Alterations in protein kinase C isozymes alpha and beta 2 in activated Ha-ras containing papillomas in the absence of an increase in diacylglycerol.

The levels of protein kinase C (PKC) activity, PKC isozymes, as well as the level of endogenous diacylglycerols (DAG) were examined in early emergence mouse skin papillomas and compared to the levels in the epidermis. The papillomas were derived from a two-stage carcinogenesis protocol in which mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice weekly for only 12 weeks with 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected, greater than 90% of these early emergence papillomas contained an activated Ha-ras gene with an A----T transversion in the 61st codon. There was a TPA-independent, irreversible decrease in total PKC activity (70%) in the early emergence papillomas compared to that in the epidermis. Immunoblot analysis of epidermis and papillomas taken 4 weeks following the cessation of TPA treatment, a time when PKC catalytic activity has completely recovered to control level in epidermis but not in papillomas, revealed that the levels of PKC-alpha and PKC-beta 2 were dramatically decreased in the cytosol of the papillomas, while the levels of these two isozymes in the particulate fraction were approximately equal to the epidermis. PKC-delta, -epsilon and -zeta immunoreactive proteins were present in both epidermis and papillomas and only minor changes were observed in the papillomas. PKC-delta and PKC-epsilon displayed a particulate fraction localization in both the epidermis and papillomas, while PKC-zeta was found in both subcellular fractions. We were unable to detect PKC-gamma in mouse epidermis or papillomas. Since the level of DAG has been shown to be elevated in some ras-transformed cells, we examined DAG levels in the papillomas, as an increased DAG level could explain the constitutive decreases in the levels of PKC. Measurements of cellular DAG indicated that there was no elevation in the total pool of DAG in the early emergence papillomas. These data demonstrate an irreversible decrease in and alteration of the subcellular distribution of PKC-alpha and beta 2 in DMBA-initiated/TPA-promoted papillomas. These changes are TPA-independent, and occur in the absence of an elevation in the total pool of endogenous DAG. These alterations of PKC isozymes may be important early events in multistage tumorigenesis.

Regulation of phosphatidic acid phosphohydrolase activity during stimulation of human polymorphonuclear leukocytes.

Phosphatidic acid phosphohydrolase (PPH) activity was determined in human polymorphonuclear leukocytes (PMNs) by measuring the hydrolysis of [32P]phosphatidic acid (PA) added to cell sonicates. Enzyme activity was localized primarily to a soluble fraction. Soluble and particulate activities required magnesium and were inhibited by calcium, N-ethylmaleimide, sphingosine, and propranolol. The activity in unstimulated PMNs was 0.64 +/- 0.11 nmol of PA hydrolyzed.mg protein-1.min-1 in particulate and 4.20 +/- 0.42 in soluble fractions. Stimulation of PMNs with 1 microM f-Met-Leu-Phe (FMLP) for 10 min caused a slight decrease in soluble activity and a small increase in the activity of particulate fractions. Preincubation with 10 microM cytochalasin B for 5 min before FMLP stimulation markedly enhanced both of these changes. The effect of FMLP plus cytochalasin B was rapid (less than 10 s), whereas the calcium ionophore A23187 (1 microM) and phorbol myristate acetate (100 ng/ml) caused slower and smaller changes in enzyme activity. These results indicate that after chemoattractant stimulation; PPH activity decreases in the soluble fraction and increases in the particulate fraction suggesting that PPH may participate in signal transduction in the PMN.

Regulation of phosphoinositide and phosphatidylcholine phospholipases by G proteins.

Two G proteins that regulate phosphoinositide phospholipase C in liver plasma membranes have been purified to homogeneity in both the heterotrimeric and dissociated forms. The heterotrimers contain a 42 kDa or 43 kDa alpha subunit and a 35 kDa beta subunit. The alpha subunits are not ADP-ribosylated by pertussis toxin and are closely related immunologically to members of the recently identified Gq class of G proteins. The specific phosphoinositide phospholipase C isozyme that responds to the G proteins has been determined to the beta 1 isozyme. GTP analogues stimulate phosphatidylcholine hydrolysis in rat liver plasma membranes. The nucleotide specificity and Mg2+ dependency of the response indicate that it is mediated by a G protein. Phosphatidic acid, diacylglycerol, choline and phosphorylcholine are the products, indicating that both phospholipase D and C activities are involved. Activation of phospholipase D is also indicated by the enhanced production of phosphatidyl-ethanol in the presence of ethanol.

Activation of phospholipase D: a signaling system set in motion by perturbation of the T lymphocyte antigen receptor/CD3 complex.

A number of cellular signaling systems are called into play by interaction of the T lymphocyte antigen receptor/CD3 complex with its cognate antigen. Well-described signaling systems include phosphoinositide turnover, tyrosine phosphorylation, protein kinase C activation, and increased cytosolic calcium. We have explored the possibility that another recently described signaling system, activation of phospholipase D, may be operative. Data presented here demonstrate that stimulation of Jurkat T cells with anti-CD3 antibodies or phorbol esters resulted in activation of phospholipase D, as measured by production of phosphatidylethanol and phosphatidic acid. The combination of anti-CD3 antibody plus phorbol ester led to a greater than additive production of phosphatidylethanol and to the additive production of phosphatidic acid (in the absence of ethanol). Phorbol esters as a second stimulus with anti-CD3 antibody led to a additive increase in cellular diacylglycerol content but provided no increased production of inositol phosphates, suggesting that diacylglycerol production in these cells results from hydrolysis of noninositol containing lipids as well as from phosphinositides. Exogenous addition of phosphatidic acid led to increases in cytosolic calcium that, depending on the concentration used, resulted from release of an intracellular store of calcium and influx of extracellular calcium. Changes in cytosolic calcium occurred in the absence of inositol phosphates production. These studies establish a role for increased phospholipase D activity in T lymphocyte activation.

Phosphatidate-dependent protein phosphorylation.

Phosphatidate-dependent protein phosphorylation was observed in soluble extracts from rat liver, brain, lung, and testis. The phosphorylation was stimulated by free Ca2+ in the range of 360-800 nM. Incubation mixtures containing phosphatidate provided markedly different profiles of protein phosphorylation from those with phosphatidylserine plus 1,2-diolein. Phosphatidate-dependent phosphorylation of a 30-kDa protein in the soluble fraction from heart was also observed. This phosphorylation did not require Ca2+. Soluble fractions from liver, testis, brain, and lung phosphorylated the 30-kDa heart protein in a phosphatidate-dependent Ca(2+)-independent manner. We propose that part of the action of phosphatidate in cells may be mediated by a protein kinase(s).

Cell signalling through phospholipid breakdown.

There is much evidence that G-proteins transduce the signal from receptors for Ca(2+)-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein alpha-subunits. It is proposed that this protein is the alpha-subunit of the G-protein that regulates the phospholipase in this tissue. Ca(2+)-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca2+, protein kinase C and perhaps growth factor receptor tyrosine kinases.

Purification of the hepatic vasopressin receptor using a novel affinity column.

A vasopressin receptor was purified, using a novel affinity column, from rat liver plasma membranes treated with guanosine 5'-(3-O-thio)triphosphate and solubilized with 0.8% cholate. Incubation of the membranes with the GTP analogue resulted in a dissociation of the receptor-guanine nucleotide regulatory protein complex. This manipulation, although resulting in a low-affinity state of the receptor, facilitated purification. The solubilized receptor was assayed using a new reconstitution procedure in which the soluble extracts were inserted into lipid vesicles composed of phosphatidylcholine and phosphatidylinositol. The receptor was purified by sequential chromatography on Q-Sepharose and hydroxyapatite. The use of a novel affinity column, a V1-vasopressin antagonist-agarose, resulted in a near-homogeneous preparation of a protein which exhibited an Mr = 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of purified receptor, as well as crude membrane preparations cross-linked to [125I]arginine vasopressin, also revealed a protein band with an approximate Mr = 58,000. These findings indicate that V1-antagonist affinity chromatography should be useful for purifying adequate amounts of the receptor for studies of structure and function.

Hormonal stimulation of diacylglycerol formation in hepatocytes. Evidence for phosphatidylcholine breakdown.

The molecular species of 1,2-diacylglycerol in control and agonist-stimulated rat hepatocytes were analyzed by high performance liquid chromatography. Twelve species were identified which were increased nonuniformly by 100 nM vasopressin. Most species were increased 2-3-fold, but some (C16:0/C20:4 and C18:0/C20:4) were increased 3-6-fold. Selectively greater increases in the latter two species were also induced by ATP, angiotensin II, and A23187 ionophore, however, phorbol ester caused uniform increases. Calcium depletion of the cells with chelator resulted in a uniform 2-fold effect of vasopressin on 1,2-diacylglycerol species, with greater increases in C16:0/C20:4 and C18:0/C20:4 being restored by Ca2+ readdition. Comparison of the increases in 1,2-diacylglycerol species caused by the Ca2+-mediated agents with the molecular species present in rat hepatocyte phospholipids supports the concept that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates. In hepatocytes incubated for 5 min to 2 h with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine, the label was incorporated mainly into phosphatidylcholine, and subsequent incubation with vasopressin, angiotensin II, ATP, epinephrine, A23187, and phorbol ester caused formation of [3H]alkyl-acylglycerol, but not [3H]alkyl-phosphatidic acid. The time course and concentration dependence of the vasopressin effect were similar to those reported previously for total 1,2-diacylglycerol (Bocckino, S. B., Blackmore, P. F., and Exton, J. H. (1985) J. Biol. Chem. 260, 14201-14207). Calcium depletion induced by chelator inhibited the effect of vasopressin, and readdition of Ca2+ largely restored the effect. In cells incubated with [14C]lyso-phosphatidylcholine, [3H]phosphatidylcholine, or [14C]phosphatidylethanolamine for 5 or 30 min to label hepatocyte phosphatidylcholine, vasopressin also induced the formation of labeled 1,2-diacylglycerol, but not phosphatidic acid. In contrast, in hepatocytes prepared from rats injected intraportally with [3H]alkyl-lyso-glycerophosphocholine 20 h previously, the hormone induced the rapid formation of both labeled 1,2-diacylglycerol and phosphatidic acid. In summary, these isotopic data indicate that a rapidly labeled pool of phosphatidylcholine is hydrolyzed to 1,2-diacylglycerol and a slowly labeled pool is broken down to both 1,2-diacylglycerol and phosphatidic acid in hepatocytes stimulated by Ca2+-mobilizing agents. It is concluded from both the analyses of molecular species of 1,2-diacylglycerol and the labeling experiments that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates in hepatocytes stimulated with Ca2+-mobilizing agonists and that the mechanisms responsible may involve both Ca2+ and protein kinase C.

An early elevation of diacylglycerol and phosphatidate in regenerating liver.

Liver diacylglycerol and phosphatidate are elevated following partial hepatectomy. These increases precede those in DNA synthesis and triacylglycerol accumulation. Possible factors involved in the increase in the lipids and the possible role of the lipids in liver regeneration are discussed.

An enzymatic assay for picomole levels of phosphatidate.

An assay for phosphatidate that is sensitive, specific, and highly reproducible is described. Phosphatidate is transacylated with methylamine and the glycerol 3-phosphate produced is measured enzymatically. The assay is linear from 50 to 1500 pmol of phosphatidate and can be used to quantitate phosphatidate in small amounts of biological materials.

Activation of phospholipase D by the fucose-sulfate glycoconjugate that induces an acrosome reaction in spermatozoa.

We report for the first time that phospholipase D activity in sea urchin spermatozoa can be regulated by a component of egg jelly known to induce an acrosome reaction. The fucose-sulfate glycoconjugate (FSG) of egg jelly that induces an acrosome reaction in spermatozoa caused Ca2+-dependent increases in 1,2-diacylglycerol and phosphatidic acid. Diacylglycerol concentrations were increased 2-fold, and phosphatidic acid concentrations were elevated up to 10-fold 2 min after the addition of FSG to spermatozoa. FSG also caused increases in choline, but not in choline phosphate concentrations. Neither phosphorylation of diacylglycerol nor de novo synthesis from glycerol were significant routes of synthesis of phosphatidic acid during the acrosome reaction. When spermatozoa were incubated with FSG in the presence of ethanol, phosphatidylethanol was produced. As ethanol concentrations in the extracellular medium were increased from 0.1 to 2.5%, the amount of phosphatidylethanol increased, whereas phosphatidic acid concentrations decreased, suggesting a competitive transphosphatidylation reaction catalyzed by phospholipase D. Furthermore, when a phosphatidylcholine pool in spermatozoa was radiolabeled using [3H]1-O-alkyl-2-lyso-glycerol-3-phosphorylcholine, the subsequent addition of FSG caused a 4-fold accumulation of [3H]phosphatidic acid. FSG-induced elevations in [3H]phosphatidic acid were positively correlated with the percent of cells that had undergone an acrosome reaction.

Role of guanine nucleotide regulatory proteins and inositol phosphates in the hormone induced mobilization of hepatocyte calcium.

Treatment of isolated hepatocytes with F- produced a concentration-dependent activation of phosphorylase, efflux of Ca2+, rise in [Ca2+]i, increase in Ins 1,4,5-P3 levels, decrease in PI-4,5-P2 levels, and increase in DAG levels. The levels of intracellular cAMP were decreased by NaF. The effects of NaF were potentiated by AlCl3. This potentiation was abolished by the Al3+ chelator deferoxamine. These results illustrate that AlF4- can mimic the effects of Ca2+-mobilizing hormones in hepatocytes and suggest that the coupling of the receptors for these hormones to the hydrolysis of PI-4,5-P2 is through a guanine nucleotide-binding regulatory protein. This is because AlF4- is known to modulate the activity of other guanine nucleotide regulatory proteins (Gi, Gs, and transducin). Calcium-sensitive inositide release in a purified rat liver plasma membrane preparation was increased by calcium-mobilizing hormones in the presence of guanine nucleotides. Vasopressin-stimulated inositide release was evident in the presence of GTP or GTP gamma S. The guanine nucleotide and hormonal stimulation was evident on both inositide production and PI 4,5-P2 degradation. Treatment of plasma membranes with cholera toxin or islet activating protein or prior injection of animals with islet activating protein did not affect stimulation of inositide release by GTP gamma S or GTP gamma S plus vasopressin. The results suggest that calcium-mobilizing hormones stimulate polyphosphoinositide breakdown in rat liver plasma membranes through a novel guanine nucleotide binding protein. The GTPase activity of rat liver plasma membranes was stimulated 20% by 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, most of the protein-bound [3H]vasopressin migrated as a single band, also, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased 90% when the sucrose gradient centrifugation was run in the presence of 10 M GTP gamma S. Direct evidence that a GTP-binding protein was present in the [3H]vasopressin peak was obtained by the immuno-detection of a 35 kDa beta subunit of a GTP-binding protein and a 40 kDa alpha subunit. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium-mobilizing hormones and phorbol myristate acetate mediate heterologous desensitization of the hormone-sensitive hepatic Na+/K+ pump.

The Na+/K+ pump in rat hepatocytes is stimulated in response to Ca2+-mobilizing hormones such as [arginine]vasopressin (AVP), angiotensin II and adrenaline, as well as tumour promoters such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The ability of these agents to increase cellular contents of diacylglycerol and activate protein kinase C may be necessary to observe this response. In the present work, ouabain-sensitive 86Rb+ uptake was studied in isolated rat hepatocytes to help to explain why stimulation of the Na+/K+ pump by Ca2+-mobilizing hormones and tumour promoters is not temporally sustained relative to other hormone responses. A transient stimulation (3-4 min) of the Na+/K+ pump was observed in hepatocytes exposed to high (10 nM), but not low (0.1 nM), concentrations of AVP. Experiments with the Ca2+ chelator EGTA and the Na+ ionophore monensin indicate that the rapid secondary decrease in Na+/K+-pump activity which occurs after AVP stimulation is not due to changes in cytosolic Ca2+ and Na+ concentrations. When added after the stimulation and rapid decrease in Na+/K+-pump activity induced in hepatocytes by a high concentration of AVP, a second challenge with AVP or PMA failed to stimulate the pump. Similarly, previous exposure of hepatocytes to angiotensin, adrenaline or PMA attenuated the subsequent Na+/K+-pump responses to AVP and PMA. In contrast, previous exposure to AVP had no significant effect on subsequent stimulation of the Na+/K+-pump by monensin, glucagon, forskolin or 8-p-chlorophenylthio cyclic AMP. In addition, exposure to monensin had no effect on subsequent responses to AVP and PMA. These data indicate that high concentrations of Ca2+-mobilizing hormones and PMA result in heterologous desensitization of the hepatic Na+/K+ pump to subsequent stimulation by Ca2+-mobilizing hormones and PMA, but not by cyclic-AMP-dependent agonists or monensin.

Ca2+-mobilizing hormones elicit phosphatidylethanol accumulation via phospholipase D activation.

Vasopressin, angiotensin II and epinephrine elicited the accumulation of phosphatidylethanol in rat hepatocytes exposed to ethanol and of phosphatidate in the absence of ethanol. When isolated liver plasma membranes were exposed to ethanol, GTP gamma S stimulated the production of phosphatidylethanol whereas phosphatidate was formed in the absence of ethanol. With increasing ethanol concentrations, phosphatidate formation declined whereas phosphatidylethanol production increased. These findings suggest that rat hepatocytes possess a hormone-dependent phospholipase D activity that can also catalyze the formation of phosphatidylethanol.

Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism.

Isolated rat hepatocytes responded to a variety of Ca2+-mobilizing agents (vasopressin, angiotensin II, epinephrine, epidermal growth factor, ATP, and ADP) with a rapid increase in phosphatidate mass, as measured by a sensitive new method. When hepatocytes were incubated with vasopressin (10(-8) M), phosphatidate levels increased 2-3-fold in 2 min, but there was no significant increase in diacylglycerol at this time. Changes in the fatty acid composition of phosphatidate also preceded those in diacylglycerol. De novo synthesis of phosphatidate from [3H]glycerol was unaffected by vasopressin in short-term incubation. Incubation of washed rat liver plasma membranes with GTP gamma S caused a time-dependent increase in phosphatidate. When membranes were incubated with GTP gamma S and [gamma-32P]ATP, no incorporation of 32P into phosphatidate was observed. This excludes the phospholipase C-diacylglycerol kinase pathway and suggests that a phospholipase D activity produced the phosphatidate. At submaximal concentrations of GTP gamma S, ATP and ADP stimulated membrane phosphatidate formation, presumably by acting through P2-purinergic receptors. Only phosphatidylcholine, among the major phospholipids, decreased in the membranes in response to GTP gamma S. The fatty acid composition of the phosphatidate produced in response to vasopressin in hepatocytes also suggests that phosphatidylcholine may be the source of hormonally elicited phosphatidate. We conclude that Ca2+-mobilizing hormones mainly increase phosphatidate levels in hepatocytes by a mechanism that does not involve phosphorylation of diacylglycerol or de novo synthesis but involves a guanine nucleotide-binding protein coupled to phospholipase D.

Perturbation of the human T-cell antigen receptor-T3 complex leads to the production of inositol tetrakisphosphate: evidence for conversion from inositol trisphosphate.

Antibodies directed against the T-cell antigen receptor-T3 complex mimic antigen and lead to cellular changes consistent with activation. When cells of the human T-cell line Jurkat were stimulated with a monoclonal antibody directed against T3, inositol phosphates were produced. In addition to inositol trisphosphate, which is the product of phosphatidylinositol bisphosphate cleavage, a second inositol polyphosphate was formed. This compound was more polar than inositol trisphosphate but less polar than inositol pentakisphosphate. It cochromatographed with inositol tetrakisphosphate from ostrich erythrocytes. In permeabilized Jurkat cells, this compound was shown to be formed from inositol 1,4,5-trisphosphate, but only in the presence of ATP, and 32P was incorporated into it from [gamma-32P]ATP. There also was coincident formation of inositol 1,3,4-trisphosphate. We conclude that the more polar compound is inositol tetrakisphosphate, which is formed by phosphorylation of inositol 1,4,5-trisphosphate and may be the precursor of inositol 1,3,4-trisphosphate.

Mechanism of hepatic glycogen synthase inactivation induced by Ca2+-mobilizing hormones. Studies using phospholipase C and phorbol myristate acetate.

Incubation of hepatocytes with the protein kinase C activator and tumour promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) produced a time- and concentration-dependent inactivation of glycogen synthase, but no change in phosphorylase. The same rate and extent of inactivation occurred in hepatocytes depleted of Ca2+ by treatment with the Ca2+ chelator EGTA. When hepatocytes were treated with the Ca2+-mobilizing hormone vasopressin (10 nM), the rate of glycogen synthase inactivation was similar to that observed with PMA (1 microM). Depletion of intracellular Ca2+ stores with EGTA abolished the ability of vasopressin to mobilize Ca2+ and activate phosphorylase without abolishing its ability to inactivate glycogen synthase and increase 1,2-diacylglycerol (DAG), the endogenous activator of protein kinase C. Protein kinase C, either in membranes or after partial purification, was shown to be activated in vitro by PMA in the presence of very low concentrations of Ca2+. Exogenous phospholipase C from Clostridium perfringens, at low concentrations, inactivated glycogen synthase and increased DAG without affecting cell Ca2+ or phosphorylase. It is proposed that the inactivation of glycogen synthase elicited by the Ca2+-mobilizing hormones is due, at least in part, to generation of DAG and activation of protein kinase C.

Regulation of protein synthesis in isolated hepatocytes by calcium-mobilizing hormones.

The incorporation of leucine into protein was studied in Ca2+-depleted and Ca2+-restored preparations of normal liver cells isolated from fed, adult male rats. Ca2+-restored cells incorporated amino acid 5-10-fold more rapidly than did Ca2+-depleted cells for incubation periods up to 1 hr. Readdition of Ca2+ at supraphysiologic concentrations (3 mM) to depleted cells restored the rate of incorporation within 8-10 min, whereas lesser concentrations of the cation acted more slowly. Vasopressin and alpha-adrenergic agonists rapidly (in minutes) inhibited amino acid incorporation to variable degrees in liver cells, with pronounced inhibitions (40-75%) occurring at moderate (0.1-1 mM) extracellular Ca2+ concentrations and smaller inhibitions (10-30%) occurring at supraphysiologic concentrations of the cation. Hormonally produced inhibitions were more intense at acid pH than at alkaline pH. The effects of epinephrine were mediated through alpha 1-adrenergic receptors and were not additive with those of vasopressin at saturating concentrations. It is proposed that these hormones, which are known to mobilize sequestered Ca2+ within liver cells, inhibit amino acid incorporation by influencing a Ca2+ requirement associated with protein synthesis.

Stimulation of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II.

1,2-Diacylglycerol (DAG) was measured in neutral lipid extracts from isolated hepatocytes using high pressure liquid chromatography followed by refractive index detection. Maximally effective doses of epinephrine, angiotensin II, and vasopressin increased DAG by approximately 65, 80, and 180-250%, respectively, with maximal increases being observed at 8-10 min. Depletion of cellular Ca2+ resulted in a 50% decrease in DAG accumulation elicited by vasopressin. Other agents which increased DAG levels were the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (120% increase at 10(-6) M), the Ca2+ ionophore A23187 (385% increase at 10(-5) M), and ATP (180% increase at 1 mM). The concentration dependence of DAG accumulation in response to epinephrine, angiotensin II, and vasopressin was similar to that found for myoinositol triphosphate accumulation (Charest, R., Prpic, V., Exton, J. H., and Blackmore, P.F. (1985) Biochem. J. 227, 79-90), which was approximately 5-10 times less sensitive to hormone than was phosphorylase activation. Fatty acid analysis revealed that hormonally induced DAG was partially derived from sources other than inositol phospholipids. It is proposed from these studies that Ca2+-mobilizing hormones elicit a prolonged increase in the levels of hepatocyte DAG, which may activate protein kinase C.