• Biomedical Science Letters
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• v.20 no.3
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• pp.129-138
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• 2014

In this study, we investigated the effect of water extract from rice bran (RB) on ADP ($20{\mu}M$)-stimulated platelet aggregation. RB dose-dependently inhibited ADP-induced platelet aggregation, and its $IC_{50}$ value was $224.0{\mu}g/mL$, which was increased by adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS. RB elevated the phosphorylation of VASP ($Ser^{157}$) which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that RB-elevated cAMP contributed to the phosphorylation of VASP ($Ser^{157}$) to inhibit ADP-induced platelet aggregation. Therefore, we demonstrate that RB has an antiplatelet effect via cAMP-dependent phosphorylation of VASP ($Ser^{157}$), and RB may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.5
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• pp.435-440
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• 2015

This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive $K^+$ channel blocker). However, neither $N^G$-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-${\alpha}$]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive $K^+$ channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.

• Biomedical Science Letters
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• v.21 no.2
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• pp.103-114
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• 2015

In this study, we investigated the effect of rice bran water extract fermented with Lactobacillus plantarum KCCM-12116 (RBLp) on ADP ($20{\mu}M$)-, collagen ($10{\mu}g/mL$)-, and thrombin (0.2 U/mL)-stimulated platelet aggregation. RBLp dose-dependently inhibited ADP-, collagen-, and thrombin-induced platelet aggregation, with $IC_{50}$ values of 501.1, 637.2, and > $2,000{\mu}g/mL$, respectively. The platelet aggregation induced by ADP plus RBLp ($750{\mu}g/mL$) was increased by the adenylate cyclase inhibitor, SQ22536, and the cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS. Treatment with RBLp increased the phosphorylation of VASP ($Ser^{157}$), an A-kinase substrate, which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that the RBLp-induced increases in cAMP contributed to the phosphorylation of VASP ($Ser^{157}$), which in turn resulted in an inhibition of ADP-induced platelet aggregation, thereby indicating that RBLp has an antiplatelet effect via cAMP-dependent phosphorylation of VASP ($Ser^{157}$). Thus, RBLp may have therapeutic potential for the treatment (or prevention) of platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.3
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• pp.275-282
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• 1999

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.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.3
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• pp.227-237
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• 2021

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca2+-activated K+ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents (IBK). The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-NG-monomethyl arginine citrate and L-NG-nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with N-ethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DL-dithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.

• Journal of Life Science
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• v.23 no.5
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• pp.609-615
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• 2013

Glyceollin I has gained attention as a useful therapy for various dermatological diseases. However, the binding property of glyceollin I to the mammalian adenylyl cyclase (hereafter mAC), a critical target enzyme for the down-regulation of skin melanogenesis, has not been fully explored. To clarify the action mechanism between glyceollin I and mAC, we first investigated the molecular docking property of glyceollin I to mAC and compared with that of SQ22,536, a well-known mAC inhibitor, to mAC. Glyceollin I showed superiority by forming three hydrogen bonds with Asp 1018, Trp 1020, and Asn 1025, which exist in the catalytic site of mAC. However, SQ22,536 formed only two hydrogen bonds with Asp 1018 and Asn 1025. Secondly, we confirmed that glyceollin I effectively inhibits the formation of forskolin-induced cAMP and the phosphorylation of PKA from a cell-based assay. Long term treatment with glyceollin I had little effect on the cell viability. The findings of the present study also suggest that glyceollin I may be extended to be used as an effective inhibitor of hyperpigmentation.

• Molecules and Cells
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• v.26 no.2
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• pp.181-185
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• 2008

The effects of calcitonin gene-related peptide (CGRP) on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine were investigated using the whole-cell patch clamp technique at $30^{\circ}C$. Under voltage clamping at a holding potential of -70 mV, CGRP decreased the amplitude and frequency of pacemaker currents and activated outward resting currents. These effects were blocked by intracellular $GDP{\beta}S$, a G-protein inhibitor and glibenclamide, a specific ATP-sensitive $K^+$ channels blocker. During current clamping, CGRP hyperpolarized the membrane and this effect was antagonized by glibenclamide. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor) or naproxen (a cyclooxygenase inhibitor) did not block the CGRP-induced effects, whereas pretreatment with ODQ (a guanylate cyclase inhibitor) or L-NAME (an inhibitor of nitric oxide synthase) did. In conclusion, CGRP inhibits pacemaker currents in ICC by generating nitric oxide via G-protein activation and so activating ATP-sensitive $K^+$ channels. Nitric oxide- and guanylate cyclase-dependent pathways are involved in these effects.

• Korean Journal of Veterinary Research
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• v.43 no.4
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• pp.563-571
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• 2003

Dysfunction of mesangial cells has been contributed to the onset of diabetic nephropathy. Insulin like growth factors (IGFs) are also implicated in the pathogenesis of diabetic nephropathy. However, it is not yet known about the effect of high glucose on IGF-I and IGF-II secretion in the mesangial cells. Furthermore, the relationship between cAMP and high glucose on the secretion of IGFs was not elucidated. Thus, we examined the mechanisms by which high glucose regulates secretion of IGFs in mesangial cells. Glucose increased IGF-I secretion in a time- (>8 hr) and dose- (>15 mM) dependent manner (p<0.05). Stimulatory effect of high glucose on IGF-I secretion is predominantly observed in 25 mM glucose (high glucose), while 25 mM glucose did not affect cell viability and lactate dehydrogenase release. High glucose also increased IGF-II secretion. The increase of IGF-I and IGF-II secretion is not mediated by osmotic effect, since mannitol and L-glucose did not affect IGF-I and IGF-II secretion. 8-Br-cAMP mimicked high glucose-induced secretion of IGF-I and IGF-II. High glucose-induced stimulation of IGF-I and IGF-II secretion was blocked not by pertussis toxin but by SQ 22536 (adenylate cyclase inhibitor). Rp-cAMP (cAMP antagonist), and myristoylated protein kinase A (PKA) inhibitor amide 14-22 (protein kinase A inhibitor). These results suggest that cAMP/PKA pathways independent of Gi protein may mediate high glucose-induced increase of IGF-I and IGF-II secretion in mesangial cells. Indeed, glucose (>15 mM glucose) increased cAMP formation. In conclusion, high glucose stimulates IGF-I and IGF-II secretion via cAMP/PKA pathway in mesangial cells.

• Biomedical Science Letters
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• v.10 no.4
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• pp.375-383
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• 2004

It has been reported that osteoporosis induced by the deficiency of estrogens in menopause is associated with the unbalance of Ca/sup 2+/ and Pi levels. Proximal tubule is very important organ to regualte Ca/sup 2+/ and Pi level in the body. However, the effect of estrogens on Ca/sup 2+/ and Pi regulation was not elucidated. Thus, we examined the effect of 17-β estradiol (E₂) on Ca/sup 2+/ and Pi uptake in the primary cultured rabbit renal proxiaml tubule cells. In the present study, E₂(> 10/sup -9/M) decreases Ca/sup 2+/uptake and stimulates Pi uptake over 3 days. E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by actinomycin D (a gene transcription inhibitor), cycloheximide (a protein synthesis inhibitor). tamoxifen, and progesterone (estrogen receptor antagonists). E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by SQ22536 (an adenylate cyclase inhibitor), Rp-cAMP (a cAMP antagonist), and PKI (a protein kinase A inhibitor). Indeed, E₂ increased cAMP formation. In addition, E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by staurosporine, H-7, and bisindolylmaleimide I (protein kinase C inhibitors) and E₂ translocated PKC from cytoslic fraction to membrane fraction. In conclusion, E₂ decreased Ca/sup 2+/ uptake and stimulated Pi uptake via cAMP and PKC pathway in the PTCs.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.3
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• pp.111-115
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• 2008

The effects of (-)-epigallocatechin gallate (EGCG) on pacemaker activities of cultured interstitial cells of Cajal (ICC) from murine small intestine were investigated using whole-cell patch-clamp technique at $30^{\circ}C$ and $Ca^{2+}$ image analysis. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. The treatment of ICC with EGCG resulted in a dose-dependent decrease in the frequency and amplitude of pacemaker currents. SQ-22536, an adenylate cyclase inhibitor, and ODQ, a guanylate cyclase inhibitor, did not inhibit the effects of EGCG. EGCG-induced effects on pacemaker currents were not inhibited by glibenclamide, an ATP-sensitive $K^+$ channel blocker and TEA, a $Ca^{2+}$-activated $K^+$ channel blocker. Also, we found that EGCG inhibited the spontaneous $[Ca^{2+}]_i$ oscillations in cultured ICC. In conclusion, EGCG inhibited the pacemaker activity of ICC and reduced $[Ca^{2+}]_i$ oscillations by cAMP-, cGMP-, ATP-sensitive $K^+$ channel-independent manner.