Beta adrenergic receptor stimulated prostacyclin synthesis in rabbit coronary endothelial cells is mediated by selective activation of phospholipase D: inhibition by adenosine 3'5'-cyclic monophosphate

Activation of beta adrenergic receptors in the isolated rabbit heart by catecholamines stimulates prostacyclin (PGI2) synthesis, which is inhibited by adenosine 3'5'-cyclic monophosphate (cAMP). The purpose of this study was to determine if activation of beta adrenergic receptors in cultured coronary endothelial cells (CEC) of rabbit heart with isoproterenol (ISOP) stimulates PGI2 synthesis and if cAMP inhibits the synthesis of this prostanoid and to investigate the underlying mechanism. Incubation of CEC with ISOP increased production of cAMP and PGI2, measured as immunoreactive cAMP and 6-keto-prostaglandin F1alpha, (6-keto-PGF1alpha), respectively. Forskolin, an activator of adenylyl cyclase, increased cAMP accumulation and inhibited ISOP-stimulated 6-keto-PGF1alpha synthesis. 8-(4-chlorophenyl-thio) cAMP also inhibited ISOP-induced 6-keto-PGF1alpha production. However, miconazole, an inhibitor of adenylyl cyclase, reduced cAMP accumulation and enhanced ISOP-stimulated 6-keto-PGF1alpha synthesis in CEC. ISOP-induced 6-keto-PGF1alpha synthesis was attenuated by C2-ceramide, an inhibitor of phospholipase D (PLD) by propranolol, a beta-AR antagonist that also inhibits phosphatidate phosphohydrolase and by the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267). Acetylcholine (ACh) induced 6-keto-PGF1alpha synthesis was also inhibited by these agents. Both ISOP and ACh increased PLD activity, which was inhibited by C2-ceramide but not by RHC 80267 or propranolol. ACh but not ISOP increased phospholipase A2 activity in CEC. ISOP- but not ACh-induced increase in PLD activity was attenuated by forskolin and 8-(4-chlorophenyl-thio)-adenosine 3'-5'-cyclic monophosphate and augmented by miconazole. These data suggest that beta adrenergic receptors activation promotes PGI2 synthesis in the CEC by selective activation of PLD and that cAMP decreases PGI2 synthesis by decreasing PLD activity. Moreover, beta adrenergic receptors activated PLD appears to be distinct from that stimulated by ACh.     

Selective effects of ethanol on the generation of cAMP by particular members of the adenylyl cyclase family

A selective action of ethanol on major signal transduction proteins, such as adenylyl cyclase, has been considered to be important for certain actions of ethanol, and alcoholics have been demonstrated to differ from controls in measures of platelet adenylyl cyclase activity. Recent advances in identification and characterization of isoforms of adenylyl cyclase have demonstrated that there exists at least eight different forms of this enzyme. To examine whether the effect of ethanol on generation of cAMP is modified by the presence of particular isoforms of adenylyl cyclase within a cell, we transiently expressed each of six adenylyl cyclases in human embryonic kidney (HEK293) cells and measured cAMP accumulation in whole cells in the presence and absence of ethanol. The treatment of cells expressing the various adenylyl cyclases with ethanol alone did not enhance cAMP generation. In the presence of prostaglandin E1, cAMP generation by type I and type III adenylyl cyclases was insensitive to ethanol. cAMP accumulation generated by the other adenylyl cyclases was, however, increased by incubation of cells with ethanol in the presence of stimulatory agonists (e.g., prostaglandin E1). Stimulation by ethanol of cAMP generation by type VII adenylyl cyclase was 2- to 3-fold greater than that seen with the other tested adenylyl cyclases. The noted stimulation of cAMP generation by ethanol was dose-dependent and required concurrent activation of adenylyl cyclase through the stimulatory G protein. The effects of ethanol were reversible and mimicked by butanol but not by chloroform.     

A novel adenylyl cyclase detected in rapidly developing mutants of Dictyostelium

Disruption of either the RDEA or REGA genes leads to rapid development in Dictyostelium. The RDEA gene product displays homology to certain H2-type phosphotransferases, while REGA encodes a cAMP phosphodiesterase with an associated response regulator. It has been proposed that RDEA activates REGA in a multistep phosphorelay. To test this proposal, we examined cAMP accumulation in rdeA and regA null mutants and found that these mutants show a pronounced accumulation of cAMP at the vegetative stage that is not observed in wild-type cells. This accumulation was due to a novel adenylyl cyclase and not to the known Dictyostelium adenylyl cyclases, aggregation stage adenylyl cyclase (ACA) or germination stage adenylyl cyclase (ACG), since it occurred in an acaA/rdeA double mutant and, unlike ACG, was inhibited by high osmolarity. The novel adenylyl cyclase was not regulated by G-proteins and was relatively insensitive to stimulation by Mn2+ ions. Addition of the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) permitted detection of the novel adenylyl cyclase activity in lysates of an acaA/acgA double mutant. The fact that disruption of the RDEA gene as well as inhibition of the REGA-phosphodiesterase by IBMX permitted detection of the novel AC activity supports the hypothesis that RDEA activates REGA.     

Sodium saccharin inhibits adenylyl cyclase activity in non-taste cells

We have studied the in vitro effect of sodium saccharin (NaSacch) on the rat adipocyte adenylyl cyclase complex. NaSacch (2.5-50 mM) inhibited significantly in a dose-dependent manner basal and isoproterenol-stimulated cAMP accumulation on isolated rat adipocytes. Similarly, NaSacch (2.5-50 mM) inhibited forskolin-stimulated adenylyl cyclase activity measured in the presence of Mg(2+)-ATP on adipocyte, astrocyte and thyrocyte membrane fractions. In contrast, NaSacch did not inhibit but slightly increased the forskolin-stimulated adenylyl cyclase activity measured in the presence of Mn(2+)-ATP and GDP beta S, a stable GDP analogue. The effect of NaSacch was not mediated through either the A1-adenosine receptor (A1R) or the alpha 2-adrenergic receptor (alpha 2AR). The inhibitory effect of NaSacch was additive to that of A1R agonist and was not blocked by the addition of the alpha 2AR antagonist RX 821002. Pretreatment of adipocytes with pertussis toxin slightly attenuated but did not abolish the inhibitory effect of NaSacch on forskolin-stimulated adenylyl cyclase activity on membrane fractions. These data suggest that the inhibitory effect of NaSacch on forskolin stimulated-adenylyl cyclase in adipocytes does not imply only Gi protein but also other direct or indirect inhibitory pathway(s) which remain to be determined.     

Desensitization of the beta-adrenergic response in human brown adipocytes

Activation of adenylyl cyclase by beta-adrenergic receptors (betaARs) plays a major role in adipose tissue homeostasis. The increase in cAMP promotes lipolysis in white adipose tissue, activates both thermogenesis and lipolysis in brown adipose tissue (BAT), and induces BAT hypertrophy. Previous studies indicated that among the three betaAR subtypes present in adipose tissue, beta3AR could be a potential target for antiobesity treatments in humans. We studied immortalized human brown adipocytes (PAZ6 adipocytes) as a model of beta-adrenergic response in human BAT. PAZ6 adipocytes and freshly isolated mature human brown adipocytes display the same proportions of betaAR subtypes, with beta3AR being the most abundant (approximately 80% of the total). However, beta3AR was poorly coupled to the adenylyl cyclase pathway in PAZ6 cells, contributing to only 10% of the isoproterenol-induced accumulation of cAMP, whereas 20% and 70% of the signal depended on beta1- and beta2-subtypes, respectively. Upon isoproterenol stimulation, beta1- and beta2AR down-regulated with a half-life of about 3 h and the beta3AR with a half-life of 30-40 h. Long term stimulation with both saturating (micromolar) and nonsaturating (nanomolar) concentrations of beta-adrenergic agonists caused a complete desensitization of the beta-adrenergic response at the adenylyl cyclase level and loss of stimulated protein kinase A activity and CREB phosphorylation. These results suggest that cAMP-dependent processes will be desensitized upon permanent treatment with beta3AR agonists. Further studies should establish whether the beta3AR is coupled to other signaling pathways in human brown adipocytes and whether these may contribute to BAT hypertrophy and/or thermogenesis.     

Dopamine D2 receptor signaling via the arachidonic acid cascade: modulation by cAMP-dependent protein kinase A and prostaglandin E2

Recent studies have shown that, in Chinese hamster ovary cells transfected with D2-receptor cDNA, CHO(D2) cells, D2 agonists are potent in enhancing the release of [3H]arachidonic acid (AA) induced by stimulation of constitutive purinergic receptors or by application of Ca2+ ionophores. This facilitatory action is further amplified by the concomitant activation of D1 receptors, which per se have no effect on evoked [3H]AA release. Here, we review a series of experiments aimed at examining the molecular mechanism of this synergistic interaction. The results show that, in CHO(D2) cells: (a) application of 8-Br-cAMP or stimulation of constitutive prostaglandin (PG)E2 receptors augment the AA response produced by D2 agonists; (b) in CHO(D2) cells transfected with human beta 2-receptor cDNA, the beta-agonist, isoproterenol, produces a similar effect; (c) the potentiation of [3H]AA release produced by PGE2 and 8-Br-cAMP is prevented by overexpressing either a protein inhibitor of cAMP-dependent protein kinase (PKA) or a mutated form of pKA regulatory subunit incapable of binding cAMP; (d) mock-synergism is obtained in CHO(D2) cells overexpressing the catalytic subunit of PKA; (e) PGE2 is a major AA metabolite in stimulated CHO(D2) cells and its formation may contribute to the effect of D2 agonists on AA release. The results indicate that cAMP-induced activation of PKA represents a likely molecular basis for D1/D2 receptor synergism on AA release. They also suggest that additional membrane receptors, colocalized with D2 and positively linked to adenylyl cyclase, may exert a similar action. Furthermore, stimulation of PGE2 receptors by endogenously produced prostaglandin may participate in AA signaling at the D2 receptor, by providing a paracrine positive feedback loop.     

Type I and type VIII adenylyl cyclases constitute a family whose activation is coupled to cellular deformation through the action of calcium-calmodulin

In certain tissues and cells, increases in concentrations of the second messenger cAMP are seen in response to mechanical or deformational stimuli. Type I and type VIII adenylyl cyclases, representing members of a family of calcium-calmodulin-stimulated adenylyl cyclases, and type VII adenylyl cyclase were each stably expressed in human embryonal kidney (HEK) 293 cells. HEK 293 cells exogenously expressing either type I adenylyl cyclase or any one of three type VIII adenylyl cyclase splice variants respond to swelling with increases in cAMP, requiring the presence of calcium in the extracellular medium for such responsiveness. Type VII expressing HEK 293 cells failed to respond to swelling with increased cAMP but demonstrated potentiation of isoproterenol-stimulated activity. This is characteristic of the influence of protein kinase C on the activity of the type VII protein. The relative swelling responsiveness of HEK 293 cells expressing splice variants of the type VIII adenylyl cyclase is consistent with the relative EC50 values for calcium-calmodulin stimulation of these splice variants. This is consistent with the involvement of calmodulin and the requirement for increases in intracellular calcium in mediating swelling-induced acceleration of type VIII adenylyl cyclase activity.     

Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP-specific phosphodiesterase, in vitro and in vivo

The effect of A02131-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl thieno (3,2-c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131-1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131-1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131-1 suppressed both the generation of inositol 1,4,5-triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131-1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131-1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131-1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131-1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131-1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131-1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131-1 is a cGMP-specific PDE (type V) inhibitor in human platelets.     

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.     

Molecular analysis of Chs3p participation in chitin synthase III activity

Adenylyl cyclase superactivation, a phenomenon by which chronic activation of inhibitory Gi/o-coupled receptors leads to an increase in cAMP accumulation, is believed to play an important role as a compensatory response of the cAMP signaling system in the cell. However, to date, the mechanism by which adenylyl cyclase activity is regulated by chronic exposure to inhibitory agonists and the nature of the adenylyl cyclase isozymes participating in this process remain largely unknown. Here we show, using COS-7 cells transfected with the various AC isozymes, that acute activation of the D2 dopaminergic and m4 muscarinic receptors inhibited the activity of adenylyl cyclase isozymes I, V, VI, and VIII, whereas types II, IV, and VII were stimulated and type III was not affected. Conversely, chronic receptor activation led to superactivation of adenylyl cyclase types I, V, VI, and VIII and to a reduction in the activities of types II, IV, and VII. The activity of AC-III also was reduced. This pattern of inhibition/stimulation of the various adenylyl cyclase isozymes is similar to that we recently observed on acute and chronic activation of the mu-opioid receptor, suggesting that isozyme-specific adenylyl cyclase superactivation may represent a general means of cellular adaptation to the activation of inhibitory receptors and that the presence/absence and intensity of the adenylyl cyclase response in different brain areas (or cell types) could be explained by the expression of different adenylyl cyclase isozyme types in these areas.     

Stimulation of cAMP accumulation by the cloned Xenopus melatonin receptor through Gi and Gz proteins

The Xenopus melatonin receptor was expressed in human embryonic kidney 293 cells and assayed for cAMP accumulation. In transfected 293 cells expressing the melatonin receptor, melatonin dose-dependently inhibited the endogenous adenylyl cyclases. In contrast, melatonin stimulated the accumulation of cAMP in cells co-expressing the type II adenylyl cyclase. Both the inhibitory and stimulatory responses to melatonin were mediated via Gi-like proteins as they were blocked by pertussis toxin. Upon co-transfection with the alpha subunit of Gz, the ability of melatonin to regulate both type II and the endogenous adenylyl cyclases became refractory to pertussis toxin, indicating that the melatonin receptor can also couple to Gz. However, other pertussis toxin-insensitive G proteins such as Gq, G12 and G13 were unable to interact with the melatonin receptor.     

Alpha 1-adrenoceptor-mediated inhibition of cellular cAMP accumulation in neonatal rat ventricular myocytes

We studied adrenergic regulation of cellular cAMP in neonatal rat ventricular myocytes. Since cAMP content depends on synthesis, breakdown and egress, the contribution of each of these mechanisms was assessed. In the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, cAMP accumulation stimulated by the beta-adrenoceptor agonist (-)-isoprenaline was diminished when the mixed alpha + beta adrenoceptor agonist (-)-noradrenaline was coincubated with (-)-isoprenaline. Moreover, adenylyl cyclase activation stimulated by (-)-isoprenaline was decreased by (-)-noradrenaline and by the selective alpha 1-adrenoceptor agonists (-)-phenylephrine and methoxamine, suggesting that alpha-adrenoceptor agonism regulates cAMP metabolism through its effect on the synthetic pathway. Evidence for alpha 1-adrenoceptor mediation of this response was enhancement of (-)-noradrenaline-induced cAMP generation by the selective alpha 1-adrenoceptor antagonist terazosin (10 nmol/l). The selective alpha 2-adrenoceptor antagonist yohimbine (10 nmol/l) had no effect. The alpha 1-adrenoceptor mediated depression of (-)-isoprenaline-stimulated cAMP generation and adenylyl cyclase activation was prevented by terazosin and in separate experiments markedly enhanced by pertussis toxin pretreatment, suggesting involvement of a guanine-nucleotide regulatory protein in this process. Occupation of the alpha 1-adrenoceptor by (-)-noradrenaline did not accelerate the rate of cAMP breakdown in the absence of phosphodiesterase inhibition. Furthermore, there was no enhancement of total phosphodiesterase activity by (-)-noradrenaline in the presence of (-)-propranolol. By contrast, pertussis toxin pretreatment augmented phosphodiesterase activity. Neither pertussis toxin nor (-)-noradrenaline increased cAMP egress. We conclude that in rat neonatal cardiac myocytes agonist occupation of the alpha 1-adrenoceptor inhibits beta-adrenoceptor stimulated cAMP accumulation most likely by coupling to a guanine nucleotide inhibitory protein.     

Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca2+

The Ca2+-sensing receptor protein and the Ca2+-inhibitable type 6 adenylyl cyclase mRNA are present in a defined segment of the rat renal tubule leading to the hypothesis of their possible functional co-expression in a same cell and thus to a possible inhibition of cAMP content by extracellular Ca2+. By using microdissected segments, we compared the properties of regulation of extracellular Ca2+-mediated activation of Ca2+ receptor to those elicited by prostaglandin E2 and angiotensin II. The three agents inhibited a common pool of hormone-stimulated cAMP content by different mechanisms as follows. (i) Extracellular Ca2+, coupled to phospholipase C activation via a pertussis toxin-insensitive G protein, induced a dose-dependent inhibition of cAMP content (1.25 mM Ca2+ eliciting 50% inhibition) resulting from both stimulation of cAMP hydrolysis and inhibition of cAMP synthesis; this latter effect was mediated by capacitive Ca2+ influx as well as release of intracellular Ca2+. (ii) Angiotensin II, coupled to the same transduction pathway, also decreased cAMP content; however, its inhibitory effect on cAMP was mainly accounted for by an increase of cAMP hydrolysis, although angiotensin II and extracellular Ca2+ can induce comparable release of intracellular Ca2+. (iii) Prostaglandin E2, coupled to pertussis toxin-sensitive G protein, inhibited the same pool of adenylyl cyclase units as extracellular Ca2+ but by a different mechanism. The functional properties of the adenylyl cyclase were similar to those described for type 6. The results establish that the co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in a same cell allows an inhibition of cAMP accumulation by physiological concentrations of extracellular Ca2+.     

Glutathione peroxidase activity in endometrium: effects of sex hormones and cancer

In the present study, we investigated the mechanism of phenylephrine (alpha-1-adrenergic receptor agonist)-induced arachidonic acid release in Japanese white rabbit aortic smooth muscle cells (SMC). When introduced into permeabilized smooth muscle cells, guanosine S-[gamma-thio] triphosphate (GTPgamma S), which activates GTP-binding proteins (G proteins), stimulated arachidonic acid (AA) release. Neomycin, an inhibitor of phosphoinositide (PI) turnover, was almost without effect on GTP[gamma S] stimulated AA release. In addition, pertussis toxin (PT) partially inhibited phenylephrine-stimulated AA release, suggesting that IAP (Islet activating protein)-sensitive G proteins mediate this process. Phenylephrine-stimulated AA release was also inhibited by decreased extracellular calcium and aristolochic acid, suggesting a role for a phospholipase A2 (PLA2). Next PLA2 is reported to be a substrate for mitogen-activated protein (MAP) kinase. We examined the effect of phenylephrine on MAP kinase and c-jun N-terminal kinase (JNK) phosphorylation. Phenylephrine didn't induce phosphorylation of MAP kinase, but did induce phosphorylation of JNK. In addition, cells which were pretreated with PT inhibited the phosphorylation of JNK. These results suggest that IAP-sensitive G protein is involved in the coupling between alpha1-adrenergic receptor (AR) and PLA2 in cultured rabbit aortic SMCs, and that the alpha1-AR-induced AA release is mediated by JNK.     

Effects of menstrual cycle on creativity

Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC50 values of 2.6, 3.8 and 76 microM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.     

Angiotensin II AT1 receptor/signaling mechanisms in the biphasic effect of the peptide on proximal tubular Na+,K+-ATPase

The present study was designed to determine the cellular signaling mechanisms responsible for mediating the effects of angiotensin II on proximal tubular Na+,K+-ATPase activity. Angiotensin II produced a biphasic effect on Na+,K+-ATPase activity: stimulation at 10(-13) - 10(-10) M followed by inhibition at 10(-7) - 10(-5) M of angiotensin II. The stimulatory and inhibitory effects of angiotensin II were antagonized by losartan (1nM) suggesting the involvement of AT1 receptor. Angiotensin II produced inhibition of forskolin-stimulated cAMP accumulation at 10(-13) - 10(-10) M followed by a stimulation in basal cAMP levels at 10(-7) - 10(-5) M. Pretreatment of proximal tubules with losartan (1nM) antagonized both the stimulatory and inhibitory effects of angiotensin II on cAMP accumulation. Pretreatment of the proximal tubules with pertussis toxin (PTx) abolished the stimulation of Na+,K+-ATPase activity but did not affect the inhibition of Na+,K+-ATPase activity produced by angiotensin II. Pretreatment of the tubules with cholera toxin did not alter the biphasic effect of angiotensin II on Na+,K+-ATPase activity. Mepacrine (10microM), a phospholipase A2 (PLA2) inhibitor, reduced only the inhibitory effect of angiotensin II on Na+,K+-ATPase activity. These results suggest that the activation of AT1 angiotensin II receptors stimulates Na+,K+-ATPase activity via a PTx-sensitive G protein-linked inhibition of adenylyl cyclase pathway, whereas the inhibition of Na+,K+-ATPase activity following AT1 receptor activation involves multiple signaling pathways which may include stimulation of adenylyl cyclase and PLA2.     

Calcitriol attenuates the basal and vasoactive intestinal peptide-stimulated cAMP production in prolactin-secreting rat pituitary (GH4C1) cells

A clonal strain of prolactin-producing rat pituitary tumour cells (GH4C1 cells) was used to study the effect of calcitriol on cyclic adenosine monophosphate (cAMP) production. Calcitriol (10 nM) attenuated both the basal and vasoactive intestinal peptide (VIP)-stimulated cAMP production after 2 days' pretreatment of the cells. The effect was detectable at 1 nM and maximal at about 10 nM. Calcitriol was at least 100 times more potent than calcidiol and 24-hydroxycalcidiol. Calcitriol (10 nM, 4 days) did not affect the specific binding of 125I-VIP, but attenuated the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-stimulated (100 microM) adenylyl cyclase activity by 25%. Calcitriol (10 nM, 4 days) also attenuated both the Mn2+ (1 mM) and the forskolin-stimulated (10 microM) adenylyl cyclase activity by 43 and 41%, respectively. In conclusion, these data suggest that calcitriol attenuates the basal and VIP-stimulated cAMP production by inhibiting the catalytic subunit of the adenylyl cyclase as well as the amount of the G protein Gs alpha.     

Dopamine D2 receptors potentiate arachidonate release via activation of cytosolic, arachidonate-specific phospholipase A2

Several G(i)-linked neurotransmitter receptors, including dopamine D2 receptors, act synergistically with Ca(2+)-mobilizing stimuli to potentiate release of arachidonic acid (AA) from membrane phospholipids. In brain, AA and its metabolites are thought to act as intracellular second messengers, suggesting that receptor-dependent potentiation of AA release may participate in neuronal transmembrane signaling. To study the molecular mechanisms underlying this modulatory response, we have now used Chinese hamster ovary cells transfected with rat D2-receptor cDNA, CHO(D2). Two antisense oligodeoxynucleotides corresponding to distinct cDNA sequences of cytosolic, AA-specific phospholipase A2 (cPLA2) were synthesized and added to cultures of CHO(D2) cells. Incubation with antisense oligodeoxynucleotides inhibited D2 receptor-dependent release of AA but had no effect on D2-receptor binding or D2 inhibition of cyclic AMP accumulation. In addition, pharmacological experiments showed that D2 receptor-dependent AA release was prevented by nonselective phospholipase inhibitors (such as mepacrine) but not by inhibitors of membrane-bound, non-AA-specific PLA2 (such as p-bromophenacyl bromide). cPLA2 is expressed in brain tissue. The results, showing that cPLA2 participates in receptor-dependent potentiation of AA release in CHO(D2) cells, suggest that this phospholipase may serve a similar signaling function in brain.     

A molecular network that produces spontaneous oscillations in excitable cells of Dictyostelium

A network of interacting proteins has been found that can account for the spontaneous oscillations in adenylyl cyclase activity that are observed in homogenous populations of Dictyostelium cells 4 h after the initiation of development. Previous biochemical assays have shown that when extracellular adenosine 3',5'-cyclic monophosphate (cAMP) binds to the surface receptor CAR1, adenylyl cyclase and the MAP kinase ERK2 are transiently activated. A rise in the internal concentration of cAMP activates protein kinase A such that it inhibits ERK2 and leads to a loss-of-ligand binding by CAR1. ERK2 phosphorylates the cAMP phosphodiesterase REG A that reduces the internal concentration of cAMP. A secreted phosphodiesterase reduces external cAMP concentrations between pulses. Numerical solutions to a series of nonlinear differential equations describing these activities faithfully account for the observed periodic changes in cAMP. The activity of each of the components is necessary for the network to generate oscillatory behavior; however, the model is robust in that 25-fold changes in the kinetic constants linking the activities have only minor effects on the predicted frequency. Moreover, constant high levels of external cAMP lead to attenuation, whereas a brief pulse of cAMP can advance or delay the phase such that interacting cells become entrained.     

ROC-analysis of detection performance by analogue and digital plain film systems in chest radiography

We have previously reported that a microcarrier-attached human hepatoma (Hep G2) cell line responds to hydrodynamic shear upon transfer to an agitated, clean, autoclaved spinner flask with a transient increase in cytochrome P450IA1 (CYPIA1) activity. Physiological changes induced by hydrodynamic stress could be problematic in the scaleup of microcarrier cultures. A better understanding of how stress alters cell physiology may assist in reactor scaleup. The induction of CYPIA1 activity was dependent on the agitation level of the cultures, and the level of CYPIA1 induction was comparable to that obtained with exposure to approximately 0.1 nM TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin). It has been well documented that hydrodynamic shear stress can cause alterations in the metabolism of phospholipid membrane-bound arachidonic acid (AA) in adherent cells in a parallel plate system. The present study was carried out to determine if either AA or a metabolite of AA was involved in the induction of CYPIA1 activity in the microcarrier cultures of Hep G2 cells. Addition of exogenous AA followed by initiation of the stress resulted in an increase in the level of CYPIA1 activity. Pretreatment of the cultures with quinacrine, an inhibitor of phospholipase A2, reduced the stress-induced CYPIA1 activity. Furthermore, addition of propranolol, an inhibitor of phosphatidic acid phosphohydrolase, resulted in an increase in the response in addition to sustaining the induced enzyme activity. Pretreatment with the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, caffeic acid, had no effect on the response, suggesting that the cyclooxygenase and lipoxygenase pathways were not involved in generating AA metabolites that alter CYPIA1 activity. The agent, nordihydroguaiaretic acid, blocks the monooxygenase pathway and blocks CYPIA1 activity increases. These observations suggest a possible mechanism where the stress on the cells induces phospholipase D, resulting in the formation of phosphatidic acid which then activates phospholipase A2, resulting in the release of AA. Further, these results are consistent with a mechanism in which the metabolism of AA, most likely through the monooxygenase pathway, results in a metabolite that by a yet unknown mechanism induced CYPIA1.