Direct correlation between level of morphine and its biochemical effect on monoamine systems in mouse brain. Evidence for involvement of dopaminergic neurons in the pharmacological action of acute morphine

The pharmacokinetics (uptake and elimination) and pharmacodynamics (biochemical effects on monoamine systems) of morphine in the CNS were investigated concurrently. ICR mice, weighing about 25 g, were injected intravenously with several doses (2.5-80 mg/kg) of morphine. The animals were killed by microwave irradiation (5 kW, 0.6 sec) at 10 and 30 min, and 1, 2, 4, 8 and 24 hr after the injection. The intracerebral levels of morphine and metabolically related substances consisting of monoamines [noradrenaline, dopamine (DA), 5-hydroxytryptamine (5-HT), 3, 4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylacetic acid [homovanillic acid (HVA)], 5-hydroxyindoleacetic acid (5-HIAA), tyrosine and tryptophan] were determined in identical samples by a combination of organic extraction and high-performance liquid chromatography with electrochemical detection. The intracerebral level of morphine was found to depend on the dose injected, and the biological half-life of the drug was estimated to be about 1 hr. The morphine injection (2.5-80 mg/kg) caused significant increases in monoamine metabolites although only slight changes occurred in the concns of parent transmitters. The intracerebral level of morphine was significantly correlated with the ratios DOPAC/DA and HVA/DA (r = 0.7033, P less than 0.0001; and r = 0.6455, P less than 0.0001, respectively). On the other hand, the correlation between the morphine level and 5-HIAA/5-HT was lower than those for DOPAC/DA and HVA/DA. These results suggest that monoamine systems, especially DA, are closely involved in the biochemical effects of morphine. Furthermore, the proposed procedure is demonstrated to be useful as a new approach in biochemical pharmacology, where the direct correlation between the distribution of a drug (pharmacokinetics) and the biochemical effects of the drug (pharmacodynamics) can be measured.     

Partial purification and properties of cytochrome P450 from homogenates of human fetal livers.

Using ω-amino-n-octyl Sepharose 4B and hydroxylapatite columns, cytochrome P450 was purified to approx. 6.7 nmoles per mg of protein from the 105,000 g precipitate of homogenates of human fetal livers. The partially purified preparation of cytochrome P450 was free of detectable amounts of cytochrome b₅, NADPH-cytochrome P450 reductase and NADH-cytochrome b₅ reductase. The absolute spectrum of the preparation exhibited a peak at 417 nm in the Soret region, indicating that this cytochrome P450 is a low spin species. Binding of aniline and SKF 525-A to this partially purified preparation of cytochrome P450 produced a modified type II and a type I difference spectra, respectively. As judged by Ouchterlony double diffusion analysis, the cytochrome P450 preparation did not cross react with antibody against cytochrome P450 isolated from the livers of phenobarbital-pretreated rats. In reconstituted systems, the cytochrome exhibited considerable activity for aniline hydroxylation but only a low ethlymorphine N-demethylation activity compared to cytochrome P450 isolated from the livers of phenobarbital-pretreated rats.     

Metabolic activation and cytotoxicity of cyclophosphamide in primary cultures of postnatal rat hepatocytes

We have developed a model of primary cultures of postnatal rat hepatocytes to characterize the metabolic activation of xenobiotics to toxic intermediates and to study the mechanism(s) by which these chemicals produce cellular injury. This model was employed to investigate the cytochrome P-450 mediated biotransformation of cyclophosphamide (CP) to cytotoxic metabolites that nonspecifically alkylate DNA and cellular proteins. The parenchymal cells were isolated by an in situ collagenase perfusion technique and cultured for 24 hr prior to drug treatment. The cultures were then exposed to CP concentrations ranging from 1 × 10^?4 M to 1 × 10^?3 M for 24 hr. Initial studies indicated minimal toxicity to non-replicating parenchymal hepatocytes maintained in ornithine-supplemented, arginine-deficient medium. The addition of arginine permitted the overgrowth of fibroblasts in the same culture system. These fibroblasts then became the target of alkylating CP metabolites produced by the par-enchymal cells. By day 3 after CP administration, cell number and total protein per dish decreased by over 40 percent. The morphology of the cultures changed dramatically because of fibroblast destruction. The cytotoxicity to dividing fibroblasts was eliminated by administering 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride (SKF 525-A), an inhibitor of the cytochrome P-450 monooxygenase system, to the co-cultures treated with CP. The alkylating metabolites of CP produced by the parenchymal cells and released into the culture medium were quantitated by reacting aliquots of medium from CP-treated cells with 4-(p-nitrobenzyl)pyridine. These results provide both direct and indirect evidence of drug metabolism in cultured cells and suggest that this co-culture system can be utilized to evaluate the metabolic activation of xenobiotics.     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

Metabolism of 15N-labelled N-nitrosodimethylamine and N-nitroso-N-methylaniline by isolated rat hepatocytes

The N-demethylation of ¹⁵N-labeled N-nitrosodimethylamine (DMN) and N-nitroso-N-methylaniline (NMA) by isolated rat hepatic cells has been investigated. The values obtained in this system for molecular nitrogen formed during metabolism, compared with substrate consumed, were DMN 47%, NMA 23%, and N-nitroso-N-methylurea (NMU) 105%. The results for DMN are roughly halfway between those previously determined with rat liver S-9 fraction in vitro (33%) and in vivo (67%). For NMA, the hepatocyte data are closer to those obtained from S-9 in vitro (19%), rather than the in vivo (52%). No mixed nitrogen (¹⁵N¹⁴N) or labeled nitrogen oxides were found.     

Chlorphentermine binding in rat lung subcellular fractions and its displacement by desmethylimipramine.

An examination of possible mechanisms of binding interactions between drugs accumulated in the lung has been made by equilibrium dialysis using [¹⁴C]chlorphentermine (CP) in rat lung subcellular fractions (microsomal and 15,000 g and cytosol). By far the greatest CP binding was in the microsomal and 15,000 g fractions and with all isotherms, CP binding was shown to consist of a specific, saturable component and a non-specific partitioning component. Desmethylimipramine (DMI) was the most potent inhibitor of a series of compounds and was selected for detailed study. Generally, as DMI concentration was increased, CP binding decreased, however this relationship was not in accordance with simple direct competitive theory. At 5 × 10^?4 M DMI in the microsomal fraction, CP binding was initially increased with increasing CP concentration. In the 15,000 g fraction, the apparent CP affinity constant did not decrease regularly with increasing DMI concentration, the affinity constant of DMI increased with increasing DMI concentration, a Hill coefficient significantly less than unity was obtained and the binding model became increasingly untenable for higher DMI levels. It is likely that DMI and CP interact in rat lung via a cooperative mechanism involving binding-induced conformational transitions. The possible role of phospholipids in the binding of basic amines in lung is discussed.     

Involvement of alpha 1-adrenergic receptors in stimulation of phosphatidylinositol metabolism by catecholamines in mouse thyroids

The effects of alpha-adrenergic agonists and thyroid stimulating hormone on the incorporation of radioactive phosphate into phosphatidylinositol were investigated in mouse thyroids in vitro. The incorporation of 32P orthophosphate into phosphatidylinositol was stimulated by thyroid stimulating hormone, norepinephrine (a mixed alpha 1- and alpha 2-adrenergic agonist), methoxamine and phenylephrine (alpha 1-agonists) and slightly by clonidine and oxymetazoline (alpha 2-agonists) but not by isoproterenol (beta-agonist). Prazosin (alpha 1-antagonist) inhibited the stimulation by norepinephrine of 32P incorporation into phosphatidylinositol, but yohimbine (alpha 2-antagonist) was less effective. Although norepinephrine inhibits the thyroid stimulating hormone-induced release by activating alpha-, especially alpha 1-adrenoceptors in mouse thyroids [M. L. Maayan et al., Metabolism 26, 473 (1977); M. L. Maayan et al., Endocrinology 101, 284 (1977); T. Muraki et al., Endocrinology 110, 51 (1982)] alpha 1-agonists did not decrease the stimulation of turnover elicited by thyroid stimulating hormone and did not have additive action with it. These results suggest that (1) the stimulation of phosphatidylinositol turnover of mouse thyroids elicited by adrenergic agonists is mediated by activation of alpha 1-adrenoceptors and (2) the inhibitory effect of norepinephrine on the thyroid stimulating hormone-induced release of thyroxine is not mediated by norepinephrine-inhibition of phosphatidylinositol-turnover stimulated by thyroid stimulating hormone.     

Alloxan toxicity in isolated rat hepatocytes and protection by sugars

Suspensions of isolated rat hepatocytes incubated in the presence of the diabetogenic agent alloxan exhibit time- and concentration-dependent damage. At concentrations of 3.5 mM and above, alloxan caused an increase in lactate dehydrogenase (LDH), glutamate-pyruvate transaminase (GPT) and intracellular potassium (K⁺) leakage, all of which are indices of plasma membrane damage, and decreased the intracellular reduced glutathione content (GSH) of the cells. Preincubation (10 min) in d-glucose (50 or 100 mM, but not 10 mM) partially protected the hepatocytes from LDH, GPT and K⁺ leakage and the decrease in GSH produced by alloxan (7mM) during a 60-min incubation period. Other sugars (d-galactose, 2-deoxy-d-glucose, d-fructose, d-mannoheptulose and d-mannitol) were also found to protect hepatocytes against damage caused by alloxan. d-Fructose was found to be the most potent protective sugar. These results indicate that alloxan is not selectively toxic to the pancreatic (β-cell and that sugars can protect against alloxan-induced cytotoxicity in hepatocytes.     

Carbon tetrachloride-dependent inhibition of lipid secretion by isolated hepatocytes. Characterization and requirement for bioactivation

Secretion of lipid as very low density lipoprotein (VLDL) by isolated hepatocytes was studied in a system in which the cells were exposed to a constant concentration of carbon tetrachloride (CCl4) throughout the duration of the incubation. Inhibition of secretion was characterized in terms of CCl4 concentration and duration of incubation. Half-maximum inhibition of VLDL secretion occurred at 80 microM CCl4. At 390 microM CCl4, VLDL secretion was inhibited 40% in 2 min and was suppressed completely in 5 min. No CCl4-dependent release of cellular glutamic oxaloacetic transaminase occurred under the conditions studied. Evidence is presented indicating that the metabolism of CCl4 is required for the expression of CCl4-dependent inhibition of lipid secretion. With respect to the parameters studied, the isolated hepatocyte system closely mimics the hepatic response of the intact animal to CCl4.     

Hepatic drug transport in the rat: A comparison between isolated hepatocytes,the isolated perfused liver and the liver in vivo

The hepatic transport of three different drugs, the organic anion dibromosulphophthalein, the organic cation d-tubocurarine and the uncharged compound ouabain was studied in vivo in the isolated perfused rat liver and isolated hepatocytes. The respective clearances by uptake were determined for the various substrates and corrected for differences in hepatic blood flow and extracellular protein binding in the three liver preparations. The corrected uptake values in the intact organ, in vivo and in the isolated perfused liver were highly comparable; for dibromosulphophthalein a clearance of 2.1ml/minper 10⁶ hepatocytes was found in vivo, whereas in perfusion a value of 2.4 ml/min per 10⁶ cells was calculated. For d-tubocurarine, the values were 34 × 10^?4 and 55 × 10^?4ml/min per 10⁶ cells obtained in vivo and in the isolated perfused organ, respectively. With ouabain as the substrate, the in vivo clearance amounted to 5.1 × 10^?2, whereas in the isolated perfused liver a value of 4.8 × 10^?2ml/min per 10⁶ cells was calculated. The clearance by uptake obtained for dibromosulphophthalein and ouabain in the isolated hepatocytes appeared to be a factor of 2–3 lower than in the intact organ. In the case of d-tubocurarine however the clearance was identical to that in vivo and the isolated perfused liver.The rate of secretion from isolated hepatocytes was, for dibromosulphophthalein identical to, and for d-tubocurarine and ouabain lower than that in the intact organ, especially as compared with the in vivo preparation.It is concluded that transport function is well preserved in the isolated perfused liver and isolated hepatocytes. For certain substrates freshly isolated hepatocytes may exhibit a somewhat lower uptake and/or secretion rate, in spite of a good cell quality as judged by generally accepted criteria for cell viability. Whether this is due to changes in membrane composition (not detected by our viability tests) or a selection of a subpopulation of hepatocytes, is discussed.     

Evidence against involvement of calcium in carbon tetrachloride-dependent inhibition of lipid secretion by isolated hepatocytes

Carbon tetrachloride (CCl4)-induced inhibition of very low density lipoprotein (VLDL) secretion was studied in isolated hepatocytes. The hypothesis that inhibition of secretion is due to altered calcium homeostasis following CCl4-dependent inhibition of endoplasmic reticulum calcium sequestration was investigated. Inhibition of VLDL secretion by CCl4 was not dependent on extracellular calcium, since inhibition occurred when extracellular calcium was reduced to 0.1 microM. CCl4 inhibited hepatocyte VLDL secretion more rapidly than it inhibited microsomal calcium sequestration. Further, the concentration of CCl4 that produced half-maximal inhibition of VLDL secretion was about one-half the concentration required to produce half-maximal inhibition of microsomal calcium sequestration. The calcium ionophore A23187 did not mimic the action of CCl4 in inhibiting VLDL secretion under conditions in which A23187 altered cellular calcium homeostasis. The results that an alteration of calcium homeostasis is not involved in inhibition of VLDL secretion by carbon tetrachloride.     

Metabolic effects of acarbose administration in normal and diabetic rats

The effect of acarbose on cardiac and hepatic metabolism was investigated in normal and diabetic rats. Groups of rats were fed one of the three following diets for 7 days: (1) ground Purina chow, (2) ground Purina chow fortified with raw corn starch and sucrose, and (3) the above high carbohydrate diet, with added acarbose (40 mg/100 g food). At the end of the dietary period the rats were decapitated, and a sample of liver tissue was removed and frozen in liquid nitrogen. The heart was extirpated for subsequent perfusion by the Langendorff technique. Increases in liver and heart glycogen produced by the high carbohydrate diet in the normal rats were prevented completely when acarbose was incorporated into the food. In diabetic animals, liver glycogen was uniformly lower than normal, irrespective of the diet or the presence of acarbose. With animals fed the control diet, cardiac glycogen was higher in diabetic than in normal rats. The high carbohydrate diet caused a lowering of heart glycogen in diabetic rats and this reduction in glycogen content was reversed by including acarbose in the diet. Effects of isoproterenol on myocardial phosphorylase a activity were determined in hearts from normal and diabetic rats given one of the three diets. The high carbohydrate diet decreased the enzymatic response to the catecholamine in hearts from both normal and diabetic animals, and this phenomenon was prevented by the presence of acarbose in the diet. In diabetic rats fed any of the three diets, the activation of cardiac phosphorylase by isoproterenol was greatly accentuated. Measurements of heart uridine kinase showed that the activity of this enzyme was lower than normal in hearts from diabetic rats given either the control or the high carbohydrate diet. The presence of acarbose in the latter diet resulted in a significant decrease in cardiac uridine kinase activity in hearts from normal rats. The results of this study demonstrate the effectiveness of acarbose in modulating tissue metabolism in normal and diabetic animals.     

Activation of coronary arterial guanylate cyclase by nitric oxide,nitroprusside, and nitrosoguanidine—Inhibition by calcium,lanthanum, and other cations,enhancement by thiols

Although reports that certain vasodilate s activate soluble guanylate cyclase, especially in the presence of thiols, and elevate cyclic GMP levels in smooth muscle suggest that cyclic GMP is involved in vascular smooth muscle relaxation, earlier reports that Ca²⁺ activates guanylate cyclase and that Ca²⁺ -dependent contractile agents elevate cyclic GMP levels are seemingly at odds with this hypothesis. The objective of this study was to examine the effects of Ca²⁺ related cations, and thiols on bovine coronary arterial soluble guanylate cyclase. Guanylate cyclase activity was detected in the presence of Mg²⁺ or Mn²⁺ but not of other cations. Basal activity was greater in the presence of Mn²⁺ than of Mg²⁺. Activity of guanylate cyclase stimulated by nitroprusside, nitric oxide, or nitrosoguanidine, however, was greater with Mg²⁺, although the requirement of activated enzyme for Mn²⁺ was reduced about 10-fold. Ca²⁺ markedly inhibited guanylate cyclase activation in the presence of Mg²⁺ but not of Mn²⁺. La²⁺ inhibited enzyme activation in the presence of Mg²⁺ or Mn²⁺. Neither Ca²⁺ nor La³⁺ altered basal enzymatic activity. Results that were qualitatively similar to those indicated above were observed with partially purified, heme-free, coronary arterial soluble guanylate cyclase. Nitric oxide and nitroso compounds activated partially purified enzyme, and thiols enhanced enzyme activation by nitroprusside and nitrosoguanidine without appreciably altering basal activity. Irreversible sulfhydryl binding agents such as ethacrynic acid and gold inhibited both basal and activated guanylate cyclase. These results suggest that changes in intracellular concentrations of free Ca²⁺ and sulfhydryl groups could influence the rate of formation of cyclic GMP by vasodilators and that this, in turn, could alter smooth muscle tone.     

Effects of propranolol and a number of its analogues on sodium channels

To assess the relative contributions that the sodium channel blocking activity of propranolol may play in a variety of its therapeutic applications, its effects were examined in vitro with a sodium channel specific 22Na+ uptake system, using rat brain membranes. Propranolol inhibited 22Na+ uptake in the rat brain membrane preparation by acting as a competitive inhibitor of the binding of the sodium channel opening agent veratridine, with an IC50 for this action of 6.5 microM. This is approximately one order of magnitude higher in concentration than that necessary for expression of the beta-adrenergic antagonism of propranolol. The binding of propranolol and its action to block sodium channels were demonstrably different from those of the neurotoxins tetrodotoxin and saxitoxin. Propranolol had effects on sodium channels that are similar, although not identical to those of the local anesthetics procaine and lidocaine. The concentrations of propranolol and a number of its analogues which produced 50% inhibition of 22Na+ uptake (IC50 values ranging from 4 to greater than 100 microM) were similar to the concentrations of these same analogues which were required to produce negative inotropic and antiarrythmic effects (ED40) on isolated rabbit atria [D. O. Rauls and J. K. Baker, J. med. Chem, 22, 81 (1979)]. These effects showed correlations of 0.945 and 0.936, respectively, with the 22Na+ uptake inhibition. It is concluded from this information that a substantial proportion of the negative inotropic and antiarrythmic effects of propranolol is due to its action on sodium channels.     

The uptake and secretion of 2-acetylaminofluorene by the rat and dog prostate

In unanesthetized rats examined 4–140 hr after the ip administration of 1.8 mg/kg of [9-¹⁴C]2-acetylaminofluorene ([¹⁴C]2-AAF), detectable amounts of radioactivity were found in the plasma and in the ventral and dorsolateral lobes of the prostate. Radioactivity was also found in the prostatic fluid collected from anesthetized rats between 25 and 29 hr after doses of 2 mg/kg. When three unanesthetized dogs with surgically prepared fistulas allowing the collection of prostatic fluid were given intraperitoneal doses of 0.16–0.25 mg/kg of [¹⁴C]2-AAF and followed for 6 hr (two dogs) or 166 hr (one dog), there was a relatively rapid rise in the amount of radioactivity in plasma followed by a slow decline and an appearance of radioactivity in the prostatic fluid. Radioactivity was also present in the prostate glands of each of two dogs examined 6 hr after treatment. Thus 2-AAF and/or metabolites were found to enter both the prostate gland and the prostatic secretion of both the rat and dog.     

Teratogenicity in vitro of two deacetylated metabolites of N-hydroxy-2-acetylaminofluorene

In previous studies [E. Faustman-Watts, J. C. Greenaway, M. J. Namkung, A. G. Fantel, and M. R. Juchau (1983) Teratology 27, 19–28] an embryo culture system was utilized to investigate the role of biotransformation in the embryotoxicity of 2-acetylaminofluorene. For this investigation, the capacity of two deacteylated metabolites of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to produce malformations in cultured whole rat embryos is reported. The relative capacities of N-hydroxy-2-aminofluorene (N-OH-AF) and 2-nitrosofluorene (NF) to elicit embryotoxic effects, including embryolethality, malformations, growth retardation, and alterations in macromolecular content, were assessed and compared with effects produced by N-OH-AAF and bioactivated 2-acetylaminofluorene (AAF). Qualitatively similar patterns of malformations were produced by NF and N-OH-AF. At initial concentrations greater than 60 μm, both deacetylated compounds caused abnormalities in axial rotation (flexure), decreased viability, and decreases in embryonic DNA and protein content. Both chemicals were active in the absence of a bioactivating system. AAF produced a different spectrum of defects, and was active only in the presence of a complete monooxygenase system. The malformations produced by bioactivated AAF included abnormally open neural tubes; flexure abnormalities were rarely observed. The primary defect elicited by N-OH-AAF was prosencephalic hypoplasia. This chemical was active without an added bioactivating system. Temporal studies demonstrated that exposure of embryos to NF (128 μm) for as little as 2 hr was sufficient to elicit embryotoxic effects. None of the individual metabolites appeared to be solely responsible for the interruptions of neural tube closure produced by bioactivated AAF.     

Activation of glycogenolysis by the reduction in the extracellular calcium concentration in verapamil-perfused rat liver

In an attempt to elucidate the role of Ca2+ flux in the initial events of hepatic glycogenolysis, extracellular Ca2+ concentration was manipulated in rat liver perfused with Ca2+ antagonistic drugs. After the liver had been perfused with a buffer containing verapamil and 1 mM CaCl2, either the addition of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid to the perfusate or the replacement of the perfusate with Ca2+-free buffer caused a rapid increase in glucose output as well as 45Ca2+ efflux. Substitution of diltiazem, but not 5-20 mM LaCl2, for verapamil also stimulated glucose output and 45Ca2+ efflux. However, when Ca+-free buffer was used throughout the experiment, any modes of verapamil or diltiazem perfusion were without significant effects on glucose output or Ca2+ efflux. The increases in glucose output and 45Ca2+ efflux were not affected by either 20 microM phentolamine or 300 microM ouabain, but they were inhibited significantly by 10-100 microM trifluoperazine. These results indicate that rapid decline in the extracellular Ca2+ concentration in verapamil- or diltiazem-perfused liver initiates the change in Ca2+ equilibrium on or across plasma membrane and activates glycogenolysis through a Ca2+-dependent mechanism.     

Increased formation of phosphatidic acid induced with vasopressin or Ca2+ ionophore A23187 in rat hepatocytes

The effects of vasopressin and Ca2+ ionophore A23187 on phospholipid metabolism were investigated in rat hepatocytes. Vasopressin stimulated the incorporation of [32P]Pi into phosphatidic acid within 2 min but then it returned to control level after 10 min. On the other hand, the stimulation of the incorporation of [32P]Pi into phosphatidylinositol continued with incubation times up to 20 min. The Ca2+ ionophore A23187 also increased the 32P-labeling in phosphatidic acid, although it had no effect on [32P]Pi incorporation into phosphatidylinositol. Concerning the incorporation of [3H]glycerol, vasopressin did not enhance its incorporation into phosphatidic acid and phosphatidylinositol. The Ca2+ ionophore A23187 increased the incorporation into phosphatidic acid without significant effects on that into phosphatidylinositol. In the hepatocytes prelabeled with [3H]arachidonic acid, stimulated degradation of phosphatidylinositol with the addition of vasopressin and resultant formation of phosphatidic acid were observed within 5 min. The transient accumulation of diacylglycerol, the product of phosphatidylinositol hydrolysis, also occurred within 5 min with vasopressin. On the other hand, with the Ca2+ inophore A23187, stimulated degradation of triacylglycerol to diacylglycerol and the consequent formation of phosphatidic acid were observed. The Ca2+ ionophore A23187 caused a significant release of free [3H]arachidonic acid, although vasopressin had no effect.