Diacylglycerol Protein Kinase C Research Articles

Apoptosis of beta cells in diabetes mellitus.

Diabetes mellitus is a multifactorial metabolic disorder characterized by hyperglycemia. Apoptosis in beta cells has been observed in response to diverse stimuli, such as glucose, cytokines, free fatty acids, leptin, and sulfonylureas, leading to the activation of polyol, hexosamine, and diacylglycerol/protein kinase-C (DAG/PKC) pathways that mediate oxidative and nitrosative stress causing the release of different cytokines. Cytokines induce the expression of Fas and tumor necrosis factor-alpha (TNF-α) by activating the transcription factor, nuclear factor-κb, and signal transducer and activator of transcription 1 (STAT-1) in the β cells in the extrinsic pathway of apoptosis. Cytokines produced in beta cells also induce proapoptotic members of the intrinsic pathway of apoptosis. The genetic alterations in apoptosis signaling machinery and the pathogenesis of diabetes include Fas, FasL, Akt, caspases, calpain-10, and phosphatase and tensin homolog (Pten). The other gene products that are involved in diabetes are nitric oxide synthase-2 (NOS2), small ubiquitin-like modifier (SUMO), apolipoprotein CIII (ApoCIII), forkhead box protein O1 (FOXO1), and Kruppel-like zinc finger protein Gli-similar 3 (GLIS3). The gene products having antiapoptotic nature are Bcl-2 and Bcl-XL. Epigenetic mechanisms play an important role in type I and type II diabetes. Further studies on the apoptotic genes and gene products in diabetics may be helpful in pharmacogenomics and individualized treatment along with antioxidants targeting apoptosis in diabetes.

Effect of intervention in the diacylglycerol-protein kinase C signaling pathway on JNK1 expression and its downstream signaling in diabetic cardiomyopathy

This study aimed to investigate the expression of signaling molecules, such as c‑Jun N‑terminal kinase 1 (JNK1) and insulin receptor substrate 1 (IRS1), in the myocardium of diabetic rats following intervention in the diacylglycerol‑protein kinase C (DAG‑PKC) signal transduction pathway. The rats were divided into three groups, the diabetic model, control and breviscapine‑treated diabetes (intervention) group. Following modeling and drug treatment, hematoxylin and eosin (HE) and Masson staining and electron microscopy were used to observe the pathological changes in the rat myocardium. The expression of PKC‑β2, JNK1, and IRS1 was assessed in rat myocardium by immunohistochemistry and quantitative polymerase chain reaction (qPCR). The expression levels of PKC‑β2, JNK1, phosphorylated JNK (p‑JNK) and IRS1 in the diabetic model group were significantly higher than those in the control group. Furthermore, compared with the diabetic model group, expression levels of PKC‑β2, JNK1, p‑JNK and IRS1 were significantly reduced following intervention in the DAG‑PKC signal transduction pathway. The DAG‑PKC pathway may affect downstream signaling through JNK1 (the common signal point of the G‑protein receptor pathway and insulin receptor pathway at the cell membrane) to result in the occurrence and development of diabetic cardiomyopathy (DCM). The series of signal points DAG‑PKC‑JNK1‑IRS1‑Akt/PKB‑mTOR‑p70S6K1 is a potential pathway for inducing DCM by DAG‑PKC signal transduction. Enhanced expression of JNK1, p‑JNK and IRS1 may accelerate diabetic myocardial fibrosis.

Diacylglycerol kinase α exacerbates cardiac injury after ischemia/reperfusion

Early coronary reperfusion of the ischemic myocardium is a desired therapeutic goal for the preservation of myocardial function. However, reperfusion itself causes additional myocardium injuries. Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade has been implicated in the cardioprotective effects occurring after ischemia/reperfusion (I/R). DAG kinase (DGK) controls cellular DAG levels by converting DAG to phosphatidic acid, and may act as an endogenous regulator of DAG-PKC signaling. In the present study, we examined the functional role of DGKα in cardiac injury after I/R in in vivo mouse hearts. We generated transgenic mice with cardiac-specific overexpression of DGKα (DGKα-TG). The left anterior descending coronary artery was transiently occluded for 20min and reperfused for 24h in DGKα-TG mice and wild-type littermate (WT) mice. The levels of phosphorylation activity of PKCε, extracellular-signal regulated kinase (ERK) 1/2, and p70 ribosomal S6 kinase (p70S6K) were increased after I/R in WT mouse hearts. However, in DGKα-TG mice, activation of PKCε, ERK1/2, and p70S6K was attenuated compared to WT mice. After 24h, Evans blue/triphenyltetrazolium chloride double staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that DGKα-TG mice had significantly larger myocardial infarctions and larger numbers of TUNEL-positive cardiomyocytes than WT mice. Echocardiography and cardiac catheterization revealed that left ventricular systolic function was more severely depressed in DGKα-TG mice than in WT mice after I/R. These findings suggest that DGKα exacerbates I/R injury by inhibiting the cardioprotective effects of PKCε, ERK1/2, and p70S6K activation.

Regulatory mechanisms of melanogenesis in human skin

Melanin in human skin is synthesized in the melanosomes of melanocytes,and there are many modes of regulation of melanogenesis,such as transcriptional regulation,regulation by intracellular signaling pathway,feedback regulation by products,and other positive(stimulation)or negative(inhibitory)regulation by nuclear receptors and cytokines.During the process of melanogenesis,microphthalmia transcription factor(MITF)plays a fundamental role in the regulation of tyrosinases and other tyrosinase-related proteins,with cyclic adenosine monophosphate/protein kinase A(cAMP/PKA)pathway,mitogen-activated protein kinase(MAPK)pathway,nitric oxide/cyclic guanosine monophosphate/protein kinase G(NO/cGMP/PKG)pathway and diacylglycerol/protein kinase C(DG/PKC)pathway as the four major intracellular signaling pathways.Some molecules and their receptors play positive(stimulating)roles in melanogenesis,such as G protein-coupled receptors and their ligands,stem cell growth factor and its receptor,nuclear receptors and their ligands,while some molecules and receptors play negative(inhibiting)roles,such as the Agouti signaling protein and its receptor,G protein-coupled receptors and their ligands,as well as cytokines,growth factors and their receptors. Key words: Melanins; Genes,regulator; Monophenol monooxygenase

Changes in the inositol lipid signal system and effects on the secretion of TNF-α by macrophages in severely scalded mice

Aim In order to study the mechanism of abnormal macrophage (Mϕ) function in pro-inflammatory cytokine changes after burn, the inositol lipid signal system and its role in tumour necrosis factor-alpha (TNF-α) secretion by peritoneal Mϕs was observed in severely scalded mice. Methods Eighteen percent total body surface area (TBSA) full-thickness scalded mice were used as animal model in this experiment. Peritoneal Mϕs stimulated by lipopolysaccharide in vitro were collected at different time intervals (0, 2, 6, 12, 24 and 48 after burn hour (PBH)), The activities of phosphatidylinositol-phospholipase C (PI-PLC), inositol-1, 4,5, -triphosphate (IP 3), protein kinase C (PKC), diacylglycerol (DAG) and TNF-α and the level of Ca 2+ concentration in peritoneal Mϕs were measured, and the effects of specific PKC inhibitor H-7 and calmodulin antagonist W-7 on the production of TNF-α were also observed. Results After scald, increased activities of TNF-α and PLC of Mϕ were observed and peaked at 12 PBH. The activities of DAG and IP 3 and the concentration of Ca 2+ were markedly increased and reached their peaks at 24 PBH simultaneously. Membrane PKC activity was up-regulated after scald and showed a positive correlation with the change of DAG ( r = 0.83, P < 0.05). There was also positive correlation between IP 3 and Ca 2+ activity ( r = 0.946, P < 0.01). When 12 PBH was chosen as the time point for in vitro intervention with the pre-treatment by H-7, both membrane PKC and TNF-α activity decreased significantly. There was no obvious change of TNF-α activity with the application of W-7. Conclusions These results indicated that the abnormal activity of TNF-α of Mϕs might be regulated by the inositol lipid signal system following severe burn. The DAG–PKC signal pathway showed closer relationship than IP 3-Ca 2+ in TNF-α production and could be the optimal target in the prevention and treatment of the systemic inflammatory response syndrome.

Reversal of redox-dependent inhibition of diacylglycerol kinase by antioxidants in mesangial cells exposed to high glucose

Activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is one of the pathomechanisms of diabetic nephropathy. We previously reported that d-α-tocopherol, well known as an antioxidant, enhances diacylglycerol kinase (DGK) activity, leading to the reduction of excess DAG accumulation and PKC activation in the glomeruli of streptozotocin-induced diabetic rats. However, it remains to be determined whether the effect of d-α-tocopherol on DGK activity is exerted through its antioxidative action. DAG contents, PKC and membranous DGK activity were measured in cultured human glomerular mesangial cells under normal (5.5 mM) or high glucose (27.5 mM) conditions in the presence or absence of two antioxidants (50 μM d-α-tocopherol and probucol). Mesangial cells were exposed to hydrogen peroxide (H2O2) (10-1000 μM) in the presence or absence of 300 U/ml catalase, followed by measurement of DGK activity. Both antioxidants restored the high glucose-induced decrease in DGK activity, resulting in the reduction of high glucose-induced activation of the DAG-PKC pathway. In mesangial cells exposed to H2O2 at various concentrations, DGK activity decreased in a dose-dependent manner. The addition of antioxidative enzyme catalase to the cells reversed the H2O2-mediated down-regulation of DGK activity. In conclusion, DGK activity is reduced by oxidative stress in human mesangial cells cultured under high glucose conditions. Antioxidants, including d-α-tocopherol and probucol may improve hyperglycemia-induced DAG-PKC activation by enhancing DGK activity.

Role of protein kinase C in diabetic complications

Hyperglycemia is an important factor in the development of macrovascular and microvascular complications in all diabetic patients. Several hypotheses have been postulated to explain the adverse effect of hyperglycemia on the vasculature; and one of these hypotheses is the activation of specific isoforms of protein kinase C (PKC) by diabetes. In this review, we summarize the molecular mechanisms of PKC activation and its relationship to diabetic complications. PKC activity regulates vascular permeability, contractility, extracellular matrix synthesis, hormone receptor turnover and proliferation, cell growth, angiogenesis, cytokine activation and leukocyte adhesion. All of these properties are abnormal in diabetes and are correlated with increased diacylglycerol–PKC pathway and PKCα, β1/2 and δ isoforms activation in the retina, aorta, heart and renal glomeruli.

PKC Inhibition and Diabetic Complications

Hyperglycemia activates several signaling pathways associated with vascular complications in diabetes. Hyperfunction of the diacylglycerol - protein kinase C (DAG-PKC) pathway can lead to enhanced vascular permeability, endothelial cell activation and expression of leukocyte adhesion molecules, altered blood flow and abnormal growth factor signaling. One of the most important approaches aimed at modulating these adverse effects involves the inhibition of PKC, and several inhibitory compounds have been proposed including ruboxistaurin (Eli Lilly & Co.), PKC412 and PKC412-A (Novartis Pharmaceuticals). Other patents with similar function has also been proposed such as substituted pyrrolines kinases inhibitors (US200698710B2; US2006020576A1; WOO4094422A1; US2006987110) modulation of angiogenesis (W007035782A2; US20070141040A1). This present approach intends to modulate of angiogensis by the inhibition of PKC beta as an monotherapy or in combination with other antioangiogenic compounds. Some of these compounds are currently tested in a clinical trial while other are on intensive investigation. This brief review considers therapeutic strategies with new drugs and/or new approaches using PKC inhibitors to treat diabetic complications. Keywords: Diabetes, vascular complications, angiogenesis, PKC inhibitors, VEGF inhibitors, ruboxistaurin, PKC 142, PKC 142-A

Abstract 165: Diacylglycerol kinase α Exacerbates Cardiac Injury After Ischemia/reperfusion In Transgenic Mouse Hearts

Background: Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade has been implicated in cardiac dysfunction after ischemia/reperfusion (I/R). DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid, and thus may act as a regulator of DAG-PKC signaling. It has been reported that α, ϵ, and &amp;#03B6; isoforms of DGK have been identified in the heart. We examined the functional role of DGKα in cardiac injury after I/R. Methods and Results: We generated transgenic mice with cardiac specific overexpression of DGKα (DGKα-TG) using α-myosin heavy chain promoter. Left anterior descending coronary artery was transiently occluded for 30 min and reperfused for 24 hr in DGKα-TG and wild type littermate (WT) mice. After 24 hr, echocardiography was performed, mice were sacrificed, and the hearts were removed. Areas at risk and infarct size were assessed by Evans blue/TTC double staining. Left ventricular chamber dilatation after I/R was more pronounced in DGKα-TG mice than in WT mice (left ventricular end-diastolic dimension: 3.44 +/− 0.41 mm vs. 3.05 +/−0.24 mm, P &lt; 0.01). Left ventricular systolic function was more severely depressed in DGKα-TG mice than in WT mice (left ventricular fractional shortening: 34.3 +/− 3.6% vs. 42.2 +/− 4.0%, P &lt; 0.01). The ratio of infarct size/area at risk at 24 hr after I/R was larger in DGKα-TG mice than in WT mice (17.9 +/− 4.2% vs. 11.3 +/− 3.3%, P &lt; 0.01). The phosphorylation activity of extracellular-signal regulated kinase (ERK) was increased after I/R in WT mouse hearts (2.58 +/−0.52-fold increase, P&lt;0.0001). However in DGKα-TG mice, activation of ERK after I/R was abolished (1.77+/−0.67-fold increase, P&lt;0.01 vs. WT). Conclusion: DGKα exacerbates cardiac injury by inhibiting cardio-protective effect of ERK activation.

Effect of cholecystokinin octapeptide on diacylglycerol-PKC signaling pathway in rat pulmonary interstitial macrophages stimulated by lipopolysaccharide1

To investigate the effect of cholecystokinin octapeptide (CCK-8) on the diacylglycerol-protein kinase C (DAG-PKC) signaling pathway in rat pulmonary interstitial macrophages (PIM) stimulated by lipopolysaccaride (LPS). The PIM from rat lung tissues were isolated using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. DAG content and PKC activity were measured by radioenzymatic assay. The translocation of PKCzeta was determined by semi-quantitative immunoblot analysis. CCK-8, at high concentrations (1 x 10(-6) - 1 x 10(-5) mol/L), decreased DAG content and inhibited PKC activity and PKCzeta translocation compared with that in rat resting PIM of a control group (P< 0.01). LPS increased DAG content, and promoted PKC activity and PKCzeta translocation (P< 0.01). CCK-8 decreased LPS-induced DAG content and inhibited LPS-induced PKC activity and PKCzeta translocation significantly at 1 x 10(-8) - 1 x 10(-5) mol/L (P< 0.01). This inhibitory effect of CCK-8 could be abrogated partly by proglumide (non-selective CCK receptor antagonist), CR-1409 (selective CCK-A receptor antagonist), and CR-2945 (selective CCK-B receptor antagonist) in a concentration-dependent manner (P< 0.01). CCK-8 was a negative modulator of the DAG-PKC signaling pathway in rat resting PIM, which is very important for maintaining body homeostasis. It significantly inhibited LPS-induced DAG content, PKC activity and PKCzeta translocation in a concentration-dependent manner. The CCK receptor, especially the CCK-A receptor, might play a major role in this process.

Effect of CD40–CD40 ligand interaction on diacylglycerol–protein kinase C and inositol trisphosphate-Ca 2+ signal transduction pathway in human umbilical vein endothelial cells

Background: Increasing evidence shows that CD40–CD40L interaction plays a crucial role in the pathogenesis of atherosclerosis and coronary artery disease. The mechanism of CD40–CD40L interaction might be related to signal transduction via receptor. The transduction pathway of the CD40 receptor may involve the activation of phospholipase C (PLC) which induces the production of inositol trisphosphate (IP 3) leading to the increase of the intracellular free calcium on one hand, and of diacylglycerol (DAG) which stimulates the translocation to the membrane of protein kinase C (PKC). Methods: Endothelial cells were isolated from human umbilical vein and incubated with indicated concentrations of CD40 ligand (CD40L) for various periods. The DAG levels in HUVEC were studied with radioenzymatic assay. Quantitative measurements of 32P phosphatidic acid were performed by thin-layer chromatography and autoradiography. IP 3 was quantitatively measured by the radioreceptor binding assay. The activity of PKC and [Ca 2+]i induced by CD40L were measured by its ability to transfer phosphate from [γ- 32P]ATP to lysine-rich histone and flow cytometric analysis loading with the Ca 2+ dye fluo3/Am, respectively. Results: The DAG levels were raised by CD40L in a dose-dependent, biphasic manner. The early phase was rapid and transient, peaking at 20 s; and the late phase reached the maximal level at 10 min and then decayed slowly. CD40L increased the PKC total activity in a dose-dependent manner with phase peaking at 12 min, then decreased slowly and maintained for at least 20 min. The results also showed that CD40L induced PKC activity translocation from the cytosolic to membrane. Similarly, the CD40L-induced transient IP 3 formation was coincident with the first peak of DAG formation. Moreover, CD40L also induced biphasic [Ca 2+]i responses including the rapid initial transient phase and the sustained phase. Anti-CD40 monoclonal antibody can significantly suppress CD40L-induced DAG–PKC and IP 3–[Ca 2+]i signal pathway activation in HUVEC. Conclusions: CD40–CD40 ligand interaction can induce a robust stimulation of the DAG–PKC and inositol trisphosphate–Ca 2+ signal transduction pathway in HUVEC.

Diacylglycerol production and protein kinase C activity are increased in a mouse model of diabetic embryopathy.

Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade by excess glucose has been implicated in vascular complications of diabetes. Its involvement in diabetic embryopathy has not been established. We examined DAG production and PKC activities in embryos and decidua of streptozotocin (STZ)-diabetic or transiently hyperglycemic mice during neural tube formation. STZ diabetes significantly increased DAG and total PKC activity in decidua (1.5- and 1.4-fold, respectively) and embryos (1.7- and 1.3-fold, respectively) on day 9.5. Membrane-associated PKC alpha, betaII, delta, and zeta were increased in decidua by 1.25- to 2.8-fold. Maternal hyperglycemia induced by glucose injection on day 7.5, the day before the onset of neural tube formation, also increased DAG, PKC activity, and PKC isoforms (1.1-, 1.6-, and 1.5-fold, respectively) in the embryo on day 9.5. Notably, membrane-associated PKC activity was increased 24-fold in embryos of diabetic mice with structural defects. These data indicate that hyperglycemia just before organogenesis activates the DAG-PKC cascade and is correlated with congenital defects.

Drug-induced and postnatal hypothyroidism impairs the accumulation of diacylglycerol in liver and liver cell plasma membranes.

Thyroid hormones are well known modulators of signal transduction. The effect of hyper- and hypo-thyroidism on diacylglycerol/protein kinase C (DAG/PKC) signaling in cardiomiocytes has been determined. Triiodothyronine (T3) has been shown to prevent the alpha1-adrenoreceptor-mediated activation of PKC but does not alter the stimulation of enzyme and hepatic metabolism by phorbol ethers. It has been suggested that the elevation of endogenous DAG in senescent or hypothyroid cells changes the PKC-dependent response of cells to phorbol esters and hormones. In the present study, was examined the formation of DAG and activation of PKC in liver cells from rats of different thyroid status. The results obtained provide the first demonstration of DAG accumulation in liver and cell plasma membranes at age- and drug-dependent thyroid gland malfunction. The experiments were performed in either the [14C]CH3COOH-labeled rat liver, liver slices or hepatocytes labeled by [14C] oleic acid and [3H]arachidonic acid or [14C]palmitic acid as well as in the isolated liver cell plasma membranes of 90- and 720-day-old rats of different thyroid status. The decrease of T4 and T3 levels in blood serum of 720-day-old rats and mercazolil-treated animals was associated with increases of both the DAG mass in liver and liver cell plasma membranes and newly synthesized [14C]DAG level in liver and isolated hepatocytes. Hypothyroidism decreased PKC activity in both membrane and cytosol as well as phospholipid and triacylglycerol synthesis in liver. These hypothyroidism effects were restored in liver by injection of T4. T4 administration to the intact animals of different ages decreased the DAG level in liver and isolated plasma membranes and the content of newly synthesized DAG in liver. The reduction of DAG level in liver was not associated with increasing free fatty acid level. DAG labeling ratio 14C/3H in liver slices of rats of different thyroid state sharply differed from PL. DAG was relatively enriched in [14C]oleic acid whereas PL were enriched in [3H]arachidonic acid. The above data have indicated that thyroid hormones are important physiological modulators of DAG level in rat liver and cell plasma membranes. Age- and drug-induced malfunction of thyroid gland resulted in a prominent decrease of glycerolipid synthesis which may promote DAG accumulation in liver.

Tissue and isoform-selective activation of protein kinase C in insulin-resistant obese Zucker rats - effects of feeding.

The mechanisms of insulin resistance in the obese Zucker rat have not been clearly established but increased diacylglycerol-protein kinase C (DAG-PKC) signalling has been associated with decreased glucose utilisation in states of insulin resistance and non-insulin-dependent diabetes mellitus. The purpose of this study was to characterise tissue- and isoform-selective differences in DAG-PKC signalling in insulin-sensitive tissues from obese Zucker rats, and to assess the effects of feeding on DAG-PKC pathways. Groups of male obese (fa/fa, n=24) and lean (fa/-, n=24) Zucker rats were studied after baseline measurements of fasting serum glucose, triglycerides, insulin and oral glucose tolerance tests. Liver, epididymal fat and soleus muscle samples were obtained from fed and overnight-fasted rats for measurements of DAG, PKC activity and individual PKC isoforms in cytosol and membrane fractions. Obese rats were heavier (488+/-7 vs 315+/-9 g) with fasting hyperglycaemia (10.5+/-0.8 vs 7.7+/-0.1 mM) and hyperinsulinaemia (7167+/-363 vs 251+/-62 pM) relative to lean controls. In fasted rats, PKC activity in the membrane fraction of liver was significantly higher in the obese group (174+/-16 vs 108+/-12 pmol/min/mg protein, P<0.05) but there were no differences in muscle and fat. The fed state was associated with increased DAG levels and threefold higher PKC activity in muscle tissue of obese rats, and increased expression of the major muscle isoforms, PKC-theta and PKC-epsilon: e.g. PKC activity in the membrane fraction of muscle from obese animals was 283+/-42 (fed) vs 107+/-20 pmol/min/mg protein (fasting) compared with 197+/-27 (fed) and 154+/-21 pmol/min/mg protein (fasting) in lean rats. In conclusion, hepatic PKC activity is higher in obese rats under basal fasting conditions and feeding-induced activation of DAG-PKC signalling occurs selectively in muscle of obese (fa/fa) rats due to increased DAG-mediated activation and/or synthesis of PKC-theta and PKC-epsilon. These changes in PKC are likely to exacerbate the hyperglycaemia and hypertriglyceridaemia associated with obesity-induced diabetes.

Protein Kinase C and Exocytosis in Mammalian Spermatozoa

The recent papers by O'Toole et al. (1996)and Liu and Baker (1997)are further steps towards ascribing a role for protein kinase C (PKC) in the acrosome reaction (Breitbart et al., 1992, Rotem et al., 1992). The source of Ca2+ (intracellular versus extracellular pools) and diacylglycerol (DAG) [phosphatidylinositol (4,5)-bisphosphate (PIP2) versus phosphatidylcholine (PC)] during sperm activation requires extensive and accurate measurements of lipid messenger molecules and inositol (1,4,5)-trisphosphate (IP3) formation, which are under investigation. Indeed, it was demonstrated that IP3-sensitive Ca2+ pools are present in activated mammalian sperm (Walensky and Snyder, 1995). We still favour the model that phosphoinositide turnover is an early event in sperm activation that provides IP3 for Ca2+ mobilization and DAG for PKC activation (Naor and Breitbart, 1997). Concerning the signal transduction events leading to phospholipase A2 (PLA2) activation, we favour the DAG–PKC–mitogen activated protein kinase–PLA2 pathway during sperm activation, and studies are in progress to confirm this pathway. Experiments are also underway to identify further mammalian sperm PKC subspecies, their endogenous substrates, the modes of their respective activation and their relative physiological roles in sperm–egg interaction.

Inositol lipids and signal transduction in the nervous system: an update.

Abstract: The role that inositol lipids play in cellular signaling events in eukaryotic cells remains one of the most intensively investigated areas of cell biology. In this respect, phosphoinositide‐mediated signal transduction in the CNS is no exception; major advances have been made since a previous review on this subject (Fisher and Agranoff, 1987). Not only have stimulated phosphoinositide turnover and its physiological sequelae been demonstrated repeatedly in a variety of neural preparations, but, in addition, the detailed molecular mechanisms underlying these events continue to unfold. Here we review the progress that has occurred in selected aspects of this topic since 1987. In the first two sections of this article, emphasis is placed on novel functional roles for the inositol lipids and on recent insights into the molecular characteristics and regulation of three key components of the phosphoinositide signal transduction system, namely, the inositol lipid kinases, phospholipases C (PLCs), and the inositol 1,4,5‐trisphosphate[I(1,4,5)P3] receptor. The metabolic fate of I(1,4,5)P3 in neural tissues, as well as its control, is also detailed. Later we focus on identification of the multiple receptor subtypes that are coupled to inositol lipid turnover and discuss possible strategies for intervention into phosphoinositide‐mediated signal transduction. Due to space limitations, an extensive evaluation of the diacylglycerol/protein kinase C (DAG/PKC) limb of the signal transduction pathway is not included (for reviews, see Nishizuka, 1988; Kanoh et al., 1990).

Erratum

Retinal capillary pericyte is a cell type selectively lost in early diabetic retinopathy. The physiological function of pericytes is not yet clearly identified, although it probably has contractile properties. We determined the specific binding of endothelin 1, a 21-amino acid peptide with potent vasoconstrictive action, and the stimulation of diacylglycerol/protein kinase C (DAG/PKC) pathway in cultured retinal capillary pericytes by endothelin. A single specific binding site for 125I-labeled endothelin was identified, with an apparent Kd of 1.3 nM and a maximal binding capacity of approximately 1-2 x 10(5) sites/cell. Endothelin (100 nM) increased total cellular DAG content by 15% at 5 min and 24% at 10 min. When pericytes were labeled isotopically with [3H]glycerol, endothelin stimulated [3H]DAG formation by 100% at 10 min and 88% at 30 min. After 10 min of endothelin treatment, PKC activities were increased by 60 and 100% in the membranous and cytosolic pools, respectively. We conclude that bovine retinal capillary pericytes possess numerous high-affinity specific binding sites for endothelin that mediate the action of endothelin by the stimulation of the DAG/PKC pathway in pericytes. These findings suggest that endothelin is a regulator of the contractile properties of pericytes, which may be adversely affected in diabetic retinopathy.

Second messenger imbalance hypothesis of schizophrenia

Abstract Based on the results of studies on intracellular signaling in platelets of schizophrenics, an imbalance of the second messenger system is proposed: Diacylglycerol (DG), which activates protein kinase C (PKC), was increased, while adenylate cyclase (AC)-cAMP function was decreased. It is proposed that the increased DG PKC function may entail a decrease in inositol 1,4,5-trisphosphate (IP 3 ) Ca 2+ function and lowering of phosphoinositide turnover. If such a pathological intracellular signaling takes place in the brain, it may cause a distorted balance of protein activation via phosphorylation in neurons, resulting in some of the deficits of schizophrenia. Neuroleptics have been reported to antagonize the above-mentioned pathological processes of intracellular signaling. The imbalance hypothesis of the DG PKC pathway and AC-cAMP pathway is not inconsistent with the dopamine hypothesis of schizophrenia, because dopamine receptor stimulation is indirectly related to reduction in IP 3 Ca 2+ function and lowering of phosphoinositide metabolism.

Protein kinase C activity and contractile responsiveness in senescent blood vessels

To examine Ca 2+-signaling receptor function in the aging vasculature, the status of the diacylglycerol/protein kinase C (DAG/PKC) arm of the signal transduction pathway was assessed. Contractile responsiveness to the PKC activator, phorbol 12-myristate 13-acetate (PMA), is substantially reduced in aortae from 24-month-old Fischer 344 rats compared to 6-month-old rats. PKC activity is reduced in all senescent vessels yet studied including aorta, renal artery, iliac artery and vena cava. This may account for the reduced contractile responsiveness. Additionally, PMA-stimulated PKC translocation is substatially reduced in senescent aorta and this may also contribute to reduced contraction.

Effects of Insulin on Diacylglycerol–Protein Kinase C Signaling in Rat Diaphragm and Soleus Muscles and Relationship to Glucose Transport

Insulin was found to provoke rapid increases in diacylglycerol (DAG) content and [3H]glycerol incorporation into DAG and other lipids during incubations of rat hemidiaphragms and soleus muscles. Insulin also rapidly increased phosphatidic acid and total glycerolipid labeling by [3H]glycerol, suggesting that insulin increases DAG production at least partly through stimulation of the de novo pathway. Increased DAG production may activate protein kinase C (PKC) as reported previously in the rat diaphragm. We also observed apparent insulin-induced translocation of PKC from cytosol to membrane in the rat soleus muscle. The importance of insulin-induced increases in DAG-PKC signaling in the stimulation of glucose transport in rat diaphragm and soleus muscles was suggested by 1) PKC activators phorbol esters and phospholipase C stimulation of [3H]-2-deoxyglucose (DOG) uptake and 2) PKC inhibitors staurosporine and polymixin B inhibition of insulin effects on [3H]-2-DOG uptake. Although phorbol ester was much less effective than insulin in the diaphragm, phospholipase C provoked increases in [3H]-2-DOG uptake that equaled or exceeded those of insulin. In the soleus muscle, phorbol ester, like phospholipase C, was only slightly but not significantly less effective than insulin. Similar variability in effectiveness of phorbol ester has also been noted previously in rat adipocytes (weak) and BC3H1 myocytes (strong), whereas DAG, added exogenously or generated by phospholipase C treatment, stimulates glucose transport to a degree that is quantitatively more comparable to that of insulin in each of the four tissues. Differences in effectiveness of phorbol ester and DAG could not be readily explained by postulating that the latter acts independently of PKC, because DAG provoked the apparent translocation of the enzyme from cytosol to membranes in rat adipocytes, and effects of DAG on [3H]-2-DOG uptake were blocked by inhibitors of PKC in both rat adipocytes and BC3H1 myocytes. Collectively, our findings provide further support for the hypothesis that insulin increases DAG production and PKC activity, and these processes are important in the stimulation of glucose transport in rat skeletal muscle and other tissues.