Phorbol ester inhibition of the hormone-stimulated phosphoinositide cycle in WRK-1 cells.

WRK-1 rat mammary tumour cells respond to vasopressin with increased accumulation of inositol phosphates as well as increased precursor incorporation into phosphatidylinositol. The phorbol ester, phorbol 13-myristate 12-acetate (PMA) inhibits by 80% both inositol phosphate accumulation and increased precursor incorporation. This inhibition is much less evident at early times (2 min) than at later times (25 min). The vasopressin-induced rise in cytosolic free Ca2+ is inhibited in a similar manner. Oleoylacetylglycerol is inactive with respect to inhibition of vasopressin-induced increases in incorporation of 32P into phosphoinositides. PMA has no effect on vasopressin binding at saturating concentrations of the hormone and does not affect the binding affinity.     

Effect of dual agonists on phosphoinositide pools in WRK-1 cells.

Both vasopressin and bradykinin activate the phosphoinositide cycle in WRK-1 rat mammary tumour cells. When the two agonists are added simultaneously, partial additivity is observed with respect to disappearance of prelabelled phosphoinositides and accumulation of inositol phosphates; no additivity is observed with respect to resynthesis of phosphatidylinositol as assessed by monitoring [32P]Pi incorporation. Lack of complete additivity can be explained, at least in part, by heterologous desensitization. In order to determine whether the two agonists were accessing a common or individual hormone-sensitive phosphoinositide pools, cells were incubated with [32P]Pi in the presence of either vasopressin or bradykinin and subsequently restimulated with the alternative agonist. The lipid pool labelled in the presence of either agonist was sensitive to subsequent treatment by the other ligand, suggesting a common phosphoinositide pool. However, when cells were incubated with [32P]Pi in the absence of agonists, the time course of labelling of the hormone-sensitive pool was different for bradykinin and vasopressin, with that for bradykinin becoming labelled within a much shorter time. Thus although there is a significant overlap between the phosphoinositide pools responding to vasopressin and bradykinin, there is a small fraction of the hormone-sensitive lipid which responds only to bradykinin.     

A new model system for studying the phosphatidylinositol cycle

An early manifestation of the response of WRK-1 rat mammary tumor cells to vasopressin is an increase in incorporation of (³²P)P_i into phospholipids. Incorporation into all classes of phospholipids is stimulated; however, incorporation into phosphatidylinositol (PI) is increased to the greatest degree (3- to 10-fold as compared with 1.3- to 2-fold for the other phosholipids). Furthermore, increased incorporation into PI is accompanied by an increased rate of PI turnover; turnover rates of the other phospholipids are unaffected by vasopressin.     

The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.     

Phospholipid synthesis in isolated alveolar type II cells exposed in vitro to paraquat and hyperoxia.

Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.     

Characterization of the vasopressin receptor on WRK-1 cells

Specific vasopressin binding to WRK-1 rat mammary tumor cells was assessed and compared with vasopressin-induced alterations in phosphatidylinositol metabolism. Scatchard analysis revealed the presence of two binding sites: a saturable, high affinity site with a dissociation constant of 1 X 10(-9) M and an n of 2700 sites per cell, and a nonsaturable, apparent lower affinity site. The higher affinity site appeared to have V1a specificity and to correlate with vasopressin's ability to stimulate phosphatidylinositol turnover in the cells.     

Effect of kinase C inhibitor, gossypol, on the actions of prolactin in cultured mouse mammary tissues

Gossypol, a drug which has been shown to be an inhibitor of kinase C activity in mouse mammary tissues, is shown to abolish several of the actions of prolactin in cultured mouse mammary gland explants. The prolactin effects that are abolished include its stimulatory effects on ornithine decarboxylase activity, the rate of [3H]uridine incorporation into RNA, the rate of [3H]leucine incorporation into a casein-rich phosphoprotein fraction, and the rate of [14C]acetate incorporation into lipids. Since the inhibitory concentrations of gossypol employed in these studies correspond well with the gossypol concentrations required to inhibit kinase C activity, we conclude that ongoing kinase C activity is essential for prolactin to express its differentiative actions in mammary tissues.     

Improved maintenance of adult rat alveolar type II cell differentiation in vitro: effect of hydrocortisone and cyclic AMP

We have examined the effect of hydrocortisone and cyclic AMP on the maintenance of lipid synthesis in primary cultures of adult rat alveolar type II cells. These hormones were tested in the presence of either 1% or 5% charcoal-stripped rat serum (CS-rat serum). The effect of substratum on responsiveness to these hormones was evaluated by comparing cells cultured for 4 days on tissue culture plastic, on floating type I collagen gels, on rat lung fibroblast feeder layers on floating collagen gels (floating feeder layers), and on Engelbreth-Holm-Swarm (EHS) tumor basement membrane gels. Type II cells cultured on floating feeder layers in medium containing 1% CS-rat serum and 10(-5) M hydrocortisone plus 0.5 mM dibutyryl cyclic AMP exhibited significantly increased incorporation of [14C]acetate into total lipids (238% of control). The hormone combination also increased the relative percentage of acetate incorporated into phosphatidylglycerol (PG; 7.3% versus 1.9%) and saturated phosphatidylcholine (PC; 43.6% versus 37.6%). The percentage of acetate incorporated into neutral lipids was significantly decreased by the addition of hormones (28.6% versus 70.0%). The addition of hydrocortisone and cyclic AMP to medium containing 5% CS-rat serum resulted in an increase in the relative incorporation of acetate into saturated PC (51.2% versus 46.4%), but had no effect on the relative incorporation of acetate into PG or on the incorporation of acetate into total lipids. Type II cells cultured on EHS gels in medium containing 1% CS-rat serum plus hydrocortisone and cyclic AMP showed increased acetate incorporation into total lipids (204% of control) and a relative decrease in the percentage of acetate incorporated into neutral lipids (16.9% versus 47.0%). The hormone combination also increased the relative incorporation of acetate into PG (4.4% versus 2.5%) and saturated PC (49.9% versus 42.1%). Hydrocortisone and cyclic AMP added to medium containing 5% CS-rat serum concentration increased the relative incorporation of acetate into saturated PC by type II cells on EHS gels, but these additions had no effect on acetate incorporation into PG. No responses to these soluble factors were seen when type II cells were cultured on floating type I collagen gels without feeder layers or on tissue culture plastic. These data indicate that there are positive interactions between substratum, soluble factors and serum in the maintenance of differentiated function of adult rat alveolar type II cells in vitro.     

Acetate assimilation by the fission yeast, Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe utilizes acetate at subinhibitory concentrations in the presence of D-glucose. The nonionized form of acetate is preferentially utilized, oxidized to 14CO2, and assimilated into lipids and proteins. Acetyl CoA synthetase activity greatly increases in the yeast cells grown in media containing acetate. However, glyoxylate cycle enzymes are not detectable in Schizosaccharomyces pombe. [1-14C]Acetate is incorporated into stereols, sterol esters, neutral lipids, and phospholipids. Assimilation of [1-14C]acetate into the peptide structure of proteins was confirmed by a proteolytic digestion experiment.     

Induction of lipid synthesis in mammary organ cultures from mature virgin and pregnant goats.

The hormones needed to induce lipogenesis in mammary organ cultures from mature virgin and pregnant goats were studied. In tissues from both mature virgin goats and goats at week 10 of pregnancy, cultivated in Waymouth medium without hormones, the rate of the incorporation of (1-(14C))-acetate into the lipids was low and decreased throughout culture. In the presence of insulin, the rate of acetate incorporation was maintained at a higher level. Cortisol acted synergistically with insulin, to produce a rate of lipid synthesis higher than that using insulin alone. The further addition of prolactin had little effect on the incorporation of acetate into the lipids of mammary explants from mature virgin goats, but markedly stimulated it in tissue from animals at weeks 9--10 of pregnancy. The maximum increase in the rate of lipid synthesis was achieved in the presence of 0.5 microgram prolactin/ml, whereas with growth hormone 50 microgram/ml was needed for the maximum effect. The initial rate of acetate incorporation into mammary explants from goats at weeks 13 and 18 of pregnancy was high. It was not stimulated by the hormones during culture, however, and decreased more rapidly in the absence of hormones than in their presence. The rate of acetate incorporation into the lipids was in agreement with the histological evaluation of the secretory response of the mammary explants after cultivation. The secretory response to prolactin and the rate of the incorporation of acetate into the lipids were highest in goats at weeks 9--10 of pregnancy while in tissues from goats at weeks 13 and 18 were not stimulated and decreased during culture.     

Hormonal regulation of the tricarboxylic acid cycle in the isolated perfused rat liver

The effect of Ca2+-mobilizing hormones, vasopressin, angiotensin II and the alpha-adrenergic agonist phenylephrine, on the metabolic flux through the tricarboxylic acid cycle was investigated in isolated perfused rat livers. All three Ca2+-mobilizing agonists stimulated 14CO2 production and gluconeogenesis in livers of 24-h-fasted rats perfused with [2-14C]pyruvate. Prazosin blocked the phenylephrine-elicited stimulation of 14CO2 and glucose production from [2-14C]pyruvate whereas the alpha 2-adrenergic agonist, BHT-933, did not affect the rates of 14CO2 and glucose production from [2-14C]pyruvate indicating that the phenylephrine-mediated response involved alpha 1-adrenergic receptors. Phenylephrine, vasopressin and angiotensin II stimulated 14CO2 production from [2-14C]acetate in livers derived from fed rats but not in livers of 24-h-fasted rats. In livers of 24-h-fasted rats, perfused with [2-14C]acetate, exogenously added pyruvate was required for an increase in the rate of 14CO2 production during phenylephrine infusion. This last observation suggests increased pyruvate carboxylation as one of the mechanisms involved in stimulation of tricarboxylic acid cycle activity by the Ca2+-mobilizing agonists, vasopressin, angiotensin II and phenylephrine.     

Stimulation of [1-14C]oleate oxidation to 14CO2 in isolated rat hepatocytes by the catecholamines, vasopressin and angiotensin. A possible mechanism of action.

Adrenaline, noradrenaline, vasopressin and angiotensin increased 14CO2 production from [1-14C]oleate by hepatocytes from fed rats but not by hepatocytes from starved rats. The hormones did not increase 14CO2 production when hepatocytes from fed rats were depleted of glycogen in vitro. Increased 14CO2 production from ]1-14C]oleate in response to the hormones was observed when hepatocytes from starved rats were incubated with 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase. 3-Mercaptopicolinate inhibited uptake and esterification of [1-14C]oleate, slightly increased 14CO2 production from [1-14C]oleate and greatly increased the [3-hydroxybutyrate]/[acetoacetate] ratio. In the presence of 3-mercaptopicolinate 14CO2 production in response to the catecholamines was blocked by the alpha-antagonist phentolamine and required extracellular Ca2+. The effects of vasopressin and angiotensin were also Ca2+-dependent. The actions of the hormones of 14CO2 production from [I-14C]oleate by hepatocytes from starved rats in the presence of 3-mercaptopicolinate thus have the characteristics of the response to the hormones found with hepatocytes from fed rats incubated without 3-mercaptopicolinate. The stimulatory effects of the hormones on 14CO2 production from [1-14C]oleate were not the result of decreased esterification (as the hormones increased esterification) or increased beta-oxidation. It is suggested that the effect of the hormones to increase 14CO2 production from [1-14C]oleate are mediated by CA2+-activation of NAD+-linked isocitrate dehydrogenase, the 2-oxoglutarate dehydrogenase complex, and/or electron transport. The results also demonstrate that when the supply of oxaloacetate is limited it is utilized for gluconeogenesis rather than to maintain tricarboxylic acid-cycle flux.     

Characterization of specific V1a vasopressin-binding sites on a rat mammary-tumour-cell line.

WRK 1, a cloned cell line derived from a rat mammary tumour, carries specific vasopressin-binding sites. Specific binding of 2-tyrosine-3H-labelled [8-lysine]vasopressin ([3H]vasopressin) was time-dependent, saturable and reversible. Scatchard-plot analysis of hormone binding indicated the presence of a single class of receptors with an equilibrium dissociation constant of 12.7 +/- 0.2 nM. The maximal binding capacity was 75 +/- 6 fmol/10(6) cells, which corresponds to approx. 45,000 sites per cell. Oxytocin and a highly potent oxytocin analogue were able to inhibit completely [3H]vasopressin binding, but, in this respect, they were far less potent than vasopressin. This clearly demonstrates the vasopressinergic nature of this receptor. Pharmacological studies using a series of 14 vasopressin or oxytocin analogues indicated that the ligand selectivity of the vasopressin receptor found on WRK 1 cells resembles that of the rat hepatocyte. This signifies that this vasopressin receptor is of the V1a subtype. This conclusion was confirmed by the observation that vasopressin did not influence the production of intracellular cyclic AMP in WRK 1 cells.     

Insulin increases the synthesis of phospholipid and diacylglycerol and protein kinase C activity in rat hepatocytes

The effects of insulin on phospholipid metabolism and generation of diacylglycerol (DAG) and on activation of protein kinase C in rat hepatocytes were compared to those of vasopressin and angiotension II. Insulin provoked increases in [3H]glycerol labeling of phosphatidic acid (PA), diacylglycerol (DAG), and other glycerolipids within 30 s of stimulation. Similar increases were also noted for vasopressin and angiotensin II. Corresponding rapid increases in DAG mass also occurred with all three hormones. As increases in [3H]DAG (and DAG mass) occurred within 30-60 s of the simultaneous addition of [3H]glycerol and hormone, it appeared that DAG was increased, at least partly, through the de novo synthesis of PA. That de novo synthesis of PA was increased is supported by the fact that [3H]glycerol labeling of total glycerolipids was increased by all three agents. Increases in [3H]glycerol labeling of lipids by insulin were not due to increased labeling of glycerol 3-phosphate, and were therefore probably due to activation of glycerol-3-phosphate acyltransferase. Unlike vasopressin, insulin did not increase the hydrolysis of inositol phospholipids. Insulin- and vasopressin-induced increases in DAG were accompanied by increases in cytosolic and membrane-associated protein kinase C activity. These findings suggest that insulin-induced increases in DAG may lead to increases in protein kinase C activity, and may explain some of the insulin-like effects of phorbol esters and vasopressin on hepatocyte metabolism.     

Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in 32Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of 32Pi into this pool is slow. Results are quite different when [3H]inositol is the precursor utilized. Incorporation of [3H]inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of [3H]phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the [3H]inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of [3H]inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing [3H]inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of [3H]inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.     

Characterization of the hormone-sensitive phosphatidylinositol pool in WRK-1 cells

WRK-1 rat mammary tumor cells respond to vasopressin with an increase in the rate of phosphatidylinositol turnover. Evidence derived from a series of experiments performed under various prelabeling conditions suggests that the hormone-sensitive phosphatidylinositol resides in a distinct pool within the cell, accounting for approximately 17% (8-37%) of the total cellular phosphatidylinositol. The possibility that two distinct cell types might explain this finding is unlikely since neither newly cloned nor thymidine-blocked cells exhibit any alteration in the nature of their response. This hormone-sensitive phosphatidylinositol moiety has the following characteristics. 1) Under equilibrium labeling conditions, it is completely turned over within 5 min of hormone addition. 2) It is both synthesized and degraded even in the absence of hormone, although at a much slower rate. 3) Under the conditions employed, there does not appear to be transfer of phosphatidylinositol from the insensitive to the sensitive pool. A model of these events is outlined.     

A mevalonate requirement for maintenance of fatty acid and protein synthesis during hormonally stimulated development of mammary gland in vitro

The effect of compactin on hormonally induced lipogenesis and protein synthesis was studied in vitro in explants of mammary gland from mid-pregnant rabbits. Compactin blocks mevalonate synthesis by the specific inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, and in this system, culture with 10 microM compactin for 24, 48, and 72 h inhibited incorporation of [1-14C]acetate (but not [2-14C]mevalonate) into sterol by 98, 95, and 86%, respectively. Removal of compactin prior to assay rapidly reversed this effect and was associated with increased tissue 3-hydroxy-3-methylglutaryl-CoA reductase activity. Fatty acid synthesis (measured by incorporation of [1-14C]acetate or [4,5-3H]leucine) and protein synthesis (measured by incorporation of [4,5-3H]leucine) were both inhibited by around 50% after culture with compactin. This inhibition was not rapidly reversed by removal of compactin prior to assay, but it was prevented by inclusion of 1 mM mevalonolactone in the culture medium. After removal of compactin and continued culture in its absence for 24 h with hormones, the normal tissue capacity for fatty acid and protein synthesis was restored, indicating no permanent cell damage. The results suggest a specific requirement for mevalonate (or derived products) for the hormonal maintenance of the increased fatty acid and protein synthesis characteristic of the development of the mammary gland.     

Effects of probucol on lipid metabolism and secretion in long-term cultures of adult rat hepatocytes

To study the effects of probucol on hepatic lipid metabolism, we used adult rat hepatocytes cultured on a feeder layer of 3T3 cells lethally treated with mitomycin C. These cultures synthesize and secrete for at least 2 weeks various lipids from [14C]acetate and [14C]oleate precursors. Treatment with 20 micrograms/ml of probucol for 7 and 14 days decreased the secretion of various radiolabeled lipid species to the culture medium and produced an intracytoplasmic accumulation of triacylglycerol droplets. The lipids whose secretion was most decreased were free and esterified cholesterol (50-70% reduction). Secretion of triacylglycerols and phospholipids was also reduced but to a lower extent. Intracytoplasmic triacylglycerols accumulated and the activity of glycerol phosphate dehydrogenase, a marker enzyme of glycerolipid synthesis, also increased (35-56%). The total incorporation of both radioactive precursors into free and esterified cholesterol and phospholipids was reduced 20-60%. Our data show that 2-week treatment of 3T3-hepatocyte cultures with pharmacological concentrations of probucol reduces significantly lipid secretion and suggest that at least part of the in vivo hypolipidemic effect of probucol could be attributed to a decrease in the secretion of lipids (i.e., lipoproteins) by hepatocytes.     

Hormonal regulation of fat esterification & oxidation in hepatocytes from fed rats: modulation by 3-mercaptopicolinate

3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), decreased esterification of [1-14C] oleate and [1-14C] myristate in hepatocytes from fed rats. In the absence of 3-mercaptopicolinate, adrenaline, noradrenaline, vasopressin or angiotensin II increased esterification to triacylglycerol of [1-14C] oleate but not [1-14C] myristate. Cyclic AMP decreased esterification of both oleate and myristate. In the presence of 3-mercaptopicolinate, stimulation of oleate esterification by the catecholamines, vasopressin or angiotensin II was increased, and stimulatory effects of these hormones on myristate esterification were observed. Adrenaline, noradrenaline, vasopressin or angiotensin II increased 14CO2 production from both [1-14C] oleate and [1-14C] myristate but the degree of stimulation was similar in the absence or presence of 3-mercaptopicolinate. The results indicate a role for the catecholamines and angiotensin II in the regulation of liver fat metabolism and emphasize the potential importance of changes in activity of PEPCK as determinants of hepatic carbon flux.     

Organization of the receptor-mediated phosphoinositide cycle: Relationship between receptor occupancy and accession of phosphatidylinositol

We have previously reported the existence of separate hormone-responsive and -unresponsive pools of inositol phospholipids in WRK-1 cells. In order to further explore this concept, we have performed experiments to examine the relationship between the plasma membrane receptor and the pool of phosphatidylinositol (Ptdlns) that is metabolized in response to hormonal stimulation. The results support the following conclusions. 1) The amount of Ptdlns metabolized in WRK-1 cells in response to vasopressin is proportional to the number of receptors occupied; neither prolonged activation with nor readdition of a submaximal concentration of vasopressin induced the same degree of Ptdlns metabolism as a maximal concentration of vasopressin. 2) Dissociation of cytoskeletal structures by incubation with cytochalasin D did not alter the amount of Ptdlns accessed during hormonal stimulation. 3) Accession of Ptdlns from internal membranes does not depend on internalization and recycling of the receptor; cells incubated in potassium-free medium failed to internalize receptor-ligand complexes, yet they accessed the same amount of Ptdlns in response to vasopressin as did control cells. 4) Golgi-mediated phosphatidylinositol transport is not involved in hormone-stimulated phosphoinositide turnover, since brefeldin A, which interferes with Golgi-mediated transport processes, had no effect on the amount of Ptdlns accessed during vasopressin stimulation. 5) Phosphoinositide breakdown and compensatory resynthesis is not a closed process; newly synthesized Ptdlns is not preferentially localized to a hormone-responsive pool but is generally redistributed between responsive and unresponsive pools. J. Cell. Biochem. 64:382–389. © 1997 Wiley-Liss, Inc.

1 This article is a U.S. Government work and, as such, is in the public domain in the United States of America.