Studies on the characteristics of purinoceptor in ureteral smooth muscle

의학과/박사[한글] 세포내에서 여러가지 능동적 과정의 일차적인 에너지원으로 이용되는 ATP(adenosine triphosphate)를 비롯한 퓨린성 물질들은 비록 낮은 농도 이기는 하나 세포외액에도 존재하여 여러 가지 다양한 반응을 나타낸다. 이와 같은 효과는 여러 종류 세포의 세포막에 존재하는 퓨린 수총체에 의해 매개 되는것으로 알려지고 있다. 퓨린 수용체는 이 수용체에 대한 여러가지 agonist의 효과 및 길항제의 선택성등을 기준으로 크게 P^^1 및 P^^2 수용체로 나누어지고, 다시 P^^1 수용체는 A^^1 , A^^2 및 A^^3 로, P^^2 수용체는 P^^2X , P ^^2Y , P^^2T , 및 P^^2Z등으로 세분되고 있다. 현재 퓨린 수용체의 성질은 완전히 밝혀지지 않고 있으나, 여러가지 agonist 혹은 길항제를 사용하여 그 특성의 일부를 파악하고자 하는 연구가 시도되고 있다. 몇몇 평활근에 대한 이들 agonist의 효과는 adenosine tetraphosphate(ATPP)가 가장 컸으며, ATP, ADP(adenosine diphosphate), AMP(adenosine monophosphate) 및 adenosine의 순으로, adenine기에 붙어있는 phosphate의 수와 밀접한 관련이 있는 것으로 보고되고 있다. 그러므로 퓨린 수용체의 활성에 있어 phosphate group 이 중요한 역할을 하리라 생각하고 있다. 한편 여러 평활근에서 퓨린수용체의 존재가 확인되고 있으나 수뇨관 평활근에서는 아직 보고된 바가 없다. 따라서 본 연구에서는 i) ATPP를 비롯한 adenine nucleotide 및 adenosine이 돼지 수뇨관 평활근의 수축빈도 및 수축력 변화에 미치는 영향과, ii) 수뇨관 평활근의 안정막 전압 및 활동 전압 변화에 미치는 영향을 관찰하고, iii) 수용체 길항제를 사용하는등 여러 가지 방법으로 그 작용기전을 밝힘으로써 수뇨관 평활근에서의 퓨린 수용체 존재유무와 그 특성을 밝히고자 하였다. 실험 재료로는 신우 요관 이행부위로 부터 대략 2cm 이내의 돼지 수뇨관을 사용하였다. 수뇨관 평활근의 수축력 변화는 등척성 조건하에서 장력 측정기를 이용하여 측정하였으며 막전압은 30∼50㏁의 저항을 갖는 유리 미세 전극을 수뇨관 평활근 세포내로 삽입하여 측정하였다. 모든 실험은 37℃의 조건하에서 95% O^^2 -5% CO^^2 의 혼합기체로 포화시킨 KRB 용액에서 시행하였다. 중요한 실험결과는 아래와 같다. 1. ATPP이외의 adenine nucleotide나 adenosine은 수뇨관 평활근의 수축빈도 및 수축력에 큰 영향을 미치지 못하였다. 2. ATPP는 낮은 농도에서는 자발적 수축 빈도를 증가시켰다. 높은 농도에서는 일시적으로 긴장성 수축의 증가와 동시에 위상성 수축을 감소시켰다. 이와같은 효과는 일시적이었으며, 전기장 자극을 할때도 수축력의 탄화는 같은 결과를 보였다. 3. ATPP에 의한 긴장성 수축은 verapamil을 투여하거나 KRB 용액내의 칼슘이온을 제거했을때 거의 완전히 억제되었다. 그러나 ryanodine은 ATPP의 효과에 영향을 주지 않았다. 4. 고농도의 K**+ 이온은 초기에 위상성 수축을 증가시키나, 이어서 나타나는 긴장성 수축과 함께 위상성 수축은 감소하였다. 그러나 이경우 ATPP투여시와는 달리 발생된 긴장성 수축이 오래 유지 되었다. 5. 수뇨관 평활근 세포의 안정막 전압은 -51.7 ± 10.9㎷ 이었으며, ATPP투여시 안정막 전압의 탈분극 현상이 나타났다. 또한 ATPP는 활동전압의 빈도를 중가시켰다. 6. 여러가지 퓨린 수용체 길항제 중에서 P^^2X 수용체 길항제인 α, β-methylene ATP는 ATPP의 작용을 완전히 봉쇄하였다. 본 실험을 통해 돼지 수뇨관 평활근에서 P^^2X 퓨린 수용체의 존재를 확인하였으며, 이 수용체 활성에 있어 ATPP는 다른 어떠한 adenine nucleotide보다 강력한 agonist였다. ATPP에 의한 수축빈도의 증가나 긴장성 수축기전은 주로 세포막을 퉁한 Ca**++ 유입 증가 에 의한 것으로 생각되며, 위상성 수축 감소 및 소실은 막전압의 탈분극이나 세포질내의 Ca**++ 농도 증가로 인한 C**++ 통로의 비활성화에 의한 것으로 사료된다 Studios on the characteristics of purinoceptor in ureteral smooth muscle In Deok Kong Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Joong Woo Lee) Intracellular ATP(adenosine 5-'triphosphate) acts as a free energy source for most active processes in a cell. However, extracellular ATP and adenine nucleotides also affect various biological responses, namely those which are mediated by purinoceptors on the plasma membrane. Purinoceptors have been classified into P^^1 -and P^^2 -receptors, mainly on the basis of the relative potencies of adenine derivatives and selectivity of antagonists in a wide variety of tissues. The structure of the nucleotides, particularly the number of phosphates attached to the adenine nucleotide, plays an important role in purinoceptor activation. The effects of adenosine tetraphosphate(ATPP) on some smooth muscle appear to be more potent than those of any other adenine nucleotides. Purinoceptors have been identified at various sites of smooth muscle, but not yet in urethral smooth muscle. The present study was designed to investigate i) the effects of ATPP and other adenine nucleotides on mechanical contractility and membrane potential; ii ) the mochanisms of action of ATPP and other nucleotides: and iii) the characteristics of purinoceptors in ureteral smooth muscle. The effects of exogenous adenine nucleotides and adenosine were examined in isolated preparations of porcine urethral smooth muscle. The changes in tension were measured by a force transducer under isometric conditions. Membrane potentials were recorded by using glass microelectrodes filled with 3 M KCI (30∼50 ㏁). All experiments were performed in KRB solutions equilibriated with 95% O^^2 -5% CO^^2 gas mixtures at 37℃. The results were as follows: 1. The effects of ATPP on ureteral contractions were potent, while those of ATP, ADP, AMP and adenosine were minimal in the range of micromolar concentrations. 2. The effects of ATPP an spontaneous contraction were excitatory in ureteral smooth muscle. ATPP slightly increased tonic contractions, but markedly decreased phasic contractions. Contractions induced by electrical field stimulation showed similar results. These responses to ATPP were transient and disappeared in a few seconds and/or minutes. 3. In the presence of verapamil and EGTA, the ATPP-induced tonic contractions were inhibited significantly but, in the case of ryanodine, a blocker against Ca**++ releases from the intracellular store site, there was no influence on the contractions. 4. Under depolarization by high concentrations of K', phasic contractions increased initially then quickly faded, and then tonic tension developed in a dose dependent manner. These depolarization-induced tonic contractions relaxed slowly, lasting longer than 30 minutes. 5. The resting membrane Potential in urethral smooth muscle was -51.7± 10.9 mV. On application of ATPP, spontaneous action potentials were superimposed and the membrane potential depolarized markedly. 6. APCPP(α, β·methylene ATP), a P^^2x, purinoceptor blocker, completely inhibited the responses of ATPP on ureteral contractions. In conclusion, the existence of P^^2x purinoceptors in porcine ureteral smooth muscle was confirmed, and ATPP was seen as a potent purinoceptor agonist. ATPP-induced tonic contractions were mainly mediated by Ca**++ influx through the plasma membrane. The diminution of phasic contractility by ATPP may be caused by secondary effects such as Ca**++ channel inactivation. [영문] Intracellular ATP(adenosine 5-'triphosphate) acts as a free energy source for most active processes in a cell. However, extracellular ATP and adenine nucleotides also affect various biological responses, namely those which are mediated by purinoceptors on the plasma membrane. Purinoceptors have been classified into P^^1 -and P^^2 -receptors, mainly on the basis of the relative potencies of adenine derivatives and selectivity of antagonists in a wide variety of tissues. The structure of the nucleotides, particularly the number of phosphates attached to the adenine nucleotide, plays an important role in purinoceptor activation. The effects of adenosine tetraphosphate(ATPP) on some smooth muscle appear to be more potent than those of any other adenine nucleotides. Purinoceptors have been identified at various sites of smooth muscle, but not yet in urethral smooth muscle. The present study was designed to investigate i) the effects of ATPP and other adenine nucleotides on mechanical contractility and membrane potential; ii ) the mochanisms of action of ATPP and other nucleotides: and iii) the characteristics of purinoceptors in ureteral smooth muscle. The effects of exogenous adenine nucleotides and adenosine were examined in isolated preparations of porcine urethral smooth muscle. The changes in tension were measured by a force transducer under isometric conditions. Membrane potentials were recorded by using glass microelectrodes filled with 3 M KCI (30∼50 ㏁). All experiments were performed in KRB solutions equilibriated with 95% O^^2 -5% CO^^2 gas mixtures at 37℃. The results were as follows: 1. The effects of ATPP on ureteral contractions were potent, while those of ATP, ADP, AMP and adenosine were minimal in the range of micromolar concentrations. 2. The effects of ATPP an spontaneous contraction were excitatory in ureteral smooth muscle. ATPP slightly increased tonic contractions, but markedly decreased phasic contractions. Contractions induced by electrical field stimulation showed similar results. These responses to ATPP were transient and disappeared in a few seconds and/or minutes. 3. In the presence of verapamil and EGTA, the ATPP-induced tonic contractions were inhibited significantly but, in the case of ryanodine, a blocker against Ca**++ releases from the intracellular store site, there was no influence on the contractions. 4. Under depolarization by high concentrations of K', phasic contractions increased initially then quickly faded, and then tonic tension developed in a dose dependent manner. These depolarization-induced tonic contractions relaxed slowly, lasting longer than 30 minutes. 5. The resting membrane Potential in urethral smooth muscle was -51.7± 10.9 mV. On application of ATPP, spontaneous action potentials were superimposed and the membrane potential depolarized markedly. 6. APCPP(α, β·methylene ATP), a P^^2x, purinoceptor blocker, completely inhibited the responses of ATPP on ureteral contractions. In conclusion, the existence of P^^2x purinoceptors in porcine ureteral smooth muscle was confirmed, and ATPP was seen as a potent purinoceptor agonist. ATPP-induced tonic contractions were mainly mediated by Ca**++ influx through the plasma membrane. The diminution of phasic contractility by ATPP may be caused by secondary effects such as Ca**++ channel inactivation.restrictio