The aim of present study is to investigate the effects of cGMP on hyperpolarization activated inward current (If), pacemaker current of the heart, in rabbit sino-atrial node cells using the whole-cell patch clamp technique. When sodium nitroprusside (SNP, 80 muM), which is known to activate guanylyl cyclase, was added, If amplitude was increased and its activation was accelerated. However, when If was prestimulated by isopreterenol (ISO, 1 muM), SNP reversed the effect of ISO. In the absence of ISO, SNP shifted activation curve rightward. On the contrary in the presence of ISO, SNP shifted activation curve in opposite direction. 8Br-cGMP (100 muM), more potent PKG activator and worse PDE activator than cGMP, also increased basal If but did not reverse stimulatory effect of ISO. It was probable that PKG activation seemed to be involved in SNP-induced basal If increase. The fact that SNP inhibited ISO-stimulated If suggested cGMP antagonize cAMP action via the activation of PDE. This possibility was supported by experiment using 3-isobutyl-1-methylxanthine (IBMX), non-specific PDE inhibitor. SNP did not affect If when If was stimulated by 20 muM IBMX. Therefore, cGMP reversed the stimulatory effect of cAMP via cAMP breakdown by activating cGMP-stimulated PDE. These results suggest that PKG and PDE are involved in the modulation of If by cGMP: PKG may facilitate If and cGMP-stimulated PDE can counteract the stimulatory action of cAMP.
1-Methyl-3-isobutylxanthine ; Guanylate Cyclase ; Heart ; Nitroprusside ; Sinoatrial Node*

To evaluate the effects of kerationocytes on the growth of melanocytes, keratinocyte conditioned media (K-CM) with different molecular weight obtained by dialysis were added to melanocyte growth medium (M-GM). In addition, K-CM only, and K-CM mixed with each component of M-GM, such as TPA(12-tetradecanoyl- phorbol-13-acetate), IBMX(isobutylmethylxanthine) and CT(cholera toxin), were used for the culture of melanocytes. 1) The proliferation of melanocytes was incresased to 2.86 x 10(5)+/-0.87 x 10(5) cells/ well and 2.87 x 10(5)+/-0.71 x 10(5) cells/well in 25% K-CM with a cut-off molecular weight of 2,000 and 25% K-CM with a cut,-off molecular weight between 6000 8000 respectively, as compared to 1.88 x 10(5)+/-0.45 x 10(5) cells/well in the control group (p < 0.05). 2) The amount of melanin was increased to 0.2987+/-0.0830ng/mlin 25% un- dialyzed K-CM, as compared to 0.2264+/-0.0643ng/ml in the control group, but this differnce was not statistically significant. 3) Maximum proliferation of melanocytes was observed in 35% concentration of K-CM with a cut-off molecular weight of 6000 8000. 4) Maximum of melanin production was observed in 35% concentration of undialyzed K-CM 5) As compared to 7.86 x 10+/-1.74 x 10(5) cells/well in M-GM,proliferation of melanocytes in 35% K-CM with a cut-off molecular weight of 6000 8000 was de- creased to 1.38 X 10(5)+/-0.97 X 10(5) cells/well. 6) There was no difference in melanocyte proliferation between 6.81 x 10(5)+/-2.19 x 10(5) cells/well in 35% 6,000 8,000 M.W. cut-off dialyzed K-CM, with IBMX only, and 7.86 x 10(5)+/-1.74 x 10(5) cells/well in M-GM. 7) Compared to 0.2303+/-0.0700ng/well cell in M-GM, the amount of melanin was increased to 0.3227+/-0.0900ng/cell, 0.3624+/-0.0900ng/cell and 0.2928+/-0.0500ng/cell, respectively, when TPA, IBMX, CT was added to 35% undialyzed K-CM. It also increased to 0.3176+/-0.1100 in 35% undialyzed K-CM(p<0.05). In summary, the results proved that cellular activating substances released from keratinocytes affect the proliferation of melanocyte and the synthesis of melain. It is also expected that methods used in this study can be clinically utilized because melanocyte culture is possible on K-CM without adding tumor promotors.
1-Methyl-3-isobutylxanthine ; Culture Media, Conditioned ; Dialysis ; Keratinocytes* ; Melanins ; Melanocytes* ; Molecular Weight

Ms report shows that hydroxyl radical, generated by a Fenton reaction involving adenosine 5'-diphosphate/Fe2+ complex (5-15 micrometer) and H2O2 (2 micrometer), induced differentiation of HL-60 cells in a dose- and time-dependent manner. This is evidenced by the increases in 12-O-tetradecanoylphorbol 13-acetate- and fMLP-stimulated superoxide production capability. The cells exposed to hydroxyl radical for defined periods (24~96 hr) continued to differentiate even after the hydroxyl radical generating system had been removed. The differentiated cells displayed fMLP-stimulated calcium mobilization and increased expression of myeloid-specific antigen CD11b and CD14. The extent of the differentiation was markedly reduced by desferrioxamine (100micrometer), dimethylthiourea (5 mM), N,N'-diphenyl-1,4-phenylenediamine (2 micrometer), and N-acetyl-L-cysteine (5 mM). The induction of differentiation by hydroxyl radical was enhanced by 3-isobutyl-1-methylxanthine (200 micrometer) and Ro-20-1724 (8 micrometer), and inhibited by dipyridamole (2 micrometer). These results suggest that hydroxyl radicals may induce commitment of HL-60 cells to differentiate into more mature cells of myelomonocytic lineage through specific signal-transduction pathway that is modulated by phosphodiesterase inhibitors.
1-Methyl-3-isobutylxanthine ; Acetylcysteine ; Adenosine ; Calcium ; Deferoxamine ; Dipyridamole ; HL-60 Cells* ; Humans ; Hydroxyl Radical ; Phosphodiesterase Inhibitors* ; Superoxides

Obesity occurs when a person's calorie intake exceeds the amount of energy burns, which may lead to pathologic growth of adipocytes and the accumulation of fat in the tissues. In this study, the effect and mechanism of pear pomace extracts on 3T3-L1 adipocyte differentiation and apoptosis of mature adipocytes were investigated. The effects of pear pomace extract on cell viability and the anti-adipogenic and proapoptotic effects were investigated via MTT assay, Oil red O staining, western blot analysis and apoptosis assay. 3T3-L1 preadipocytes were stimulated with DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 microg/ml insulin and 1 microM dexamethasone for differentiation to adipocytes. 3T3-L1 cells were cultured with PBS or water extract of pear pomace. Water extract of pear pomace effectively inhibited lipid accumulations and expressions of PPAR-gamma and C/EBPalpha in 3T3-L1 cells. It also increased expression of p-AMPK and decreased the expression of SREBP-1c and FAS in 3T3-L1 cells. The induction of apoptosis was observed in 3T3-L1 cells treated with pear pomace. These results indicate that pear pomace water extract inhibits adipogenesis and induces apoptosis of adipocytes and thus can be used as a potential therapeutic substance as part of prevention or treatment strategy for obesity.
1-Methyl-3-isobutylxanthine ; 3T3-L1 Cells ; Adipocytes* ; Adipogenesis* ; Apoptosis* ; Blotting, Western ; Burns ; Cell Survival ; Dexamethasone ; Insulin ; Obesity ; Pyrus* ; Sterol Regulatory Element Binding Protein 1 ; Water*

OBJECTIVETo investigate the optimal conditions for establishing insulin-resistant 3T3-L1 adipocytes.

METHOSDexamethason (DEX), 3-isobutyl-methylxanthine (IBMX) and different concentrations of insulin (10(-8), 10(-7), and 10(-6) mol·L(-1)) were used to induce 3T3-L1 preadipocytes into mature adipocytes identified by oil red O staining. We established insulin- resistant 3T3-L1 adipocytes cell model (IR-3T3-L1) by exposing the cells to 1µmol·L(-1) DEX, and the changes of glucose concen- tration in the cell culture were determined by glucose oxidase-peroxidase (GOD-POD) assay.

RESULTSTreatment of 3T3-L1 cells with DEX, IBMX and 10(-6) mol·L(-1)) insulin for 9 days resulted in the differentiation of >90% of the cells into mature adipocytes. IR-3T3-L1 cells cultured for 96 h in the culture media containing 1 µmol·L(-1) DEX showed significantly increased glucose consumption (P=0.0003) as compared with the control group at 36 h (P<0.001).

CONCLUSION3T3-L1 cells can be induced into mature adipocytes by exposure to 1 µmol·L(-1) DEX, 0.5 mmol·L(-1) IBMX and 10(-6) mol·L(-1)) insulin. A 96 h exposure to 1 µmol·L(-1) DEX can induce 3T3-L1 adipocytes to acquire insulin resistance that can be maintained for 36 h.

1-Methyl-3-isobutylxanthine ; chemistry ; 3T3-L1 Cells ; Adipocytes ; cytology ; Animals ; Cell Differentiation ; Culture Media ; chemistry ; Dexamethasone ; chemistry ; Glucose ; chemistry ; Insulin ; chemistry ; Insulin Resistance ; Mice

Substantia gelatinosa (SG) neurons receive synaptic inputs from primary afferent Adelta- and C-fibers, where nociceptive information is integrated and modulated by numerous neurotransmitters or neuromodulators. A number of studies were dedicated to the molecular mechanism underlying the modulation of excitability or synaptic plasticity in SG neurons and revealed that second messengers, such as cAMP and cGMP, play an important role. Recently, cAMP and cGMP were shown to downregulate each other in heart muscle cells. However, involvement of the crosstalk between cAMP and cGMP in neurons is yet to be addressed. Therefore, we investigated whether interaction between cAMP and cGMP modulates synaptic plasticity in SG neurons using slice patch clamp recording from rats. Synaptic activity was measured by excitatory post-synaptic currents (EPSCs) elicited by stimulation onto dorsal root entry zone. Application of 1 mM of 8-bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP) or 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) for 15 minutes increased EPSCs, which were maintained for 30 minutes. However, simultaneous application of 8-Br-cAMP and 8-Br-cGMP failed to increase EPSCs, which suggested antagonistic cross-talk between two second messengers. Application of 3-isobutyl-1-methylxanthine (IBMX) that prevents degradation of cAMP and cGMP by blocking phosphodiesterase (PDE) increased EPSCs. Co-application of cAMP/cGMP along with IBMX induced additional increase in EPSCs. These results suggest that second messengers, cAMP and cGMP, might contribute to development of chronic pain through the mutual regulation of the signal transduction.
1-Methyl-3-isobutylxanthine ; Adenosine ; Animals ; Chronic Pain ; Guanosine ; Myocytes, Cardiac ; Neurons ; Neurotransmitter Agents ; Plastics ; Rats ; Second Messenger Systems ; Signal Transduction ; Spinal Nerve Roots ; Substantia Gelatinosa

BACKGROUND: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current (ICa) in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of ICa by cGMP. However, there is no direct evidence that cGMP-PK can stimulate ICa in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK an ICa in rabbit ventricular myocytes. METHODS AND RESULTS: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of ICa by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. ICa was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal ICa. cGMP-PK also increased basal ICa. The stimulation of basal ICa by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal ICa by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When ICa was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of ICa. In the presence of cGMP-PK, already increased ICa was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. CONCLUSIONS: We present evidence that cGMP-PK stimulated basal ICa by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.
1-Methyl-3-isobutylxanthine ; Adenosine Triphosphate ; Baths ; Calcium Channels, L-Type ; Calcium* ; Colforsin ; Heart ; Hot Temperature ; Isoproterenol ; Muscle Cells* ; Phosphorylation ; Protein Kinases*

BACKGROUND/OBJECTIVE: This study was designed to investigate how a Portulaca oleracea L. extract (POE) stimulates insulin secretion in INS-1 pancreatic β-cells. MATERIALS/METHOD: INS-1 pancreatic β-cells were incubated in the presence of various glucose concentrations: 1.1 or 5.6, 16.7 mM glucose. The cells were treated with insulin secretagogues or insulin secretion inhibitor for insulin secretion assay using an insulin ELISA kit. In order to quantify intracellular influx of Ca2+ caused by POE treatment, the effect of POE on intracellular Ca2+ in INS-1 pancreatic β-cells was examined using Fluo-2 AM dye. RESULTS: POE at 10 to 200 µg/mL significantly increased insulin secretion dose-dependently as compared to the control. Experiments at three glucose concentrations (1.1, 5.6, and 16.7 mM) confirmed that POE significantly stimulated insulin secretion on its own as well as in a glucose-dependent manner. POE also exerted synergistic effects on insulin secretion with secretagogues, such as L-alanine, 3-isobutyl-1-methylxanthine, and especially tolbutamide, and at a depolarizing concentration of KCl. The insulin secretion caused by POE was significantly attenuated by treatment with diazoxide, an opener of the K+ ATP channel (blocking insulin secretion) and by verapamil (a Ca2+ channel blocker). The insulinotropic effect of POE was not observed under Ca2+-free conditions in INS-1 pancreatic β-cells. When the cells were preincubated with a Ca2+ fluorescent dye, Fluo-2 (acetoxymethyl ester), the cells treated with POE showed changes in fluorescence in red, green, and blue tones, indicating a significant increase in intracellular Ca2+, which closely correlated with increases in the levels of insulin secretion. CONCLUSIONS: These findings indicate that POE stimulates insulin secretion via a K+ ATP channel-dependent pathway in INS-1 pancreatic β-cells.
1-Methyl-3-isobutylxanthine ; Adenosine Triphosphate ; Alanine ; Calcium Channels ; Diabetes Mellitus ; Diazoxide ; Enzyme-Linked Immunosorbent Assay ; Fluorescence ; Glucose ; Insulin* ; Portulaca* ; Tolbutamide ; Verapamil

In order to investigate the effect of ANP on surfactant secretion from alveolar type II cell(AT II cell) during circulatory derangement in adult respiratory distress syndrome (ARDS), the secretion of surfactant from AT II cells was evaluated in purely isolated AT II cultures from rat lungs. For the simulation of sympathetic stimulation during circulatory derangement, primary AT II cultures were incubated with isoproterenol and IBMX. In this isoproterenol stimulated AT II cells, ANP were added in the media for the investigation of effect of ANP on surfactant secretion from AT II cells. For the evaluation of surfactant secretion,(3H)-methylcholine was incorporated and the level of radiolabelled choline chloride secreted from the cells was determined. As previously reported, isoproterenol and IBMX stimulated surfactant secretion from AT II cells. Isoproterenol showed synergistic increase of surfactant secretion with IBMX in AT II cells. In isoproterenol stimulated AT II cells, physiological level of ANP inhibited the secretion of surfactant in primary cultures of AT II cells. On the basis of these experimental it is suggested that, in association with circulatory change during ARDS, increased secretion of ANP by the pulmonary edema, hypoxia and congestive heart heart failure might aggravate the symptoms of ARDS by reduction of surfactant secretion from AT II cells.
1-Methyl-3-isobutylxanthine ; Animals ; Anoxia ; Atrial Natriuretic Factor* ; Choline ; Estrogens, Conjugated (USP) ; Heart ; Heart Failure ; Isoproterenol* ; Lung ; Pulmonary Edema ; Rats ; Respiratory Distress Syndrome, Adult

BACKGROUND: Endotoxins play important roles in the pathophysiologic alterations associated with sepsis so we examined the effects of volatile anesthetics on vascular smooth muscle contractile function in LPS-treated rat aorta. METHODS: Fifty male Sprague-Dawley rats(250~300 gm) were made septic by intraperitoneal injection of lipopolysaccharide(1.5 mg/kg). Cumulative doses of phenylephrine and norepinephrine(10 -8~10 -5M) were added to construct a contraction response curve. Two percent of volatile anesthetics, IBMX (3-isobutyl-1-methylxanthine, phosphodiesterase inhibitor) or L-NAME(Ng-nitro-L-arginine-methylester, Nitric oxide synthase inhibitor) was added and those contractile responses were observed respectively. We also measured nitric oxide synthase (NOS) activity of liver, lung and adrenal gland after 18 hours in the LPS-treated rats. Individual values between the control rats and LPS-treated rats were compared by unpaired t-test. A p-value less than 0.05 was considered statistically significant. RESULTS: Contractile response of 2% halothane to norepinephrine was significantly decreased both in the control rats and LPS-treated rats. The NOS inhibitor enhanced the contractile responses to phenylephrine and norephinephrine in the vessels from LPS-treated rats more significantly than those of control rats. CONCLUSIONS: These results suggest that LPS-treatment impairs vasopressor-induced contractility and doesn't alter the contractile responses during administration of volatile anesthetics.
1-Methyl-3-isobutylxanthine ; Adrenal Glands ; Anesthetics* ; Animals ; Aorta ; Endotoxins ; Halothane ; Humans ; Injections, Intraperitoneal ; Liver ; Lung ; Male ; Muscle, Smooth, Vascular ; Nitric Oxide ; Nitric Oxide Synthase ; Norepinephrine ; Phenylephrine ; Rats* ; Rats, Sprague-Dawley ; Sepsis