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PubMed1999

Identification, cloning, and expression of the CAMP factor gene (cfa) of group A streptococci.

The CAMP reaction is a synergistic lysis of erythrocytes by the interaction of an extracellular protein (CAMP factor) produced by some streptococcal species with the Staphylococcus aureus sphingomyelinase C (beta-toxin). Group A streptococci (GAS [Streptococcus pyogenes]) have been long considered CAMP negative, and this reaction commonly has been used to distinguish GAS from Streptococcus agalactiae. We here provide evidence that GAS possess this gene and produce an extracellular CAMP factor capable of participating in a positive CAMP reaction. The S. pyogenes CAMP factor is specified by a 774-bp open reading frame homologous to the CAMP factor genes from S. agalactiae and Streptococcus uberis. This gene, designated cfa, was isolated on a 1,256-bp fragment and cloned in Escherichia coli. Recombinant clones of E. coli expressing cfa secreted an active CAMP factor. The deduced 28.5-kDa protein encoded by cfa consists of 257 amino acids, with a predicted 28-amino-acid signal peptide. The cfa gene is widely spread among GAS: 82 of 100 clinical GAS isolates produced a positive CAMP reaction. Of the CAMP-negative strains, 17 of the 18 GAS strains contained the cfa gene. Additionally, CAMP activity was detected in streptococci from serogroups C, M, P, R, and U. The cfa gene was cloned and actively expressed in Escherichia coli and gene fusions were made, placing the beta-galactosidase gene (lacZ) under control of the cfa promoter. These cfa promoter-lacZ fusions were introduced into S. pyogenes via a bacteriophage-derived site-specific integration vector where they showed that the cfa gene has a strong promoter that may be subject to as-yet-unidentified regulatory factors. The results presented here, along with previous reports, indicate that the CAMP factor gene is fairly widespread among streptococci, being present at least in groups A, B, C, G, M, P, R, and U.

Amino Acid Sequence↗
PubMed1997

cAMP and forskolin inhibit potassium currents in rat taste receptor cells by different mechanisms.

In gustatory transduction, adenosine 3',5'-cyclic monophosphate (cAMP) has been suggested to close potassium channels when elevated by sweet stimuli or to open cAMP-gated cation channels when depressed by bitter stimuli. These experiments examine the effect of cAMP on whole cell currents from posterior taste receptor cells with standard patch-clamp techniques. Elevating cytosolic cAMP by pipette administration, membrane-permeant analogs [8-(4-chlorophenylthio)-cAMP (CPT-cAMP) and dibutyryl-cAMP], or by phosphodiesterase inhibition [3-isobutyl-1-methylxanthine (IBMX)] produced poorly reversible inhibitions of outward potassium currents by up to 33%. Unexpectedly, middle to high concentrations of forskolin (> 5 microM) profoundly and reversibly inhibited these currents (95%) with greatly accelerated inactivation kinetics. 1,9-Dideoxyforskolin, an ineffective activator of adenylate cyclase, was similarly potent. Kinase inhibitors effectively blocked the effects of cAMP elevations produced by IBMX or CPT-cAMP but did not block these forskolin actions. However, at low concentrations (5 microM), forskolin reduced potassium currents in a phosphorylation-dependent manner. Collectively, these data suggest that cAMP produces a phosphorylation-dependent inhibition of outward potassium currents but that forskolin's actions are independent of cAMP or phosphorylation except at low concentration. cAMP was also effective in altering the waveform of the gustatory action potential, implying it may modify transmission of gustatory information to the brain.

1-Methyl-3-isobutylxanthine↗
PubMed2005

cAMP increases surface expression of NKCC2 in rat thick ascending limbs: role of VAMP.

NaCl absorption by the thick ascending limb of Henle's loop (TAL) is mediated by the apical Na-K-2Cl cotransporter NKCC2. cAMP increases NaCl absorption in the TAL by stimulating NKCC2. In oocytes, cAMP increases NKCC2 activity by regulating its trafficking. However, the mechanism by which cAMP stimulates NKCC2 in TALs is not clear. We hypothesized that cAMP increases surface expression of NKCC2 and NaCl absorption in TALs and that vesicle-associated membrane protein (VAMP) is involved in this mechanism. We used surface biotinylation of rat medullary TALs (mTAL) to examine surface and total NKCC2 levels. When mTAL suspensions were treated with dibutyryl cAMP (db-cAMP) or forskolin plus IBMX for 20 min, surface NKCC2 expression increased by 126 +/- 23 and 92 +/- 17% above basal, respectively (P < 0.03). No changes in total NKCC2 expression were observed, suggesting that cAMP increased translocation of NKCC2. We studied the role of VAMP in NKCC2 translocation and found that incubating mTALs with tetanus toxin (30 nM), which inhibits vesicle trafficking by inactivating VAMP-2 and -3, completely blocked the stimulatory effect of db-cAMP on surface NKCC2 expression (tetanus toxin = 100% vs. tetanus toxin + db-cAMP = 102 +/- 21% of control; not significant). We studied VAMP-2 and -3 expression and localization in isolated perfused TALs by confocal microscopy and found that both of them were located in the subapical space of the TAL. Finally, in isolated perfused mTALs, db-cAMP increased net Cl absorption by 95.0 +/- 34.8% (P < 0.03), and pretreatment of TALs with tetanus toxin blocked the stimulation of Cl absorption (from 110.9 +/- 15.9 to 109.7 +/- 15.6 pmol.min(-1).mm(-1); not significant). We concluded that cAMP increases NKCC2 surface expression by a mechanism involving VAMP and that NKCC2 trafficking to the apical membrane is involved in the stimulation of TAL NaCl absorption by cAMP.

1-Methyl-3-isobutylxanthine↗
PubMedDate not supplied

P2U-purinergic receptor activation mediates inhibition of cAMP accumulation in cultured renal mesangial cells.

Extracellular ATP has been reported to exert mitogenic and contractile effects on cultured renal mesangial cells (MCs). Since it is possible that these actions involve changes in the cAMP second messenger system, we examined the effect of extracellular nucleotides on the accumulation of cAMP in rat MCs. ATP, UTP and adenosine 5'-0-(3-thio)triphosphate (ATP gamma S) (100 microM) had no significant effects on baseline cAMP levels, but inhibited forskolin-stimulated accumulation of cAMP by 21-75% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Maximal inhibitory effects were observed at 100 microM of ATP gamma S with a threshold dose of 1 microM. ATP gamma S, ATP and UTP were the most potent inhibitors indicating stimulation of the P2u receptor. The P2x agonists adenosine 5'-(alpha, beta-methylene) triphosphate and adenosine 5'-(beta, gamma-methylene) triphosphate, and the P2y agonist 2-methylthio-ATP did not affect cAMP accumulation. Treatment with the P2 receptor antagonist suramin (200 microM) reduced the inhibition by 58%. The inhibitory effects of the nucleotides were significantly attenuated by preincubation with pertussis toxin (10-100 ng/ml). Inhibition of phospholipase C and protein kinase C did not prevent the inhibitory effect of the nucleotides. Inhibitors of forskolin-stimulated cAMP accumulation had different effects on DNA synthesis in cultured MCs as measured by 3H-thymidine uptake at 48 h: ATP, ATP gamma S and the inhibitor of adenylyl cyclase, SQ 22536, stimulated DNA synthesis in MCs, while UTP showed no significant mitogenic effect. Agents which increased baseline levels of intracellular cAMP (forskolin, IBMX, dibutyryl-cAMP) significantly diminished DNA synthesis in MCs. The results indicate that the P2u-purinergic receptor mediates inhibition of forskolin-induced cAMP accumulation which is likely due to inhibition of adenylyl cyclase. This effect appears to be partially mediated by PTX-sensitive G proteins. While the increase in cAMP accumulation is anti-mitogenic, inhibition of cAMP accumulation by P2u receptors is not correlated with MC growth control. Thus, additional mechanisms other than inhibition of cAMP accumulation by P2u receptors are likely to be involved in the mitogenesis of extracellular ATP.

Adenosine Triphosphate↗
PubMed2005

Identification of the cAMP-responsive enhancer of the murine ABCA1 gene: requirement for CREB1 and STAT3/4 elements.

OBJECTIVE: To determine the mechanism by which expression of the murine ABCA1 gene is highly induced by cAMP analogues. METHODS AND RESULTS: ABCA1 mRNA turnover cannot account for its induction by cAMP. Thus cAMP induction of ABCA1 mRNA occurs at a transcriptional level. Shotgun cloning DNA fragments from the murine ABCA1 locus identified a strong cAMP responsive enhancer located in the first intron, which led to 25- to 100-fold cAMP-mediated induction of reporter gene activity. Deletions and mutations of this enhancer led to the identification a cAMP-responsive element (CRE) that was essential for the cAMP induction. Furthermore, the capacity of this CRE site to mediate the cAMP induction required the presence of a STAT3/4 element located 81 bp away. A dominant-negative CREB expression vector inhibited the cAMP induction of ABCA1, demonstrating that CREB was required for cAMP induction of ABCA1 expression in RAW264.7 cells. CONCLUSIONS: Phospho-CREB1 controls the cAMP-mediated induction of murine ABCA1 gene expression through a CRE site acting in cooperation with a nearby STAT element. This CRE site is not conserved in the human ABCA1 gene, explaining why human ABCA1 is not strongly stimulated by cAMP analogs.

ATP Binding Cassette Transporter 1↗
PubMed1991

Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients.

Isolated adult canine ventricular myocytes were used to study the role of compartmentation of cAMP in the diverse functional responses to various drugs that elevate cAMP. Myocytes presented with the beta-agonist isoproterenol accumulated cAMP with a half maximally effective concentration (EC50) of 3.55 x 10(-8) M. Approximately 45% of the total cAMP was recovered in the particulate fraction of digitonin-lysed myocytes under these conditions. With phosphodiesterase inhibition (10 microM isobutylmethylxanthine), isoproterenol-stimulated cAMP production was up to 3.4-fold greater, but the proportion of total cAMP residing in the particulate fraction declined to less than 20%. Similar results were obtained with forskolin, a direct activator of adenylate cyclase. Treatment with isoproterenol shortened the duration at 50% maximum peak height (T 1/2) and increased the peak fluorescence ratio of electrically triggered single-cell free Ca2+ transients in fura-2-loaded canine myocytes. Isoproterenol dose-response curves gave EC50 values of 1.7 x 10(-9) and 4.4 x 10(-9) M for effects on T 1/2 and peak height, respectively. Alterations in peak height and T 1/2 of Ca2+ transients also showed a dose dependency on isobutylmethylxanthine and forskolin. Comparison of myocyte cAMP content with the corresponding changes in free Ca2+ transients demonstrated a close correlation between particulate cAMP and the extent of shortening or increase in peak height of the fura-2 Ca2+ transients (r = 0.92 for each). However, when these two parameters were plotted as a function of total cAMP, the resulting curves were nonlinear and divergent for each agent tested. The results support the hypothesis that differences in responses to agents that augment cAMP can be explained in part by compartmentation of cAMP. Furthermore, Ca2+ mobilization seems to be most affected by cAMP located in the particulate compartment of canine cardiac myocytes.

Adrenergic beta-Agonists↗
PubMed1992

Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-beta subunit gene expression.

cAMP regulation of the human thyrotropin-beta (TSH beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH beta gene.

Animals↗
PubMed2002

Gonadotropins, via cAMP, negatively regulate GATA-1 gene expression in testicular cells.

We and others demonstrated that the mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, were detected in mouse and rat testis, and in isolated rat Sertoli cells and testicular tumor cell lines derived from Leydig and Sertoli cells. In this study, we investigated the possible effects of gonadotropins and cAMP on the expression of GATA-binding protein genes in testicular cells. Unexpectedly, FSH negatively regulated GATA-1 (but not GATA-4) mRNA in a dose-dependent manner in primary cultures of rat Sertoli cells isolated from 21-d-old animals. GATA-1 mRNA was also negatively regulated by cAMP in a dose- and time-dependent manner in MA-10, a mouse Leydig tumor cell line. When 0.3 mM cAMP was administered to MA-10 cell cultures for 4 h, more than 95% of the GATA-1 mRNA and protein was abolished. The reduction of GATA-1 mRNA by cAMP can be mimicked by treatment with forskolin, which elevates intracellular cAMP levels. The inhibitory effect of cAMP was specific to the GATA-1 gene, given that GATA-4 and alpha-tubulin mRNA levels were not changed by any of the cAMP treatments. Inhibin alpha-subunit mRNA, on the other hand, was evidently increased by cAMP treatment in both MA-10 and Sertoli cells. However, inhibin alpha-subunit mRNA levels were elevated at 60-90 min before the suppression of GATA-1 mRNA detected. The inhibitory effect of cAMP on GATA-1 mRNA and protein was shown to be specific to testicular cells. The GATA-1 mRNA expressed in MEL, a mouse erythroid leukemia cell line, was not affected by cAMP. The reduction of GATA-1 mRNA by cAMP can be prevented when a translational inhibitor, cycloheximide, is added. In summary, we demonstrated that gonadotropins via cAMP negatively regulate the mRNA and protein levels of GATA-1, but not GATA-4, in testicular cells. The inhibitory effect on GATA-1 gene expression was specific to testicular cells and was not observed in erythroid cells.

Animals↗
PubMed2001

cAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel.

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca(2+)](i), as well as pulsatile release of GNRH: We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca(2+) inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca(2+) oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca(2+) signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca(2+) oscillations. The effects of Sp-cAMPS or NE on Ca(2+) signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca(2+) reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca(2+) oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.

Adrenergic alpha-Agonists↗
PubMed2001

Varicella outbreak at a summer camp for human immunodeficiency virus-infected children.

OBJECTIVES: Varicella can result in severe, persistent, or recurrent disease in human immunodeficiency virus (HIV)-infected children. In the summer of 1997, we were notified of a suspected varicella outbreak among attendees of a summer camp for HIV-infected children. We investigated this outbreak to determine the extent and sequelae of the outbreak, and to identify factors that contributed to the outbreak to identify measures for preventing such outbreaks at the camp in the future. DESIGN: To identify varicella-susceptible persons and those developing varicella after camp and to evaluate the camp's varicella prevention measures, we reviewed camp records for the 110 campers and 96 staff at the implicated camp session, mailed questionnaires to the campers' parents/guardians and physicians, and interviewed susceptible staff. We defined a case as varicella in a person who attended the session with onset </=21 days after the session ended. RESULTS: Eleven of 31 susceptible children (36%) and 2 of 4 susceptible adults developed varicella. Two children were hospitalized. One developed cellulitis. Cases occurred among children in 5 of 15 cabins. The most likely index case was a child with active zoster at camp, reported to the camp after the session ended. The camp had varicella-prevention measures in place, but the varicella-susceptibility and exposure information provided to the camp was often incomplete or inaccurate. Staff with no varicella history underwent serologic testing, but susceptible staff members were not vaccinated. CONCLUSIONS: Widespread varicella transmission occurred at the camp. A case of zoster was the most likely source. The risk for such outbreaks can be minimized through vaccinating susceptible staff members, considering vaccination for asymptomatic or mildly symptomatic HIV-infected children according to Advisory Committee on Immunization Practices and American Academy of Pediatrics guidelines, rigorously collecting recent varicella and zoster exposure information, excluding anyone with active varicella or zoster or with recent varicella or zoster exposure, and considering varicella and zoster exposures at camp to be potentially camp-wide.varicella, human immunodeficiency virus infections, disease outbreaks, intravenous immunoglobulin.

Adolescent↗
PubMed2006

Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-dependent cystic dilation.

Metanephric organ culture has been used to determine whether embryonic kidney tubules can be stimulated by cAMP to form cysts. Under basal culture conditions, wild-type kidneys from embryonic day 13.5 to 15.5 mice grow in size and continue ureteric bud branching and tubule formation over a 4- to 5-d period. Treatment of these kidneys with 8-Br-cAMP or the cAMP agonist forskolin induced the formation of dilated tubules within 1 h, which enlarged over several days and resulted in dramatically expanded cyst-like structures of proximal tubule and collecting duct origin. Tubule dilation was reversible upon withdrawal of 8-Br-cAMP and was inhibited by the cAMP-dependent protein kinase inhibitor H89 and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR(inh)172. For further testing of the role of CFTR, metanephric cultures were prepared from mice with a targeted mutation of the Cftr gene. In contrast to kidneys from wild-type mice, those from Cftr -/- mice showed no evidence of tubular dilation in response to 8-Br-cAMP, indicating that CFTR Cl(-) channels are functional in embryonic kidneys and are required for cAMP-driven tubule expansion. A requirement for transepithelial Cl(-) transport was demonstrated by inhibiting the basolateral Na(+),K(+),2Cl(-) co-transporter with bumetanide, which effectively blocked all cAMP-stimulated tubular dilation. For determination of whether cystic dilation occurs to a greater extent in PKD kidneys in response to cAMP, Pkd1(m1Bei) -/- embryonic kidneys were treated with 8-Br-cAMP and were found to form rapidly CFTR- and Na(+),K(+),2Cl(-) co-transporter-dependent cysts that were three- to six-fold larger than those of wild-type kidneys. These results suggest that cAMP can stimulate fluid secretion early in renal tubule development during the time when renal cysts first appear in PKD kidneys and that PKD-deficient renal tubules are predisposed to abnormally increased cyst expansion in response to elevated levels of cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

A prospective study of psychological distress related to refugee camp experience.

OBJECTIVE: Previous reports have suggested a direct relationship between refugee camp experience and levels of psychological distress among refugees. Specifically, it has been postulated that refugee camps with harsh conditions and low levels of social support foster high levels of psychological distress. The present study was designed to assess the relationship between camp conditions and social support within a refugee camp and refugees' levels of psychological distress. Unlike previous reports, which were based on retrospective data, the present study evaluated psychological distress among a group of Vietnamese refugees both prior to departure from Vietnam and during their refugee camp experience. METHOD: A group of 101 Vietnamese Amerasians was assessed at a transit centre in Vietnam and subsequently at a refugee camp in the Philippines. Assessment instruments were the Hopkins Symptom Checklist-25 and a Camp Comparison Questionnaire. RESULTS: There were significant decreases in symptom levels of anxiety and depression between the transit centre in Vietnam and the refugee camp in the Philippines. However, these changes were not related to changes in refugee camp conditions or social support within the camp. CONCLUSIONS: Contrary to previous reports, levels of psychological distress among this group of Vietnamese refugees were not related to either refugee camp conditions or levels of social support within the camp.

Adolescent↗
PubMed2000

Identification of streptococci isolated from various sources by determination of cfb gene and other CAMP-factor genes.

In the present study, the CAMP-factor (cfb) gene of streptococci of serological group B (Streptococcus agalactiae) and the CAMP-factor (cfu) gene of S. uberis could be amplified by polymerase chain reaction. A cfb specific amplicon could be observed for all 128 phenotypically CAMP-positive S. agalactiae, for the phenotypically CAMP-negative S. agalactiae strain 74-360, and for 2 S. difficile reference strains. A cfu specific amplicon could be observed for all 7 phenotypically CAMP-positive S. uberis. Four S. agalactiae strains isolated from 4 cows with mastitis appeared to be phenotypically CAMP-negative and negative in the cfb gene PCR. The CAMP-positive and CAMP-negative isolates, including both S. difficile, could be identified as S. agalactiae by amplification of a S. agalactiae specific part of the V2 region of the 16S rRNA and a species-specific part of the 16S-23S rRNA intergenic spacer region. Amplification of an internal fragment of the cfb gene with a reduced annealing temperature yielded positive reactions not only for CAMP-positive S. agalactiae, but also for phenotypically CAMP-positive S. pyogenes (n = 4), S. canis (n = 28), and S. uberis (n = 7), indicating a close relation of the CAMP genes of these 4 species. The relation could be further demonstrated by sequencing the internal fragment of the CAMP-factor (cfg) gene of S. canis and comparing the sequence with those of S. agalactiae, S. pyogenes, and S. uberis.

Animals↗
PubMed2001

8-chloro-cAMP and 8-chloro-adenosine act by the same mechanism in multiple myeloma cells.

Previous work with 8-chloro-cAMP (8-Cl-cAMP) has raised questions as to whether it works as a cAMP analogue or as a nucleoside analogue after its conversion to 8-chloro-adenosine (8-Cl-Ado). Although degradation of 8-Cl-cAMP to 8-Cl-Ado in culture medium or plasma has been shown, cellular pharmacology data are missing. The purpose of the present study was to identify the cellular metabolism of these drugs and their actions in a human multiple myeloma cell line. The cells were incubated with either 8-Cl-Ado or 8-Cl-cAMP to follow the cellular metabolism of these agents. Both 8-Cl-cAMP and 8-Cl-Ado incubation resulted in the accumulation of 8-Cl-Ado mono-, di-, and tri-phosphate (8-Cl-ATP), however, the triphosphate was the major cytotoxic metabolite. Accumulation of 8-Cl-ATP was dependent on both the exogenous concentration of 8-Cl-Ado and incubation time. At the 10 microM level of 8-Cl-Ado, >400 microM 8-Cl-ATP accumulated in multiple myeloma cells after continuous incubation for 12 h. Similar incubation with 8-Cl-cAMP also resulted in accumulation of 8-Cl-ATP in the cells, albeit at a lower level. The formation of 8-Cl-ATP from 8-Cl-cAMP was inhibited by >80% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in the medium, suggesting extracellular conversion of 8-Cl-cAMP to 8-Cl-Ado. Cells lacking Ado kinase did not accumulate 8-Cl-ATP, either from 8-Cl-Ado or 8-Cl-cAMP, and were resistant to these agents. There was also a decline in the endogenous level of the cellular ATP pool parallel to the accumulation of 8-C1-ATP. The elimination of 8-Cl-ATP was biphasic and slow from the cells. The accumulation of 8-Cl-ATP and a decline in the ATP pool inhibited RNA synthesis but did not affect DNA synthesis for up to 12 h of incubation. Taken together, these data demonstrate that the cytotoxic metabolite of 8-Cl-Ado and 8-Cl-cAMP is 8-Cl-ATP. Hence, 8-Cl-cAMP serves as a prodrug and is converted to 8-Cl-Ado in medium with subsequent phosphorylation to accumulate as 8-Cl-ATP in cells. At the cellular level, 8-Cl-ATP is associated with a decrease in the endogenous ATP pool; at the nuclear level, it inhibits RNA synthesis.

2-Chloroadenosine↗
PubMed2005

Effect of cAMP analogues on glomerular inulin space of isolated rats renal glomeruli.

Cyclic AMP has been generally recognised as activator of cAMP-dependent protein kinases. However, there is little evidence about role of cAMP-dependent protein kinase (PKA), in particular izoenzymes PKA-I and PKA-II, in glomeruli contractility. We measured changes of glomerular inulin space (GIS) as a marker of its contractility in the presence of phosphodiesterase resistance cAMP analogues; activators and inhibitors of PKA. Activator of PKA i.e. (Sp) 8-Cl-cAMPS (0.1-100 microM) decreased GIS. (Rp) 8-Cl-cAMPS (0.1-100 microM), inhibitor of PKA, was ineffective but shifted concentration-response curve of (Sp) 8-Cl-cAMPS to right at 50 microM. Specific A site activation by N6-benzoyl-cAMP decreased GIS with maximum at 0.1 microM. Activation of B site by 8-aminobutyloamino-cAMP (0.1-100 microM) had no effect. However, specific activation of both sites on PKA-I or PKA-II by site-selective analogue pairs e.g. 8-aminobutyloamino-cAMP plus 8-piperidino-cAMP or N6-benzoyl-cAMP plus 8-piperidino-cAMP respectively, significantly increased sensitivity of glomeruli to analogues. Our data suggest that activation of PKA-I or PKA-II might have an important role in the regulation of glomerular contractility.

Animals↗
PubMed1991

Inhibition of prostaglandin E2-stimulated cAMP accumulation by lipopolysaccharide in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages with 100 nM prostaglandin E2 (PGE2) produced a rapid biphasic increase in intracellular cAMP that was maximal at 1 min and sustained through 20 min. Pretreatment of macrophages with 100 ng/ml of lipopolysaccharide (LPS) for 60 min prior to PGE2 decreased the magnitude of cAMP elevation by 50%, accelerated the decrease of cAMP to basal levels, and abolished the sustained phase of cAMP elevation. The effect of LPS was concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in the presence of 5% fetal calf serum and at 1 microgram/ml in the absence of fetal calf serum. LPS also inhibited cAMP accumulation in cells treated with 100 microM forskolin, but the decrease was about half that seen in cells treated with PGE2. LPS concentrations that inhibited cAMP accumulation produced a 30% increase in soluble low Km cAMP phosphodiesterase activity while having no effect on particulate phosphodiesterase activity. The nonspecific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram and Ro-20-1724 were effective in inhibiting soluble phosphodiesterase activity in vitro, producing synergistic elevation of cAMP in PGE2-treated cells, and blocking the ability of LPS to inhibit accumulation of cAMP. Separation of the phosphodiesterase isoforms in the soluble fraction by DEAE chromatography indicated that LPS activated a low Km cAMP phosphodiesterase. The enzyme(s) present in this peak could be activated 6-fold by cGMP and were potently inhibited by low micromolar concentrations of Ro-20-1724 and rolipram. Using both membranes from LPS-treated cells and membranes incubated with LPS, no decrease in adenylylcyclase activity could be attributed to LPS. Although effects of LPS on the rate of synthesis of cAMP cannot be excluded, the present evidence is most consistent with a role for phosphodiesterase activation in the inhibitory effects of LPS on cAMP accumulation in murine peritoneal macrophages.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Regulation of jun-B expression by a cyclic AMP (cAMP)-dependent mechanism in human myeloid cells.

The present studies have examined the regulation of the jun-B early response gene by cyclic AMP (cAMP)-dependent signaling pathways. The 2.0-kb jun-B transcript was at low but detectable levels in uninduced human HL-60 myeloid leukemia cells. In contrast, treatment with 1 mmol/L8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP) in the presence of isobutylmethylxanthine, an inhibitor of cAMP-dependent phosphodiesterase, was associated with increases in jun-B transcripts that were maximal by 1 hour and then decreased to near pretreatment levels by 6 hours. Similar findings were obtained with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP) and N6,2'-0-dibutyryladenosine 3',5'-cyclic monophosphate (dBt-cAMP). jun-B transcripts were also increased with other agents that increase intracellular cAMP levels, such as prostaglandin E2 (PGE2) and forskolin. Moreover, inhibition of cAMP-dependent protein kinase by the isoquinolinesulfonamide H-8 blocked 8-Br-cAMP-induced increases in jun-B expression. The results of nuclear run-on assays demonstrate that treatment of HL-60 cells with PGE2, forskolin, 8-Br-cAMP, and dBt-cAMP is associated with increases in the rate of jun-B transcription. The present findings also demonstrate that the related jun-D gene is similarly regulated by a cAMP-dependent pathway. Taken together, these findings suggest that stimulation of cAMP-dependent protein kinase is involved in the induction of jun gene expression in myeloid leukemia cells.

Bone Marrow↗
PubMed1990

Role of cAMP in interleukin-1-induced kappa light chain gene expression in murine B cell line.

The murine lymphoid cell line 70Z/3 has been extensively used to study the intracellular mechanisms of interleukin-1 (IL-1) action. In these cells IL-1 is known to induce kappa gene expression but the signal transduction pathway has yet to be defined. IL-1-induced kappa expression is associated with stimulation of Na+/H+ exchange and activation of protein kinase C, but these events are not sufficient to trigger kappa expression. Thus, other signals must be present. Because cAMP is a well recognized second messenger, we sought to determine whether cAMP is the signal that triggers IL-1-induced kappa expression. To that end we first measured intracellular levels of cAMP following IL-1 treatment. The results showed that exposure of 70Z/3 cells to IL-1 alpha induced a rapid and a transient increase in cAMP, it peaked at 5 min and was back to base-line level at 20 min. Prostaglandin E2 (PGE2) also increased cAMP with similar kinetics to IL-1 alpha but the increased levels were far greater. IL-1 alpha-induced increase in cAMP proved not be a sufficient signal because an increase in intracellular cAMP by N6,O2'-dibutyryl cAMP (Bt2cAMP) or PGE2 failed to increase surface IgM or to increase kappa mRNA level. Although when used alone they had no effect, Bt2cAMP and PGE2 were found to amplify the IL-1 alpha-induced kappa expression. IL-1 alpha transiently activated NF-kappa B transcription factor. But this effect could not be simulated by Bt2cAMP or PGE2. This observation provides further evidence that cAMP is not a trigger of kappa expression. Although Bt2cAMP or PGE2 when used alone had no effect, they did consistently modify the level of NF-kappa B activity induced by IL-1 alpha. Results of this study show that cAMP is not sufficient to induce NF-kappa B or kappa expression. Therefore, the role of cAMP may not be trigger but rather to modulate the IL-1 alpha-induced kappa expression. Regulation of the response could occur at one or a number of points along the signal pathway. Such a regulatory role is supported by the observation that cAMP modulates the IL-1 alpha-induced NF-kappa B activity.

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