Effects of radioprotectors on the cAMP and cGMP systems

Summary The sulphur-containing radioprotectors mercaptoethylamine (MEA), aminoethylisothiourea (AET), 2-aminothiazoline, 4-oxo-2-aminothiazoline, and S-S-3-oxapentane-1,5-diisothiourea, and the radioprotective biogenic amines serotonin, histamine, and dopamine, caused the elevation of cAMP content and intensified the rate of cAMP-dependent protein phosphorylation in tissues of animals following intraperitoneal injection at radioprotective doses. Biogenic amines stimulated the adenylate cyclase activity in membrane preparations from liver, spleen, and small-intestine mucosa; sulphur-containing radioprotectors caused no such effects. None of the radioprotectors affected cAMP and cGMP phosphodiesterases in vitro. AET and MEA inhibited guanylate cyclase in vitro, whereas serotonin and dopamine stimulated the enzyme. A biphasic change in the level of cGMP was observed in tissues after the administration of MEA and AET (more than 2-fold fall by 1–3 min after the administration of drug and 1.4-fold rise after 15–20 min); serotonin and dopamine caused a slow rise in the cGMP level; the cAMP/cGMP ratio in liver showed biphasic changes in level during the 20 min following injection of serotonin.The data obtained support the conclusion that the action of radioprotectors on cellular metabolism in animals may be mediated by the cAMP system. The reciprocal regulation of radioresistance by cAMP and cGMP is unlikely to exist.     

Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity

The effect of insulin on the state of phosphorylation of hormone-sensitive lipase, cellular cAMP-dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone-sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP-dependent protein kinase activity ratio was greater than or equal to 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone-sensitive lipase. Insulin (1 nM) reduced cAMP-dependent protein kinase activity and also reduced lipolysis with both cAMP-dependent and cAMP-independent antilipolytic effects up to an activity ratio of approximately 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone-sensitive lipase at all levels of cAMP-dependent protein kinase activity. Under basal conditions, with cAMP-dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone-sensitive lipase in a manner not mediated by cAMP. When the cAMP-dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone-sensitive lipase, the insulin-induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP-dependent protein kinase activity ratios (0.05-0.1), dephosphorylation of the regulatory site correlated with reduced cAMP-dependent protein kinase activity, but not at higher activity ratios (greater than 0.1). Stimulation of cells with isoproterenol produced a transient (1-5 min) peak of cAMP-dependent protein kinase activity and of phosphorylation of hormone-sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2 approximately 1 min) dephosphorylation of hormone-sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2 approximately 5 min). These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone-sensitive lipase and lipolysis through a cAMP-dependent pathway, involving reduction of cAMP, and through a cAMP-independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone-sensitive lipase.     

Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast

In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.     

Minor participation of cAMP on the protein kinase phosphorylation of mitochondrial and cytosolic fractions from Ascaris suum: a comparative study with porcine heart muscle

In contrast to porcine heart muscle in which cAMP effectively activated the phosphorylation of cytosolic proteins, cAMP exerted a minor effect on the phosphorylation of proteins from the soluble fraction of Ascaris suum muscle. Similarly, cAMP did not enhance the kinase activity in the mitochondrial membranes from porcine heart and A. suum, although major differences in protein phosphorylation were observed between both fractions. However, cAMP-dependent protein kinases (PKA) were evidenced in the parasitic soluble mitochondrial fraction, since the phosphorylation of histone IIA and kemptide was augmented in this fraction, in the presence of cAMP. An increase in the phosphorylation of exogenously added A. suum phosphofructokinase was also obtained when cAMP was added to the parasite soluble mitochondrial fraction. The phosphorylation of phosphofructokinase by this fraction was inhibited when kemptide and cAMP were included in the reaction mixture, suggesting substrate competition for the same PKA. Although PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent PKAs, did not affect the endogenous phosphorylation of proteins in the various A. suum fractions, an inhibition on the phosphorylation of exogenously added kemptide and phosphofructokinase was observed when PKI (6-22) was incubated with the parasite mitochondrial soluble fraction.     

Substrates for adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase in the rat pituitary gland

1. Substrates for cAMP-dependent protein kinase were investigated in anterior, intermediate, and neural lobes of the rat pituitary gland. In a cell-free assay system, cAMP increased phosphorylation of 17 K, 33 K, and 60 K macromolecules of the anterior lobe, 17 K, 33 K, 60 K, and 80 K macromolecules of the intermediate lobe, and 60 K, 80 K, and 85 K macromolecules of the neural lobe. 2. Other nucleotides were tested in the intermediate lobe; 8 Br-cAMP mimicked cAMP, cGMP was much less effective than cAMP or 8 Br-cAMP, and 5'-AMP showed no significant effect. The purified catalytic subunit of cAMP-dependent protein kinase evoked the same phosphorylation pattern as the endogenous kinase. 3. Maximum cAMP-dependent phosphorylation occurred at between 1 and 2 min of incubation; after 20 min, phosphorylation was reduced by 80%. This suggests the presence of phosphatase activity in the intermediate lobe. 4. When tested upon dispersed intermediate lobe cells permeabilized by high-voltage electrical discharges, cAMP increased phosphorylation of the 17 K and 33 K macromolecules.     

Stimulation of in vitro motility of Chlamydomonas axonemes by inhibition of cAMP-dependent phosphorylation

When demembranated axonemes of Chlamydomonas were reactivated with Mg-ATP, the proportion of motile axonemes was significantly increased by the presence of either phosphodiesterase (PDE) or protein inhibitor of cAMP-dependent kinase (PKI). The effect of PDE was cancelled by the addition of cAMP. These findings strongly suggest that the axoneme samples have endogenous cAMP, which can reduce the proportion of motile axonemes via phosphorylation. This inhibitory effect of cAMP on Chlamydomonas axonemes is opposite to its stimulatory effect on the axonemal motility in other organisms so far reported. PKI or PDE activated the motility either in the absence of Ca2+, when the axonemes beat with an asymmetric waveform, or in 10(-5) M Ca2+, when the axonemes beat with a symmetric waveform. This cAMP-dependent regulation of motility was observed with the axonemes from which detergent-soluble material had been removed, indicating that the proteins responsible for the regulation still remained in the axonemes. Preliminary in vitro phosphorylation studies have implicated two polypeptides as candidates for the target protein of cAMP-dependent protein kinase: one with a molecular weight of 270 kD and the other with a much larger molecular weight.     

Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases

Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.     

Phosphorylation of the carotenoid droplet protein p57 by the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase and the effect of fluoride

We have previously shown that the dispersion and aggregation of carotenoid droplets in goldfish xanthophores are regulated, respectively, by phosphorylation and dephosphorylation of a carotenoid droplet protein p57. There is a basal level of p57 phosphorylation of p57 in unstimulated cells, which is greatly stimulated by adrenocorticotropic hormone (ACTH) or cyclic adenosine monophosphate (cAMP) acting via cAMP-dependent protein kinase. We have also observed that, in permeabilized xanthophores, pigment dispersion can be induced when cAMP is replaced by fluoride. Since p57 has multiple phosphorylation sites, there is the question of whether all p57 phosphorylation is by cAMP-dependent protein kinase or whether phosphorylation by cAMP-independent protein kinase coupled with inhibition of phosphatase activity by fluoride can replace cAMP-dependent protein kinase and that the ability of fluoride to replace cAMP for pigment dispersion in permeabilized cells is probably due to activation of adenylcyclase. We also show that ACTH causes an approximately threefold increase in the level of cAMP in these cells.     

Regulation of NAD-dependent glutamate dehydrogenase by protein kinases in Saccharomyces cerevisiae

The active NAD-dependent glutamate dehydrogenase of wild type yeast cells fractionated by DEAE-Sephacel chromatography was inactivated in vitro by the addition of either the cAMP-dependent or cAMP-independent protein kinases obtained from wild type cells. cAMP-dependent inhibition of glutamate dehydrogenase activity was not observed in the crude extract of bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase. The cAMP-dependent protein kinase of CYR3 mutant cells, which has a high K alpha value for cAMP in the phosphorylation reaction, required a high cAMP concentration for the inactivation of NAD-dependent glutamate dehydrogenase. An increased inactivation of partially purified active NAD-dependent glutamate dehydrogenase (Mr = 450,000) was observed to correlate with increased phosphorylation of a protein subunit (Mr = 100,000) of glutamate dehydrogenase. The phosphorylated protein was labeled by an NADH analog, 5'-p-fluorosulfonyl[14C]benzoyladenosine. Activation and dephosphorylation of inactive NAD-dependent glutamate dehydrogenase fractions were observed in vitro by treatment with bovine alkaline phosphatase or crude yeast cell extracts. These results suggested that the conversion of the active form of NAD-dependent glutamate dehydrogenase to an inactive form is regulated by phosphorylation through cAMP-dependent and cAMP-independent protein kinases.     

The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis

The mitochondria, the microsomes and the cystosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.     

Control of growth and differentiation by cyclic AMP in fungi

InSaccharomyces cerivisiae intracellular cAMP mediates environmental signals that regulate cellular metabolism and growth. The studies on the cAMP-requiring mutants and their suppressors in the yeast revealed that cAMP-dependent protein phosphorylation is involved in the G1 phase of the cell cycle, stimulation of the phosphoinositide pathway and the post-meiotic stage of spourlation, and that inhibition of cAMP-dependent protein phosphorylation is required to go into the GO stage of and to induce meiotic division. Growth of some filamentous fungi was observed with significantly reduced levels of cAMP, suggesting that cAMP may not be essential for growth in some species of fungi. Germination of fungal spores, yeast-mycelium dimorphism and hyphal morphogenesis of several species of fungi were affected by cAMP. cAMP was involved in extension of hyphae, formation of hyphal aggregates and fruit body formation. Phosphorylation of cellular proteins is required in these processes, and the nature of these proteins phosphorylated by cAMP-dependent protein kinase is important to the understanding of the role of cAMP for growth and differentistion in fungal cells.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Phosphopeptide patterns of the ribosomal protein S6 following stimulation of guinea pig parotid glands by secretagogues involving either cAMP or calcium as second messenger

Stimulation of secretion in exocrine cells is associated with the incorporation of up to 3 to 4 phosphates into the ribosomal protein S6. This occurs with secretagogues involving either cAMP or free calcium as second messenger. An analysis of the phosphorylation pattern of S6 from stimulated guinea pig parotid glands reveals 3 phosphopeptides (termed A,B,C). The phosphopeptide pattern was identical for cAMP- or calcium-mediated stimulation, whereas phosphorylation of the S6 protein in vitro with catalytic subunit of cAMP-dependent protein kinase resulted only in the formation of phosphopeptides A and C. Therefore, secretagogue-mediated phosphorylation is not or not exclusively catalyzed by cAMP-dependent protein kinase even when cAMP is the second messenger.     

The effect of various protein kinase inhibitors on the response of snail neurons to the intracellular injection of cAMP

Drugs preventing cAMP interaction with regulatory subunit of cAMP-dependent protein kinase, tolbutamide and db-cAMP injected into neurons of Helix lucorum decreased the cell response to cAMP, but H-8-a potent inhibitor of this enzyme catalytic subunit did not produce such effect. It is suggested that the neuron electric response to cAMP injection is not caused by protein phosphorylation.     

Impact of various glycosaminoglycans upon cAMP-dependent protein kinase

The physiological effects of the second messenger cAMP are displayed by cAMP-dependent protein kinase-medicated phosphorylation of specific target proteins which in turn control diverse cellular functions. We have determined this enzyme substrate phosphorylation in the presence of various glycosaminoglycans using a cAMP-dependent protein kinase isolated from rat liver. The results indicate that sulfated and unsulfated polysaccharides are able to inhibit phosphorylation of histone type IIa catalysed by cAMP-dependent protein kinase. Based on their impact upon substrate phosphorylation, glycosaminoglycans can be divided into three groups: group I with the highest inhibitory effect: dermatan sulfate and heparan sulfate; group II: chondroitin 4-sulfate and group III with the lowest inhibitory effect: chondroitin 6-sulfate, keratan sulfate and hyaluronic acid.     

Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility

cAMP and calcium are two important regulators of sperm flagellar motility. cAMP stimulates sperm motility by activating cAMP-dependent protein kinase and catalyzing the phosphorylation of sperm proteins. The stimulation of sperm motility by cAMP appears to be at two different levels. Evidence has been presented to suggest that cAMP-dependent phosphorylations may be required in order for motility to be initiated. In addition, cAMP-dependent phosphorylation appears to modulate specific parameters of motility resulting in higher beat frequency or greater wave amplitude. Calcium, on the other hand, when elevated intracellularly to 10(-6) M or higher, inhibits flagellar motility. The calcium-binding protein, calmodulin, appears to mediate a large number of effects of calcium on motility. Evidence suggests that calcium-calmodulin may be involved at the level of the membrane to pump calcium out of the flagellum. In addition, calcium-calmodulin may be involved in the control of axonemal function by regulating dynein ATPase and myosin light chain kinase activities. The identification of cAMP-dependent protein kinase, calmodulin and myosin light chain kinase in the sperm head suggests that cAMP and calcium-dependent phosphorylations are also involved in the control of the fertilization process, i.e., the acrosome reaction, in a manner similar to that known for the control of stimulus/secretion coupling. Finally, the effects of cAMP on flagellar motility are mediated by protein phosphorylation while the effects of calcium on motility are also in part, mediated by effects on protein phosphorylation.     

Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.     

Endothelial nitric-oxide synthase enhances lipopolysaccharide-stimulated tumor necrosis factor-alpha expression via cAMP-mediated p38 MAPK pathway in cardiomyocytes

The purpose of this study was to investigate the role of endothelial nitric-oxide synthase (eNOS), cAMP, and p38 MAPK in tumor necrosis factor-alpha (TNF-alpha) expression induced by lipopolysaccharide (LPS). LPS dose- and time-dependently induced phosphorylation of p38 MAPK and TNF-alpha expression in neonatal mouse cardiomyocytes. TNF-alpha expression was preceded by p38 MAPK phosphorylation, and selective inhibition of p38 MAPK abrogated LPS-induced TNF-alpha expression. Deficiency in eNOS decreased basal and LPS-stimulated TNF-alpha expression in cardiomyocytes. NOS inhibitor l-NAME attenuated LPS-induced p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes, whereas NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (DETA-NO) (2 microm) or overexpression of eNOS by adenoviral gene transfer restored the response of eNOS(-/-) cardiomyocytes to LPS. These effects of NO were mediated through cAMP-dependent pathway based on the following facts. First, deficiency in eNOS decreased basal levels of intracellular cAMP, and DETA-NO elevated intracellular cAMP levels in eNOS(-/-) cardiomyocytes. Second, a cAMP analogue 8-Br-cAMP mimicked the effect of NO in eNOS(-/-) cardiomyocytes. Third, either inhibition of cAMP or cAMP-dependent protein kinase attenuated LPS-stimulated p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes. In conclusion, eNOS enhances LPS-stimulated TNF-alpha expression in cardiomyocytes. Activation of p38 MAPK is essential in LPS-stimulated TNF-alpha expression. Moreover, the effects of NO on LPS-stimulated TNF-alpha expression are mediated through cAMP/cAMP-dependent protein kinase-dependent p38 MAPK pathway in neonatal cardiomyocytes.