• The Korean Journal of Physiology and Pharmacology
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• 제1권5호
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• pp.547-553
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• 1997

It has been known that activation of tyrosine kinases is involved in signal transduction. Role of the tyrosine kinase in vascular smooth muscle contraction was examined in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy, one week after which they were subcutaneously implanted with DOCA (200 mg/kg) and supplied with 1% NaCl and 0.2% KCl drinking water for $4{\sim}6$ weeks. Control rats were treated the same except for that no DOCA was implanted. Helical strips of carotid arteries were mounted in organ baths for measurement of isometric force development. Genistein was used as a tyrosine kinase inhibitor. Concentration-response curves to 5-hydroxytryptamine (5-HT) shifted to the right by genistein in both DOCA-salt hypertensive and control rats. Although the sensitivity to genistein was similar between the two groups, the maximum force generation by 5-HT was less inhibited by genistein in arteries from DOCA-salt hypertensive rats than in those from controls. Genistein-induced relaxations were attenuated in arteries from DOCA-salt rats. Genistein affected the contraction to phorbol 12, 13-dibutyrate (PDBu) neither in DOCA-salt nor in control arteries. These observations suggest that tyrosine kinase is involved in 5-HT-induced vascular contraction, of which role is reduced in DOCA-salt hypertension.

• The Korean Journal of Physiology and Pharmacology
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• 제9권1호
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• pp.9-15
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• 2005

We attempted to analyze the mechanism of polychlorinated biphenyl (PCB)-induced neurotoxicity and identify the target molecules in the neuronal cells for PCBs.Since the developing neuron is particularly sensitive to PCB-induced neurotoxicity, we isolated cerebellar granule cells derived from 7-day old Sprague Dawley (SD) rats and grew cells in culture for additional 7 days to mimic PND-14 conditions. Only non-coplanar PCBs at a high dose showed a significant increase of total protein kinase C (PKC) activity at phobol 12,13-dibutyrate ([$^3M$]PDBu) binding assay, indicating that non-coplanar PCBs are more neuroactive than coplanar PCBs in neuronal cells. PKC isozymes were immunoblotted with the selected monoclonal antibodies. PKC-${\alpha}$, ${\delta}$, and ε were activated with non-coplanar PCB exposure. Receptor for activated C kinase-1 (RACK-1), anchoring protein for activated PKC, was more induced with exposure to coplanar PCBs than non-coplanar PCBs. Reverse transcription PCR (RT-PCR) analysis showed induction of neurogranin (RC-3) and growth associated protein-43 (GAP-43) mRNA with non-coplanar PCBs. The results indicate that these factors may be useful biomarkers for differentiating non-coplanar PCBs from coplanar PCBs. The present study demonstrated that non-coplanar PCBs are more neuroactive congeners than coplanar PCBs.

• Archives of Pharmacal Research
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• 제21권4호
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• pp.452-457
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• 1998

Three compounds, 5-(acetoxymethyl)-5-(hydroxymethyl)-3-tetradecyl-2,5-dihydro-2-furanone (3), 5-(acetoxymethyl)-5-(hydroxymethyl)-3,3-dihexyltetrahydro-2-furanone (4) and 5-(acetoxymethyl)-5-(hydroxymethyl)-3,3-dioctyltetrahydro-2-furanone (5), were designed and synthesized as surrogates of the ultrapotent DAG analogue, 5-(acetoxymethyl)-5-(hydroxymethyl) 3-[(Z)-tetradecylideneltetrahydro-2-furanone (1), a compound that showed high affinity for PKC-$\alpha$ ($K_1$=35 nM) in a competition binding assay with [$^3H$-20]phorbol-12,13-dibutyrate (PDBU). In an attempt to overcome the problem of generating geometrical E- and Z- isomers, as encountered with 1, the double bond was moved to an endocyclic location as in 3, or an additional alkyl chain was appended to C3 to give the corresponding 3,3-dialkyl saturated lactones (4 and 5). The lactone was constructed from glycidyl-4-methoxyphenyl ether in 5 steps. The target compounds showed reduced binding affinities for PKC-.alpha. with $K_{i}$ values of 192 nM (3), 4,829 nM (4), and 2,812 nM (5), respectively. These results indicate that constrained DAG analogues having a tetrahydro-2-furanone template are effectively discriminated by PKC-(X in terms of the direction of the long alkyl chain connected to the 3-position.n.

• Environmental Analysis Health and Toxicology
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• 제22권3호
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• pp.279-285
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• 2007

The present study attempted to analyze the mechanism of PCB-induced neurotoxicity with respect to the PKC signaling. Since the developing neuron is particularly sensitive to PCB-induced neurotoxicity, we isolated cerebellar granule cells derived from 7-day old SD rats and grew cells in culture for additional 7 days to mimic PND-14 conditions. Only non-coplanar PCBs at a high dose showed a significant increase of total PKC activity at $[^3H]PDBu$ binding assay, indicating that non-coplanar PCBs are more neuroactive than coplanar PCBs in neuronal cells. PKC isoforms were immunoblotted with respective monoclonal antibodies. PKC-alpha and-epsilon were activated with non-coplanar PCB exposure. The result suggests that coplanar PCBs have a PKC pathway different from non-coplanar PCBs. Activation of PKC with exposure was dampened with treatment of high molecular weight of chitosan. Chilean (M.W. > 1,000 kDa) inhibited the total activity of PKC induced by the non-coplanar PCBs. Translocation of PKC isoforms was also inhibited by the high molecular weight of chitosan. The study demonstrated that non-coplanar PCBs are more potent neurotoxic congeners than coplanar PCBs and the alteration of PKC activities by PCB exposure can be blocked with the treatment of chitosan. The results suggest a potential use of chitosan as a means of nutritional intervention to prevent the harmful effects of pollutant-derived diseases.

• The Korean Journal of Physiology and Pharmacology
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• 제5권2호
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• pp.139-146
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• 2001

L-type $Ca^{2+}$ channels play an important role in regulating cytosolic $Ca^{2+}$ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type $Ca^{2+}$ channel currents in rat pituitary $GH_3$ cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the $Ba^{2+}$ current through the L-type $Ca^{2+}$ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the $Ba^{2+}$ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced $Ba^{2+}$ currents. The above results show that the L-type $Ca^{2+}$ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in $GH_3$ cells may be attributable, at least in part, to their effect on L-type $Ca^{2+}$ channels.

• The Korean Journal of Physiology and Pharmacology
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• 제15권3호
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• pp.143-147
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• 2011

Defensins, cysteine-rich cationic polypeptides released from neutrophils, are known to have powerful antimicrobial properties. In this study, we sacrificed 30 rats to investigate the effects of ${\alpha}$-defensin 1 on detrusor muscle contractions in isolated rat bladder. From the experiments we found relaxing effects of ${\alpha}$-defensin 1 on the contractions induced by phenylephrine (PE) but not by bethanechol (BCh) in the detrusor smooth muscles. To determine the mechanisms of the effects of ${\alpha}$-defensin 1, the changes of effects on PE-induced contraction by ${\alpha}$-defensin 1 pretreatment were observed after pretreatment of Rho kinase inhibitor (Y-27632), protein kinase C (PKC) inhibitor (Calphostin C), potent activator of PKC (PDBu; phorbol 12,13-dibutyrate), and NF-${\kappa}B$ inhibitors (PDTC; pyrrolidinedithiocarbamate and sulfasalazine). The contractile responses of PE ($10^{-9}{\sim}10^{-4}$ M) were significantly decreased in some concentrations of ${\alpha}$-defensin 1 ($5{\times}10^{-9}$ and $5{\times}10^{-8}$ M). When strips were pretreated with NF-kB inhibitors (PDTC and sulfasalazine; $10^{-7}{\sim}10^{-6}$ M), the relaxing responses by ${\alpha}$-defensin 1 pretreatment were disappeared. The present study demonstrated that ${\alpha}$-defensin 1 has relaxing effects on the contractions of rat detrusor muscles, through NF-${\kappa}B$ pathway. Further studies in vivo are required to clarify whether ${\alpha}$-defensin 1 might be clinically related with bladder dysfunction by inflammation process.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제23권4호
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• pp.295-305
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• 2001

구강은 흡연이나 음주와 같은 화학적 발암물질이 쉽게 접촉할 수 있는 화학적 발암물질의 표적장기이며 구강암을 포함한 대부분의 암 발생의 근원이 되는 세포는 상피세포이다. 따라서 본 연구는 인체상피세포를 화학적 발암물질인 MNING에 노출시켜 발암화를 유도하고 이에 따른 작용 기전을 분석함으로써 구강암과 같은 상피세포 기원의 종양 발생기전을 이해하는 데 기여하고자 하였다. 인체 상피세포에 $0.001{\mu}g/ml$에서 $1{\mu}g/ml$ 용량의 MNNG를 투여한 결과 용량 의존적인 세포발암성을 나타내었으며 $0.01{\mu}g/ml$ 투여군이 가장 높은 암세포의 지표를 보였다. MNNG투여후 TPA를 처리한 결과 발암세포의 지표인 saturation density, soft agar colony formation, cell aggregation 등에서 MNNG의 단독 투여시보다 높은 발암성을 나타내었으며 최초의 foci출현시기도 단축되었다. 이와같은 결과는 Phorbol ester binding assay에서도 나타나 세포 발암화 촉진에 PKC활성이 관여함을 추정할 수 있다. PKC translocation 현상은 세포외 칼슘이 있을 경우에만 나타나 MNNG에 의한 PKC활성에 classical PKC가 관여함을 추정할 수 있었다. MNNG에 대한 초기반응으로 cPKC의 경우 $PKC-{\alpha}$와 $PKC-{\gamma}$가 고농도에서 활성의 증가를 보였으며 nPKC의 경우 $PKC-{\varepsilon}$가 뚜렷한 활성을 보여 이들 isoform이 MNNG에 의한 발암화 초기단계에 관여함을 암시하였다. 반면 aPKC는 어느 형태도 MNNG에 반응하지 않아 화학적 발암화 과정에 isoform의 특이성이 존재함을 입증하였다. MNNG에 의해 발암화 특성을 나타낸 세포는 $PKC-{\alpha}$ 및 $PKC-{\gamma}$의 지속적인 활성증가를 나타내어 발암의 초기단계부터 지속적이 활성을 유지하고 있는 isoform으로 추정된다. 본 연구결과 인체상피 세포의 모든 PKC isoform에 대한 발현을 분석하고 화학적 발암화에 관여하는 isoform을 선별해냄으로써 특정한 inhibitor 등을 상요한 발암화 억제제의 개발에 필요한 기초자료를 제공하였을 뿐만 아니라 구강암과 같은 상피세포 기원의 암발생 기전을 이해하는 데 기여할 것으로 사료된다.