Modulation of positive inotropy and metabolic coronary dilatation by myocardial alpha-adrenoceptors

Cardiac hyperactivity and its consequent metabolically induced coronary vasodilation (MCD) were studied in isolated, perfused, electrically paced rat hearts. The alpha-adrenoceptor agonists, phenylephrine and methoxamine, produced a concentration-dependent inhibition of the inotropic responses to noradrenaline, dobutamine, isoprenaline, tyramine, and glucagon, while relatively potentiating their MCD reactions. This inhibition was unrelated to the alpha-agonists' known inotropic action and was not affected by catecholamine depletion of the heart. Withdrawal of the alpha-agonists or administration of the alpha-adrenoceptor antagonists phentolamine, phenoxybenzamine, or prazosin returned the inotropic and MCD reactions to normal. Neither the MCD response to electrically induced tachycardia nor the inotropic reactions produced by calcium chloride were affected by alpha-adrenoceptor agonists or antagonists. Alone, alpha-adrenoceptor antagonists were shown to potentiate the inotropic responses to noradrenaline and isoprenaline while the MCD was relatively diminished. The responses to glucagon were unaltered by alpha-antagonists. We postulate that myocardial reactivity to sympathetic stimulation can be modulated through alpha-adrenoceptors by the inhibition of processes that mediate cardiostimulation at post-beta-adrenoceptor sites, together with facilitation of those leading up to MCD. Accordingly, this modulation would act to prevent ischaemic damage to the heart by acting to limit the inotropic responses to increasing sympathetic stimulation while maximizing the blood supply to the myocardium.     

Evidence that catecholamines stimulate renal gluconeogenesis through an alpha 1-type of adrenoceptor.

1. Noradrenaline stimulates gluconeogenesis through an alpha-adrenoceptor in renal cortical tubule fragments from fed rats incubated with 5 mM-lactate. 2. The selective alpha 1-adrenoreceptor agonist methoxamine stimulated gluconeogenesis, but the selective alpha 2-adrenoceptor agonist clonidine was ineffective. 3. The selective alpha 1-adrenoceptor antagonist thymoxamine blocked the stimulatory effects on gluconeogenesis of noradrenaline and of oxymetazoline (a synthetic alpha-agonist). The selective alpha 2-adrenoceptor antagonist yohimbine was ineffective in this respect. 4. It is concluded that noradrenaline and oxymetazoline stimulate gluconeogenesis in rat kidney via an alpha 1-rather than an alpha 2-type of adrenoceptor.     

Increase of phosphoinositide hydrolysis and diacylglycerol production by PAF in isolated rat liver nuclei.

When isolated rat liver nuclei were treated with platelet-activating factor (PAF), a rapid increase in the mass of diacylglycerol (DAG) occurred. This effect was dose- and time-dependent. The maximum effect was observed after 1 min of 10(-7) M PAF treatment. A concomitant decrease of polyphosphoinositides and phosphatidic acid (PA) levels was observed. PAF-induced DAG accumulation was inhibited by the treatment with WEB 2086 or PCA-4248, specific PAF-receptor antagonists. This result may suggest that PAF exerts its action in the nucleus through specific nuclear PAF binding sites. The findings described herein are due to the activation of phospholipase C, as the results from experiments using U73122, a phospholipase C inhibitor, indicate. These are the first data on the action of     

1983 Upjohn Award lecture. Endocrine dysfunction and cardiac performance

Studies were carried out to study the effect of endocrine changes on rat cardiac performance, biochemistry, and responses to drugs. Hyperthyroidism increased contractility in rat hearts and enhanced the phosphorylase response to catecholamine. The inotropic response may be due to an increase in cardiac mass while the enzyme changes may be due to several factors. Hypothyroidism decreased force of contraction, enhanced alpha-adrenergic inotropic and chronotropic responses, and decreased beta-adrenergic responses in isolated atrial preparations. An interaction between cyclic AMP and cyclic GMP is suggested as a possible explanation. Diabetes induced by alloxan or streptozotocin produced a decrease in cardiac performance after 42 days which was correlated with a decrease in sarcoplasmic reticulum (SR) Ca2+ uptake. Insulin treatment reversed or prevented both SR and functional changes; other treatments were not as successful. Responses to cardiotonic drugs were altered by the diabetic state. The phosphorylase response to isoproterenol was enhanced while the inotropic response was not affected. An initial subsensitivity to carbachol at 30-100 days of diabetes subsequently converted to a supersensitivity to the muscarinic agent. Ouabain responses were decreased in atrial and papillary preparations from diabetic animals. Studies are continuing to elucidate the mechanisms involved in the altered pharmacological responses seen in hearts from diabetic animals.     

alpha-Adrenergic stimulation of trans-sarcolemma electron efflux in perfused rat heart. Possible regulation of Ca2+-channels by a sarcolemma redox system

The role of trans-sarcolemma membrane electron efflux in the alpha-adrenergic control of Ca2+ influx in perfused rat heart was examined. Electron efflux was measured by monitoring the rate of reduction of extracellular ferricyanide and compared with changes in contractility, as an indirect assessment of changes in cytoplasmic Ca2+ concentration. Methoxamine and phenylephrine each increased the rate of ferricyanide reduction from 80 to approx. 114 nmol/min per g wet wt. of heart, with half-maximal activation occurring at 10 microM for each agonist. Activation of the rate of ferricyanide reduction by both 10 microM methoxamine and 10 microM phenylephrine was blocked by the alpha-adrenergic antagonist, phenoxybenzamine, but not by the beta-antagonist, propranolol. Stimulation of the rate of ferricyanide reduction by the alpha-agonist coincided with the increase in contractility, each reaching maximum values at approx. 80 s. Removal of the alpha-agonists led to parallel decreases in contractility and the rate of reduction, each returning to pre-stimulation values in approx. 400 s. In addition, the relationship between Ca2+ and ferricyanide reduction was examined. Perfusion of the heart with medium containing 6 mM CaCl2 significantly increased contractility and the rate of ferricyanide reduction. Perfusion of the heart with low Ca2+ diminished contractility, did not affect the rate of ferricyanide reduction, but amplified the stimulatory effect of methoxamine on this rate. The increase in ferricyanide reduction by alpha-adrenergic agonists resulted from a change in the apparent Vmax, indicative of an increase in electron efflux sites in the plasma membrane. It is concluded that alpha-adrenergic control of electron efflux closely parallels changes in contractility and therefore changes in the cytoplasmic concentration of Ca2+. The data suggest that alpha-agonist-mediated changes in electron efflux may lead to Ca2+ influx.     

Diacylglycerol: Formation and function in phospholipid-mediated signal transduction

1. Properties, distribution and multiplicity of phosphoinositidases (phospholipase C, PLC) are investigated.2. Generation of diacylglycerol (DAG) by a variety of enzymes such as phosphoinositide and phosphatidylcholine specific PLC, by a combination of phospholipase D and phosphatidic hydrolase, and by triglyceride lipase is examined.3. Ca²⁺ and phospholipid-dependent protein kinase C act as the target of DAG messenger action.4. There are differences in the formation of DAG in normal and transformal cell.     

Synergistic Action of Postsynaptic α-Adrenergic Receptor Stimulation on Vasoactive Intestinal Polypeptide-Induced Increases in Pineal N-Acetyltransferase Activity

The alpha-adrenergic agonists phenylephrine and methoxamine, at concentrations that have little effect on pineal N-acetyltransferase activity, markedly enhance stimulation of this enzyme by vasoactive intestinal polypeptide (VIP). This augmentation can be blocked by the alpha 1-adrenergic antagonists phenoxybenzamine and prazosin and, at 10 but not 1 microM, by the alpha 2-antagonist yohimbine. The time course for VIP stimulation is not altered by concomitant alpha-adrenergic stimulation. Augmented activity does not require concomitant alpha-adrenergic stimulation, but alpha-adrenergic agonists must be present for augmentation to be maintained. Phorbol 12,13-diacetate or -dibutyrate but not 4 alpha-phorbol can substitute for phenylephrine, a finding suggesting that protein kinase C is involved in the augmentation. These results are, in general, analogous to alpha-adrenergic magnification of N-acetyltransferase induction by beta-adrenergic agonists.     

Arachidonate activation of protein kinase C may be involved in the stimulation of protein synthesis by insulin in L6 myoblasts

Insulin stimulated protein synthesis in L6 myoblasts but did not increase the labelling of DAG or the release of phosphocholine from phosphatidylcholine. The DAG lipase inhibitor, RHC 80267, more than doubled the amount of label appearing in DAG but did not stimulate protein synthesis. Even in the presence of the DAG lipase inhibitor insulin failed to have any effect on DAG labelling, and conversely RHC 80267 did not modify the insulin-induced increase in protein synthesis. These results suggest that endogenous DAG production is not involved in the stimulation of protein synthesis by insulin. However, exogenous diacylglycerols (1-oleoyl-2-acetyl glycerol and 1-stearoyl-2-arachidonoyl glycerol) both stimulated protein synthesis in L6 myoblasts. The efficacy of the former (arachidonatefree) DAG suggested that their action was by activation of protein kinase C rather than by arachidonate release and prostaglandin formation. Ibuprofen, an inhibitor of cyclo-oxygenase failed to block the effects of insulin whereas a second cyclo-oxygenase inhibitor, indomethacin had only a partial inhibitory effect. The protein kinase C (PKC) inhibitor, RO-31-8220, totally blocked the effect of insulin. Since indomethacin is also recognised to inhibit phospholipase A₂, the data suggests that insulin acts on protein synthesis in myoblasts by arachidonate activation of PKC.     

Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

Calcium-dependent inhibition of renin secretion: TMB-8 is a non-specific antagonist

Intracellular Ca (Cai) is an inhibitory second messenger in renin secretion, and it has been hypothesized that some first messengers--especially angiotensin II [A-II] and antidiuretic hormone [ADH], and possibly A1-adenosine receptor antagonists as well--increase Cai and thereby inhibit renin secretion by causing the release or mobilization of Ca from intracellular sites of sequestration. The present experiments were designed to test this hypothesis, by using 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8), a putative antagonist of Ca release from intracellular sequestration sites. The rat renal cortical slices preparation was used. Basal renin secretory rate was unaffected by 1 and 10 microM TMB-8, but more than doubled in response to 100 microM TMB-8. Basal renin secretory rate was inhibited by A-II (1 microM), by ADH (200 units/1), by an A1-adenosine receptor agonist (N6-cyclohexyladenosine, or CHA; 0.5 microM), and by an alpha-adrenergic agonist (methoxamine; 10 microM). Only the inhibitory effect of methoxamine was blocked by 1 and 10 microM TMB-8, but these concentrations had no effect on basal secretory rate. At 100 microM, TMB-8 blocked the inhibitory effects of ADH as well as of methoxamine, but failed to block the inhibitory effects of CHA and A-II. However, these observations cannot be taken as evidence that methoxamine and ADH, but not CHA and A-II, inhibit renin secretion by a mechanism involving release of Ca from intracellular sequestration sites, because 100 microM TMB-8 clearly had non-specific effects. Among them, it completely blocked the inhibitory effect of K-depolarization on renin secretion. Collectively, at least three separate actions of TMB-8 must be invoked to explain the present results. Likely candidates are an Na-channel blocking effect and a Ca channel blocking effect in addition to antagonism of the release of Cai.     

A fundamental temperature-dependent difference between beta-adrenoceptor agonists and antagonists

Positive inotropic and chronotropic responses of guinea-pig isolated left and right atria to sympathomimetic amines were examined at bath temperatures of 38, 30 or 25 degrees C. The concentration-response curves to isoproterenol and orciprenaline were displaced to the left by cooling, indicating hypothermia-induced supersensitivity. The affinities of isoproterenol and orciprenaline were determined as their dissociation constants (pKA) from antagonism of their responses by either the functional antagonist carbachol or Ro 03-7894 which is reported to be an irreversible beta-adrenoceptor antagonist. By both methods of calculation, the affinities of isoproterenol and orciprenaline for the beta-adrenoceptors mediating inotropic and chronotropic responses were increased by lowering the temperature. In contrast, the affinity of practolol, measured as the pA2 for competitive antagonism of the isoproterenol- and orciprenaline-induced inotropic and chronotropic responses, did not increase with cooling. Thus hypothermia-induced supersensitivity is associated with an increase in agonist affinity only, which indicates a fundamental temperature-dependent difference between agonist and antagonist interactions with the beta-adrenoceptor.     

Angiotensin stimulates phosphatidylcholine synthesis via a pathway involving diacylglycerol, protein kinase C, ERK1/2, and CTP:phosphocholine cytidylyltransferase

We are probing the regulation of phosphatidylcholine (PC) synthesis by angiotensin II. In the accompanying paper, we showed that manipulation of the lipid second messengers, arachidonic acid or hydroxyeicosatetraenoic acid, produced downstream of the angiotensin AT1a receptor did not affect the PC synthesis rates in a manner consistent with direct activation of the rate limiting enzyme in the pathway, CTP:phosphocholine cytidylyltransferase (CCT). However, suppression of diacylglycerol (DAG) production with an inhibitor of phospholipase C-beta reduced angiotensin-dependent PC synthesis as well as ERK1/2 phosphorylation. Here, we show that the stimulation of PC synthesis and activation of CCT by angiotensin requires a signaling pathway that involves protein kinase C and ERK1/2. The inhibitors bis-indolylmaleimide I and PD98059 blocked ERK1/2 phosphorylation and completely eliminated angiotensin stimulation of the CCT-catalyzed reaction and PC synthesis. Exogenous addition of DAG using a lipid vesicle delivery system exactly mimicked the kinetics of angiotensin-promoted PC synthesis, suggesting that this mode of DAG delivery can effectively substitute for the DAG generated downstream of the activated AT1a receptor. Moreover, exogenous DAG activated ERK1/2, and the activation of PC synthesis by DAG was blocked by inhibition of protein kinase C and MEK. These data suggest that angiotensin-dependent DAG and the exogenously supplied DAG stimulate PC synthesis, not solely by direct action on CCT, but via a signaling pathway involving protein kinase C and ERK1/2. Angiotensin did not alter the net phosphorylation state of CCT as probed by immunoprecipitation of 32P-labeled CCT. Angiotensin stimulation of ERK1/2 likely mediates effects on CCT via a process other than CCT dephosphorylation.     

Adrenergic control of myocardial contractile function in rats with incorporated 137Cs]

It was shown on isolated myocardium preparations that internal gamma irradiation of rats with 137Cs (1.6 MBq/kg) decreased the contractile function of ventricles and had no essential effect on the auricle contractility. The inotropic response of myocardium to activation of alpha-adrenergic receptors decreased, and of beta-adrenergic receptors decreased.     

Alpha-adrenergic inhibition of cyclic AMP accumulation in hamster adipocytes. Similarity of receptor with alpha-2 adrenergic receptors

The present communication shows the effects of several alpha-adrenergic agonists and antagonists on cyclic AMP levels in hamster epididymal adipocytes. In response to ACTH (30 mU/ml) in combination with 1-methyl-3-isobutylxanthine (0.10 mM) or adenosine deaminase (1.0 micrograms/ml), cyclic AMP levels increased to a maximum by 10 min and this level was maintained for another 20 min. Elevated cyclic AMP levels were partially suppressed by the alpha-adrenergic agents clonidine, methoxamine, methyl norepinephrine and phenylephrine. The lowest effective concentration of each of these agonists required to suppress cyclic AMP levels was 10 nM clonidine; 3 microM methoxamine; 10 microM methyl norepinephrine; 10 microM phenylephrine. Clonidine and methoxamine suppressed cyclic AMP levels by nearly 65% while phenylephrine and methyl norepinephrine caused only a 30% decline. Studies of the relative potencies of alpha-adrenergic blocking drugs on prevention of the inhibitor effect of clonidine on cyclic AMP levels disclosed that phentolamine and yohimbine were more potent blockers of clonidine action than phenoxybenzamine and prazosin. The rank order of potencies of agonists at causing suppression of cyclic AMP levels and the rank order of potencies of antagonists of clonidine action suggest similarity of the alpha-adrenergic receptors present on hamster adipocytes, which affect cyclic AMP accumulation to alpha-2 adrenergic receptors.     

The effect of alpha-adrenergic agonists on the membrane potential of fat-cell mitochondria in situ.

1. The accumulation of [3H]methyltriphenylphosphonium by isolated fat-cells was used to estimate the membrane potential of mitochondria in situ. 2. An alpha-adrenergic receptor-mediated decrease in the apparent accumulation of [3H]methyltriphenylphosphonium was observed. Methoxamine, clonidine and low concentrations of phenylephrine decreased the calculated mitochrondrial membrane potential without significantly raising cyclic AMP levels, adenylate cyclase activity or stimulating lipolysis. The agonist potency order was phenylephrine greater than methoxamine greater than clonidine. 3. The decrease in the calculated mitochondrial membrane potential caused by phenylephrine, clonidine and methoxamine was blocked by the alpha-adrenergic antagonist prazosin but not by yohimbine nor by the beta-antagonist propranolol. This suggests that the effect on the calculated mitochondrial membrane potential may be mediated by alpha 1-like receptors.     

Sensitivity changes to inotropic agents in rabbit atria after chronic experimental diabetes

The effect of experimental diabetes on the sensitivity of isolated left atrial strips to inotropic agents was investigated in rabbits made diabetic with alloxan. After 4 weeks of diabetes no change in sensitivity was detected in response to isoproterenol or ouabain. In contrast, 15 weeks of diabetes induced a decreased sensitivity to beta-adrenergic stimulation, exhibited as a shift to the right in concentration-response curves obtained in response to isoproterenol and noradrenaline. In addition, after 15 weeks of diabetes the inotropic response to ouabain was depressed, and a small decrease in sensitivity was detected in response to forskolin. In contrast, no significant changes in the concentration-response curves obtained from alpha-adrenergic stimulation by phenylephrine or calcium chloride were detected. Unlike the streptozotocin diabetic rat, which exhibits low serum thyroid hormone levels, no changes in serum thyroid hormones were detected in the alloxan diabetic rabbit. It is suggested that the increased inotropic sensitivity to alpha-adrenergic agonists observed in the diabetic rat, but not in the rabbit, may be due to low serum thyroid hormone levels. In contrast, the deleterious effects of diabetes on beta-adrenergic and ouabain sensitivity occur independently of changes in serum thyroid hormones.     

The diacylglycerol and protein kinase C pathways are not involved in insulin signalling in primary rat hepatocytes.

Diacylglycerol (DAG) and protein kinase C (PKC) isoforms have been implicated in insulin signalling in muscle and fat cells. We evaluated the involvement of DAG and PKC in the action of insulin in adult rat hepatocytes cultured with dexamethasone, but in the absence of serum, for 48 h. Our results show that although insulin stimulated glycolysis and glycogen synthesis, it had no effect on DAG mass or molecular species composition. Epidermal growth factor showed the expected insulin-mimetic effect on glycolysis, whereas ATP and exogenous phospholipase C acted as antagonists and abolished the insulin signal. Similarly to insulin, epidermal growth factor had no effect on DAG mass or molecular species composition. In contrast, both ATP and phospholipase C induced a prominent increase in several DAG molecular species, including 18:0/20:4, 18:0/20:5, 18:0/22:5 and a decrease in 18:1/18:1. These changes were paralleled by an increase in phospholipase D activity, which was absent in insulin-treated cells. By immunoblotting or by measuring PKC activity, we found that neither insulin nor ATP translocated the PKCalpha, -delta, -epsilon or -zeta isoforms from the cytosol to the membrane in cells cultured for six or 48 h. Similarly, insulin had no effect on immunoprecipitable PKCzeta. Suppression of the glycogenic insulin signal by phorbol 12-myristate 13-acetate, but not by ATP, could be completely alleviated by bisindolylmaleimide. Finally, insulin showed no effect on DAG mass or translocation of PKC isoforms in the perfused liver, although it reduced the glucagon-stimulated glucose output by 75%. Together these results indicate that phospholipases C and D or multiple PKC isoforms are not involved in the hepatic insulin signal chain.     

Contribution of lipid second messengers to the regulation of phosphatidylcholine synthesis during cell cycle re-entry

During entry into the cell cycle a phosphatidylcholine (PC) metabolic cycle is activated. We have examined the hypothesis that PC synthesis during the G(0) to G(1) transition is controlled by one or more lipid products of PC turnover acting directly on the rate-limiting enzyme in the synthesis pathway, CTP: phosphocholine cytidylyltransferase (CCT). The acceleration of PC synthesis was two- to threefold during the first hour after addition of serum to quiescent IIC9 fibroblasts. The rate increased to approximately 15-fold above the basal rate during the second hour. The production of arachidonic acid, diacylglycerol (DAG), and phosphatidic acid (PA) preceded the second, rapid phase of PC synthesis. However, an increase in the cellular content of these lipid mediators was detected only for DAG. CCT activation and translocation to membranes accompanied the second phase of the PC synthesis acceleration. Bromoenol lactone (BEL), an inhibitor of calcium-independent phospholipase A(2) and PA phosphatase, blocked production of fatty acids and DAG, inhibited both phases of the PC synthesis response to serum, and reduced CCT activity and membrane affinity. The effect of BEL on PC synthesis was partially reversed by in situ generation of DAG via exogenous PC-specific phospholipase C to generate approximately 2-fold elevation in PC-derived DAG. Exogenous arachidonic acid also partially reversed the inhibition by BEL, but only at a concentration that generated a supra-physiological cellular content of free fatty acid. 1-Butanol, which blocks PA production, had no effect on DAG generation, or on PC synthesis. We conclude that fatty acids and DAG could contribute to the initial slow phase of the PC synthesis response. DAG is the most likely lipid regulator of CCT activity and the rapid phase of PC synthesis. However, processes other than direct activation of CCT by lipid mediators likely contribute to the highly accelerated phase during entry into the cell cycle.     

Phospholipase C may be involved in norepinephrine-induced cardiac hypertrophy

Cardiac hypertrophy is characterized by increased cardiomyocyte size, mRNA levels for atrial natriuretic factor (ANF), and protein synthesis. Although activation of the phosphoinositide-specific phospholipase C (PLC) leads to the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, the involvement of PLC in hypertrophic response remains to be fully understood. The present study was therefore undertaken to examine if the inhibition of PLC activity is associated with a decrease in ANF expression and protein synthesis in cardiomyocytes, due to norepinephrine (NE), a known hypertrophic agent. NE resulted in an increase in ANF gene expression and protein synthesis in adult rat cardiomyocytes, these effects of NE were attenuated by a PLC inhibitor, U73122. The NE-induced increase in ANF gene expression and protein synthesis was also inhibited by an alpha-adrenoceptor blocker, prazosin. Both U73122 and prazosin depressed the NE-induced increase in DAG production in cardiomyocytes. These results indicate that the alpha-adrenoceptor mediated PLC activation may be involved in the process of NE-induced cardiac hypertrophy.     

Modulation of the biologic activities of IgE-binding factors. VII. Biochemical mechanisms by which glycosylation-enhancing factor activates phospholipase in lymphocytes

Cells of the T cell hybridoma 23A4 produce IgE-binding factors lacking N-linked oligosaccharides (unglycosylated form) when they are incubated with IgE alone. In the presence of glycosylation-enhancing factor (GEF) or bradykinin, however, the same cells produce IgE-binding factors with N-linked oligosaccharides (glycosylated form). Switching the cells from the formation of unglycosylated IgE-binding factors to the formation of glycosylated factors was accompanied by the release of both glycosylation-inhibiting factor (GIF) in its phosphorylated form, i.e., phosphorylated lipomodulin, and arachidonate from the cells. Analysis of the biochemical processes for the release of GIF from 23A4 cells showed that affinity-purified GEF or bradykinin induced transient phospholipid methylation and diacylglycerol (DAG) formation, and enhanced 45Ca uptake into the cells. Inhibitors of methyltransferases, i.e., 3-deaza-adenosine plus L-homocysteine thiolactone, inhibited not only phospholipid methylation but also DAG formation and GIF release. Exogenously added 1-oleoyl-2-acetyl glycerol, i.e., a DAG that is permeable to the plasma membrane, induced the release of GIF from the cells. It was also found that 12-O-tetradecanoyl-phorbol 13-acetate (TPA) switched 23A4 cells and normal lymphocytes to the selective formation of N-glycosylated IgE-binding factor, and induced the release of GIF from the cells. 32PO4-labeled lipomodulin was detected in the extract of 23A4 cells 3 to 5 min after the addition of GEF, bradykinin, or TPA. These results indicate that GEF and bradykinin induced the activation of methyltransferases and phospholipase C for the formation of DAG, which in turn activated Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) for the phosphorylation of lipomodulin. Because lipomodulin loses phospholipase inhibitory activity after phosphorylation, increased phospholipase A2 activity would be expressed by this process.