Addition Of Rat Serum Research Articles

Age-dependent decline in bone nodule formation stimulating activity in rat serum is mainly due to the change in the corticosterone level

The replacement of fetal bovine serum with rat serum in a culture medium brought about a marked increase in the formation of mineralized bone nodules (BN) in primary cultures of rat calvarial cells. These effects of rat serum were most prominent when added during the early phase of the culture, indicating that the serum factor mainly acts on the cells during the growing phase. A significant increase in BN formation was observable at final rat serum concentration as low as 1%, and the effect was dependent on serum concentration, at least up to 10%. The addition of rat serum also increased alkaline phosphatase (ALP) activity, collagen synthesis, and DNA synthesis in calvarial cells. BN formation stimulating activity was extractable with ethyl acetate. The ethyl acetate extract was purified by TSK-GEL OH-120 column chromatography by monitoring the stimulation of ALP activity in ROS 17/2.8 cells. The chromatographic behavior of the ALP activity was found to be identical to that of corticosterone, the major glucocorticoid in rodents and the preincubation of the purified fraction with anticorticosterone antibody abolished the ALP stimulating activity. These results suggest that BN formation stimulating activity in rat serum is mainly attributable to corticosterone. The concentration of serum corticosterone decreased with age in parallel with BN formation stimulating activity, which suggests that the physiological level of corticosterone may have a regulatory role in the maintenance of osteoblast function.

Effect of serum from partially hepatectomized cirrhotic and noncirrhotic rats on liver regeneration with primary hepatocyte cultures.

The effects of serum from cirrhotic and noncirrhotic rats after 33% and 70% partial hepatectomy on the hepatocyte culture were studied. Liver cirrhosis was produced by weekly intragastric feeding with carbon tetrachloride (CCl4) for 12-16 weeks. The serum was prepared with blood withdrawn from the rats 2 and 48 h after partial hepatectomy or sham operation. The incorporation of [3H]thymidine in the hepatocyte culture after addition of rat serum (1.0 +/- 10(5) cells/ml) was assessed. We found that the mitogenic effect of the serum at 2 and 48 h after partial hepatectomy was not significant. In the 70% hepatectomy group, the serum from cirrhotic rats had a stronger stimulatory effect to induce hepatocytes in primary culture entering DNA synthesis than that from noncirrhotic rats; but the difference was not statistically significant. The mitogenic effect of the serum from noncirrhotic rats after 33% hepatectomy was significantly lower than that from cirrhotic rats after 33% hepatectomy (p < 0.05). In this series, the serum from the cirrhotic rats after 33% hepatectomy had the strongest mitogenic effect; the serum from the partially hepatectomized rats with cirrhotic and non-cirrhotic livers increased [3H]thymidine uptake into hepatocyte DNA significantly more than control serum or plasma.

Rat heart organ culture for the study of trophic substances involved in the maintenance of normal sensitivity to norepinephrine: possible involvement of cAMP and search for other factors

In organ culture conditions, in the absence of in vivo factors, the newborn rat right atria acquire a high sensitivity to agonists similar to that seen before sympathetic innervation and after denervation. In the present study, we examined the effects of various extracts and substances on the development of supersensitivity to norepinephrine (NE) to obtain information on the in vivo factors that regulate myocardial sensitivity. Addition of rat serum, right atrial extract, superior cervical ganglionic extract, vas deferens extract, carbachol, insulin, cortisone, thyroxin, and neuropeptide Y in the culture medium did not prevent the development of supersensitivity. Addition of NE completely inhibited the development of supersensitivity. This effect of NE was blocked by sotalol but not by phentolamine. Addition of calcitonin gene related peptide, forskolin, and 8-bromo-cAMP partially inhibited the development of supersensitivity. These results are consistent with the view that NE released from sympathetic nerve terminals in the newborn atria maintains myocardial sensitivity at normal level by acting on beta-adrenergic receptors, and that the effect may be partially mediated by a rise in intracellular cAMP concentration.

Interactions Between Lymphoid Tumor Cells and Isolated Liver Endothelial Cells

Interactions were studied between highly metastatic murine MB6A lymphosarcoma cells and rat liver endothelial cells that had been isolated by collagenase perfusion and purified by unit gravity sedimentation. Experiments were performed on the day of isolation. MB6A cells were observed to adhere to the endothelial cells. Addition of rat serum had a striking effect: The endothelial cells spread over the MB6A cell surface, engulfing the tumor cells. The factor involved was nondialyzable and also was present in rat plasma. Similar interactions were seen with highly metastatic ESb and MDAY-D2 lymphoma cells and with nonmetastatic Eb cells. Low-metastatic GRSL 34 leukemia and TA3/Ha ascites mammary carcinoma cells did not adhere to the endothelial cells. With this in vitro model the molecular mechanisms of adhesion to liver endothelium were studied. As a first step, univalent antibodies against MB6A cells were found to inhibit adhesion, indicating involvement of specific cell surface molecules.

Effect of phospholipids on lipase activity in rat arterial wall homogenate.

Triolein hydrolyzing activity in homogenates of the rat arterial wall was studied. Two optimal pH values were observed around 5.0 (acid lipase) and 7.0 (neutral lipase). Neutral lipase activity was enhanced by serum and was inhibited by 1 M NaCl but was not inhibited by protamine. Heparin decreased the acid lipase activity. A 1-mM solution of Cu2+ ion and Zn+2 ion decreased both lipase activities. The activities of both acid and neutral lipases were increased by the addition of rat serum (2 micrograms protein serum/ml) and were decreased by the addition of more than 5 micrograms protein serum/ml. The activities of neutral and acid lipases were increased by the addition of 0.25 mM phosphatidylcholine and phosphatidylserine. Both lipase activities were decreased by the addition of 0.25 mM phosphatidylethanolamine. The addition of 0.25 mM sphingomyelin increased neutral lipase activity and decreased acid lipase activity. At concentrations higher than 2 mM, these phospholipids decreased the activities of both lipases. These results suggest that at least acid and neutral lipases exist in the arterial wall cells, and their activities might be modified by phospholipids.

Progesterone and prostaglandin secretion by ovulated rat cumulus cell-oocyte complexes.

Hormone production (progesterone and prostaglandin) by oocyte-cumulus cell complexes was studied in the postovulatory period. Cumulus-oocyte complexes were collected from oviducts of prepuberal rats after the synchronization of follicle development and ovulation with PMS gonadotropin and hCG injections. Cumulus masses from individual animals were cultured in roller vessels containing Eagles' Minimal Essential Media with or without added heat-inactivated rat sera (5% CO2-air; 37 C). Culture media were collected after 4.5 h of incubation and analyzed by RIA for prostaglandin E2 (PGE), PGF, and progesterone. Extensive progesterone secretion occurred during the culture period and was markedly stimulated by the addition of rat serum; however, cumulus masses did secrete some progesterone in the absence of rat serum and without exogenous gonadotropins. The addition of aminoglutethimide, a steroidogenesis inhibitor, suppressed progesterone secretion but did not alter the secretion of PGF or PGE. Both PGF and PGE were detected in the culture medium, and in all cases, PGE was the predominant PG detected. Indomethacin, an inhibitor of PG synthesis, suppressed PGE and PGF secretion but had no effect on progesterone synthesis. Small amounts of PGs were also detected in nonincubated cumulus-oocyte masses. Hormone secretion in culture was measured after cumulus mass dissociation by enzymatic digestion. Somatic cumulus cells, rather than germ cells, were the primary cellular source of progesterone and PGs. The results demonstrate that cumulus-oocyte complexes can synthesize and secrete steroid and PG hormones subsequent to ovulation, and thus, these masses should be considered functional endocrine tissues. The physiological significance of the endocrine activities of the cumulus-oocyte complex are discussed in terms of oviductal and gametic processes. (Endocrinology 108: 457, 1981)

Effect of rat serum on glycerol metabolism in adipose tissue in vitro.

It has been proposed that the lipolytic effect of serum is based on the presence of either lipoproteins or catecholamines. To test these hypotheses, pieces of epididymal fat pads from fed rats were incubated in the presence of albumin and glucose for 120 min. The addition of rat serum (5 mul/vial) enhanced the rates of both glycerol release to the media and [U-14C] glycerol utilization by the tissue. Heparin did not alter these parameters or the response produced by serum. VLDL from rat plasma also enhanced glycerol release and utilization for the formation of CO2 and lipids, and heparin significantly augmented these effects. Neither of the conditions studied affected the percentual distribution of 14C-lipid fractions in the tissues. It is known that in similar conditions to those used in the present study, adrenaline produces a decrease in the utilization of glycerol. Thus our findings do not support the proposed hypothesis explaining the fat-mobilizing action of serum, the mechanism of which remains to be determined.

Improved response of isolated liver cells to glucagon in the presence of rat serum

The stimulation by glucagon of gluconeogenesis from 10 mM lactate and urea formation in intact isolated rat liver cells is enhanced from about 33 % to 61 % and 92 %, respectively, by the addition of rat serum (30 % v/v) to the incubation medium. This effect is exerted by (a) dialysable, heat-labile serum component(s), different from known stimulators of gluconeogenesis, such as fatty acids, lysine, ammonium chloride or calcium, which do not improve the response of hepatocytes to glucagon. The factor(s) could provide a useful tool for the study of glucagon action(s) on liver metabolism.

Regulation of gingival collagenase: a possible role for a mast cell factor.

Serum free media from rat gingival tissue cultures contained active collagenase which was inhibited by the addition of rat serum or fetal bovine serum. The inhibitory action of serum on gingival collagenase was blocked by the addition of mast cell extract. The mast cell factor was non-dialyzable and heat stable but was inactivated by exposure to 20% TCA. Neither heparin, histamine and 5-hydroxytryptamine nor extracts from a variety of other tissues could substitute for the mast cell factor in the activation of gingival collagenase. A mast cell factor may participate in the in vivo regulation of collagenase activity.

Activation of lipoprotein lipase. Effects of rat serum lipoprotein fractions and heparin.

Guinea pig serum is deficient in high density lipoproteins (HDL); post-heparin lipoprotein lipase (LPL) from it hydrolyzes a triglyceride (TG) emulsion very slowly. The rate is markedly increased by the addition of rat serum and even further by rat serum plus heparin. We have studied further the activation of LPL in this system. Rat serum lipoprotein fractions were isolated by ultracentrifugation and added to guinea pig postheparin serum in the presence or absence of heparin. When added in proportion to their original serum concentrations, HDL caused the greatest increase in TG hydrolysis. When each fraction was added at equal protein concentrations, purified low density lipoproteins had almost no effect; both very low density lipoproteins and HDL were very effective in increasing the rate of hydrolysis. Rat serum or rat HDL, added to the assay system in increasing amounts, appeared to increase the effective substrate concentration. In the absence of heparin, increasing the concentration of HDL increased the reaction rate which approached a limiting velocity (V max ) and produced a hyperbolic curve which conformed to Michaelis-Menten kinetics. In the presence of heparin, increasing the concentration of HDL produced an S-shaped curve and increased the V max . These data conformed to the sigmoidal kinetics described by the Hill equation. Our results suggest that (1) heparin may function as a specific ligand which acts as an allosteric modifier of LPL and alters the kinetics of interaction of LPL with the effective substrate and (2) the rate of hydrolysis of the effective TG substrate is regulated by the concentration of this substrate.

A factor in the serum of tumour-bearing rats which is deleterious to cells in tissue culture.

IN attempts to improve the growth of a transplantable rat carcinoma (S.C.K.1)* in tissue culture by the addition of rat serum, it was observed that sera from rats bearing S.C.K.1 did in fact exert a deleterious effect on the cells. This observation was investigated.

Hormones and protein biosynthesis in isolated rat diaphragm.

SUMMARY 1. With rat diaphragm in vitro, addition of insulin to the medium so as to give a concentration as low as 0·05 mu./ml. of the hormone, stimulated the incorporation of [14C]glycine into protein of tissue. Simultaneous addition of glucose to the medium did not affect either the minimal amount of insulin required to produce a significant stimulation of incorporation of glycine, or the magnitude of the effect of the small concentration of insulin used. 2. Addition of a mixture of oxidized A and B chains of the insulin molecule did not affect incorporation of a mixture of labelled amino acids into the protein of isolated diaphragm, but a degraded insulin (DHA-insulin), which has about 15% of the activity of insulin in stimulating glucose uptake by diaphragm, was found to stimulate incorporation of [14C]glycine to an extent comparable with its effect in stimulating glucose-uptake. 3. Addition of rat serum, or the dipping of diaphragm in a medium containing insulin, stimulated incorporation of [14C]glycine into protein of diaphragm. Both these effects and the stimulation produced by insulin in vitro were abolished when the medium contained an antiserum to insulin. 4. Addition in vitro of growth hormone (GH) stimulated incorporation of [14C]glycine into protein of diaphragm from the hypophysectomized rat but had no effect on diaphragm from the normal rat, whether or not a small dose of insulin was also added in vitro. The action of GH in promoting incorporation of [14C]glycine into protein of diaphragm from the hypophysectomized rat was not neutralized by insulin antiserum. 5. Corticotrophin, cortisol, thyroxine, vitamin B12, vitamin D2 and linoleic acid all had no observable effect on incorporation of labelled amino acids into diaphragm. Glucagon stimulated incorporation, but the stimulation was abolished by the in vitro addition of antiserum to insulin and was probably attributable to the presence of a trace of insulin in the glucagon. 6. Anaerobiosis, and the addition of various metabolic inhibitors, were found to suppress incorporation of [14C]glycine into diaphragm protein almost entirely.