Opiate receptor antagonism by l-prolyl-l-leucyl-glycinamide, MIF-I

Chronic administration of l-prolyl-l-leucyl-glycinamide (MIF-I) to mice slightly reduced morphine's antinociceptive activity in the hot-plate test and modified the biphasic motor activity response to morphine. MIF-I antagonized the initial depression of activity and potentiated the increased motor activity phase. Chronic treatment of rats with MIF-I prevented morphine's antinociceptive activity in the tail flick test. MIF-I partly antagonized the inhibition by morphine of the coaxially stimulated guinea-pig ileum preparation. The inhibition of the ileum produced by ethylketocyclazocine was weakly antagonized by MIF-I. In contrast, MIF-I had no effect on the inhibition of the stimulated mouse vas deferens produced by Leu-enkephalin. The findings show that MIF-I weakly and selectively inhibits μ-type opiate receptors which suggests that MIF-I could be an endogenous inhibitor of opiate receptors.     

Somatostatin inhibits adrenergic and cholinergic neurotransmission in smooth muscle.

Somatostatin reduced the response to field stimulation in the guinea pig ileum and reduced the spontaneous contractions in the rabbit jejunum, an effect that was blocked by tetrodotoxin. Somatostatin also inhibited field stimulated alpha adrenergic contractions in the rat vas deferens and rabbit ear artery. However, the responses to direct application of either acetylcholine in the ileum or to norepinephrine in the ear artery or vas deferens were not affected by somatostatin. These results strongly suggest that somatostatin inhibits neuronal release of cholinergic and adrenergic transmitter substances in smooth muscle.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

Immortalization of murine primary spleen cells by v-myc, v-ras, and v-raf.

Growth factor-independent cell lines, including four lines characterized as macrophages, were isolated by infection of BALB/c mouse primary spleen cells with combinations of three retroviruses encoding v-myc, v-ras, and v-myc/v-raf. Proliferating cell lines were isolated only rarely, and after long crisis periods, following the introduction of myc and raf by infection with J2 virus, or of myc and ras by coinfection with myc309 and raszip6 viruses. However, sequential infections with all three viruses--myc plus ras cells reinfected with J2, or J2 followed by myc plus ras coinfection--resulted in rapid outgrowth of cell lines which grew at high growth rates to high densities. When cells were treated with anti-IgG F(ab')2/IL-4/IL-5 to specifically stimulate B cells, cell lines were isolated readily by infection with myc plus ras alone, J2 alone, or all three viruses. These cell lines arose after shorter crisis times and all grew at high growth rates and to high densities. Analysis of cell surface markers and immunoglobulin gene arrangement revealed no lymphoid characteristics in any of the lines. Four cell lines express all three macrophage markers analyzed (F4/80, Mac1, FcR), and many others are Mac1+ and/or FcR+. Out of 20 immortalized cell lines tested, 13 show clonal growth in soft agar, and 3/6 of these produced tumors in BALB/c mice, indicating that fully transformed cells may be isolated by these procedures. In at least one of the cell lines, integration of all three infecting viruses has occurred.     

Effect of insulin pump therapy on blood pressure and the renin-angiotensin system of diabetic rats

Insulin therapy, administered by continuous subcutaneous infusion with osmotic pumps over a 28 day period at doses of 2.5 and 5.0 units/day, resulted in a statistically significant increase in body weight of diabetic rats. The concentration of blood glucose was reduced by 68% to 109 mg/dl blood sugar by the higher dose of insulin and only partial control of diabetes was achieved by the lower dose (185 mg/dl blood sugar, -39%). Blood pressure was normalized by both doses of insulin. Elevated serum angiotensin converting enzyme activity and plasma renin activity, expressed as generated angiotensin I, were unaffected by the lower dose of insulin, but were reduced by 26% and 40%, respectively at the higher dose. These data suggest that elevated serum ACE and plasma renin activity, commonly found in the streptozotocin-diabetic rat, may not be primarily responsible for hypertension in this model.     

Receptor-independent catabolism of low density lipoprotein. Involvement of the reticuloendothelial system

In this study we examine the effects of intravenous ethyl oleate emulsions on the metabolism of native and cyclohexanedione-modified human low density lipoprotein in rabbits. Treatment produced a highly significant fall in receptor-independent catabolism as measured by the fractional clearance rate of cyclohexanedione-modified low density lipoprotein. Receptor-dependent catabolism (the difference between the fractional clearance rates of native and cyclohexanedione-modified low density lipoprotein) was variably affected with some animals showing a decrease in receptor activity. These data suggest that the reticuloendothelial system makes a substantial contribution to receptor-independent low density lipoprotein catabolism in the rabbit.     

Epidermal growth factor induces tyrosine hydroxylase in a clonal pheochromocytoma cell line, PC-G2

We have previously described the isolation of a clonal cell line (PC-G2) in which the level of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of the catecholamine neurotransmitters, is induced by nerve growth factor (NGF). We now report that epidermal growth factor (EGF) also induces TH in the PC-G2 cell line. Although EGF has been shown to be mitogenic for many cultured cells, no neuronal function has been previously reported for this protein. The TH response to EGF is elicited in a dose-dependent fashion at concentrations as low as 0.1 ng/ml and is maximal at 10 ng/ml EGF. The maximal response is observed after 3--4 d of exposure to 10 ng/ml EGF. The induction by NGF and EGF is inhibited by their respective antisera. Dexamethasone, a synthetic glucocorticoid which we have previously shown modulates the response of PC-G2 cells to NGF, also modulates the TH induction elicited by EGF.     

Binding of ADP to beef-heart mitochondrial ATPase (F1)

1. ADP binding to beef-heart mitochondrial ATPase (F₁), in the absence of Mg²⁺, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure.2. Since during the binding experiments the ‘tightly’ bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters.3. The binding data show that only one tight binding site (K_d about 0.5 μM) for ADP is present.4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 μM.