Sue C. Liu

Arachidonic acid, bradykinin and phospholipase A2 modify both prolactin binding capacity and fluidity of mouse hepatic membranes

Abstract The objective of this study was to determine if arachidonic acid, a precursor of prostaglandin synthesis, bradykinin, a decapeptide known to stimulate membrane phospholipid methylation, arachidonic acid release and prostacyclin synthesis, and enzyme phospholipase A 2 , capable of liberating arachidonic acid, alter the fluidity of hepatic membranes which could in turn modify the functionality of prolactin receptors. Liver homogenates of adult C 3 H female mice incubated at 28°C for various times with 1–20 μg/ml arachidonic acid, 1–100 μg/ml bradykinin or 0.26–0.00026 U/ml phospholipase A 2 provided the 100,000 × g membrane pellets for subsequent ovine prolactin binding and membrane fluidity studies. Membrane microviscosity was determined by fluorescence polarization techniques using the lipid probe 1,6 diphenylhexatriene. Arachidonic acid, bradykinin and phospholipase A 2 stimulated specific oPRL binding, in a dose-related fashion, with maximum increases of 73%, 21% and 46%, at 4 μg/ml arachidonic acid, 5 μg/ml bradykinin and 0.026 U/ml PLA 2 , respectively. This induction, occurring within 30 min of incubation, was found to be due to an increase in the number of receptor sites. Under the same conditions, arachidonic acid, bradykinin and PLA 2 induced 22%, 16%, and 18% decreases in membrane microviscosity, respectively. These data suggest that prostaglandin synthesis modifying agents may modulate the number of prolactin receptors in vivo by changing the lipid fluidity of the target cell membranes by either of their known effects: arachidonic acid release from the phospholipid matrix, synthesizing appropriate prostaglandins at correct concentration or methylation of membrane phospholipids.

Dietary Essential Fatty Acids Are Required for Maintenance and Induction of Prolactin Receptors

Summary C3H mice maintained on diets lacking the essential fatty acids exhibited a progressive decrease with time in the number of prolactin receptors detectable in their hepatic microsomal membranes. These deficient animals were also insensitive to induction of receptor by treatment with exogenous prolactin. These data show that the quality of dietary lipids modifies the prolactin receptor in vivo. We thank E. B. Gould, R. H. Lewis, R. L. Graeter, J. R. Mayer, P. C. Wright, J. A. Keller, K. E. Nickols, M. J. Gillogley, D. Barrett, D. L. Whitcomb, Dr. M. Wicha, Dr. W. R. Kidwell, Dr. P. Dragsten, and Dr. P. M. Gullino for their assistance and suggestions.

Prolactin modifies the fluidity of rat liver membranes.

Summary The objective of this study was to determine the effect of prolactin upon the fluidity of hepatic membranes and subsequent modification of the prolactin receptors. Hypophysectomized, immature, female rats (HIFR) having subcutaneous diethyl stillbesterol implants were treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). In addition, graded doses of 0, 0.25, 0.5, 1.0, 2.5 or 50 μg oPRL were injected hourly for the 48 hours prior to sacrifice at 28 days of age. Both the lipid fluidity as determined by 1,6-diphenylhexatriene (DPH) fluorescence polarization and phospholipid/cholesterol (P/C) ratio were measured in hepatic microsomal membranes. A biphasic response was observed in both of these parameters as the dose of oPRL was increased. P/C values were 91,113, 117, 88, and 93% of control values for the 0.25, 0.5, 1.0, 2.5 and 50 μg oPRL groups, respectively, the maximal value being significantly different from the saline injected controls. These ratios were inversely proportional to the corresponding values of fluidity which also varied in a biphasic manner, with fluorescence polarization equal to 97, 90, 82, 100 and 101% of control values. The value for the 1.0 μg group was, again, significantly different from control. No significant specific 125 I-oPRL binding was detectable in any of the groups after treating the membranes with distilled water, however, pretreatment of membrane preparations with 4 M MgCl 2 for 5 min resulted in a significant increase in the oPRL-binding in the 0.5 and 1.0 μg oPRL groups. The values being 273 and 521% of control, respectively in these two groups. These data demonstrate that prolactin modifies the viscosity of hepatic membranes and suggests that this phenomenon may be responsible for the autoregulation of detectable prolactin receptors.

Prostaglandin synthesis and binding is increased in regressing NMU mammary carcinoma.

Mammary tumors induced in Buffalo rats by treatment with nitrosomethyl urea will regress after oophorectomy. Their ability to synthesize and bind prostaglandins E and F2 alpha was studied in the growing and regressing states. Prostaglandins present in suspension of 100,000 xg tumor membranes after 2 hr incubation at 37 degrees C +/- 5x10(-4)M indomethacin were partially purified by silica gel column chromatography before assay by specific PG RIA. The amounts of PGE and F2 alpha synthesized rose from 0.13 and 10.5 ng/mg protein in the growing tumors to a maximum of 1.2 and 26.5 ng/mg protein 5 days after oophorectomy. Specific binding of 3H-PGE2 ad 3H-PGF2 alpha to 15,000 xg tumor membranes was achieved during a 45 min incubation at 23 degrees C +/- excess unlabelled PG. Free and bound prostaglandins were separated by filtration. Binding reached equilibrium after 30 min, was saturable and reversible. Scatchard analysis revealed high affinity binding of PGF2 alpha but only low affinity PGE2 binding in membranes obtained from growing tumors. A 2-3-fold increase in specific binding of PGE2 and PGF2 alpha was noted at 4 days after oophorectomy which represented an increase in the number of PGF2alpha receptors. PGE2 binding retained a low affinity character. The elevated PGF2 alpha synthesis rates observed in the regressing tumors coupled with a regression-associated increase in receptor number suggests that PGF2 alpha plays a significant role in hormone-dependent mammary tumor regression.

Prostaglandin synthesis by murine mammary gland is modified by the state of the estrus cycle

Mammary glands were excised from female C3H mice at various stages of their estrus cycle. Homogenates incubated at 37 degrees C +/- 10(5) M indomethacin synthesized prostaglandins (PG) E and f2 alpha at rates that varied as a function of the stage at estrus. The rate of PGF2 alpha synthesis was maximal at 1330 pg per mg protein per 2 hr in early diestrus and fell to undetectable levels in early estrus. PGE synthesis exhibited a similar pattern, being maximal at 83 pg per mg protein per 2 hr in early diestrus. These observations suggest that prostaglandins play a role in the cyclic changes observed within the mammary gland.

The effect of copper on the binding of prostaglandin E1 to NMU-induced rat mammary carcinoma membranes.

Mammary carcinomata were induced by administration of nitrosomethyl urea to female Buffalo rats. Specific binding of PGE1 to the 15,000 xg membranes obtained from these tumors was maximal after approximately 60 minutes of incubation at 15 degrees C. An excess of unlabeled PGE1 dissociated 50% of the [3H]PGE1 from the membrane within 15 minutes. The binding of [3H]PGE1 was inhibited by various prostaglandins in a concentration-dependent manner, the order of potency being PGE1 greater than PGE2 greater than PGA2 greater than PGF2 alpha greater than PGB2. High affinity receptor sites were not definable by Scatchard analysis until 5mM CuCl2 was added. This resulted in the detection of both high and low affinity receptors (Kd = 1.01 nM and 21 microM) having binding capacities of 29 and 357 fmole/mg protein, respectively. Addition of CaCl2 or MgCl2 did not result in a significant increase in the specific binding of PGE1 to the receptors. These studies provide a means by which high affinity prostaglandin E1 receptors can be detected in neoplastic tissue and suggest a mechanism by which copper ions can increase the effect of PGE1 in tissues.