreasing dienoic eicosanoids in the aging rat.

Characteristics of hepatic receptors for somatomedin-C/insulin-like growth factor I and insulin in the developing human.

Insulin and the somatomedins (Sms) are putative regulators of cell proliferation and metabolism in the fetus. Since the liver is a potential target tissue of these hormones during fetal life, we characterized the hepatic receptors for Sm-C/insulin like growth factor I (Sm-C/IGF-I) and insulin during the second trimester of human fetal development. Membrane-enriched fetal liver homogenates specifically bound 8.9 +/- 1.5% (+/- SD) of added [125I]insulin and 5.1-7.2% of [125I]Sm-C/IGF-I. Binding of both hormones was constant from 12-20 weeks gestation and was much greater than that in adult liver membranes. Analysis of dose-response data indicated high affinity between each receptor and its respective ligand (Kd for the Sm-C/IGF-I receptor, 2.2 X 10(-10) M; Kd for insulin receptor, 5.2 X 10(-10) and 7.7 X 10(-9) M). Limited cross-reactivity (approximately 1:1,000) of insulin with the Sm-C/IGF-I receptor and Sm-C/IGF-I with the insulin receptor was found. Affinity labeling studies showed that each receptor possessed an approximately 135,000-dalton subunit which was a part of a larger disulfide-linked complex. Thus, the human fetal liver has specific receptors for Sm-C/IGF-I and insulin that are similar to those described for other tissues in terms of both hormone-binding characteristics and subunit structure, suggesting that these receptors mediate important cellular functions at this stage of fetal development.

Early effect of myo-inositol deficiency on fatty acid synthetic enzymes of rat liver.

Young rats (100 g) were fed either a purified myo-inositol-deficient balanced diet or a control diet containing 0.5% by weight myo-inositol, ad libitum, for up to 2 weeks following a 48 h fast. Weight gain was the same for animals in both groups. Liver triacylglycerol levels in the deficient animals were 1.8-, 3.5- and 3.0-fold higher than the corresponding levels in the control animals after 4, 8 and 14 days of feeding, respectively. In the myo-inositol-deficient group the specific activities of liver fatty acid synthetase and acetyl-CoA carboxylase were elevated 1.5-2.0-fold over controls, reaching a maximum after 3-4 days of feeding. Subsequently, activities declined to control levels. Rates of fatty acid synthetase synthesis in the deficient group, as measured by [3H]leucine incorporation into immunoprecipitable fatty acid synthetase polypeptide, were significantly higher (1.5-2.0-fold) than controls after 12-18 h of feeding and then declined to control levels by 1 day. No difference was noted between groups in either the rate of total, soluble liver protein synthesis or the half-life of fatty acid synthetase over this time period. These results suggest that the liver lipodystrophy observed during myo-inositol deficiency in rats may be due in part to elevated levels of lipogenic enzymes in this tissue in the early stage of the deficiency.