Cl - CFTR Cl - 3. (DPC) 52nitro222(32phenylpropylamino)2benzoate (NPPB) [3 ],, , Na + Ca 2 + [2

Transcription

1 , , 51 (3), Acta Physiologica Sinica Cl - CFTR Cl - 3 (, ), Cl - (CFTR) Cl -, 92AC ( ISO) CFTR Cl -, 52nitro222( 32phenylpropylamino) 2benzoate (NPPB) (DPC) ISO CFTR Cl - NPPB ISO, Cl -, : ; Cl - ; ; : Q445 (cystic fibrosis transmembrane conductance regulator, CFTR) Cl -, camp (CPKA)2 CFTR Cl -, Cl - Cl -,, [1 ],, Na + Ca 2 + [2 ] CFTR Cl -,, Cl - CFTR Cl -, 4,4 2dinitrostilbene22,2 2disulfon2 ic acid(dnds) 42acetamido24 2isothiocyanostilbene22,2 2disulfonic acid (SITS) 4,4 2diisothio2 cyanostilbene22,2 2disulfonic acid (DIDS) [3 ] ; 92 (92AC) [4 6 ] (DPC) 52nitro222(32phenylpropylamino)2benzoate (NPPB) [3 ],, [7 Harvey SITS DIDS ( ISO), ] Hwang DPC CFTR Cl - [8 ] Vandenberg, CFTR Cl - 92AC [9 ], CFTR Cl -, 92AC, 92AC CFTR Cl -, ISO CFTR Cl - ], CFTR Cl -, Cl - CFTR Cl -, (No )

2 ] ( g), (35 mg/ kg),, : (3 min) (3 min) 011 mg/ ml (yakult) 017 mg/ ml BSA (6 min) KB (5 min), KB, KB 112 CFTR Cl - (mmol/ L) : (1) NaCl 150, MgCl 2 015, CdCl 2 1, glucose 515, HEPES2NaOH 5 (ph 714) (2) aspartic acid 85, EG2 TA 10, TEA2Cl 20, MgCl 2 015, Na 2 2creatinephosphate 5, MgATP 10, NaGTP 012, glucose 515, HEPES2CsOH 10 (ph 7135), ( Cl mmol/ L) CFTR Cl -, NaCl (47 mmol/ L), CsCl 113 ISO 92AC DPC NPPB Sigma 114 ] 115 mm,, M,, ] (Axopatch 200A), Bessel (2 khz), TA240 ( Gould Instru2 ment Systems Inc1),, A/ D (PCM 50lES, Sony), (PC8901 DX, NEC) (ramp pulse) 1915 (NF Electronic Instruments) CFTR Cl - 0 mv, 5 s, mv, 715 Hz 115 ( x s x ), t AC NPPB DPC CFTR Cl - CFTR Cl -, Cl -, Cl - I2V [3 ] 1A, CFTR Cl - ISO, NPPB (50 mol/ L), ( ), mv, NPPB ISO CFTR Cl - ( ) % ( n = 8, P < 0105), ( ) NPPB, DPC (100 mol/ L) ( 1B), 2 min, 92AC, 92AC (500 mol/ L) ISO CFTR Cl na/ nf na/ nf ( n = 9, P < 0101) NPPB DPC CFTR Cl 1

3 3 : Cl - CFTR Cl NPPB DPC 92AC ISO CFTR Cl Fig11 Effects of extracellular application of 92AC, NPPB and DPC on ISO2activaed CFTR Cl - 2currents The Cl - current was activated by ISO in guinea pig ventricular Myocytes and recorded at asymmetri2 cal (153 mmol L - 1 [ Cl - ] O / 21 mmol L - 1 [ Cl - ] i ) chloride concentrations during repetitive appli2 cation of ramp pulses from 0 to 100 mv. A and C, representative chart records of whole2cell cur2 rents before and after exposure to ISO, NPPB ( A), DPC ( C) and 92AC1 B and D, I2V curves from the data in panel A and C ( b through f) respectively after subtraction of the basal current ( a) NPPB CFTR Cl NPPB, DPC 92AC,, 92AC CFTR Cl - ], NPPB, NPPB (50 mol/ L)

4 300 51, ISO CFTR Cl -, 10 min CFTR Cl - ( ) na/ nf ( ) na/ nf ( mv, n = 5, P < 0105),, NPPB, CFTR Cl NPPB DPC CFTR Cl Table 1 Effects of extracellular NPPB and DPC on CFTR Cl - n currents (at mv) CFTR Cl - 2current density (na/ nf) Control (1 mol/ L ISO) NPPB (50 mol/ L) First phase Second phase Control (1 mol/ L ISO) DPC (100 mol/ L) First phase # Second phase # # 3 P < 0105 vs control, 3 3 P < 0101 vs control ; # P < 0105 vs control, # # P < 0101 vs control. 2 NPPB CFTR Cl - Fig12 Effects of intracellular application of NPPB on ISO2activated CFTR Cl - cur2 rents ISO2activated Cl - currents of ventricular myocytes in guinea pig was recorded at symmetrical (106 mmol L - 1 [ Cl - ] O / 106 mmol L - 1 [ Cl - ] i ) chloride concentrations during repetitive application of ramp pulses (0 100 mv). A. The whole2cell currents were observed before and after intracellular perfusion with NPPB and bath ap2 plication of ISO and NPPB ( indicated by horizontal bars). B. I2V curves from the data in panel A ( b through d) after subtraction of the basal current ( a).

5 3 : Cl - CFTR Cl CFTR Cl - [4,12 ], Cl -, ( ),, Cl - NPPB DPC ISO CFTR Cl -, NPPB ISO, NPPB NPPB 92AC ],,,, Cl -, NPPB DPC,,, Hwang, 2 min, DPC Forskolin Cl - [8 ],, DPC, 2 min, CFTR Cl - Mg 2 +, Mg 2 + CFTR Cl - ( ), Mg 2 +,, 92AC CFTR Cl - ], NPPB DPC,, Cl -, [1 ] Gogelein H. Chloride channels in epithelia. Biochim Biophys Acta, 1988, 947 : [2 ] Conforti L, Sumii K, Sperelakis N. Diphenylamine222carboxylate blocks voltage dependent Na + and Ca 2 + chan2 nels in rat ventricular cardiomyocytes. Eur J Pharmacol, 1994, 259 : [3 ] Gadsby DC, Nagel G, Hwang TC. The CFTR chloride channel of mammalian heart. Annu Rev Physiol, 1995, 57 : [4 ] Harvey RD, Hume JR. Autonomic regulation of a chloride current in heart. Science, 1989, 244 : [5 ] Harvey RD, Clark CD, Hume JR. Chloride current in mammalian cardiac myocytes. Novel mechanism for auto2 nomic regulation of action potential duration and resting membrane potential. J Gen Physiol, 1990, 95 : [6 ] Levesque PC, Clark CD, Zakarov SI, et al. Anion and cation modulation of the guinea2pig ventricular action po2 tential during beta2adrenoceptor stimulation. Pfluegers Arch, 1993, 424 : [7 ] Harvey RD. Effects of stilbenedisulfonic acid derivatives on the camp2regulated chloride current in cardiac my2 ocytes. Pfluegers Arch, 1993, 422 : [8 ] Hwang TC, Horie M, Dousmanis A, et al. Regulation of PKA activated Cl - 2conductance in guinea pig ventri2 cular myocytes : whole cell studies. J Gen Physiol, 1992, 100 : 69a. [9 ] Vandenberg J I, Yoshida A, Kirk K, et al. Swelling2activated and isoprenaline2activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology. J Gen Physiol, 1994, 104 :

6 ] Zhou SS, Takai A, Tominaga M, et al. Phosphatase2mediated enhancement of cardiac camp2activated Cl - con2 ductance by a Cl - 2channel blocker, anthracene292carboxylate. Circ Res, 1997, 81 : [11 ] Zhou SS ( ), Zang YM ( ). A convenient and practicable device for intracellular perfusion. Chin J Appl Physiol ( ), 1997, 14 : (in Chinese with English abstract). [12 ] Bahinski A, Nairn AC, Greengard P, et al. Chloride conductance regulated by cyclic AMP2dependent protein kinase in cardiac myocytes. Nature, 1989, 340 : Acta Physiologica Sinica Jun. 1999, 51 (3), EFFECTS OF MONOCARBOXYL IC ACID DERIVATIVES ON CARDIAC VENTRICULAR CFTR Cl - CHANNELS IN GUINEA PIG ZHOU SHI2SHENG, ZANG YI2MIN ( Department of Physiology, Fourth Military Medical University, Xi an ) ABSTRACT Using the whole2cell recording technique, the effects of monocarboxylic acid deriva2 tives on cystic fibrosis transmembrane conductance regulator (CFTR) Cl - 2channel were examined in guinea pig ventricular myocytes. Anthracene292carboxylic acid ( 92AC) added to the bath solution further enhanced the outward component of isoproterenol2in2 duced currents in a reversible manner, whereas 52nitro222(32phenylpropylamino) benzoic acid (NPPB) or diphenylamine222carboxylic acid (DPC) induced a biphasic effect on the currents. Either NPPB or DPC first produced a transient increase in the outward compo2 nent of current before ensuing inhibition. Intracellular NPPB was found to potentiate iso2 proterenol2activated currents. It is concluded that these monocarboxylic acid derivatives have different binding sites in cardiac ventricular myocytes, which might partially account for the varied effects in blocking anion channels. Key words : isoproterenol ; chloride channel blocker ; ventricular myocyte ; guinea pig 3 Supported by the National Natural Science Foundation of China (No )