Effects of second messengers on serine/threonine protein phosphatases in insulin-secreting cells

Reversible protein phosphorylation is an important and versatile mechanism by which cells transduce external signals into biological responses. Cellular levels of protein phosphorylation are determined by the balanced actions of both protein kinases and protein phosphatases (PPases). Compared with protein kinases, however, serine/threonine PPases have received less attention. In the present study, the effects of certain insulin secretagogues and intracellular second messengers, known to stimulate or inhibit insulin secretion, on the activities of cation-independent serine/threonine PPases were investigated in insulin-secreting RINm5F insulinoma cells. Raising cellular cAMP through adenylyl cyclase activation and phosphodiesterase inhibition in intact cells, evoked inhibitory effects on PPase activities. The addition of a nitric oxide donor, cyclic nucleotides, or proinflammatory prostaglandins to RINm5F cell homogenates at widely different concentrations did not affect type-1 or -2A PPase activities. Phosphatidyl serine seemingly activated PPase-1, while inactivating PPase-2A. A protein kinase C-activating phorbol ester produced the opposite results when added to RINm5F cell homogenates. These studies suggest that several known intracellular second messengers are without effect on beta-cell PPase activities. However, phosphatidyl serine and protein kinase C activation, whose activity is transiently increased by glucose, may promote insulin release through PPase inactivation, likely contributing to the increase in phosphorylation state that occurs after stimulation of insulin release. Thus, inhibition of protein dephosphorylation may be a novel regulatory mechanism, assisting in activation of the stimulus-secretion coupling in insulin-producing cells.     

Signalling in taste receptor cells: cAMP-dependent protein kinase causes depolarization by closure of 44 pS K-channels

1. In whole-cell patch-clamp recordings cytosolic cAMP causes a substantial depolarization of taste receptor cells isolated from the frog tongue. The depolarization requires the presence of ATP in the cell and is suppressed by protein kinase inhibitor. 2. The depolarization also develops in the absence of cAMP while the catalytic subunit of the kinase is allowed to diffuse into the cell in the presence of ATP. 3. In membrane patches excised from these cells the catalytic subunit is found to inactivate K-channels of 44 pS conductance, presumably by phosphorylation. 4. It appears that cAMP is one of the intracellular messengers in gustatory chemoreception, and that it causes membrane depolarization through activation of a protein kinase which controls the activity of one set of K-channels.     

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease

Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.     

Receptor-mediated endocytosis of asialoglycoproteins and diferric transferrin is independent of second messengers.

The intracellular concentrations of cyclic AMP, polyphosphoinosides and free Ca2+ were unaffected during receptor-mediated endocytosis of the neoglycoprotein beta-D-galactosyl-bovine serum albumin (D-Gal-BSA) by isolated hepatocytes. Elevation of either intracellular cyclic AMP by glucagon or inositol phosphates and Ca2+ by vasopressin were without effect on the binding and internalization of D-Gal-BSA. The normal response of this cell to glucagon- and vasopressin-mediated mobilization of these second messengers was not modified in the presence of saturating concentrations of D-Gal-BSA. Receptor-mediated endocytosis of diferric transferrin (Fe3+-TRF) by both hepatocytes and HL60 cells was also shown to be independent of second messengers, although the unequivocal expression of the transferrin receptor by hepatocytes could not be satisfactorily demonstrated. The results of the present study are at variance with a suggested regulatory role for second messengers in receptor-mediated endocytosis of serum-derived ligands such as asialoglycoproteins and Fe3+-TRF. Receptor phosphorylation by protein kinase C in particular has been proposed to regulate the distribution and recycling of these receptors in actively endocytosing cells. We would suggest that if receptor phosphorylation has a regulatory role during endocytosis, it is likely to be mediated by a second-messenger-independent protein kinase analogous to casein kinase II. An alternative interpretation is that phosphorylation has no physiological significance and receptor-mediated endocytosis is a constitutive event coupled to membrane turnover.     

Evidence That Receptor-Linked G Protein Inhibits Exocytosis by a Post-Second-Messenger Mechanism in AtT-20 Cells

In AtT-20 cells somatostatin inhibits the secretion of adrenocorticotropic hormone (ACTH) through the activation of GTP binding proteins (G proteins) linked to second messengers such as calcium and cyclic AMP (cAMP). Recently, it has been proposed that there may be G proteins that regulate directly the exocytotic machinery. We have investigated whether somatostatin could inhibit secretion at a step distal to second messengers through a GTP binding protein. For these studies two experimental paradigms were used: (1) intact cells stimulated by calcium ionophores and (2) digitonin-permeabilized cells exposed to buffers of increasing Ca2+ concentrations. Somatostatin inhibited by 70% the ACTH release caused by the calcium ionophore ionomycin without modifying the ionophore-induced elevation in cytosolic [Ca2+]. This effect was cAMP independent because (1) it was observed in the presence of high concentrations of membrane-permeant cAMP analogues, and (2) it was not accompanied by a change in cAMP levels. The effect was also independent of the levels of activators of protein kinase C because it could be produced in the presence of high concentrations of phorbol esters. The action of somatostatin was prevented by pertussis toxin. In digitonin-permeabilized AtT-20 cells somatostatin inhibited release induced by calcium buffers in a GTP-dependent manner. These two observations indicate the involvement of a G protein. It is proposed that a G protein coupled to somatostatin receptors inhibits the intracellular machinery of secretion at a step distal to second messengers, perhaps at the exocytotic site.     

Ba2+ current oscillations modulated by cyclic AMP and phorbol esters in ras-transformed fibroblasts.

An oscillatory influx of divalent cations was measured as Ba2+ inward currents (Ba2+ current oscillations) by voltage-clamp recording in v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts after activation with bradykinin or serum. Application of forskolin or dibutyryl cyclic AMP onto DT cells initiated Ba2+ current oscillations. Increasing intracellular cyclic AMP reduced the amplitude but increased the frequency of the Ba2+ current oscillations. Activation of protein kinase C by phorbol esters terminated Ba2+ current oscillations. No inhibition of Ba2+ current oscillations by phorbol esters was observed in down-regulated cells that had been pretreated with phorbol esters for 24 hrs. The results suggest that Ba2+ current oscillations are regulated by intracellular second messengers.     

Correlation between changes in intracellular level of cyclic AMP, activation of cyclic AMP-dependent protein kinase, and the morphology of Chinese hamster ovary cells in culture.

The morphological conversion of Chinese hamster ovary cells induced by treatment with dibutyryl cyclic AMP is correlated with increases in the intracellular level of cyclic AMP and the activation of cyclic AMP-dependent protein kinase. When cholera toxin is used to induce the increase in intracellular cyclic AMP, a similar correlation is obtained. Treatment of cells with prostaglandin E₁, which causes a transient increase in intracellular cyclic AMP and a transient activation of protein kinase activity, does not result in the morphology change. From these studies we conclude that a stable activation of the cyclic AMP-dependent protein kinase, which results from an increase in intracellular cyclic AMP, induces the morphological conversion of Chinese hamster ovary cells through phosphorylation of one or more cellular components.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts.

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743-745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L6 myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system. We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f2b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10(-5) M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase. Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems. The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane phosphorylation as a regulator of membrane functions including those that may control cellular differentiation.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743–745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L₆ myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system.We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f₂b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10^?5 M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase.Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems.The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane missing data     

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.     

Phosphorylation-mediated activation and translocation of the cyclic AMP-specific phosphodiesterase PDE4D3 by cyclic AMP-dependent protein kinase and mitogen-activated protein kinases. A potential mechanism allowing for the coordinated regulation of PDE4D activity and targeting

In this study, we describe a novel mechanism by which a protein kinase C (PKC)-mediated activation of the Raf-extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade regulates the activity and membrane targeting of members of the cyclic AMP-specific phosphodiesterase D family (PDE4D). Using a combination of pharmacological and biochemical approaches, we show that increases in intracellular cAMP cause a protein kinase A-mediated phosphorylation and activation of the two PDE4D variants expressed in vascular smooth muscle cells, namely PDE4D3 and PDE4D5. In addition, we show that stimulation of PKC via the associated activation of the Raf-MEK-ERK cascade results in the phosphorylation and activation of PDE4D3 in these cells. Furthermore, our studies demonstrate that simultaneous activation of both the protein kinase A and PKC-Raf-MEK-ERK pathways allows for a coordinated activation of PDE4D3 and for the translocation of the particulate PDE4D3 to the cytosolic fraction of these cells. These data are presented and discussed in the context of the activation of the Raf-MEK-ERK cascade acting to modulate the activation and subcellular targeting of PDE4D gene products mediated by cAMP.     

Formation of second messengers in response to activation of ion channels in excitable cells

1. Depolarization of excitable cells of the central nervous system results in the formation of the second messengers cyclic AMP, cyclic GMP, inositol phosphates, and diacylglycerides. 2. Depolarization-evoked accumulation of cyclic AMP in brain preparations can be accounted for mainly by the release of adenosine, which subsequently interacts with stimulatory adenosine receptor linked to adenylate cyclase. 3. Depolarization-evoked formation of cyclic GMP in brain preparations is linked to activation of voltage-dependent calcium channels, presumably leading to activation of guanylate cyclase by calcium ions. 4. In brain slices depolarization-evoked stimulation of phosphoinositide breakdown and subsequent formation of inositol phosphates and diacylglycerides are linked to activation of voltage-dependent calcium channels, which are sensitive to dihydropyridines, presumably leading to activation of phospholipase(s) C by calcium ions. 5. In the synaptoneurosome preparation depolarization-evoked stimulation of phosphoinositide breakdown does not involve activation of dihydropyridine-sensitive calcium channels and, instead, appears to be regulated primarily by the intracellular concentration of sodium ions. Thus, agents that induce increases in intracellular sodium--such as toxins that open or delay inactivation of voltage-dependent sodium channels; ouabain, an inhibitor of Na+/K+ ATPase that transports sodium outward and a sodium ionophore--all stimulate phosphoinositide breakdown. Mechanistically, increases in intracellular sodium either might directly affect phospholipase(s) C or might lead to influx of calcium ions through Na+/Ca2+ transporters. 6. Depolarization-evoked stimulation of cyclic AMP formation and phosphoinositide breakdown can exhibit potentiative interactions with responses to receptor agonists, thereby providing mechanisms for modulation of receptor responses by neuronal activity. 7. Since all these second messengers can induce phosphorylation of ion channels through the activation of specific kinases, it is proposed that depolarization-evoked formation of second messengers represents a putative feedback mechanism to regulate ion fluxes in excitable cells.     

环鸟苷酸依赖的蛋白激酶对心血管功能的调节作用

环鸟苷酸(cGMP)依赖的蛋白激酶(PKG)是一氧化氮-cGMP的主要细胞内受体,在哺乳动物细胞中分为PKG-I和PKG-II两型。在PKG介导的血管平滑肌舒张作用中,其主要通过活化细胞膜上的钙活化的钾通道(BK通道),磷酸化肌质网上的受磷蛋白(phospholamban,PLB)和三磷酸肌醇受体相关的PKG-I底物(IP3receptor-associated PKG-I substrate,IRAG),降低细胞内Ca2 浓度。PKG还可通过活化肌球蛋白轻链磷酸酶及抑制Rho激酶降低肌球蛋白对Ca2 敏感性。PKG调节血管平滑肌细胞的基因表达和表型调变,调节细胞增生。PKG活化以后还具有抑制血小板聚集,抑制心肌细胞肥大等功能。最近的研究证明,PKG的表达水平和活性改变与动脉粥样硬化和再狭窄、高血压、糖尿病心血管病变以及硝酸盐耐受等的发病机制有密切关系。     

Phospholipids and electrolyte transport.

Our understanding of the role of phospholipids in ion transport processes is only beginning to be appreciated. Although the role of polyphosphoinositide and its derived second messenger molecules IP3, diacylglycerol, and arachidonic acid are well studied, we are still not certain as to how changes in the lipid bilayer structure influence the status of ion channels. This review focused on those studies which show a strong correlation with ion conductance changes and the status of the membrane phospholipids. In addition, a number of observations point to a major role of lipid second messengers that activate enzymes involved in protein phosphorylations, i.e., protein kinase C, as major regulators of a variety of ion channels and transporters. Such lipid second messengers provide a cellular mechanism whereby hormones, neurotransmitters, and pharmacologic agents functionally control the ionic environment and intracellular pH of target cells. Some of these pathways still remain to be elucidated; however, an appreciation for the participation of membrane phospholipids in these actions has been presented.     

Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol.

The interaction of high density lipoproteins (HDL) with the HDL receptor stimulates the translocation of cholesterol from intracellular pools to the plasma membrane where the cholesterol becomes available for removal by appropriate acceptors. The role of signal transduction through protein kinase C in HDL receptor-dependent cholesterol translocation and efflux was examined using cholesterol-loaded cultured human skin fibroblasts. Treatment of cells with HDL3 activated protein kinase C, demonstrated by a transient increase in membrane associated kinase activity. Kinase activation appeared to be dependent on binding of HDL3 to the HDL receptor, since tetranitromethane-modified HDL3, which does not bind to the receptor, was without effect. Translocation of intracellular sterol to the plasma membrane was stimulated by treatment of cells with the protein kinase C activators, dioctanoylglycerol and phorbol myristic acetate, and the calcium ionophore A23187. Conversely, treatment of cells with sphingosine, a protein kinase C inhibitor, reduced HDL3-mediated translocation and efflux of intracellular sterols. However, sphingosine had no effect on efflux of labeled cholesterol derived from the plasma membrane. Down-regulation of cellular protein kinase C activity by long term incubation with phorbol esters also inhibited HDL3-mediated efflux of intracellular sterols and abolished the ability of sphingosine to further inhibit HDL3-mediated efflux. These studies support the conclusion that HDL receptor-mediated translocation and efflux of intracellular cholesterol occurs through activation of protein kinase C.     

G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.     

Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells

In quiescent cultures of Swiss 3T3 cells, platelet-derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c-fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane-permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c-fos mRNA, and this action was mimicked by 8-bromo-cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c-fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells.     

Inhibitors of intracellular cyclic AMP accumulation affect differentiation of sporogenous mutants of Dictyostelium discoideum

Abstract Sporogenous mutants of Dictyostelium discoideum strain V12M2 were used to determine whether the intracellular levels of cyclic AMP or other second messengers regulate differentiation. Increasing external concentrations of cyclic AMP promoted spore formation. Caffeine and progesterone, which lower intracellular cyclic AMP levels by different mechanisms, blocked spore formation and favored stalk cell formation. In contrast, differentiation of both spore and stalk cells occurred normally in the presence of agents that disrupt calcium/calmodulin or protein kinase C-based second messenger systems. The data are in accord with the view that (1) intracellular cyclic AMP is essential for terminal differentiation of both cell types, and (2) higher levels are required for formation of spores than for stalk cells.     

The role of inositol lipid metabolism in the ovary

Two major signal transduction systems operate within ovarian cells to control their function. Gonadotropins, such as follicle-stimulating hormone and luteinizing hormone, primarily utilize the cyclic adenosine 3',5'-monophosphate (cAMP) pathway to stimulate steroid hormone biosynthesis. On the other hand, an inositol lipid metabolism pathway is used by other effector molecules such as gonadotropin-releasing hormone or prostaglandin F2 alpha, as well as gonadotropins, to alter ovarian hormone production. Membrane polyphosphoinositides are hydrolyzed to inositol phosphates and diacylglycerol, resulting in alterations of intracellular free calcium concentration, activation of protein kinase C, and liberation of arachidonic acid. Some or all of these intracellular messengers may interact with the gonadotropin-induced cAMP pathway to control ovarian function.     

A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts

Endothelin, a novel vasoactive peptide derived from endothelial cells (Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature 332, 411-415), acts as a potent mitogen in Swiss 3T3 fibroblasts. The effect is dose-dependent with a half-maximal effect obtained at approximately 3 x 10(-11) M and is synergistically enhanced by a low concentration of insulin-like growth factor-I. Endothelin specifically binds to a single class of high affinity receptors in intact Swiss 3T3 cells and stimulates phospholipase C with the production of second messengers inositol trisphosphate and 1,2-diacylglycerol, leading to biphasic increases in the intracellular free Ca2+ concentration, as measured with a fluorescent indicator fura-2, phosphorylation of a putative cellular substrate of 80 kDa for protein kinase C, and transient expression of cellular protoonocogenes, c-fos and c-myc. Mitogenic effect of endothelin is markedly attenuated in phorbol ester-pretreated, protein kinase C-depleted cells. Endothelin-induced inositol phosphates production is not affected by removal of extracellular Ca2+, suggesting that endothelin-induced phospholipase C activation is not the result of stimulation of Ca2+ influx across the plasma membrane. These composite results indicate that the inositol lipid signaling pathway plays an important role in endothelin-induced mitogenesis in Swiss 3T3 fibroblasts. The mitogenic effect of endothelin is considerably smaller than that of bombesin, another well characterized mitogen acting through the inositol lipid pathway, despite comparable potencies in eliciting initial second messenger signals. In endothelin-treated cells, an increase in cellular 1,2-diacylglycerol content is transient, and cellular cyclic AMP content is reduced. By contrast, bombesin induces a more prolonged increase in cellular 1,2-diacylglycerol content and a slight increase in cellular cyclic AMP content. Because both 1,2-diacylglycerol and cyclic AMP are thought to serve as signals for promoting DNA synthesis in Swiss 3T3 fibroblasts, these differences in the signal generation may contribute to the differences in potencies between the two mitogens.