Original Articles Korean Circulation J 1999;29 2 : Protein Kinase C 에의한막전압의존성 K + 전류억제효과가 Histamine 에의한토끼관동맥긴장도증가에미치는효과 배상욱 1 하미영 2 안덕선 2 강복순 2

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1 Original Articles Korean Circulation J 1999;292: Protein Kinase C 에의한막전압의존성 K + 전류억제효과가 Histamine 에의한토끼관동맥긴장도증가에미치는효과 배상욱 1 하미영 2 안덕선 2 강복순 2 Effect of PKC-dependent Change of K Current Activity on Histamine-induced Contraction of Rabbit Coronary Artery Sang-Wook Bai, MD 1, Mi-Young Ha, MD 2, Duck-Sun Ahn, MD 2 and Bok-Soon Kang, MD 2 1 Department of Obsterics and Gynecology, 2 Physiology, College of Medicine, Yonsei University, Seoul, Korea ABSTRACT BackgroundHistamine, released from mast cells in atheromatous plaque, has been known to cause cardiac ischemia or sudden cardiac death in atherosclerosis patient. Previous reports have suggested that histamine induced coronary vasoconstriction was due to increase in IP 3 and DAG, which induce release of Ca 2 from SR and increase the Ca 2 sensitivity of contractile element via activation of PKC. Recently, it was reported that application of histamine cause depolarization of intestinal smooth muscle, which may contribute to histamineinduced contraction via augmenting Ca 2 influx through activation of Ca 2 channels. However, the underyling mechanism of histamine-induced depolarization and its contribution to the magnitude of coronary vasoconstriction are still uncertain. MethodTo elucidate the underlying mechanism of Ca 2 influx change during histamineinduced vasoconstriction, we examined the effect of Ca 2 channel antagonist and PKC blocker on histamineinduced contractions, and then measured the effect of PKC antagonist on whole cell K current using patch clamping method in rabbit coronary smooth muscle cells. ResultsApplication of histamine induced phasic and tonic constraction of coronary rings via activation of H 1 receptors. Pretreatment of Ca 2 channel antagonist nifedipine, 1 M or PKC blockers 10 nm staurosporine and 10 M Gö6976 markedly inhibited histamineinduced tonic contraction, which suggest that the magnitude of tonic contraction depend on the Ca 2 influx. Application of 4-AP, a blocker of voltage-dependent K channels, increased resting tone of coronary rings, and combined treatment of nifedipine blocked this 4-AP induced increase of resting tone. Application of active analoge of DAG 1,2-DiC 8 significantly inhibited the activity of voltage-dependent K current in single smooth muscle cell, meanwhile the inactive analogue of DAG 1,3-DiC 8 has no apparent effect on the activity of voltage-dependent K current. Furthermore, pretreatment of calphostin C 1 M, a blocker of PKC, diminished the 1,2-DiC 8 -induced inhibition of K current. ConclusionsPKC dependent inhibition of voltage-dependent K current may be responsible for the maintaining of histamine-induced tonic contraction in rabbit coronary artery. Korean Circulation J 1999;292: KEY WORDSHistamine Voltage dependent K current Protein kinase C Rabbit coronary artery. 192

2 서론 193

3 대상및방법 장력측정 194 이온전류의측정 Korean Circulation J 1999;292:

4 약물 ö 결과분석 결 Histamine에의한혈관수축반응에미치는 histamine 수용체길항제의효과 Fig. 1. Histamine-induced contractile response in endothelium denuded rabbit coronary artery. A Application of histamine to the bath increased contractile response of rabbit coronary strips in a dose dependent manner from 10 8 M to 10 5 M. B Concentrationresponse curve of histamine in rabbit coronary artery. Contractions induced by histamine were expressed as a % of 70 mm K -induced contraction. Each data points were expressed as mean±s.e and half maximal concentration of histamine is 254.3±1.1 nm, n=6. 과 195

5 PKC antagonist가 histamine에의한혈관수축반응에미치는효과 ö Fig. 2. Effect of pyrilamine, H1 receptor antagonist, on histamine-induced contraction in endothelium-denuded rabbit coronary artery. Representative tracing of tension to histamine 10 6 M in control A and after 1 μm pyrilamine application to the bath B. CPretreatment of pyrilamine significantly blocked the histamine-induced contraction in rabbit coronary strips p0.05, n=4. The amplitude of tonic contraction was expressed as % contraction of 70 mm K + -induced contraction. 196 Korean Circulation J 1999;292:

6 Fig. 3. Effect of cimetidine, H2 receptor antagonist, on histamine-induced contraction in endothelium denuded rabbit coronary artery. A Representative tracing of tension to histamine in control A and in the presense of 10 M cimetidine B. C Pretreatment of cimetidine shows no apparent change in histamine-induced tension response n=4. The amplitude of histamine-induced contraction was expressed as % contraction of 70 mm K -induced contraction. Fig. 4. Effect of staurosporine, a nonspecific PKC antagonist, on tension response to histamine in rabbit coronary artery. A Representative tracing of tension to histamine without A and with staurosporine 10 nm treatment B. Staurosporines were added to the bath 10 minutes before exposure to histamine 10 6 M. C Summary of staurosporine-induced inhibition of phasic and tonic contraction developed by histamine. Staurosporine significantly inhibited tonic contraction. Filled rectangle represents staurosporine-treated group, and open rectangle represents control group. Asterisks denote p0.05, n=5. The amplitude of histamine-induced contraction was expressed as % contraction of 70 mm K + -induced contraction. 197

7 ö ö ö ö 세포외로부터의 Ca 2+ 유입이 histamine에의한혈관수축반응에미치는효과 ö Fig. 5. Effect of G6976, specific PKC antagonist, on tension response to histamine in rabbit coronary artery. Representative tracing of tension to histamine 10 6 M without A and with G μm treatment B. G 6976 was added to the bath 10 minutes before exposure to histamine. C Summary of G6976-induced inhibition of phasic and tonic contraction developed by histamine. G6976 significantly inhibited tonic contraction, and shows no significant change on phasic contraction Filled rectangle represents G6976-treated group, and open rectangle represents control group. Asterisk denotes p0.05, n=5. The amplitude of histamine-induced contraction was expressed as % contraction of 70 mm K -induced contraction. 198 Korean Circulation J 1999;292:

8 막전압의존성 K + 전류의활성에미치는 PKC의효과 Fig. 6. Effect of nifedipine, a Ca 2 channel antagonist, on tension response to histamine in rabbit coronary artery. A Application of 10 6 M nifedipine to the bath relax the tension developed by 10 6 M histamine, and summary of effect of nifedipine filled rectangle on histamine-induced contraction B. Data were expressed as the % of 70 mm K -induced contraction n=9. Representative tracing of tension to histamine 10 6 M in control C and pretreatment of 10 6 M nifedipine D. E Summary of nifedipine-induced inhibition of phasic and tonic contraction developed by histamine M nifedipine filled rectangle significantly inhibited tonic contraction, and shows no significant change on phasic contraction n=7. Asterisk denotes statistically significant. The amplitude of histamine-induced contraction was expressed as % contraction of 70 mm K -induced contraction. 199

9 Fig. 7. Effects of K + channel and Ca 2+ channel antagonists on resting tension in endothelium-denuded rabbit coronary artery. Representative tracing of resting tension change to 10-6 M nifedipine (A), 1 mm TEA (B), 5 mm 4- AP (C), and 5 mm 4-AP+10-6 M nifedipine (D). E) Summary of resting tone change by application of nifedipine, TEA, 4-AP, and 4-AP+nifedipine. The amplitude of resting tone change was expressed as % contraction of 70 mm K + -induced contraction (nifedipine 처치군 ;-5.0 ±1.1%, TEA 처치군 ; 17.6±8.9%, 4-AP 처치군 ; 89.5± 5.0%, 4-AP+nifedipine 처치군 ;-0.6±0.7%, n=5). 200 PKC agonist의 K + 전류감소작용에미치는 PKC 차단제의효과 Korean Circulation J 1999;292:

10 Fig. 8. Effect of PKC agonist on voltage dependent K + currents. A) Time course of 1,2-DiC8-induced change of peak K + currents measured at 0 mv steps at every 10 seconds. Application of 1,2-DiC8 significantly decreased K + currents. The 1, 2, & 3 indicated the representative current traces of control, 1,2-DiC8, and recovery. B) Representative traces of whole cell currents during control, 1,2-DiC8 (10 μm), and after washout of 1,2-DiC8. K + currents were evoked by stepping the membrane potentials from a holding potential of -80 mv to potentials ranging from -50 mv to +25 mv in 5 mv increments. C) Average current voltage relations of peak currents in control ( ), after 1,2-DiC8 ( ) treatment (n=6). The peak current amplitudes were normalized with whole cell capacitance. Application of 1,2-DiC8 signifi-cantly inhibited the K + currents (*:p<0.01). 201

11 Fig. 9. Effect of Dimethyl sulfoxide (DMSO) on voltagedependent K + currents. A) Representative traces of whole cell currents during control, and after application of 0.02% and 0.05% DMSO in the bath. K + currents were evoked by stepping the membrane potentials from a holding potential of -80 mv to potentials ranging from -50 mv to +25 mv in 5 mv increments. B) Current voltage relations of K + currents obtained before and after DMSO treatment. 202 고안 ö Korean Circulation J 1999;292:

12 Fig. 10. Effect of inactive analogue of 1,2-DiC8 on voltage-dependent K + currents. A) Time course of 1,3-DiC8 and 1,2-DiC8 induced change of peak K + currents measured at 0 mv steps at every 10 seconds. Application of 1,3-DiC8 slightly decreased K + currents, whereas 1,2- DiC8 apparently decreased K + currents. The 1, 2, & 3 indicated the representative current traces of control, 1,3-DiC8, and 1,2-DiC8. B) Representative traces of whole cell currents during control and after application of 1,3-DiC8 (10 μm), and 1,2-DiC8 (10 μm) in the bath. The current traces recorded during 195 ms step pulses from -50 mv to +25 mv in 5 mv increments. C) Average current voltage relations of peak currents in control ( ), after 1,3-DiC8 ( ) treatment (n=4). The peak current amplitudes were normalized with whole cell capacitance. 세포외로부터의 Ca 2+ 유입이 histamine에의한관동맥수축에미치는효과 203

13 Fig. 11. Inhibition of PKC-dependent change of voltage-dependent K + current by calphostin C. A) representative traces of whole cell currents during control and after application of calphostin C (1 μm), and 1,2-DiC8 (10 μm) in the bath. The current traces recorded during 195 ms step pulses from -50 mv to +25 mv in 5 mv increments. B) Average current voltage relations of peak currents in control ( ), after calphostin C treatment ( ), and calphostin C+1,2-DiC8 ( ) treatment (n=4). The peak current amplitudes were normalized with cell capacitance. C) Summary of voltage-dependent K + current amplitudes recorded at 0 mv steps. The current amplitudes were expressed as the percent of control current amplitudes. Cal-C and Cal-C+1,2-DiC8 represent calphostin C and calphostin C+1,2-DiC8 treatment, respectively. Pretreatment of calphostin C significantly attenuated the 1,2- DiC8-induced inhibition of K + current (1,2-DiC8 treated group vs calphostin C+1,2DiC8 treated group;59.4±1.9 vs 84.1±9.6% of control current amplitude*:p<0.05, n=4). 204 Korean Circulation J 1999;292:

14 Fig. 12. Effects of 1,2-DiC8 on steadystate activation and inactivation of voltage-dependent K + currents. A) Representative traces of control current elicited by pulsing from -50 mv to 25 mv in 5 mv increment from a holding potential of -80 mv. Tail currents evoked upon repolarization to -50 mv. Traces at top right show the effects of 10 μm 1,2-DiC8 on currents evoked by an identical protocol of control. B) Current traces were generated by double pulse protocol. 10 seconds preconditioning steps ranging from to +10 mv were applied from a holding potential of -80 mv. Each preconditioning pulse was followed by a constant 1 sec test pulse to +10 mv to record IdK. Traces at right show the effects of 10 μm 1,2-DiC8 on currents evoked by an identical protocol of control. C) Relative states of activation and inactivation at each voltage were fitted by boltzman functions, and their fits are shown for control (, for activation & inactivation) and 1,2-DiC8 (, ;for activation & inactivation). Half maximal activation values are ±0.4 and -16.2±0.7 in control and 1,2-DiC8 treated group (n=10). Half maximal inactivation values are -31.2± 0.6 and -35.5±0.4 in control and 1,2- DiC8 treated group (n=7). ö 205

15 PKC에의한막전압의존성 K + 통로의활성변화 206 결론 Korean Circulation J 1999;292:

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Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract