Pharmacological Investigation of Hydrogen sulfide on Interstitial Cells of Cajal from Murine Small Intestine

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Interstitial Cells of Cajal|Hydrogen sulfide|작용기전
위장관 운동성에 대한 hydrogen sulfide (H₂S)의 기능은 지금까지 평활근 수준 으로 많이 연구가 이루어져 있지만, 위장관의 향도잡이 세포의 역할을 담당하는 Interstitial cells of Cajal (ICC)에 대한 H₂S의 연구는 아직까지 이루어지지 않고 있다. 따라서 본 연구는 세포막 고정법을 이용하여 ICC의 향도잡이 활동도에 대한 H₂S의 영향에 대하여 연구하고자 하였다. 전압고정 상태에서 낮은 농도 (100 - 300 μM) 의 H₂S는 긴장성 내향성 전류를 발생시키고, 높은 농도 (1 mM)에서는 ICC에서 발생되는 향도잡이 전류를 억제하였다. 낮은 농도의 H₂S에 의해 나타나 는 내향성 전류는 비선택성 양이온 통로 억제제인 flufenamic acid와 Na+ 또는 Ca^(2+)-free 용액에 의해 억제되었다. 이와 마찬가지로, Ca^(2+)-ATPase 억제제인 thapsigargin도 H₂S의 효과를 억제하였지만 protein kinase C 억제제인 calphostin C는 영향을 주지 못하였다.
높은 농도의 H₂S에 의한 억제효과는 ATP-sensitive K+ 이온통로 억제제인 glibenclamide에 의해 약한 억제효과를 보인 것으로 보아, 부분적으로 ATP-sensitive K+ 이온통로가 그 역할을 담당하는 것으로 생각되어 진다. 이와 더불어 다른 종류의 K+ 이온통로 억제제는 H₂S의 효과에 큰 영향을 주지 못하였 다. 또한 guanylate과 adenylate cyclase 억제제인 ODQ와 SQ-22536 그리고 nitric oxide 억제제인 NAME는 H₂S의 향도잡이 전류에 대한 역할에 영향을 주지 못하였다.
Ca^(2+) 표시자인 fluo-4로 처리된 ICC는 자발적인 Ca^(2+) 진동을 보여주었고 H₂S는 이러한 자발적인 Ca^(2+)의 진동을 활성화시키는 역할을 담당하였다. 이러한 결과는 H₂S가 비선택성 양이온과 세포 내 그리고 세포외 Ca^(2+)을 이용하여 긴장성 내향전류를 발생시키는 것으로 사료되며, H₂S에 의한 억제효과는 부분적이 긴 하지만 ATP-sensitive K+ 이온통로를 통하여 그 역할을 담당하는 것으로 생각 된다.|Pharmacological investigation on the role of hydrogen sulfide in pacemaker activity and signal mechanisms in cultured interstitial cells of Cajal (ICC) from mouse was carried out using whole cell patch-clamp techniques at 30?C. Spontaneously active ICC generated pacemaker potential in the current clamp mode and pacemaker currents at a holding potential of -70 mV. Exogenously applied sodium hydrogen sulfide, a donor of hydrogen sulfide responded dose dependently to ICC. Hydrogen sulfide triggered effects on pacemaker activity by membrane polarization in lower concentration (100-300 μM) without changing pacemaker amplitudes and pacemaker frequency, and produced membrane hyperpolarization with inhibited pacemaker amplitude and pacemaker frequency of the pacemaker currents generated from ICC, and also increased resting currents in the outward direction in higher concentration (1 mM). NaHS induced inward excitatory pacemaker currents were abolished with pre-treatment of flufenamic acid, an inhibitor of voltage-independent non-selective cation channels. Sodium and calcium ion depleted bathing solution also abolished pacemaker currents as well as NaHS induced inward pacemaker currents but DIDS, an inhibitor of calcium sensitive chloride channels itself suppressed the pacemaker currents but did not response to NaHS induced pacemaker currents. Similarly, thapsigargin, an inhibitor of Ca2+-ATPase suppressed the NaHS induced effect and not responded to Calphostin C, an inhibitor of protein kinase C. NaHS induced inhibitory outward current was suppressed by pre-treatment of glibenclamide, an inhibitor of ATP- sensitive potassium channels without altering amplitudes and frequency suggest the partial involvement of ATP sensitive potassium channel but inhibitors of other potassium channels not responded to NaHS induced effect on pacemaker currents. Pre-treatment with ODQ (a guanylate cyclase inhibitor) did not change amplitudes and frequency of NaHS induced pacemaker current from ICC, but partially enhanced the inhibitory outward current. Similarly, pre-treatment of L-NAME, an inhibitor of nitric oxide also not affected the action of NaHS. But SQ 22536, an inhibitor of cAMP partly blocked the NaHS induced inhibitory outward currents. Calcium indicator (fluo-4) loaded ICC produced spontaneous calcium waves and NaHS significantly enhanced Spontaneous [Ca2+]i oscillations. These results suggest that activation of non-selective cation channels, and influx of sodium and efflux of calcium ion from internal store is responsible for inward pacemaker currents while inhibitory mechanism on pacemaker activity might be due to partly activation of ATP-sensitive K+ channels via partially dependent cAMP. NaHS regulated pacemaker activity was found independent to protein kinase C. Furthermore, RT-PCR results revealed that ICCs enriched population lack mRNA for neither CSE nor CBS, but a prominent expression of mRNA for CSE in whole dish cells could be the strong evidence to elucidate that ICC are not the target of hydrogen sulfide but the response of hydrogen sulfide may be transmitted either from smooth muscle cells or neuronal cells.
Alternative Title
생쥐 소장에서 분리된 Interstitial Cells of Cajal에 대한 Hydrogen sulfide의 작용 기전
Alternative Author(s)
Shankar Prasad Parajuli
조선대학교 대학원
일반대학원 생물신소재학과
Awarded Date
Table Of Contents
I. Introduction = 1
II. Materials and Methods = 6
2.1. Experimental animals = 6
2.2 Preparation of cells = 6
2.3 Patch clamp experiments = 7
2.4 Immunohistochemical experiments = 7
2.5 Measurement of intracellular Ca2+ concentration = 8
2.6 Molecular biological analysis = 8
2.7 Physiological Bathing Solution = 10
2.8 Internal Solution = 11
2.9 Drugs and chemicals = 11
2.10 Statistical analysis = 12
III. Results = 13
3.1 interstitial cell of Cajal expresses c-kit receptors = 13
3.2 Spontaneous activity in interstitial cells of Cajal = 13
3.3 Sodium hydrogen sulfide regulates the pacemaker current and membrane potentials generated from ICC = 16
3.4 NaHS regulates the pacemaker activity in ICC in a concentration dependent manner = 18
3.5 Role of Calphostin C on NaHS induced effects in pacemaker activity of ICC = 18
3.6 Effects of adenylyl cyclase inhibitor on NaHS induced effects inICC pacemaker activity = 18
3.7 Effects of Flufenamic acid, DIDS and depletion of sodium in bathing = 19
3.8 Effects of Thapsigargin and Calcium ion depletion in NaHS induced tonic inward currents produced from ICC = 25
3.9 Effects of guanylyl cyclase inhibitor and NAME, an inhibitor of NO on NaHS induced effects in ICC pacemaker activity = 27
3.10 Effect of ATP sensitive potassium channels on NaHS induced = 29
3.11 Effect of Ca2+ sensitive potassium channels on NaHS induced = 31
3.12 Effect of Ba+ on NaHS induced pacemaker currents = 31
3.13 Experiment on extracellular calcium mobilization in ICC = 33
3.14 Purified population of ICC expresses c-kit genes = 36
3.15 Expression of CSE and CBS enzymes in musculature of mouse small intestine = 36
IV. Discussion = 39
V. Conclusion = 50
VI. References = 52
조선대학교 대학원
산카르프라사드파라줄. (2008). Pharmacological Investigation of Hydrogen sulfide on Interstitial Cells of Cajal from Murine Small Intestine.
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General Graduate School > 4. Theses(Ph.D)
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