Effect of Calcium Activated Factor on Properties of Actin and Myosin of Rabbit Skeletal Muscle

The effect of calcium activated factor (CAF) on enzymatic properties of actin and myosin was investigated. SDS polyacrylamide gel electrophoresis revealed that CAF did not degrade actin, but a slight degradation was found in myosin during CAF digestion, which might have been due to contaminated protease (s) in CAF preparation. No influence was found in EDTA ATPase of myosin and polymerization of G-actin during CAF digestion. However, heavy meromyosin (HMM) ATPase activating ability of actin was slightly decreased during CAF digestion. Although CAF digestion slightly decreased the biological activity of myofibrillar proteins, a single sarcomere prepared by CAF digestion is a useful model for studying muscle contraction because of its almost intact contractility.     

Effect of 3-isobutyl-1-methylxanthine on prolactin actions on RNA synthesis, casein synthesis, lipid synthesis and ornithine decarboxylase activity in mouse mammary gland explants

The effect of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, was tested on several actions of prolactin in cultured mouse mammary tissues. At concentrations of 0.5 mM and above, IBMX abolished the actions of prolactin on RNA and casein synthesis. IBMX by itself, stimulated ornithine decarboxylase (ODC) activity in a dose-response fashion; but the IBMX at concentrations up to 1 mM had no effect on the magnitude of the prolactin-stimulated ODC activity. IBMX inhibited in a dose-response fashion the rate of [14C]-acetate incorporation into lipids; however, prolactin stimulated lipid biosynthesis in the presence of IBMX concentrations of up to 1 mM.     

Expression regulation and function of Pref-1 during adipogenesis of human mesenchymal stem cells (MSCs)

Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARγ (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARγ expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.     

Caffeic Acid Alkyl Amide Derivatives Ameliorate Oxidative Stress and Modulate ERK1/2 and AKT Signaling Pathways in a Rat Model of Diabetic Retinopathy

The purpose of this study was to examine the neuroprotective effects of caffeic acid hexyl ( CAF6 ) and dodecyl ( CAF12 ) amide derivatives on the early stage of retinopathy in streptozotocin‐induced diabetic rats. Animals were divided in five groups (n=8/group); one group consisted of non‐diabetic rats as control, while the other four were diabetic animals either non‐treated or treated with CAF6 , CAF12 or resveratrol intravitreally for four weeks. Retinal superoxide dismutase (SOD) activity and 8‐iso‐prostaglandin F_2α (iPF_2α) levels were evaluated by an ELISA assay. Phosphorylation of ERK1/2 and AKT was determined by immunoblotting in retinal homogenates. Retinal morphology was also examined using light microscopy. Treatment with CAF6 and CAF12 increased retinal SOD activity, while it decreased iPF_2α levels in diabetic rats. Phosphorylation of ERK1/2 was increased, while AKT phosphorylation was decreased in diabetic rats compared to normal control and these alterations were significantly reversed in diabetic rats treated with CAF6 and CAF12 . Furthermore, thickness of the whole retinal layer, outer nuclear layer, and ganglion cell count were decreased in diabetic rats compared to control and CAF6 and CAF12 treatments prevented these changes. CAF6 and CAF12 seem to be effective agents for treatment of diabetic retinopathy via attenuation of retinal oxidative stress and improvement of neuronal survival signaling.     

Steroid-induced final maturation in brook trout (Salvelinus fontinalis) oocytes in vitro: the effects of forskolin and phosphodiesterase inhibitors

The effects of phosphodiesterase inhibitors and forskolin on steroid-induced germinal vesicle breakdown (GVBD) were investigated in brook trout (Salvelinus fontinalis) oocytes using an in vitro incubation technique. Follicles were first treated with a collagenase solution to remove the follicle wall. Denuded oocytes were examined, using scanning electron microscopy. In all experiments GVBD was induced by the use of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one. Cyclic adenosine 3',5'-monophosphate (cAMP) levels were measured (by protein-binding assay) in control and forskolin-treated oocytes. Collagenase treatment removed a majority of the follicle wall, as shown by scanning electron microscopy. Partially denuded (PD) oocytes were slightly more sensitive to steroid treatment than intact follicles (IF), as shown by ED50 values; but PD oocytes did not respond to gonadotropin (GTH) stimulation. Both 3-isobutyl-1-methyl-xanthine (IBMX) and SQ20,006 (Squibb) blocked GVBD, but IBMX was more inhibitory. Forskolin also blocked steroid-induced GVBD. Kinetics of inhibition studies were performed using IBMX, forskolin, and cycloheximide. IBMX and cycloheximide inhibited GVBD if added during the first 18 h following steroid stimulation, whereas forskolin blocked GVBD if added within 12 h after steroid treatment. Forskolin, at levels that block GVBD in vitro, significantly increased cAMP in both IF and PD oocytes, but the response of IF was greater than that of PD oocytes.     

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 microM roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24 h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16 h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24 h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning.     

Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform

Tumor stroma and growth factors provide a survival environment to tumor cells and can modulate their chemoresistance by dysregulating several signal pathways. In this study, we fabricated a three-dimensional (3D) microfluidic chip using polydimethylsiloxane (PDMS) to investigate the impact of hepatocyte growth factor (HGF) from cancer-associated fibroblasts (CAF) on the Met/PI3K/AKT activation, glucose regulatory protein (GRP78) expression and the paclitaxel-induced A549 cell apoptosis. With a concentration gradient generator, the assembled chip was able to reconstruct a tumor microenvironment in vitro. We found high levels of HGF in the supernatants of CAF and the CAF matrix from the supernatants of activated HFL1 fibroblasts or HGF enhanced the levels of Met, PI3K and AKT phosphorylation and GRP78 expression in A549 cells cultured in a 3D cell chamber, which was abrogated by anti-HGF. Inhibition of Met attenuated the CAF matrix-enhanced PI3K/AKT phosphorylation and GRP78 expression while inhibition of PI3K reduced GRP78 expression, but not Met phosphorylation in A549 cells. Inhibition of GRP78 failed to modulate the CAF matrix-enhanced Met/PI3K/AKT phosphorylation in A549 cells. Furthermore, inhibition of PI3K or GRP78 enhanced spontaneous and paclitaxel-induced A549 cell apoptosis. Moreover, treatment with the CAF matrix inhibited spontaneous and medium or high dose of paclitaxel-induced A549 cell apoptosis. Inhibition of PI3K or GRP78 attenuated the CAF matrix-mediated inhibition on paclitaxel-induced A549 cell apoptosis. Our data indicated that HGF in the CAF matrix activated the Met/PI3K/AKT and up-regulated GRP78 expression, promoting chemoresistance to paclitaxel-mediated apoptosis in A549 cells. Our findings suggest that the microfluidic system may represent an ideal platform for signaling research and drug screening.     

Effects of caffeine-pretreatment on SCE and chromosome aberrations induced by monofunctional- and bifunctional-mitomycin C in bloom syndrome lymphocytes

Bloom syndrome (BS) lymphocytes, which are characterized by a high incidence (75.4 per cell) of SCE, were treated with caffeine (CAF) during the first cell cycle and with monofunctional-(M-MC) and bifunctional-(MC)mitomycin C during the second cycle. The effect on the SCE level was synergistic. The CAF-pretreated cells in combination with M-MC and MC post-treatments, had significantly higher (SCE values 152.5 and 167.9 SCE per cell, resp.) than those treated with M-MC or MC alone during the second cycle (101.1 and 116.4 SCE per cell, resp.). M-MC and MC in the presence of BrdU (without CAF) for 2 cell cycles increased SCE to 157.6 and 169.4 per cell (about twice the control level). M-MC + CAF and MC + CAF treatments for 2 cell cycles did not produce a synergistic effect on the SCE frequency in BS cells; the SCE level was not significantly greater than that with M-MC or MC alone. Normal cells treated with MC and CAF for 2 cycles had a maximum SCE frequency of 156 per cell. This suggests that cells with SCE frequencies above this level may not be able to survive, i.e., this is the “saturation” level of SCE. However, CAF alone had almost no effect on SCE in either BS or normal cells and did not produce multiple chromosome aberrations. The lack of CAF effect on BS cells suggests that the lesions in DNA strands of BS cells which lead to SCE are double-strand lesions. In normal cells CAF is known to significantly slow down DNA-chain growth; the reduced rate of DNA-chain growth in BS is an inherent defect of the cells. Therefore, though CAF enhanced SCE and chromosome aberrations (shattered chromosomes) in combination with alkylating agents, CAF alone did not significantly increase the SCE rate in either BS cells or in normal cells. Thus, processes which may induce SCE are not only related to retarded rate of DNA-chain growth, but also to breaks in the template strand permitting double-strand exchanges to occur.     

Isoproterenol produces a rapid increase in sialidase activity in rat heart tissue and cardiomyocyte-derived H9c2 cells in culture

The effects of isoproterenol on sialidase activity in rat cardiomyocytes were examined. Administration of isoproterenol to rats (0.2 or 2 mg/kg body weight) produced an increase in sialidase activity in total membrane fraction of heart tissue within 120 min (121+/-13% of the control at 120 min after administration of 0.2 mg isoproterenol/kg, n=5, P<0.05). Sialidase activity in cardiomyocyte-derived H9c2 cells was also increased by treatment with isoproterenol (10 microM) for 60 min. The effect of isoproterenol on sialidase activity was amplified by the addition of 3-isobutyl-1-methylxanthine (IBMX). Sialidase activity in H9c2 cells was elevated by treatment with dibutyryl cAMP plus IBMX without isoproterenol. The content of N-acetylneuraminic acid in cells decreased by 22% after treatment with isoproterenol plus IBMX. These results suggest that sialidase activity in rat cardiomyocytes is regulated by beta-adrenergic stimulators via a cAMP-dependent process. The increased activity of sialidase may account for the reduction of sialic acid content of cells.     

Phorbol 12-myristate-13-acetate (PMA) stimulates a differential expression of cholecystokinin (CCK) and c-fos mRNA in a human neuroblastoma cell line.

Regulation of cholecystokinin (CCK) and the proto-oncogene c-fos mRNA expression was studied in the human neuroblastoma cell line SK-N-MC. Cells were treated either with the tumor promoting phorbol-ester phorbol-12-myristate-13-acetate (PMA), the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), which results in an elevated intracellular cyclic AMP (cAMP) level, or with a combination of PMA and IBMX. The level of CCK and c-fos mRNA was determined by Northern-blot analysis with CCK and c-fos specific antisense RNA probes after 4-24 h of drug treatment. Treatment with PMA and IBMX for 4-24 hours transiently raised the CCK mRNA level approximately 1.5-3.5 times compared to the controls, and the combination PMA and IBMX had an additive effect and elevated CCK mRNA abundance 1.5-6.5 times. Under the same experimental conditions, both PMA and IBMX elevated the c-fos mRNA level approximately 3-5.5 times. The drug combination showed a pronounced synergistic effect and raised the c-fos mRNA level approximately 3-20 times as compared to controls. Apparently, CCK and c-fos mRNA expression appears to be regulated by similar protein kinase C (PKC) and cAMP-dependent mechanisms in SK-N-MC cells.     

Comparison between effects of 3-isobutyl-1-methylxanthine and FSH on gap junctional communication, LH-receptor expression, and meiotic maturation of cumulus-oocyte complexes in pigs

We investigated cAMP content, gap junctional communications (GJCs) status, and LH-receptor (LH-R) expression in porcine cumulus-oocyte complexes (COCs) during in vitro maturation treated with the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) or with FSH. COCs were cultured for 20 hr (1st culture) in M199 containing 10% FBS (basic medium, BM group) or BM supplemented with FSH (FSH group) or IBMX (IBMX group). Each COC was then transferred into BM containing both FSH and LH and cultured for an additional 24 hr (2nd culture). The proportions of metaphase-II (M-II) oocytes at the end of the 2nd culture did not differ between the FSH (75.7%) and IBMX (68.2%) groups, whereas only 10.1% of oocytes in the BM group reached the M-II stage. During the 1st culture, the cAMP content of COCs and oocytes became significantly higher in the FSH and IBMX groups than in the BM group; the FSH group had a far greater increment than did the IBMX group. GJCs in the FSH and BM groups gradually closed with increasing duration of the 1st culture, whereas a significantly higher proportion of COCs in the IBMX group still had open GJCs than in the other two groups. Furthermore, LH-R mRNA expression significantly increased in both the FSH and IBMX groups compared with the BM group. These results suggest that inhibition of PDEs in porcine COCs make the oocyte ready for release from meiotic arrest, and that maintenance of a moderate cAMP content may prolong GJCs and stimulate LH-R expression.     

Schedule dependent variation in human lymphocyte sensitivity to bleomycin and repair of chromosomal aberrations in G2.

Bleomycin (BLM) induced chromosomal damage in G2 phase and its repair kinetics in normal human lymphocytes were studied following different treatment schedules. As a first step, a dose-response curve was obtained (concentrations of 5-50 micrograms/ml). For repair kinetics studies, blood samples were treated with BLM at a concentration of 20 micrograms/ml. Continuous treatment produced equal numbers of breaks per cell (br/c) when the cells were treated 3, 4 or 5 h before fixation. If the treatment time was extended to 6 h, the level of br/c was increased 2-fold (p < 0.001) as a result of an increased number of cells with more than 3 br/c. The curves obtained after pulse treatment showed maximal chromosome damage at time 3 (45 min BLM treatment, followed by 2 h repair in drug free medium). When the time after treatment was extended to 4 h (treatment time 5), a 50% reduction in chromosome damage was measured. It was found out that at treatment points 3, 4 and 5 the differences in breaks per cell at the different schedules applied were statistically highly significant. If caffeine (CAF) was added, the continuous treatment, BLM+CAF, induced a statistically significant increase in the frequency of br/c at every treatment point, but the shape of the curve illustrating the kinetics of chromosomal damage remained unchanged. Moreover, the addition of CAF at continuous BLM treatment brings the level of br/c close to that measured at the pulse BLM treatment except for treatment time 3. When applied in a combination with BLM, CAF considerably modified the kinetics of chromosome damage for a pulse (BLM alone) treatment. The possible reasons for the changes in the level of br/c as well as a tentative scheme for assessment of chromosome damage repair capacity after BLM treatment are discussed.     

Inactivation of a macronuclear intra-S-phase checkpoint in Tetrahymena thermophila with caffeine affects the integrity of the micronuclear genome

Aphidicolin (APH), an inhibitor of DNA polymerase α, arrested cell divisions in Tetrahymena thermophila. Surprisingly, low concentrations of APH induced an increase of macronuclear DNA content and cell size in non-dividing cells. In spite of the cell size increase, most proliferation of basal bodies, ciliogenesis and development of new oral primordia were prevented by the APH treatment. The division arrest induced by APH was partly overridden by caffeine (CAF) treatment, which caused the fragmentation ("pulverization") of the chromosomes in G2 micronuclei. Somatic progeny of dividers with pulverized micronuclei (APH+CAF strains) contained aneuploid and amicronucleate cells. The amicronucleate cells, after losing their oral structures and most of their cilia, and undergoing progressive disorganization of cortical structures, assumed an irregular shape ("crinkled") and were nonviable. "Crinkled" cells were not formed after APH + CAF treatment of the amicronuclear BI3840 strain, which contains some mic-specific sequences in its macronucleus. Most of the APH +CAF strains had a typical "*"- like conjugation phenotype: they did not produce pronuclei, but received them unilaterally from their mates and retained old macronuclei. However, 4 among 100 APH+CAF clones induced arrest at meiotic metaphase I in their wt mates. It is likely that the origin of such clones was enhanced by chromosome pulverization.     

Effects of caffeine-induced defective DNA replication on SCE and chromosome aberrations produced by alkylating agents

The effect of caffeine (CAF) pretreatment (during the first cell cycle) on the frequency of sister-chromatid exchanges (SCE) and chromosome aberrations induced by bifunctional(MC)- and monofunctional(M-MC)-mitomycin C, 4-nitroquinoline N-oxide (4NQO) and ethyl methanesulphonate (EMS) were examined by using a BrdU—Hoechst staining technique. When CAF was added to the cultures during the first cell cycle in the presence of BrdU and then the cultures treated with MC, M-MC, 4NQO or EMS during the second cell cycle, the effect of the CAF was synergistic, i.e., the SCE level achieved was much higher than that expected from a simple additive effect of the agents and CAF. These results do not support the concept that the process of SCE is a manifestation of CAF-sensitive post-replication repair of DNA damage (single-strand exchanges), but, instead, point to exchanges between the double-strands of the DNA duplex present in each chromatid. CAF at certain concentrations is known to significantly slow down the rate of DNA-chain growth, but not appreciably induce strand breaks. Inasmuch as CAF alone induced only a small increase in SCE rates, possible mechanisms which may induce SCE are not only related to the slowing down of the rate of DNA-chain growth, but may also involve breaks in the template strand permitting double-strand exchanges to occur. The mechanisms responsible for chemically induced SCE are also discussed.     

The combination of coffee compounds attenuates early fibrosis-associated hepatocarcinogenesis in mice: involvement of miRNA profile modulation

Aberrant microRNA expression implicates on hepatocellular carcinoma (HCC) development. Conversely, coffee consumption reduces by ~40% the risk for fibrosis/cirrhosis and HCC, while decaffeinated coffee does not. It is currently unknown whether these protective effects are related to caffeine (CAF), or to its combination with other common and/or highly bioavailable coffee compounds, such as trigonelline (TRI) and chlorogenic acid (CGA). We evaluated whether CAF individually or combined with TRI and/or CGA alleviates fibrosis-associated hepatocarcinogenesis, examining the involvement of miRNA profile modulation. Then, male C3H/HeJ mice were submitted to a diethylnitrosamine/carbon tetrachloride-induced model. Animals received CAF (50 mg/kg), CAF+TRI (50 and 25 mg/kg), CAF+CGA (50 and 25 mg/kg) or CAF+TRI+CGA (50, 25 and 25 mg/kg), intragastrically, 5×/week, for 10 weeks. Only CAF+TRI+CGA combination reduced the incidence, number and proliferation (Ki-67) of hepatocellular preneoplastic foci while enhanced apoptosis (cleaved caspase-3) in adjacent parenchyma. CAF+TRI+CGA treatment also decreased hepatic oxidative stress and enhanced the antioxidant Nrf2 axis. CAF+TRI+CGA had the most pronounced effects on decreasing hepatic pro-inflammatory IL-17 and NFκB, contributing to reduce CD68-positive macrophage number, stellate cell activation, and collagen deposition. In agreement, CAF+TRI+CGA upregulated tumor suppressors miR-144-3p, miR-376a-3p and antifibrotic miR-15b-5p, frequently deregulated in human HCC. CAF+TRI+CGA reduced the hepatic protein levels of pro-proliferative EGFR (miR-144-3p target), antiapoptotic Bcl-2 family members (miR-15b-5p targets), and the number of PCNA (miR-376a-3p target) positive hepatocytes in preneoplastic foci. Our results suggest that the combination of most common and highly bioavailable coffee compounds, rather than CAF individually, attenuates fibrosis-associated hepatocarcinogenesis by modulating miRNA expression profile.     

Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens.

The following studies have been undertaken to compare and correlate the effects of 12-O-tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration. NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10(-4) M IBMX. TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly. Human recombinant (hr) bFGF could replace TPA in the NHM growth medium. Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10(-4) M IBMX. The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX. TPA alone in the absence of CT and IBMX caused a dose-dependent stimulation of tyrosinase activity. Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10(-4) M IBMX. Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity. In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone. Neither TPA nor hrbFGF alone could increase intracellular cAMP levels. The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF. Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose-dependent increase in cAMP concentration. Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.     

Forskolin Stimulation of Cyclic AMP Accumulation in Rat Brain Cortex Slices Is Markedly Enhanced by Endogenous Adenosine

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.     

The effects of two levels of caffeine ingestion on excess postexercise oxygen consumption in untrained women

The effects of two levels of caffeine ingestion (5 mg.kg-1, CAF1, and 10 mg.kg-1, CAF2) on postexercise oxygen consumption was investigated in six untrained women aged 20.5 (SEM 0.5) years. After a test to determine maximal oxygen consumption (VO2max) each subject underwent three test sessions at 55% VO2max either in a control condition (CON) or with the CAF1 or CAF2 dose of caffeine. During exercise, oxygen consumption was found to be significantly higher in the CAF1 and CAF2 trials, compared to CON (P < 0.05). During the hour postexercise, oxygen consumption in CAF1 and CAF2 remained significantly higher than in CON (P < 0.05). At all times throughout the exercise, free fatty acid (FFA) concentrations were significantly higher in the caffeine trials than in CON. The FFA concentrations 1 h postexercise (+60 min) were further elevated above resting values for all three trials. Caffeine ingestion caused the greatest elevation above resting levels being 1.89 (SEM 0.19) mmol.l-1 and 1.96 (SEM 0.22) mmol.l-1 for the CAF1 and CAF2 trials, respectively. This was significantly higher (P < 0.0001) than the CON level which was 0.97 (SEM 0.19) mmol.l-1. Respiratory exchange ratio (R) values became significantly lower (P < 0.05) in CAF1 and CAF2 compared to CON at the onset of exercise and continued to decrease during the activity. Throughout the recovery period, R values were significantly lower for both caffeine trials compared to CON. The results of this study would suggest that caffeine is useful in significantly increasing metabolic rate above normal levels in untrained women during, as well as after, exercising at 55% VO2max.