Cellular Cyclic AMP Levels Research Articles

The antilipolytic action of insulin on adrenocorticotrophin-stimulated rat adipocytes. The roles of adenosine 3',5'-monophosphate and the protein kinase dependent on adenosine 3',5'-monophosphate.

The extent to which a fall in cellular cyclic AMP could account for the antilipolytic action in rat epididymal adipocytes incubated with adrenocorticotrophic hormone was studied. The antilipolytic effect, measured by suppression of glycerol release, was always associated with a decrease in cyclic AMP, but the magnitude of the fall was modified by several factors. For example, it was greater when the cAMP level was high, as when it is at its peak after hormone stimulation, or when cell concentrations are low. Glucose did not modify appreciably the insulin effect on the nucleotide level. The inhibitory effects of insulin on corticotrophin-stimulated lipolysis and cyclic AMP levels were detectable at the concentrations of 1 microU/ml and were biphasic, with maximal effects at 10-100 microU/ml. Protein kinase activity ratio was similarly affected. Activity of cyclic-AMP-dependent protein kinase conformed closely to the level of cyclic AMP. There was no indication that insulin modified the sensitivity of the kinase to cyclic AMP. Insulin did not alter the relationship of cellular cyclic AMP levels to glycerol when adipocytes were incubated with various concentrations of corticotrophin. This was true, irrespective of whether measurements were made when cyclic AMP was on the upward rise after hormone stimulation, or on the decline. The curves obtained with and without insulin were superimposable. It is concluded that the inhibitory action of insulin on lipolysis in fat cells can be fully accounted for by a decrease in cyclic AMP.

Synthesis, bioactivity and specificity of glucagon-like peptide-1 (7–37)/polymer conjugate to isolated rat islets

In order to increase the functionality of islets encapsulated in a biohybrid artificial pancreas (BAP), it was proposed that co-encapsulation with insulinotropic agents would improve insulin secretion from islets. To prevent agents from leaking out, conjugation with high-molecular-weight polymers was inevitable. In this study, synthetic glucagon-like peptide-1 (GLP-1) (7–37) was conjugated to a water-soluble polymer, poly( N-vinyl-2-pyrroridone-co-acrylic acid) (5 mol% acrylic acid, M w 445 kDa), via poly(ethylene glycol, M w 3.4 kDa) spacer. The chemical conjugation was confirmed by reverse phase-HPLC and the GLP-1 content in the GLP-1/polymer conjugate (VAPG) was determined by UV spectrophotometry at 280 nm (ca. 29 wt/wt%). In a static insulin secretion test, the VAPG increased insulin secretion up to 200% over a control (no stimulation) at high glucose levels, although the insulinotropic activity of VAPG was slightly lower than that of native GLP-1. The bioactivity of VAPG was prolonged for at least 2 weeks, which was examined by co-encapsulation of the conjugate into islet microcapsules. Dose–response curve revealed that the half-maximal effective dose (ED 50) of VAPG was about 55 n m (25 n m for native GLP-1). By N-terminal analysis using aminopeptidase and RP-HPLC, it was confirmed that the lowered bioactivity of VAPG stemmed from the polymer conjugation to N-terminal histidine moieties, which actively participate in binding to GLP-1 receptors, resulting in only 16% of N-terminal histidine remaining intact after the conjugation reaction. Finally, the specific interaction of the VAPG with isolated rat islets was investigated. Total cellular cyclic AMP levels were measured and confocal microscopy was conducted using GLP-1 and VAPG labeled with fluorescent probes. It was found that VAPG effectively increased the cAMP level in islet cells in a glucose concentration-dependent manner. Moreover, the confocal microscopy study showed that the binding of VAPG occurs at the same location where GLP-1 binds but with less affinity than that of native GLP-1. In summary, a GLP-1/polymer conjugate was synthesized for the first time, and its bioactivity was examined, which must result from its specific interaction with isolated islets.

DeltaRaf-1:ER* bypasses the cyclic AMP block of extracellular signal-regulated kinase 1 and 2 activation but not CDK2 activation or cell cycle reentry.

Elevation of cellular cyclic AMP (cAMP) levels inhibits cell cycle reentry in a variety of cell types. While cAMP can prevent the activation of Raf-1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) by growth factors, we now show that activation of ERK1/2 by DeltaRaf-1:ER is insensitive to cAMP. Despite this, DeltaRaf-1:ER-stimulated DNA synthesis is still inhibited by cAMP, indicating a cAMP-sensitive step downstream of ERK1/2. Although cyclin D1 expression has been proposed as an alternative target for cAMP, we found that cAMP could inhibit DeltaRaf-1:ER-induced cyclin D1 expression only in Rat-1 cells, not in CCl39 or NIH 3T3 cells. DeltaRaf-1:ER-stimulated activation of CDK2 was strongly inhibited by cAMP in all three cell lines, but cAMP had no effect on the induction of p21(CIP1). cAMP blocked the fetal bovine serum (FBS)-induced degradation of p27(KIP1); however, loss of p27(KIP1) in response to DeltaRaf-1:ER was less sensitive in CCl39 and Rat-1 cells and was completely independent of cAMP in NIH 3T3 cells. The most consistent effect of cAMP was to block both FBS- and DeltaRaf-1:ER-induced expression of Cdc25A and cyclin A, two important activators of CDK2. When CDK2 activity was bypassed by activation of the ER-E2F1 fusion protein, cAMP no longer inhibited expression of Cdc25A or cyclin A but still inhibited DNA synthesis. These studies reveal multiple points of cAMP sensitivity during cell cycle reentry. Inhibition of Raf-1 and ERK1/2 activation may operate early in G(1), but when this early block is bypassed by DeltaRaf-1:ER, cells still fail to enter S phase due to inhibition of CDK2 or targets downstream of E2F1.

Electrophysiology of posterior, NaCl-absorbing gills of Chasmagnathus granulatus: rapid responses to osmotic variations.

In the present study, the influence of short-term osmotic variations on some electrophysiological properties related to NaCl absorption across posterior gills of Chasmagnathus granulatus was investigated. The transepithelial potential difference (V(te)) of isolated and perfused gills increased significantly when hyposmotic saline (699 mosmol l(-1)) was used instead of isosmotic solution (1045 mosmol l(-1)). A reduction of the concentration of Na(+) or Cl(-) at constant osmolarity did not produce any change in V(te). Transepithelial short-circuit current (I(sc)) and conductance (G(te)), measured with split gill lamellae mounted in a modified Ussing chamber, also increased after changing to hyposmotic salines (I(sc): from -89.0+/-40.8 microA cm(-2) to -179.3+/-37.0 microA cm(-2); G(te): from 40.5+/-16.9 mS cm(-2) to 47.3+/-15.8 mS cm(-2)). The observed effects of reduced osmolarity were fast, reversible and gradually dependent on the magnitude of the osmotic variation. The activity of the Na(+)/K(+)-ATPase increased significantly after perfusion with hyposmotic saline, from 18.73+/-6.35 micromol P(i) h(-1) mg(-1) to 41.84+/-14.54 micromol P(i) h(-1) mg(-1). Theophylline maintained part of the elevated V(te) induced by hyposmotic saline, suggesting that an increased cellular cyclic AMP level is involved in the response to reduced osmolarity. In summary, the results indicate that the hemolymph osmolarity regulates active transbranchial NaCl absorption by modulating the activity of the basolateral Na(+)/K(+)-ATPase and by changing a conductive pathway, probably at the apical membrane.

Investigation of the cellular mechanism of inhibition of formyl-methionyl-leucyl-phenylalanine-induced superoxide anion generation in rat neutrophils by 2-benzyloxybenzaldehyde

The inhibition of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O 2 −) generation by 2-benzyloxybenzaldehyde (CCY1a) was investigated in rat neutrophils, and the underlying mechanism of this inhibition was assessed. CCY1a concentration-dependently inhibited O 2 − generation ( ic 50=18.5±4.3 μM). In cell-free systems, CCY1a failed to alter O 2 − generation during dihydroxyfumaric acid autoxidation, in phorbol 12-myristate 13-acetate (PMA)-activated neutrophil particulate NADPH oxidase preparations, or during arachidonic acid-induced NADPH oxidase activation. CCY1a increased cellular cyclic AMP (cAMP) levels in a time- and concentration-dependent manner, and this cAMP-elevating effect was inhibited by the adenylyl cyclase inhibitor 9-(tetrahydro-2′-furyl)adenine (SQ22536), adenosine deaminase (ADA), and the adenosine receptor antagonist 8-( p-sulfophenyl)theophylline. In neutrophils, inhibition of O 2 − generation by CCY1a was partially reversed by the protein kinase A inhibitor (9 R,10 S,12 S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1 H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720). CCY1a did not affect fMLP-induced p38 mitogen-activated protein kinase phosphorylation, but concentration-dependently attenuated the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt ( ic 50 about 31.3 and 19.4 μM, respectively). The plateau phase, but not the initial spike, of fMLP-induced [Ca 2+] i changes was inhibited by CCY1a in a concentration-dependent manner. CCY1a inhibition of Ca 2+ entry, ERK, and Akt phosphorylation was not prevented by SQ22536 or ADA. fMLP-induced phospholipase D (PLD) activation was inhibited by CCY1a ( ic 50=13.9±2.0 μM). ADA and KT5720 did not prevent the inhibition of PLD activation by CCY1a. Collectively, these results indicate that the inhibition by CCY1a of fMLP-induced O 2 − generation in rat neutrophils can probably be attributed to the increase in cAMP levels, and to the blockade of Ca 2+ entry, suppression of Akt, and PLD activation via cAMP-independent mechanisms.

Morphological Changes in Salivary Glands Induced by Theophylline in Rats

Theophylline, a phosphodiesterase inhibitor, is known to induce enlargement of the salivary glands. This enlargement has been thought to be associated with enhanced cellular levels of cyclic AMP as a result of inhibition of phosphodiesterase. In the present study, to clarify the sequential changes in salivary glands following the administration of theophylline, a four-day repeated dose study and a single dose study were carried out in male rats, and the parotid and submaxillary glands were examined histologically. The results from the glands were almost identical. In the repeated dose study, the increased organ weight just before the last administration was reduced at 4 hr and then restored at 8 hr after the last administration. In the single dose study, a slight but significant increase in organ weight was observed at 24 hr after administration, following a transient reduction. Changes in acinus histology were consistent with these organ weight changes. Acinar cells were loaded with excessive secretory granules just before the last administration in the repeated dose study. After the last administration, granules were reduced at 4 hr, and then reaccumulated at 8 hr. Such time-course variability in the number of secretory granules corresponded well to clinically observed salivation. These results suggest that inhibition of phosphodiesterase is primarily involved in the discharge of secretory granules, and that the salivary hypertrophy previously reported is an adaptive response caused by the repeated stimulation of saliva secretion.

Forskolin as a tool for examining adenylyl cyclase expression, regulation, and G protein signaling.

1. As initially shown by Seamon and Daly, the diterpene forskolin directly activates adenylyl cyclase (AC) and raises cyclic AMP levels in a wide variety of cell types. In this review, we discuss several aspects of forskolin action that are often unappreciated. These include the utility of labeled forskolin as a means to quantitate the number of AC molecules; results of those types of studies, coupled with efforts to increase AC expression, document that such expression stoichiometrically limits cyclic AMP formation by hormones and neurotransmitters. 2. Response to forskolin is also strongly influenced by the activation of AC by the heterotrimeric G-protein, Gs. Gs-promoted enhancement of AC activity in response to forskolin occurs not only when cells are incubated with exogenously administered agonists that activate G-protein-coupled receptors but also by agonists that can be endogenously released by cells. 3. Such agonists, which include ATP and prostaglandins, serve as autocrine/paracrine regulators of cellular levels of cyclic AMP under "basal" conditions and also in response to forskolin and to agonists that promote release of such regulators. 4. The ability of forskolin to prominently activate cyclic AMP generation has proved valuable for understanding stoichiometry of the multiple components involved in "basal" cyclic AMP formation, in enzymologic studies of AC as well as in defining responses to cyclic AMP in cells within and outside the nervous system.

S-carboxymethylcysteine inhibits neutrophil activation mediated by N-formyl-methionyl-leucyl-phenylalanine

In this study, the possible mechanisms of action for the inhibitory effects of S-carboxymethylcysteine on the activation of human neutrophils by N-formyl-methionyl-leucyl-phenylalanine (FMLP) were investigated. Preincubation of neutrophils with more than 10 μg/ml of S-carboxymethylcysteine was found to impair neutrophil chemotactic activity toward FMLP, and to inhibit FMLP-mediated neutrophil adherence to pulmonary vascular endothelial cells. Preincubation of neutrophils with 10 and 100 μg/ml of S-carboxymethylcysteine decreased in the production of inositol 1,4,5-triphosphate (IP3) and diacylglycerol in neutrophils stimulated with FMLP, respectively. Preincubation of neutrophils with S-carboxymethylcysteine did not affect the cellular cyclic AMP (cAMP) levels in neutrophils stimulated with FMLP. S-carboxymethylcysteine inhibited the enzymatic activity of phosphatidyl inositol-specific phospholipase C in vitro in a concentration-dependent manner. These findings indicate that S-carboxymethylcysteine attenuates FMLP-stimulated neutrophil activation at least in part by inhibiting phosphatidyl inositol-specific phospholipase C-mediated signal transduction.

Cellular mechanisms of inhibition of superoxide anion generation in rat neutrophils by the synthetic isoquinoline DMDI

This study was undertaken to assess the cellular localization of the inhibitory effect of a chemically synthetic isoquinoline compound 1-(3′,4′-dimethoxybenzyl)-6,7-dichloroisoquinoline (DMDI) on the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils. The DMDI concentration dependently inhibited the superoxide anion (O 2 ⋅−) generation and O 2 consumption (IC 50 12.2±4.9 and 15.2±8.4 μM, respectively) of neutrophils. DMDI did not scavenge the O 2 ⋅− generated during the autoxidation of dihydroxyfumaric acid in a cell-free system. DMDI did not elevate cellular cyclic AMP levels. Inhibition of O 2 ⋅− generation by DMDI in neutrophils was not reversed by a cyclic AMP-dependent protein kinase inhibitor, (8 R,9 S,11 S)-(−)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7b,11a-triazadibenzo[ a,g]cycloocta[ cde]trinden-1-one (KT5720). The DMDI concentration dependently inhibited the late plateau phase but not the initial spike of fMLP-induced [Ca 2+] i changes in the presence of extracellular Ca 2+. However, DMDI had no effect on the fMLP-induced [Ca 2+] i changes in the absence of extracellular Ca 2+. In addition, DMDI did not affect the fMLP-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activation. DMDI produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol in the presence of ethanol from fMLP-stimulated neutrophils (IC 50 13.3±4.0 and 9.4±4.3 μM, respectively). On the basis of the immunoblot analysis of the phosphorylation of the mitogen-activated protein (MAP) kinase, DMDI attenuated the fMLP-stimulated MAP kinase phosphorylation in a similar concentration range. Collectively, these results indicate that the inhibition of the respiratory burst by DMDI in rat neutrophils is mediated through the blockade of phospholipase D and MAP kinase signaling pathways.

Cyclic AMP Inhibits Agonist-Induced Heparin-Binding EGF Gene Expression Independently of Effects on p42/p44 MAPK Activation

Heparin-binding EGF-like growth factor (HB-EGF) mRNA levels are increased up to 20-fold in RIE-1 cells by two agonists that act through distinct receptor types. We demonstrated a common requirement for p42/p44 mitogen-activated protein kinase (MAPK) in this response using the selective MAPK kinase (MEK) inhibitor, PD 098059. Agonist-mediated induction of HB-EGF mRNA was markedly suppressed in cells that had been treated with cyclic AMP-elevating agents. In contrast, the activation of p42 MAPK in response to agonists was not affected by raising cellular cyclic AMP levels. We conclude that cyclic AMP negatively regulates the HB-EGF gene, but that the inhibitory action is either independent of the p42/p44 MAPK pathway or the site of action is distal to MAPK activation.

The promoter region of human prepro-nociceptin gene and its regulation by cyclic AMP and steroid hormones.

The promoter and upstream regulatory region of the human prepro-nociceptin gene has been cloned from adaptor-ligated genomic DNA libraries by polymerase chain reaction. This 1.7 kb region contains several potential binding sites for transcription factors, among which are binding sites for TF-IID, cyclic AMP response element binding protein, glucocorticoid receptor and estrogen receptor. Multiple start points for the transcription of prepro-nociceptin are identified by an 'oligoribonucleotide-capping' method, but the major one is located at -558(G). Promoter activity assays using luciferase reporter gene constructions with the 1.7 kb fragment and a series of deletion mutations demonstrate that the core promoter is located in the region from -639 to -521 (a region surrounding the major transcription start point -558). A TATA-box motif displays weak promoter activity. An increase of cellular cyclic AMP levels by forskolin treatment up-regulates prepro-nociceptin transcription. Estrogen also up-regulates gene transcription whereas glucocorticoid down-regulates transcription, each through their corresponding receptor response elements. These regulatory effects can be blocked either by mutations of the potential cyclic AMP- or estrogen receptor response elements or by the application of antagonists for glucocorticoid and estrogen receptors. These findings provide a molecular basis for the regulatory mechanisms of human prepro-nociceptin gene expression.

2-hydroxymethyl-1-naphthol diacetate (TAC) suppresses the superoxide anion generation in rat neutrophils

We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O 2 •−) generation (IC 50 10.2 ± 2.3 and 14.1 ± 2.4 μM, respectively) and O 2 consumption (IC 50 9.6 ± 2.9 and 13.3 ± 2.7 μM, respectively) of neutrophils. TAC did not scavenge the generated O 2 •− during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca 2+] i in the presence or absence of [Ca 2+] o (IC 50 75.9 ± 8.9 and 84.7 ± 7.9 μM, respectively) and the generation of inositol trisphosphate (IP 3) (IC 50 72.0 ± 9.7 μM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-β associated to membrane was attenuated by TAC (about 80% inhibition at 30 μM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC 50 17.4 ± 1.7 μM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentration-dependent manner (IC 50 25.4 ± 2.4 and 25.9 ± 1.4 μM, respectively). TAC had no effect on the O 2 •− generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47 phox in PMA-stimulated neutrophils (about 80% inhibition at 30 μM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role.

Inhibition by magnolol of formylmethionyl-leucyl-phenyl alanine-induced respiratory burst in rat neutrophils.

The influence of the plant product magnolol on neutrophil superoxide anion (O2-*) generation has been investigated in the rat. Intraperitoneal injection of magnolol (30mg kg(-1)) significantly inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat whole blood ex-vivo. Magnolol also inhibited the 02-* generation with an IC50 (concentration resulting in 50% inhibition) of 15.4+/-1.6 microM and O2 consumption in rat neutrophils in-vitro. Magnolol weakly inhibited the O2-* generation in the xanthine-xanthine oxidase system, decreased cellular cyclic AMP level and had no effect on cyclic GMP levels. It weakly inhibited neutrophil cytosolic protein kinase C activity but did not alter porcine heart protein kinase A activity. Magnolol attenuated fMLP-induced protein tyrosine phosphorylation with an IC50 of 24.0+/-1.9 microM and the phosphorylation of mitogen-activated protein kinase p42/44 with an IC50 of 28.5+/-4.5 microM. However, magnolol alone activated neutrophil phospholipase D activity as determined by the formation of phosphatidic acid and phosphatidyl-ethanol in the presence of ethanol. In the presence of NADPH, the arachidonate-activated NADPH oxidase activity in a cell-free system was weakly suppressed by magnolol. These results suggest that the inhibition of respiratory burst in fMLP-activated neutrophils by magnolol is probably attributable mainly to the attenuation of protein tyrosine phosphorylation and p42/44 mitogen-activated protein kinase activation, and partly to the suppression of protein kinase C and NADPH oxidase activities.

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O 2 ·− release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an ic 50 value of 60.7 ± 8.2 μM. A comparable effect was also demonstrated in the inhibition of O 2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O 2 ·− generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP 3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca 2+] i, but it accelerated the rate of [Ca 2+] i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8 R,9 S,11 S)-(−)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7b,11a-triazadibenzo[ a, g]cycloocta[ cde]trinden-1-one, (KT 5720), a PKA inhibitor, the inhibition of O 2 ·− generation by YT-1, as well as by prostaglandin E 1 (PGE 1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE 1. Moreover, the ability of PGE 1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.

Effects of chronic administration of antidepressants on cerebral cortical adenylyl cyclase coupling in rat brain

The effects on the adenylyl cyclase (AC) system of chronic administration of selective serotonin reuptake inhibitors (SSRIs) and trazodone were examined in rat brain. These drugs have a structure and site of action that differ from those of tricyclic antidepressants. After 21 days of treatment with tricyclic antidepressants, trazodone, and SSRIs, GppNHp-stimulated AC activity was increased in a dose-dependent manner in rat cerebral cortex membranes. In contrast to GppNHp-stimulated AC activity, no significant changes in MnCl2 (20 mM)-stimulated activity were observed between the control and the antidepressant-treated groups. These results suggest that the increase in guanine nucleotide-stimulated AC activity is caused by the activation of Gs and that elevation of cellular cyclic AMP levels through the activation of Gs is a common mechanism of action of antidepressants.

Cellular Signal Level of Cyclic AMP and Functional Integrity of the Small Bowel after Ischemic Preservation: An Experimental Pilot Study in the Rat

The intestinal mucosa is one of the tissues most sensitive to ischemia. Anoxia of the gut is known to result in an early impairment of cellular permeability and transcapillary barrier function upon reperfusion. In vitro, an increased permeability of endothelial cell monolayers could be shown to be related to a decrese in cellular content of cyclic AMP (cAMP). Thus, the present study was aimed at investigating the role of the cellular cAMP second messenger signal in the context of intestinal ischemia/reperfusion injury after cold preservation. Segments of the upper jejunum were isolated from Wistar rats with vascular pedicle and flushed with 10 ml of UW preservation solution. The intestinal lumen was rinsed with 10–15 ml of UW solution and the organ was stored immersed in UW solution at 4°C for 4 or 18 h. After 18 h of cold ischemic storage structural and functional integrity of the preparation was tested by perfusion via the vascular system with modified Krebs-Henseleit buffer and the intestinal lumen with saline solution (containing 200 mg % of galactose) for 30 min. In half of the experiments, dibutyryl-cAMP a membrane permeable cAMP analogue, was admixed to the flush solution (2 mM). It was found that tissue levels of cAMP linearily decreased to 34% during 18 h of ischemic preservation in UW. Addition of dibutyryl cAMP significantly improved postischemic recovery of the intestinal preparations by decreasing cellular loss of lactic dehydrogenase (18.2 ± 4.6 vs. 7.6 ± 2.6 U/I) and improving intestinal absorbtion of galactose from the luminal circuit (0.18 ± 0.14 vs. 0.36 ± 0.14 mg %) after 30 min of oxygenated reperfusion, but was not effective to reduce transcapillary water loss into the gut lumen. It is concluded that the anoxia-related decrease of the cellular cAMP level may represent a codeterminator influencing postischemic recovery of the small bowel and that the control of the cAMP signal of ischemic intestines might improve the quality of cold preservation of the gut prior to transplantation.

Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1 β- and cAMP-induced group II phospholipase A 2 expression in rat renal mesangial cells

Expression of group II phospholipase A 2 (PLA 2; EC 3.1.1.4) in rat renal mesangial cells is triggered in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin 1 β (IL-1 β) or tumor necrosis factor α and agents that elevate cellular levels of cyclic AMP (cAMP) such as forskolin, an activator of adenylate cyclase. Treatment of mesangial cells with IL-1 β or forskolin for 24 h induces group II PLA 2 activity secreted into cell culture supernatants by about 15-fold and 11-fold, respectively. Platelet-derived growth factor (PDGF)-BB potently inhibits secretion of IL-1 β- and forskolin-induced group II PLA 2 activity. By Western and Northern blot analyses, we demonstrate that this is due to a reduction of PLA 2 protein levels and the corresponding PLA 2 mRNA steady-state levels. Basic fibroblast growth factor (bFGF) virtually does not inhibit IL-1 β-stimulated group II PLA 2 activity, but markedly inhibits forskolin-induced expression of group II PLA 2 activity. These effects are caused by changes in the corresponding PLA 2 protein and PLA 2 mRNA steady-state levels. Inhibition of protein kinase C (PKC) by the potent and selective PKC inhibitor calphostin C converted the inhibitory action of PDGF into a bFGF-type of response thus suggesting that PKC is a major effector in PDGF-induced inhibition of IL-1 β-stimulated group II sPLA 2 expression. In summary, our data suggest that PDGF and bFGF differentially modulate in a stimulus-specific manner the expression of group II PLA 2 in mesangial cells.

Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel.

1. On the basis of their structure, we compared the ability of 35 xanthine derivatives to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel stably expressed in chinese hamster ovary (CHO) cells using the cell-attached patch clamp and iodide efflux techniques. 2. Activation of CFTR channels was obtained with 3-mono, 1,3-di or 1,3,7-tri-substituted alkyl xanthine derivatives (enprofylline, theophylline, aminophylline, IBMX, DPMX and pentoxifylline). By contrast, xanthine derivatives substituted at the C8- or N9-position failed to open CFTR channels. 3. The CFTR chloride channel activity was blocked by glibenclamide (100 microM) but not by DIDS (100 microM). 4. Activation of CFTR by xanthines was not mimicked by the calcium ionophore A23187, adenosine, UTP, ATP or the specific phosphodiesterase inhibitors rolipram, Ro 20-1724 and milrinone. In addition, we found no correlation between the effect of xanthines on CFTR and on the cellular cyclic AMP or ATP levels. 5. We then synthesized a series of 3,7-dimethyl-1-alkyl xanthine derivatives; among them, 3,7-dimethyl-1-propyl xanthine and 3,7-dimethyl-1-isobutyl xanthine both activated CFTR channels without increasing the intracellular cyclic AMP level, while the structurally related 3,7-dimethyl-1-(2-propenyl) xanthine and 3,7-dimethyl-1-(oxiranyl methyl) xanthine were inactive. 6. Our findings delineate a novel function for xanthine compounds and identify the molecular features that enable xanthine activation of CFTR. These results may be useful in the development of new molecules for studying the pharmacology of chloride channels.

Differential effect of dexamethasone on interleukin 1beta- and cyclic AMP-triggered expression of GTP cyclohydrolase I in rat renal mesangial cells.

1. Endogenous synthesis of tetrahydrobiopterin (BH4) is an essential requirement for cytokine-stimulated nitric oxide (NO) synthesis in rat mesangial cells. GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, is expressed in renal mesangial cells in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin (IL)-1beta and agents that elevate cellular levels of cyclic AMP. 2. We examined the action of the potent anti-inflammatory drug dexamethasone on GTP cyclohydrolase I induction in response to IL-1beta and a membrane-permeable cyclic AMP analogue, N6, O-2'-dibutyryladenosine 3'-5'-phosphate (Bt2cyclic AMP). 3. Nanomolar concentrations of dexamethasone markedly attenuated IL-1beta-induced GTP cyclohydrolase I mRNA steady state level as well as IL-1beta-induced GTP cyclohydrolase I protein expression and enzyme activity. In contrast, dexamethasone did not inhibit Bt2cyclic AMP-triggered increase in GTP cyclohydrolase I mRNA level and protein expression, and low (1 nM) or high (1 and 10 microM) doses of dexamethasone consistently increased Bt2cyclic AMP-induced GTP cyclohydrolase activity. 4. In summary, these results suggest that glucocorticoids act at several levels, critically dependent on the stimulus used, to control GTP cyclohydrolase I expression.

Demonstration of a novel circulating anti-prostacyclin receptor antibody.

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI2) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI2 (Kd1 = 9.1 +/- 2.0 nM; n1 = 170 +/- 32 sites per cell vs. Kd1 = 7.2 +/- 1.1 nM; n1 = 23 +/- 8 sites per cell), with no significant change in the low-affinity receptors (Kd2 = 1.9 +/- 0.1 microM; n2 = 1,832 +/- 232 sites per cell vs. Kd2 = 1. 6 +/- 0.1 microM; n2 = 1,740 +/- 161 sites per cell) as determined by Scatchard analysis of the binding of [3H]PGE1. The loss of high-affinity PGI2 binding led to the failure of PGI2 to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI2 to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [3H]PGE1 to the high-affinity PGI2 receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [3H]PGE1 to the high-affinity platelet PGI2 receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI2 binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.