Second Messengers

Enhanced responsiveness of the pituitary gland to GnRH is a fundamental step required for secretion of the proestrous LH surge, and is achieved primarily by the actions of ovarian steroid hormones on the gonadotropes. The mechanisms involved are still unclear but the CAMP second messenger pathway can mediate some of these activities. The present study was undertaken to determine the effects of in vitro cholera toxin (CTX, increases CAMP) pretreatment of pituitary cells from proestrous and diestrous 1 rats relative to their LH secretory response to GnRH using a reverse hemolytic plaque assay. The number of gonadotropes that secrete LH in the group treated with CTX increased at low doses of GnRH and also in the absence of the peptide, but decreased at high doses, showing a dual effect: stimulation of some gonadotropes and inhibition of others. The inhibition was achieved within 3 h of pretreatment in proestrous cells but it was not seen until 20 h in diestrous 1 cells. This suggests the existence of at least two subpopulations of gonadotropes, one of which is stimulated by CAMP and another which is inhibited.

Enhanced responsiveness of the pituitary gland to GnRH is a fundamental step required for secretion of the proestrous LH surge, and is achieved primarily by the actions of ovarian steroid hormones on the gonadotropes. The mechanisms involved are still unclear but the CAMP second messenger pathway can mediate some of these activities. The present study was undertaken to determine the effects of in vitro cholera toxin (CTX, increases CAMP) pretreatment of pituitary cells from proestrous and diestrous 1 rats relative to their LH secretory response to GnRH using a reverse hemolytic plaque assay. The number of gonadotropes that secrete LH in the group treated with CTX increased at low doses of GnRH and also in the absence of the peptide, but decreased at high doses, showing a dual effect: stimulation of some gonadotropes and inhibition of others. The inhibition was achieved within 3 h of pretreatment in proestrous cells but it was not seen until 20 h in diestrous 1 cells. This suggests the existence of at least two subpopulations of gonadotropes, one of which is stimulated by CAMP and another which is inhibited.

Growth hormone (GH) directly interacts with the enterocyte stimulating ion absorption and reducing ion secretion induced by agonists of cAMP. Since nitric oxide (NO) is involved in the regulation of transepithelial ion transport and acts as a second messenger for GH hemodynamic effects, we tested the hypothesis that NO may be involved in the resulting effects of GH on intestinal ion transport. Electrical parameters reflecting transepithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers and exposed to GH and cholera toxin (CT) alone or in combination, in the presence or absence of the NO synthase (NOS) inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME). Similar experiments were conducted to determine cAMP and nitrite/nitrate concentrations. NOS expression was assayed by Western blot analysis. RESULTS: L-NAME causes total abrogation of absorptive and anti-secretory effects by GH on intestinal ion transport. In addition, L-NAME was able to inhibit the GH-effects on intracellular cAMP concentration under basal conditions and in response to CT. GH induced a Ca 2+ -dependent increase of nitrites/nitrates production, indicating the involvement of the constitutive rather than the inducible NOS isoform, which was directly confirmed by Western blot analysis. These results suggest that the GH effects on intestinal ion transport, either under basal conditions or in the presence of cAMP-stimulated ion secretion, are mediated at an intracellular level by the activity of cNOS.

The MotA and MotB proteins are thought to comprise elements of the stator component of the flagellar motor of Escherichia coli. In an effort to understand interactions among proteins within the motor, we attempted to identify extragenic suppressors of 31 dominant, plasmid-borne alleles of motA. Strains containing these mutations were either nonmotile or had severely impaired motility. Four of the mutants yielded extragenic suppressors mapping to the FlaII or FlaIIIB regions of the chromosome. Two types of suppression were observed. Suppression of one type (class I) probably results from increased expression of the chromosomal motB gene due to relief of polarity. Class I suppressors were partial deletions of Mu insertion sequences in the disrupted chromosomal motA gene. Class I suppression was mimicked by expressing the wild-type MotB protein from a second, compatible plasmid. Suppression of the other type (class II) was weaker, and it was not mimicked by overproduction of wild-type ...

Current concepts of cellular oxygen-sensing include an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial electron transport chain (ETC), heme oxygenase-2 (HO-2), and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) and HIF asparagyl hydroxylase FIH-1 (factor-inhibiting HIF). Different oxygen sensitivities, cell-specific distribution and subcellular localization of specific oxygen-sensing cascades involving reactive oxygen species (ROS) as second messengers may help to tailor various adaptive responses according to differences in tissue oxygen availability. Herein, we propose an integrated model for these various oxygen-sensing mechanisms that very efficiently regulate HIF-a activity and plasma membrane potassium-channel (PMPC) conductivity.

The aim of this study was to determine the prostaglandin E (EP) receptors and second messengers implicated in glycosaminoglycan (GAG) synthesis by human cervical fibroblasts in culture. Human cervical fibroblasts were obtained from cervical biopsies in pre-menopausal, cycling women. Cultured cells were incubated with prostaglandin E 2 (PGE 2 ) and an array of agonists and antagonists. Glycosaminoglycan synthesis was assayed after extraction by measuring the [ 3 H]glucosamine and [ 35 S]sulphate incorporated into GAG and cAMP production was determined by radioimmunoassay. PGE 2 significantly stimulated GAG synthesis. Neither 17-phenyl-trinor-PGE 2 , the EP 1 selective agonist, nor sulprostone, an EP 3 agonist, had any effect on GAG production. Butaprost, the EP 2 selective agonist, also failed to increase GAG synthesis. AH6809, an EP 2 antagonist, had no effect on PGE 2 -stimulated GAG production. AH23848, an EP 4 antagonist, inhibited the GAG synthesis provoked by PGE 2 . PGE 2 and butaprost significantly increased cAMP production. Both AH6809 and AH23848 inhibited the PGE 2 -stimulated cAMP production. H89, a cAMP-dependent protein kinase (PKA) inhibitor, did not inhibit PGE 2 -stimulated GAG synthesis and Sp-cAMPS, a selective PKA activator, failed to increase GAG production. In conclusion, both EP 4 and EP 2 receptors are present and functional in human cervical fibroblasts. Only EP 4 receptors mediate PGE 2 stimulated GAG synthesis in a PKA-independent pathway.

Cyclic nucleotide phosphodiesterases (PDE) are the enzymes catalyzing the hydrolysis and inactivation of the second messengers, cAMP and cGMP. Eleven PDE families are described to date, and selective inhibitors of some PDEs families are currently used in clinic for treating cardiovascular disorders, erectile dysfunction, and pulmonary hypertension. Isoforms of the PDE4 family are involved in smooth muscle contraction and inflammation. PDE4 selective inhibitors are currently in clinical trials for the treatment of diseases related to inflammatory disorders. Because of their myorelaxant properties, we first examined their expression in human myometrium and uncover an increased expression of one specific isoform, PDE4B2, in the near-term myometrium as compared to myometrium in the nonpregnant state. Using human myometrial cells in culture, we demonstrated that PDE4B2 can be induced by its own substrate, under the control of one of the major utero-contractile agonists, PGE 2 , itself upregulated by the proinflammatory cytokine IL-1β. Functionally, augmentation of global PDE4 activity decreases the ability of β-adrenergic agonists (the most commonly used tocolytic drugs) to inhibit myometrial contraction at the end of pregnancy and during pathophysiological situations, such as persistent intrauterine inflammation which is a major cause of very preterm delivery. Currently exploring the anti-inflammatory properties of PDE4 inhibitors in gestational tissues, we recently demonstrated the ability of these drugs to block a persistent inflammatory response of the foetal membranes in Humans and to prevent inflammation-driven preterm delivery and foetal demise in mice. These data open up a new therapeutical strategy to prevent inflammation-induced preterm delivery and its sequelae in very preterm infants.

Sodium retention and ascites are serious clinical problems in cirrhosis. Urodilatin (URO) is a peptide with paracrine effects in decreasing sodium reabsorption in the distal nephron. Our aim was to investigate the renal potency of synthetic URO on urine sodium excretion in cirrhosis patients with sodium retention and ascites. Seven cirrhosis patients with diuretics-resistant sodium retention received a short-term (90 min) infusion of URO in a single-blind, placebo-controlled cross-over study. In the basal state after rehydration the patients had urine sodium excretion < 50 mmol/24 h. URO transiently increased urine sodium excretion from 22 +/- 16 micromol/min (mean +/- SD) to 78 +/- 41 mumol/min (P < 0.05) and there was no effect of placebo (29 +/- 14 to 44 +/- 32). The increase of URO's second messenger after the receptor, cGMP, was normal. URO had no effect on urine flow or on blood pressure. Most of the patients had highly elevated plasma levels of renin, angiotensin II...

The importance of protein phosphorylation in biological regulation is widely recognized; thus, a number of extracellular signaland second messenger-linked protein kinases have been well characterized, and various substrates for these kinases have been identified. We present evidence that the glycolytic intermediate 3-phosphoglycerate can cause a marked stimulation of phosphorylation of specific proteins in mammalian tissues. In the brain, there are at least two types of 3-phosphoglycerate-dependent protein phosphorylation sys- tems, which differ in activator specificity, substrate specificity, and sensitivity to the activator. Both of these phosphorylation systems are, however, insensitive to cAMP, cGMP, Ca2+, calmodulin, and protein kinase C activators and have charac- teristics different from those of casein kinase II-catalyzed protein phosphorylation. The most prominent endogenous substrates in the bovine brain are rather minor proteins with minimal molecular masses of 72 and 155 kDa, which were not detected in "nonnervous" tissues such as heart, liver, lung, and kidney. In nonnervous tissues, 3-phosphoglycerate affected the state of phosphorylation of other proteins. Evidence provided suggests the occurrence of an as yet undescribed type of protein kinase, which may be designated 3-phosphoglycerate-dependent protein kinase, in mammalian tissues. Amassed evidence attests that protein phosphorylation and dephosphorylation play a vital role in the regulation of various enzymes, receptors, cellular processes, and neuronal functions (for reviews, see refs. 1-7). For the phosphoryl- ation to serve a regulatory function, the protein kinase involved must be properly controlled by an endogenous substance whose concentration changes effectively at the site of action in response to extracellular signals or to alterations in physiological states. For these reasons, among the various protein kinases, much attention has been focused on those that are activated by second messenger-type agents, such as cAMP (8), cGMP (9), calcium/calmodulin (10), and calcium/ Abbreviations: PMA, phorbol 12-myristate 13-acetate; synaptosol, synaptosomal cytosol.

outer segment of the cell is comprised of a stack of many hundreds of densely packed discs, each of which represents a double layer of infolded plasma membrane. It has been suggested that phospholipid moieties in these membranes are metabolically active and are closely involved in the generation of physiological mediators. Modifi cations in the metabolism of photoreceptor membrane glycerolipids have been linked to the transduction of visual stimuli ( 3 ). Rod outer segment (ROS) membranes are composed of 50% protein and 50% lipid, by weight ( 2 ). Phospholipids and cholesterol represent nearly 90-95% and 4-6% (w/w) of total lipids, respectively. Rhodopsin accounts for at least 80-85% (w/w) of the total proteins ( 4-6 ) in disc ROS membranes. The rest are mainly proteins involved in phototransduction. The composition of mammalian ROS membrane phospholipids indicates that the major phospholipids are phosphatidylethanolamine (PE) and phosphatidylcholine (PC), along with relatively large amounts of phosphatidylserine (PS) (15%). Small amounts (less than 1% each) of sphingomyelin, phosphatidylinositol (PI) and phosphatidic acid (PA) are found in these membranes ( 7-13 ). In vertebrates, cholesterol (exclusively as the free sterol) is the principal neutral lipid, comprising Abstract Rod outer segments (ROSs) are specialized lightsensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for effi cient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins infl uences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules. -Giusto, N. M., S. J. Pasquaré, G. A. Salvador, and M. G. Ilincheta de Boschero. Lipid second messengers and related enzymes in vertebrate rod outer segments.

SummaryTwo novel approaches for the study of Ca2+‐mediated signal transduction in stomatal guard cells are described. Stimulus‐induced changes in guard‐cell cytosolic Ca2+ ([Ca2+]cyt) were monitored using viable stomata in epidermal strips of a transgenic line of Nicotiana plumbaginifolia expressing aequorin (the proteinous luminescent reporter of Ca2+) and in a new transgenic line in which aequorin expression was targeted specifically to the guard cells. The results indicated that abscisic acid (ABA)‐induced stomatal closure was accompanied by increases in [Ca2+]cyt in epidermal strips. In addition to ABA, mechanical and low‐temperature signals directly affected stomatal behaviour, promoting rapid closure. Elevations of guard‐cell [Ca2+]cyt play a key role in the transduction of all three stimuli. However, there were striking differences in the magnitude and kinetics of the three responses. Studies using Ca2+ channel blockers and the Ca2+ chelator EGTA further suggested that mechan...

A family of phosphoinositide-3 kinase (PI3K) isoenzymes catalyzes the production of second messengers that recruit critical regulators of cell growth, survival, proliferation and motility. Conversely, 3′- (phosphatase and tensin homolog) and 5′-inositol polyphosphatases (SH2-containing inositol phosphatases 1/2, SHIP1/2) are recruited to sites of PI3K signaling at the plasma membrane to oppose or, in some cases, to modify and enhance PI3K signaling. A substantial and growing body of literature demonstrates that these enzymes which mediate interchange of phosphates on inositol phospholipid species at the plasma membrane have prominent roles in natural killer cell biology, including development, effector functions and trafficking. Here, we review the salient points of these recent papers with a special emphasis on the role of p110δ and SHIP1 in natural killer cells.

Heart failure (HF) is a common disease that continues to be associated with high morbidity and mortality warranting novel therapeutic strategies. Cyclic guanosine monophosphate (cGMP) is the second messenger of several important signaling pathways based on distinct guanylate cyclases (GCs) in the cardiovascular system. Both the nitric oxide/soluble GC (NO/sGC) as well as the natriuretic peptide/GC-A (NP/GC-A) systems are disordered in HF, providing a rationale for their therapeutic augmentation. Soluble GC activation with conventional nitrovasodilators has been used for more than a century but is associated with cGMP-independent actions and the development of tolerance, actions which novel NO-independent sGC activators now in clinical development lack. Activation of GC-A by administration of naturally occurring or designer natriuretic peptides is an emerging field, as is the inhibition of enzymes that degrade endogenous NPs. Finally, inhibition of cGMP-degrading phosphodiesterases, particularly phosphodiesterase 5 provides an additional strategy to augment cGMP-signaling.

It was demonstrated that salicylic acid (SA) not only binds to catalase from differentiated higher plants and plant cell suspension cultures but also to those of fungi and animals. SA bound specifically to iron-containing enzymes, such as catalase, aconitase, lipoxidase and peroxidase, while not to iron-free plant enzymes. On the grounds of these experiments, the claim is further challenged that SA is a signalling compound and second messenger in plants that activates plant defense-related genes through elevated H202 levels by specifically inhibiting catalase activity. SA may just function as a phytoalexin.

Background: Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and hostpathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery. Results: Our analyses reveal four core eukaryotic PIKs which are type III PIK4A and PIK4B, and at least one homolog each from PI3K (possibly PIK3C3 as the ancestor) and PIP5K families. We also applied evolutionary analyses to PIK disease ontology and drug discovery. Mutated PIK3CA are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. We found conservation of activating mutations of PIK3CA in paralogous isoforms suggesting specific functional constraints on these residues. By mapping published compound inhibition data (IC50s) onto a phylogeny of PI3Ks, type II PI4Ks and distantly related, MTOR, ATM, ATR and PRKDC kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. Finally, we extended the rationale for drugs targeting PIKs of malarial Plasmodium falciparum, and the parasites, Leishmania sp. and Trypanosoma sp. by identifying those PIKs highly divergent from human homologs. Our phylogenomic analysis of PIKs provides new insights into the evolution of second messenger signaling. We postulate two waves of PIK diversification, the first in metazoans with a subsequent expansion in cold-blooded vertebrates that was post-emergence of Deutrostomia\Chordata but prior to the appearance of mammals. Reconstruction of the evolutionary relationships among these lipid kinases also adds to our understanding of their roles in various diseases and assists in their development as potential drug targets.

Background: Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and hostpathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery. Results: Our analyses reveal four core eukaryotic PIKs which are type III PIK4A and PIK4B, and at least one homolog each from PI3K (possibly PIK3C3 as the ancestor) and PIP5K families. We also applied evolutionary analyses to PIK disease ontology and drug discovery. Mutated PIK3CA are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. We found conservation of activating mutations of PIK3CA in paralogous isoforms suggesting specific functional constraints on these residues. By mapping published compound inhibition data (IC50s) onto a phylogeny of PI3Ks, type II PI4Ks and distantly related, MTOR, ATM, ATR and PRKDC kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. Finally, we extended the rationale for drugs targeting PIKs of malarial Plasmodium falciparum, and the parasites, Leishmania sp. and Trypanosoma sp. by identifying those PIKs highly divergent from human homologs. Our phylogenomic analysis of PIKs provides new insights into the evolution of second messenger signaling. We postulate two waves of PIK diversification, the first in metazoans with a subsequent expansion in cold-blooded vertebrates that was post-emergence of Deutrostomia\Chordata but prior to the appearance of mammals. Reconstruction of the evolutionary relationships among these lipid kinases also adds to our understanding of their roles in various diseases and assists in their development as potential drug targets.

T cells show rapid reorganization of cytoskeleton in response to antigenic stimulation. The molecular mechanisms by which TCR-CD3 regulates actin cytoskeleton are not well defined. Here we show that a type II PtdIns 4-kinase associates with cytoskeletal fraction in splenic lymphocytes in response to Con A. Protein tyrosyl phosphorylation of type II PtdIns 4-kinase appears to be the mechanism for its association with cytoskeleton. Overlay blots suggest that the enzyme binds to TCR-CD3 chain in the cytoskeletal fraction. Anti-TCR-CD3 antibodies competitively inhibit PtdIns 4-kinase association with TCR-CD3 chain. Immunodepletion of TCR-CD3 decreases PtdIns 4-kinase activity in the cytoskeletal fraction with a concomitant increase in PtdIns 4-kinase activity in anti-TCR-CD3 immunoprecipitates. We propose that the association of type II PtdIns 4-kinase with TCR-CD3 chain may bring the enzyme into close proximity of actin and a possible regulation of actin polymerization through localized production of PtdIns4P and PtdIns(4,5)P 2 .

Membrane depolarization, or agents which increase intracellular calcium, elicit transcriptional activation of tyrosine hydroxylase (TH). In this study we analyze the factors involved in the regulation of the TH promoter by a calcium ionophore. PCI2 cells were transiently transfected with plasmids containing wild type or mutated 5' flanking sequences of the rat TH gene, fused to bacterial chloramphenicol acetyl transferase (CAT). Point mutations introduced into the consensus cAMP-regulatory element (CRE) abolished the induction of CAT by ionomycin indicating that it is essential for mediating the calcium response. An intact and functional AP1 site did not confer calcium inducibility when the CRE/CaRE sequence was mutated. The extent and kinetics of the increase in intracellular calcium as well as the induction of CAT activity under the control of TH promoter by ionomycin were similar in PCI2 cells and in the A123.7, protein kinase A (PKA) deficient cell line. In both cell lines addition of ionomycin rapidly increased the phosphorylation of transcription factor CREB at Ser-133. These results suggest that the activation of TH transcription by ionomycin does not require PKA. However, KN62 an inhibitor of Ca 2~/calmodulin dependent (CAM) kinases prevented the induction indicating possible involvement of CAM kinases in the calcium response.

Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine's antiinflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway. We observed that adenosine up-regulates the expression of the COX-2 enzyme and mRNA. Production of PGE 2 in response to exogenous arachidonic acid was also increased by adenosine and correlated with COX-2 protein levels. The potentiating effect of adenosine on COX-2 could be mimicked by pharmacological increases of intracellular cAMP levels, involving the latter as a putative second messenger for the up-regulation of COX-2 by adenosine. Specific COX-2 inhibitors were used to confirm the predominant role of the COX-2 isoform in the formation of prostanoids by stimulated PMNs. Withdrawal of extracellular adenosine strikingly emphasized the inhibitory potential of PGE 2 on leukotriene B 4 formation and involved the EP 2 receptor subtype in this process. Thus, adenosine may promote a self-limiting regulatory process through the increase of PGE 2 generation, which may result in the inhibition of PMN functions. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes, introducing the concept that this autacoid may exert its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.

The phototransduction cascade in Limulus ventral photoreceptors involves multiple second messengers, including Ca 2+ and cGMP. Light-induced Ca 2+ release from intracellular stores is an intermediate step, but the subsequent Ca 2+-activated reaction remains to be determined. The possibility that Ca 2+ /calmodulin (Ca 2+ /CaM) might be involved is suggested by the high calmodulin content of the transducing lobe. To test whether CaM can excite the transduction cascade we injected a 25 M Ca 2+ /CaM solution. This produced a rapid, brief depolarization similar to that produced by light, suggesting a role for CaM in the cascade. However, an important caveat is that Ca 2+ dissociating from the Ca 2+ /CaM complex might excite this process. Several control experiments argue against, but do not entirely eliminate this possibility. To test whether endogenous CaM has a function in excitation, trifluoperazine was pressure injected into the rhabdomeric region. The response to brief flashes was not affected, but the response to steady illumination was transiently attenuated by each injection. We conclude that calmodulin should be considered a candidate to couple intermediate and late stages of the transduction cascade.

Small chemotactic cells like Dictyostelium and neutrophils transduce shallow spatial chemoattractant gradients into strongly localized intracellular responses. We show that the capacity of a second messenger to establish and maintain localized signals, is mainly determined by its dispersion range, ϭ ͌ D m /k Ϫ1 , which must be small compared to the cell's length. Therefore, short-living second messengers (high k Ϫ1) with diffusion coefficients D m in the range of 0-5 m 2 s Ϫ1 are most suitable. Additional to short dispersion ranges, gradient sensing may include positive feedback mechanisms that lead to local activation and global inhibition of second-messenger production. To introduce the essential nonlinear amplification, we have investigated models in which one or more components of the signal transduction cascade translocate from the cytosol to the second messenger in the plasma membrane. A one-component model is able to amplify a 1.5-fold difference of receptor activity over the cell length into a 15-fold difference of second-messenger concentration. Amplification can be improved considerably by introducing an additional activating component that translocates to the membrane. In both models, communication between the front and the back of the cell is mediated by partial depletion of cytosolic components, which leads to both local activation and global inhibition. The results suggest that a biochemically simple and general mechanism may explain various signal localization phenomena not only in chemotactic cells but also those occurring in morphogenesis and cell differentiation.

The naphthalene sulfonamide calmodulin antagonists, /V-(6-aminohexyl)-S-chloro-l-naphthalenesulfonamide and jV-(4-aminobutyl)-5chloro-2-naphthalenesulfonamide, both induce limited myeloid differen tiation of the human promyelocytic cell line, HL-60. In addition, these inhibitors augment the differentiation observed when HL-60 cells are induced with retinole acid, dimethyl sulfoxide, or dibutyryl cyclic adenosine monophosphate. The dose-response curve for HL-60 differentiation was consistent with the published 50% inhibitory dose for inhibition of calmodulin-activated phosphodiesterase and with the calmodulin drug-binding potential of N(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide and A'-(4-aminobu~ tyl)-5-chloro-2-naphthalenesulfonamide and their less active congeners, yV-(6-aminohexyl)-l-naphthalenesulfonamide and A'-(4-aminobutyl)-2naphthalenesulfonamide. These effects, of the naphthalene sulfonamide calmodulin antagonists, are consistent with a regulatory role for calm...

A new way of assessing thesignificance of intracellular signals that may regulate cellular proliferation, would be to analyze possible ,second messengers' when proliferation is slowed down, rather than stimulated. Therefore, we examined proliferating mononuclear blood cells from leukaemic patients which had been exposed to an inhibitory ox leucocyte extract. The extract decreased 3H-thymidine incorporation in leukaemic cells in short-term cultures. The inhibition was not cellline specific, but was nevertheless non-toxic and not due to endotoxin. The K + flux into the leukaemic cells was assessed with 86Rb+, a K + analogue. An inverse relationship was found between 86Rb+ uptake and 3H-thymidine incorporation. The increased 86Rb+ influx was probably due to leakage or exchange mechanisms other than the Na +/K + membrane pump, as suggested by ouabain inhibition experiments. However, the long lag time (> 45 min) between addition of inhibitor and a marked increase in 86Rb+ uptake does not support a role for the K + flux as an early mediator of the inhibitory signal.

11Beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) deficiency causes sodium retention and severe hypertension by allowing glucocorticoids access to the non-selective mineralocorticosteroid receptor. Understanding regulation of the HSD11B2 gene is thus of fundamental importance in hypertension research. A number of studies have suggested that second messenger pathways may be important in this regard. In the present study we show that HSD11B2 expression in human renal epithelial P58 cells is regulated at the mRNA and protein level, and that protein kinases A (PKA) and C (PKC) are involved in this process. PKA stimulation resulted in almost twofold increase while PKC activation in almost twofold decrease in the HSD11B2 mRNA and protein level. Western blot analysis revealed a dimeric form of 11beta-HSD2 of about 80 kDa. Arginine vasopressin (AVP), acting through the AVP2 receptor, as well as 11beta-HSD2 substrates, corticosterone and dexamethasone, up-regulate HSD11B2 expression, suggesting their role as possible factors affecting blood pressure. We show that the activators of the PKA pathway induce, while activators of PKC pathway repress the expression of HSD11B2 in human renal epithelial cells. AVP, acting via the PKA pathway, might be a physiological stimulator of the HSD11B2 expression. The 11beta-HSD2 substrates, both natural (corticosterone) and synthetic (dexamethasone), might protect the mineralocorticosteroid-target cells against cortisol excess.

NMDA receptors (NMDARs) play a distinct role at excitatory glutamatergic synapses, where they are usually localized with other ionotropic glutamate receptors, including tors. Two features are essential to their specialized roles in synaptic plasticity and the excitodependent magnesium block, the removal of which requires depolarization of the membrane potential. Second, upon activation, the NMDAR channel passes sodium and, importantly, calcium into the neuron. Calcium is the universal second messenger in numerous intracellular signaling cascades and is critical in synaptic plasticity and mechanisms of neurotoxicity (288).

NMDA receptors (NMDARs) play a distinct role at excitatory glutamatergic synapses, where they are usually localized with other ionotropic glutamate receptors, including tors. Two features are essential to their specialized roles in synaptic plasticity and the excitodependent magnesium block, the removal of which requires depolarization of the membrane potential. Second, upon activation, the NMDAR channel passes sodium and, importantly, calcium into the neuron. Calcium is the universal second messenger in numerous intracellular signaling cascades and is critical in synaptic plasticity and mechanisms of neurotoxicity (288).

Dopamine (DA) and atrial natriuretic factor (ANF) share a number of physiological effects. We hypothesized that ANF and the renal dopaminergic system could interact and enhance the natriuretic and diuretic effects of the peptide. We have previously reported that the ANF-stimulated DA uptake in renal tubular cells is mediated by the natriuretic peptide type-A receptor (NPR-A). Our aim was to investigate the signaling pathways that mediate ANF effects on renal 3 H-DA uptake. Methylene blue (10 μM), an unspecific inhibitor of guanylate cyclase (GC), blunted ANF elicited increase of DA uptake. ODQ (10 μM) a specific inhibitor of soluble GC, did not modify DA uptake and did not reverse ANF-induced increase of DA uptake; then the participation of nitric oxide-dependent pathways must be discarded. The second messenger was the cGMP since the analogous 125 μM 8-Br-cGMP mimicked ANF effects. The specific inhibitor of the protein kinase G (PKG), KT 5823 (1 μM) blocked ANF effects indicating that PKG is involved. We examined if ANF effects on DA uptake were able to modify Na + , K +-adenosine triphosphatase (Na + , K +-ATPase) activity. The experiments were designed by means of inhibition of renal DA synthesis by carbidopa and neuronal DA uptake blocked by nomifensine. In these conditions renal Na + , K +-ATPase activity was increased, in agreement with the decrease of DA availability. When in similar conditions, exogenous DA was added to the incubation medium, the activity of the enzyme tended to decrease, following to the restored availability of DA. The addition of ANF alone had similar effects to the addition of DA on the sodium pump, but when both were added together, the activity of Na + , K +-ATPase was decreased. Moreover, the extraneuronal uptake blocker, hydrocortisone, inhibited the latter effect. In conclusion, ANF stimulates extraneuronal DA uptake in external cortex tissues by activation of NPR-A receptors coupled to GC and it signals through cGMP as second messenger and PKG. Dopamine and ANF may achieve their effects through a common pathway that involves reversible deactivation of renal tubular Na + , K +-ATPase activity. This mechanism demonstrates a DA-ANF relationship involved in the modulation of both decreased sodium reabsorption and increased natriuresis.

We have studied the Ca 2؉ leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca 2؉ in the endoplasmic reticulum ([Ca 2؉ ] ER). Cytosolic Ca 2؉ ([Ca 2؉ ] C) was clamped to the resting level by a BAPTA-Ca 2؉ mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca 2؉ ] ER , and the rate of Ca 2؉ release generated by 10 pM cholecystokinin is at least 3؋ as fast as the basal Ca 2؉ leak revealed by inhibition of the endoplasmic reticulum Ca 2؉-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca 2؉ ] ER , with an EC 50 of 0.98 ؎ 0.06 M. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP 3) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH 2 cyclic ADP-ribose (20 M) inhibits the action of cholecystokinin, but not of acetylcholine. The basal Ca 2؉ leak from the endoplasmic reticulum is not blocked by antagonists of the IP 3 receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with puromycin (0.1-1 mM) to remove nascent polypeptides from ribosomes increases Ca 2؉ leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca 2؉ pumps and of the receptors for IP 3 or ryanodine.

Wherever you travel through the cytoplasm of the cells you will find organelles with internal [Ca 2+ ] levels higher than in the surrounding cytosol. This is particularly true of the endoplasmic reticulum (ER) (or sarcoplasmic reticulum (SR) in muscle cells); such organelles serve as the main sources of releasable Ca 2+ for cytosolic cellular signalling. Calcium pumps of the SERCA family (sarcoplasmic and endoplasmic reticulum calcium ATP-ases) import calcium into the organelle lumen. The other mechanism that is responsible for the steady state calcium level within the lumen of ER or SR is a calcium leak that balances the influx created by the pumps. The leak remains the most enigmatic of the processes involved in calcium regulation. The molecular nature of the leak mechanism is not known. The basal leak is a relatively slow process, which is difficult to investigate and which is easily outmatched (both in the amplitude of calcium responses and in attractiveness to experimenters) by substantially faster second messenger-induced release. Nevertheless, information on the properties of the calcium leak, although thinly scattered through the pages of PubMed, has been slowly accumulating. In this review we will discuss the properties of the calcium leak and speculate about possible mechanisms, which could mediate this process.

Serotonin (SHT) is thought to play a role in a wide variety of central nervous system functions, including sleep, appetite, mood and pain perception. Understanding of this role has been hampered by the paucity of selective agonists and antagonists for the different 5HT receptor subtypes. In particular, there are few available 5HT 2 antagonists devoid of activity at a~ adrenoceptors. We now report the selective 5HT 2 antago~st action of the novel r~apht.ostdtam derivative, RP 62203. RP 62203 binds potently to the 5HT 2 receptor with a Ki value of 0.26 nM in competition experiments against [3H]-ketanserin on rat cortical membranes. Its affinity for 5HT-1A receptors is some threehundred-fold lower (Ki = 68.5 nM, measured with [3H]-8-OH-DPAT on rat hippocampal membranes). RIP 62203 displayed no measurable affinity for 5HT 3 receptors in the rat entorhinal cortex (Ki > 10-6 M versus [3H]-GR 65630). Its selectivity for 5HT 2 over a l receptors was 135x (Ki = 35 nM) versus [SH-prazosin), compared to forty times for ritanserin. RP 62203 had negfigeable affinity (Ki > 10-s M) for other monoamine receptors (ACH-m, ,8, D 2, a2) , except for histamine H l receptors in the guinea-pig cerebellum s for which a Ki versus [3H]-mepyramine of 25 nM was found, compared to 6 nM for ritanserin. RIP 62203 had no detectable affmity for the following receptors: benzodiazepine, CCK-A, CCK-B, neuroten~in~ neuropeptide Y, vasopressin (Vi) , substance P (NKl) , it, ~-or ~-opiate, PAF or somatostatin. Similarly, RP 62203 was inactive in binding assays for voltage-sensitive sodium and calcium channels. In vivo, RP 62203 displaced the binding of [3I-I]-methylspiperone in the rat frontal cortex (a marker for 5HT 2 receptors) with an IDso of 0.49 mg/kg po, whilst no displacement of the bindin 8 of this figand was observed in the rat striatum (a marker for D 2 receptors) at the highest dose tested (20 mg/kg po). Similar data were obtained in the mouse. After intraperitoneal administration to guinea-pigs, RP 62203 did not displace the binding of [3H]-mepyramine to cerebellar membranes measured ex vivo at the highest dose tested (20 mg/kg po). In the neonatal rat, serotonin stimulated the formation of [3H]-inositol phosphates from cortical sli~ prelabelled with [3H]-myo-inositol with an EC5o of 8 × 10-e M. The effect of serotonin was mimicked by the selective 5HT 2 agonist DOI, but not by the 5HT-1A agonist 8-OH-DPAT. RIP 62203 antagonised 5HT-stimulated inositol phophate formation with an ICso of 7.9 nM. This compound also blocked inositoi phosphate formation induced by the 5HT 2 agonist DOI, but not that produced by carbachol. RP 62203 had no effect on basal inositol phosphate formation. The data presented demonstrate that RP 62203 is one of the most potent and selective 5HT 2 antagonists described to date, and should prove invaluable in evaluating the role of 5HT 2 receptors in serotonin-mediated neurotransmission in the central nervous system. Furthermore, the |c~ affinity of RP 62203 for a~-adrenoceptors should lead to less pronounced cardiovascular effects than those observed for currently available 5HT 2 antagonists.

BACKGROUND: Taste receptor cells are responsible for transducing chemical stimuli into electrical signals that lead to the sense of taste. An important second messenger in taste transduction is IP3, which is involved in both bitter and sweet transduction pathways. Several components of the bitter transduction pathway have been identified, including the T2R/TRB taste receptors, phospholipase C β2, and the G

The proteolytic processing of the beta-amyloid precursor protein (APP) is regulated by neurotransmitters. Stimulation of metabotropic glutamate receptors (mGluRs) has been shown to increase the release of soluble amyloid precursor protein derivatives (APPs) from cultured cells. We examined the effects of mGluR agonists on APP processing in cortical and hippocampal slices from rat brain. Incubation of the slices in the presence of L-glutamic acid (500 microM), trans-(1S,3R)-1-amino-1,3-cyclopentane dicarboxylic acid (1-100 microM) or quisqualic acid (1-100 microM) increased APP release into the medium, relative to the amount of APPs released during incubation in normal Krebs-Ringer buffer under basal conditions. N-Methyl-D-aspartate (1-320 microM), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (1-100 microM) or kainic acid (5-500 microM) did not alter APP release. The increases in APP release induced by L-glutamic acid (500 microM), trans-(1S,3R)-1-amino-1,3-cyclopen...

A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio-and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio-and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241.82 ng/10 8 cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [ 3 H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A 2 (PLA 2 ) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels indistinguishable from that seen in cells transfected with vector alone. These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mi-togenesis and provide compelling evidence for the PLA 2 -AA-EET pathway as an important intracellular-signaling pathway in cells expressing high levels of cytochrome P450 epoxygenase.

Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca2+]i). The extremely complex calcium machinery includes plasma membrane bound channels as well as int...

Tyrosine hydroxylase (TH) is induced by nerve growth factor (NGF) as well as by elevated K' concentrations in cultures of sympathetic neurons dissociated from superior cervical ganglia of newborn rats. The two inductions were found to be mediated by different mechanisms. Cultures of sympathetic neurons required the continuous presence of 10 rig/ml of 2.5 S NGF for maximal survival. Increasing the NGF concentration in cultures grown at 10 rig/ml for 5 days to 100 to 1,000 rig/ml for an additional 2 to 3 days resulted in an elevation of the specific TH activity by 50 to 100%. Immunotitration demonstrated that the elevated TH activity was due to an increased number of enzyme molecules. The fact that the NGF-mediated TH induction was blocked by cycloheximide but not by a-amanitin suggests a regulation at the post-transcriptional level. Both dibutyryl CAMP and cholera toxin failed to elevate the activity of TH, indicating that CAMP does not act as a second messenger. The same is true for Ca'+, since the NGF-mediated TH induction was neither reduced by an inhibitor of transmembrane Ca2+ flux (D-600) nor by an inhibitor of cahnodulin (trifluoperazine) . Exposing cultures grown at 1,000 ng of NGF/ml for 1 to 3 days to medium with elevated K' concentrations (55 mM) resulted in a further increase of TH activity by about 100%. Stimulation was maximal when normal medium was replaced by medium with elevated K' concentrations 7 days after plating; no inductioin was observed when cultures were switched to elevated K' concentrations later than 14 days thereafter. As for NGF, the K+-mediated TH increase was due to an augmented number of enzyme molecules (immunotitration). In contrast to the NGF-mediated induction, the K+-mediated induction appeared to be regulated at the transcriptional level, since it was markedly reduced by a-amanitin. The extent of TH induction by increasing concentrations of K+ paralleled the reduction in membrane potential as assessed by the uptake of methyltriphenylphosphonium. Reduction of the Ca2' concentrations in the medium had no influence on TH induction. However, it was inhibited by D-600, Mg2+, and diphenylhydantoin, substances interfering with transmembrane Ca2+ fluxes. Moreover, TH induction was blocked by trifluoperazine and felodipine, drugs interfering selectively with cahnodulin-mediated processes. In electron micrographs of cells grown at elevated K+ concentrations, the incidence of mitochondria containing calcium precipitates was reduced by 40%, suggesting that the K+-induced depolarization interfered with the mitochondrial sequestration of Ca'+. Nerve growth factor (NGF) is a protein which plays a central role in the development and maintenance of function of sympathetic and sensory neurons of the peripheral nervous system. One of the most characteristic

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIPE), Can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca 2÷-and polyphosphoinostidebinding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dosedependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that oobserved with gelsolin overexpression, which in addition to capping, also severs and nucleates actin illaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than controltransfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca 2÷ signaling. These responses are consistent with enhanced PLC'y activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC'y was not due primarily to an increase in the PIP2 substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP2-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation. CapG is a new nomenclature for a protein referred to previously as gCap39 (Yu et al., 1990), macrophage capping protein (southwick and DiNubile, 1986), or mbhl (Prendergast and Ziff, 1991). The new name is adopted because cDNA and genomic analyses showed that they are identical.

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIP2), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca(2+)- and polyphosphoinositide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsi...

Macrophages (mOp) derived from mice treated in utero with chlordane show a significant delay of tumoricidal induction activity. In this study, mop from chlordane-treated animals required a 48 h in vitro period of induction with interferon-7 and lipopolysaccharide (IFN/LPS) before they could kill P815 targets. Similarly, mop from chlordane-treated animals also failed to produce an immediate H20 2 burst upon perturbation. Conversely, their stimulated control mop counterparts were tumoricidal by 2 h and exhibited a respiratory burst without any delay. Moreover, levels of the second messenger, inositol triphosphate (IP3), were significantly delayed in chlordane-treated animals following interaction with IFN/LPS. When nitrate/nitrite production was analyzed as an alternate mechanism for killing tumors, stimulated mop from both normal and chlordane-treated animals responded equally. The data show that chlordane differentially introduces defects in mop biochemical mechanisms associated with tumor killing.