A Portulaca oleracea L. extract promotes insulin secretion via a K⁺(ATP) channel dependent pathway in INS-1 pancreatic β-cells

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.

Establishment of a cell model of insulin-resistant 3T3-L1 adipocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the optimal conditions for establishing insulin-resistant 3T3-L1 adipocytes.</p><p><b>METHOS</b>Dexamethason (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.</p><p><b>RESULTS</b>Treatment 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).</p><p><b>CONCLUSION</b>3T3-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.</p>

Pear pomace water extract inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes

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.

Neuroendocrine Differentiation in Acquired Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor / 결핵및호흡기질환

BACKGROUND: Small cell lung cancer (SCLC) transformation during epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment in lung cancer has been suggested as one of possible resistance mechanisms. METHODS: We evaluated whether SCLC transformation or neuroendocrine (NE) differentiation can be found in the cell line model. In addition, we also investigated its effect on responses to conventional chemotherapeutic drugs of the SCLC treatment. RESULTS: Resistant cell lines to various kinds of EGFR-TKIs such as gefitinib, erlotinib, CL-387,785 and ZD6474 with A549, PC-9 and HCC827 lung adenocarcinoma cell lines were established. Among them, two resistant cell lines, A549/GR (resistant to gefitinib) and PC-9/ZDR (resistant to ZD6474) showed increased expressions of CD56 while increased synaptophysin, Rb, p16 and poly(ADP-ribose) polymerase were found only in A549/GR in western blotting, suggesting that NE differentiation occurred in A549/GR. A549/GR cells were more sensitive to etoposide and cisplatin, chemotherapeutic drugs for SCLC, compared to parental cells. Treatment with cAMP and IBMX induced synaptophysin and chromogranin A expression in A549 cells, which also made them more sensitive to etoposide and cisplatin than parental cells. Furthermore, we found a tissue sample from a patient which showed increased expressions of CD56 and synaptophysin after development of resistance to erlotinib. CONCLUSION: NE differentiation can occur during acquisition of resistance to EGFR-TKI, leading to increased chemosensitivity.

Inhibitory Effect of Vitamin U (S-Methylmethionine Sulfonium Chloride) on Differentiation in 3T3-L1 Pre-adipocyte Cell Lines

BACKGROUND: S-methylmethionine sulfonium chloride was originally called vitamin U because of its inhibition of ulceration in the digestive system. Vitamin U is ubiquitously expressed in the tissues of flowering plants, and while there have been reports on its hypolipidemic effect, its precise function remains unknown. OBJECTIVE: This study was designed to evaluate the anti-obesity effect of vitamin U in 3T3-L1 pre-adipocyte cell lines. METHODS: We cultured the pre-adipocyte cell line 3T3L1 to overconfluency and then added fat differentiation-inducing media (dexamethasone, IBMX [isobutylmethylxanthine], insulin, indomethacin) and different concentrations (10, 50, 70, 90, 100 mM) of vitamin U. Then, we evaluated changes in the levels of triglycerides (TGs), glycerol-3-phosphate dehydrogenase (G3PDH), AMP-activated protein kinase (AMPK), adipocyte-specific markers (peroxisome proliferator-activated receptor gamma [PPAR-gamma], CCAAT/enhancer-binding protein alpha [C/EBP-alpha], adipocyte differentiation and determination factor 1 [ADD-1], adipsin, fatty acid synthase, lipoprotein lipase) and apoptosis-related signals (Bcl-2, Bax). RESULTS: There was a gradual decrease in the level of TGs, C/EBP-alpha, PPAR-gamma, adipsin, ADD-1 and GPDH activity with increasing concentrations of vitamin U. In contrast, we observed a significant increase in AMPK activity with increasing levels of vitamin U. The decrease in bcl-2 and increase in Bax observed with increasing concentrations of vitamin U in the media were not statistically significant. CONCLUSION: This study suggests that vitamin U inhibits adipocyte differentiation via down-regulation of adipogenic factors and up-regulation of AMPK activity.

Ultrastructure and electrophysiology of astrocytes differentiated from adult adipose-derived stromal cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Adipose-derived stromal cell (ADSC) differentiation into neural cells in vitro is becoming widely studied. However, there are few reports on astrocytes following differentiation, and particularly on maturation and electrophysiology. In this study, we used various methods to determine ADSC-derived astrocyte maturity.</p><p><b>METHODS</b>Chemical induction with isobutylmethylxanthine (IBMX) was used to differentiate adult ADSCs into astrocytes followed by hematoxylin-eosin (HE) staining to observe morphology and transmission electron microscopy for cellular ultrastructure assessment. Immunofluorescence was used to detect expression of neural stem cell marker nestin as well as glial markers glial fibrillary acidic protein (GFAP) and S-100. In addition, we measured membrane potentials in bis-(1,3-dibarbituric acid) trimethine oxanol-labeled ADSCs and astrocytes by stimulation with a high potassium solution under an inverted fluorescence microscope. Finally, cell cycle distribution was detected by flow cytometry.</p><p><b>RESULTS</b>Typical astrocyte morphology was shown by HE staining after 48-hour differentiation. Glial fibril was observed with transmission electron microscopy. GFAP and S-100 were not expressed in the control group, but were expressed within 24-hour differentiation and reached a maximum at day 14 with no change up to day 28. Nestin was weakly expressed in control cells and also reached a maximum at day 14 with the percentage of positive cells constant until day 21 followed by a decrease. Differentiated cell membrane potentials after stimulation with potassium were slightly increased, and then gradually declined over time. There was no significant membrane potential change in the control group. Flow cytometry showed that the percentage of cells in G0/G1 phase was 93% and only 5% in S phase.</p><p><b>CONCLUSION</b>ADSCs were differentiated into mature astrocytes with typical characteristics including morphology, ultrastructure, marker protein expression, mature potassium channels and mitotic capacity.</p>

Involvement of Crosstalk Between cAMP and cGMP in Synaptic Plasticity in the Substantia Gelatinosa Neurons

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.

In vitro brain tyrosine hydroxylase activation in catfish Heteropneustes fossilis (Bloch): seasonal changes in involvement of cAMP-dependent protein kinase A and Ca2+ -dependent protein kinase C.

In the present in vitro study, the involvement of cAMP dependent-protein kinase A (PKA) and calcium-dependent protein kinase C (PKC) in the regulation of forebrain (telencephalon and hypothalamus) tyrosine hydroxylase (TH) activity was demonstrated during the reproductive seasons of the female catfish H. fossilis. In the concentration studies conducted in prespawning phase, cAMP (0.05 nM, 0.5 nM, 1 mM and 2.0 mM) or the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX-0.5-2.0 mM) stimulated enzyme activity. Likewise, the incubation of the enzyme preparations with the cAMP dependent-protein kinase A inhibitor H-89 (1 and 10 microM) and PKC inhibitor calphostin C (cal C; 1 and 10 microM) inhibited enzyme activity in a concentration-dependent manner. In seasonal studies, the incubation of the enzyme preparations with cAMP (1 mM), IBMX (1 mM), H-89 (10 microM) and cal-C (10 microM) produced season-dependent effects on enzyme activity. The stimulatory effect of cAMP and IBMX and the inhibitory effect of H-89 and cal C were greater in the resting and spawning phases. The results suggest the involvement of both signal transduction pathways in TH activation vis-à-vis catecholaminergic activity with a more dominant role by the cAMP-PKA pathway.

Effect of xanthohumol on melanogenesis in B16 melanoma cells

Xanthohumol (XH), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), dose-dependently inhibited isobutylmethylxanthine (IBMX)-induced melanogenesis in B16 melanoma cells, with little cytotoxicity at the effective concentrations. Decreased melanin content was accompanied by reduced tyrosinase enzyme activity, protein and mRNA expression. The levels of tyrosinase-related protein 1 and 2 mRNAs were decreased by XH. XH also inhibited alpha-melanocyte stimulating hormone- or forskolin-induced increases in melanogenesis, suggesting an action on the cAMP-dependent melanogenic pathway. XH downregulated the protein and mRNA expression of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of key melanogenic enzymes. These results suggest that XH might act as a hypo-pigmenting agent through the downregulation of MITF in the cAMP-dependent melanogenic pathway.

Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination.</p><p><b>METHODS</b>Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined.</p><p><b>RESULTS</b>Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C.</p><p><b>CONCLUSION</b>These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).</p>

Protein kinase A-mediated phosphorylation of HERG potassium channels in a human cell line / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line.</p><p><b>METHODS</b>HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.</p><p><b>RESULTS</b>Elevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.</p><p><b>CONCLUSIONS</b>PKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.</p>

A novel Na+-dependent transporter and NHE3 mediate H+ efflux in the luminal membrane of the pancreatic duct: regulation by cAMP

No abstract available.

The Effect of Local Delivery of IBMX on the Neointimal Proliferation in Rat Carotid Balloon Injury Model

BACKGROUND: Hyperplastic neointima is one of the major mechanisms of restenosis following balloon angioplasty in selected patients with symptomatic angina pectoris. Elevation of cellular cyclic nucleotide levels such as cAMP and cGMP are known to inhibit the proliferation of vascular smooth muscle cells. 3-isobutyl-1-methylxanthine (IBMX) increases intracellular cAMP and cGMP by nonselective inhibition of phosphodiesterases (PDEs). We conducted this study under the hypothesis that local delivery of IBMX could inhibit neointimal hyperplasia after balloon injury of the rat carotid artery. METHODS: Left common carotid artery of 10 week old male Sprague-Dawley rats were subjected to arterial injury by 2F Fogarty balloon catheter. After injury, animals were allocated to the control groups (control 1: injury control and control 2: pluronic gel plus DMSO control) and IBMX group, which received pluronic polymer gel, DMSO and IBMX mixture periadventitially. After 3 weeks, the rats were killed by overdose of ketamine, and the injured left arteries were pressure-fixed with 10% formalin and subjected to histomorphological analysis. RESULTS: Mean body weight of rats was not statistically different among study groups. The mean area of neointima (control group 1:0.28+/-0.05 mm2,Control group 2:0.27+/-0.08 mm2 , IBMX group:0.18+/-0.08 mm2 : p<0.05) and the mean ratio of neointima to medial area[versus (control group 1:1.89+/-0.37, control group 2:1.95+/-0.41, IBMX group: 1.41+/-0.47: p<0.05)] were significantly less in IBMX group. The mean area of external elastic lamina was significantly larger in control group 1 than IBMX group (0.57+/-0.07 mm2 versus 0.47+/-0.10 mm2 ) and mean luminal area showed no significant difference among groups (control group1:0.14+/-0.07 mm2 , control group 2: 0.10+/-0.05 mm2 , control group 3: 0.16+/-0.06 mm2). CONCLUSION: Peri-adventitial single administration of IBMX showed its effectiveness in reducing the neointimal proliferation in rat carotid balloon injury model. Furthermore we observed the positive correlation between intimal area and EELA suggesting vascular remodeling depending on the intima volume.

The Effect of Local Delivery of IBMX on the Neointimal Proliferation in Rat Carotid Balloon Injury Model

BACKGROUND: Hyperplastic neointima is one of the major mechanisms of restenosis following balloon angioplasty in selected patients with symptomatic angina pectoris. Elevation of cellular cyclic nucleotide levels such as cAMP and cGMP are known to inhibit the proliferation of vascular smooth muscle cells. 3-isobutyl-1-methylxanthine (IBMX) increases intracellular cAMP and cGMP by nonselective inhibition of phosphodiesterases (PDEs). We conducted this study under the hypothesis that local delivery of IBMX could inhibit neointimal hyperplasia after balloon injury of the rat carotid artery. METHODS: Left common carotid artery of 10 week old male Sprague-Dawley rats were subjected to arterial injury by 2F Fogarty balloon catheter. After injury, animals were allocated to the control groups (control 1: injury control and control 2: pluronic gel plus DMSO control) and IBMX group, which received pluronic polymer gel, DMSO and IBMX mixture periadventitially. After 3 weeks, the rats were killed by overdose of ketamine, and the injured left arteries were pressure-fixed with 10% formalin and subjected to histomorphological analysis. RESULTS: Mean body weight of rats was not statistically different among study groups. The mean area of neointima (control group 1:0.28+/-0.05 mm2,Control group 2:0.27+/-0.08 mm2 , IBMX group:0.18+/-0.08 mm2 : p<0.05) and the mean ratio of neointima to medial area[versus (control group 1:1.89+/-0.37, control group 2:1.95+/-0.41, IBMX group: 1.41+/-0.47: p<0.05)] were significantly less in IBMX group. The mean area of external elastic lamina was significantly larger in control group 1 than IBMX group (0.57+/-0.07 mm2 versus 0.47+/-0.10 mm2 ) and mean luminal area showed no significant difference among groups (control group1:0.14+/-0.07 mm2 , control group 2: 0.10+/-0.05 mm2 , control group 3: 0.16+/-0.06 mm2). CONCLUSION: Peri-adventitial single administration of IBMX showed its effectiveness in reducing the neointimal proliferation in rat carotid balloon injury model. Furthermore we observed the positive correlation between intimal area and EELA suggesting vascular remodeling depending on the intima volume.

Involvement of Spontaneously Formed Cyclic Nucleotides in Cat Gastric Muscle Relaxation

Muscle strips and muscle cells from cat stomach were used to investigate whether spontaneously formed cyclic nucleotides were involved in the inhibition of gastric smooth muscle contraction. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), increased the levels of both cyclic GMP (cGMP) and cyclic AMP (cAMP) in resting state cells, while decreasing acetylcholine-induced muscle contraction. Under the influence of IBMX, SQ22536, an adenylyl cyclase inhibitor and methylene blue, a guanylyl cyclase inhibitor completely blocked increases in cAMP and cGMP respectively, without any effect on contraction. However, the combination of SQ22536 and methylene blue completely blocked increases in both cAMP and cGMP levels and stimulated contractions markedly even in the presence of IBMX. Muscle contraction inhibitors such as isoprenaline, vasoactive intestinal polypeptide and sodium nitroprusside also appeared to increase cyclic nucleotide levels which decreased contraction. Which nucleotide increased the most was dependent on the agonist used. Therefore, irrespective of the cyclic nucleotide class, the spontaneous formation of cyclic nucleotides should be considered in evaluating the mechanism of gastric smooth muscle relaxation.

Increase of L-type calcium current by cGMP-dependent protein kinase regulates in rabbit ventricular myocytes

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.

Hydroxyl radical-mediated commitment of HL-60 cells to differentiation: Modulation of differentiation process by phosphodiesterase inhibitors

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.

Effect and Mechanism of Growth Factors and Their Interactions on the Expression of c-fos and c-myc Proto-oncogenes in Cultured Normal Rat Thyroid Cell Line (FRTL-5)

Oncogenes are known to be involved in normal cellular growth and proliferation as well as in carcinogenesis. It is reported that stimulation of quiescent cells with growth factors makes the oncogenes produce protein products as an early and immediate response, and these protein products induce or control the cell growth. Among those oncogenes, c-fos and c-myc are well known for its generalized expressions. An experiment was performed in order to prove the hypothesis that oncogene expressions would have the same pattern with that of cellular growth by growth factors in cultured normal rat thyroid cell line(FRTL-5). Ribonucleic acids of FRTL-5 cells were extracted time-sequentially at 15, 30, 60 and 120 minutes after administration of growth factors to the media of quiescent FRTL-5 cells. Extracted ribonucleic acids were blotted to the nitrocellulose membrane, and then hybridized with radiolabelled c-fos and c-myc oligonucleotide probes, and -actin probes. Hybridized dot blots on nitrocellulose membrane were autoradiographed on X-ray films, and the amount of radioactivity were measured by densitometry. Densitometric readings were used as the indices of oncogene expressions. The concentrations of TSH, IGF-I and IgG from patients with Graves' disease, which showed the maximum expressions of c-fos and c-myc in quiescent FRTL-5 cells, were TSH 10 mU/ml, IGF-I 100 ng/ml and IgG from patients with Graves' disease 1 mg/ml, respectively. Expressions of c-fos and c-myc were more prominent in combined administrations of TSH and IGF-I, or IgG from patients with Graves' disease and IGF-I than in case of TSH and IgG from patients with Graves' disease. IgG from patients with primary myxedema suppressed oncogene expressions provoked by TSH or IgG from patients with Graves' disease, but not by IGF-I. Expressions of c-fos and c-myc were more prominent by the combined administration of TSH with TPA which stimulated the phosphoinositide turnover-protein kinase C-calcium system than by those of TSH with dBcAMP, forskolin, IBMX or cholera toxin which stimulated adenylate cyclase system. From the above results, following conclusions were obtained. 1) Expressions of c-fos and c-myc by growth factors have similar patterns with those of cell growth by growth factors in FRTL-5 cells. 2) It is suggested that the actions of TSH and IgG from patients with Graves' disease would be manifested through the same signal transduction system, and that of IGF-I would be manifested through its own, but the oncogenes would be expressed mainly through adenylate cyclase system.

Effects of cyclic-GMP on hyperpolarization-activated inward current (I|f) in sino-atrial node cells of rabbit

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.