Gen Pharmac Vol 23, No 6, pp 1189-1192, 1992
0306-3623/92 $500+000 Copyright © 1992Pergamon Press Ltd
Pnnted m Great Britain All rights reserved
AMMONIUM ION INCREASES THE TONE OF RAT PORTAL VEIN ICHIRO WAKABAYASHI,t , KATSUHIKO HATAKE2 a n d KUNIHIRO SAKAMOTOI ~Department of Hygiene and 2Department of Legal Medicine, Hyogo College of Medlcane, 1-1 Mukogawa-cho, Nlshmomtya, Hyogo 663, Japan
(Recewed 2 March 1992) Abstract--1 The effect of ammonmm 1on on vascular tone was mvestagated using the portal veto isolated from rat 2 Ammonmm chloride at 10-90 mM reduced a contractile response 3 Spontaneous twttch contracuon of portal strips was augmented by ammomum chlonde at 10-60 mM 4 Ammomum chlortde-mduced contracUon was abohshed m calcmm-free soluUon or m the presence of 1/a M mfed~pme 5 Methylamme (60 mM) also reduced a contractde response and augmented the spontaneous twitch contraction in rat portal vein 6 After w~thdrawal of ammonmm chloride or methylamme from the organ bath solutaon, the spontaneous twttch contracUon was strongly mlub~ted 7 These results suggest that ammomum compounds mcrease vascular tone by caustng mflux of extracellular calcmm through the voltage-dependent calcium channel and mtracellular alkalunzatmn Is revolved m this process
INTRODUCTION Intracellular a l k a h n t z a t l o n has been recently suggested as a signal for cellular functions such as c o n t r a c t i o n a n d p r o h f e r a u o n o f vascular s m o o t h muscle (Berk et al, 1988, Wray, 1988) ActavaUon o f p r o t e m kanase C sUmulates N a + - H + exchange a n d subsequent mtraceUular a l k a h m z a t m n m vascular s m o o t h muscle cells (Berk et al, 1987, D a n t h u l u n et al, 1987) However, the process o f signal transductlon after mtracellular a l k a h m z a t ] o n remains unk n o w n W e a k bases, such as a m m o m u m chloride a n d m e t h y l a n u n e , are c o m m o n l y used as pharmacological tools to directly elevate mtracellular p H In some arterial preparations, these bases are k n o w n to produce c o n t r a c t d e responses ( D a n t h u l u n a n d Deth, 1989, K r a m p e t z a n d Rhoades, 1991), the m e c h a m s m o f which has n o t been precasely deterrmned F u r t h e r more, the effect o f mtracellular a l k a h m z a U o n o n venous responsiveness ~s n o t yet k n o w n Thus, we dec~ded to mvesUgate w h e t h e r a m m o n m m chloride a n d m e t h y l a m m e c a n alter the tone o f portal veto isolated f r o m rat
10 glucose, pH 7 4) mamtmned at 37°C and aerated gath 95% 02 and 5% CO2
Contractwn study Each venous strip was attached to a force transducer connected to a polygraph under a load of 5 mN After 2 hr of stabd~zatmn, the changes m ~sometnc force were recorded and expressed m terms of mN of tension In the portal vein, mechamcal acUvRy ehc~ted by ammotuum compounds was analysed as the effects on two phases (I) a component of spontaneous tvatch contracUon and (2) a component of tomc contracUon The effects on the twitch contraOaon were expressed as the percentage of twitch contracule force at 15rmn after addmon of the agomst to the basal tmtch contractde force The concentratton of each drug was expressed as the final concentraUon in the organ bath The concentraUon-response relauonslup for the contractde effect of ammomum chlonde was obtained by single-dose addmons of ammomum chloride Expenments m calcmmfree soluUon were performed as follows the vessels were nnsed qutckly 3 tunes vath calcmm-free Krebs-lhnger solution contmmng 1 mM ethyleneglycol-bts-(.8-anunoethylether)-N,N'-tetraaceUc acid (EGTA) and allowed to eqmhbrate m tl~s solutmn for an addmonal 15mm Ammotuum chlonde was then added to the vessels
Drugs MATERIALS AND METHODS
Ttssue preparatwn Male WJstar rats (400-500 g) were lolled by a blow to the head The portal veto was &ssected free from surroundmg tzssues and extracted as a 6-mm long secuon Tlus stnp was suspended vertically m organ bath containing 10 ml of Krebs--Rmger soluUon (mM I18NaC1, 4 7 KC1, 25 NaHCO 3, 1 2 KH2PO4, 1 2 MgSO4, 2 5 CaC12 and *To whom all correspondence should be addressed
Ntfedlpme (Sigma Chermcal C o , St Lotus, Mo) was dtssolved m ethanol The final concentration of ethanol m bath soluUon contmnmg I # M mfechptue was 0 1%, wluch affected neither the spontaneous twRch contracUon nor contraction m response to 60 mM ammomum dalonde Other chemicals were obtmned from Wako Pure Cbenucal Industry (Osaka, Japan) and chssolved m chsttlled water
Data analyszs Data are expressed as means + SEM StaUsUcal analyses were performed w~th Student's t-test and a P value o f less than 0 05 was taken as stgmficant
1189
IcmRo WAKABAYASHIet al
1190 RESULTS
Effects o f ammonmm chiorMe and methylamme on spontaneous twttch contractton o f rat portal vem
Rat portal strips showed spontaneous twRch contraction under the basal condmon Figure IA shows the effects of various concentrations of ammomum chloride on the twitch contraction m portal veins Ammonium chloride (10-60raM) augmented the twitch contractaon Methylarmne (60 raM) also augmented it [384 0 + 60 6% (n = 6), P < 0 01, when the effect was compared with the basal spontaneous actmty (Fig 2B)] A bagh concentration of ammonmm chloride (90 raM), however, strongly inhibited the twitch contraction (Figs 1A, 2E) Basal spontaneous twitch contraction was abohshed m calcmm-free solution or m the presence of nlfedipme (1/tM) Ammonium chloride (60 raM) did not show any effect on the basal tone of the strips m calciumfree solution or m the presence of mfedipme (1/zM) (Figs 2C, D) After wRhdrawal of ammonium chloride (60 raM) or methylarmne (60 raM), the twitch contraction was strongly mlubRed temporartly (about 10-20 nun) (Figs 2A, B) Ammomum chlortde and methylamme-mduced contractde responses m rat portal veto Figure I B shows the concentration-response relationship for ammonium chloride-reduced contraction m the portal veins Ammomum chloride at 10-90raM produced a tonic contractile response
~
A
§00
400
oy lOO o
1o
30
8o
90
Ammonium Chloride (mM)
O.
10
$0 eO Ammonium Chloride (mM)
90
Fig 1 A~Effect ofammomum chloride on the spontaneous twitch contracUon m rat portal veins Data are expressed as the percentage of the twitch contractile force at 15 nun after addition of ammomum chlonde to the basal spontaneous twRch contractile force at the resting condmon Asterisks denote statistically slgmficant chfferences (P
0306-3623/92 $500+000 Copyright © 1992Pergamon Press Ltd
Pnnted m Great Britain All rights reserved
AMMONIUM ION INCREASES THE TONE OF RAT PORTAL VEIN ICHIRO WAKABAYASHI,t , KATSUHIKO HATAKE2 a n d KUNIHIRO SAKAMOTOI ~Department of Hygiene and 2Department of Legal Medicine, Hyogo College of Medlcane, 1-1 Mukogawa-cho, Nlshmomtya, Hyogo 663, Japan
(Recewed 2 March 1992) Abstract--1 The effect of ammonmm 1on on vascular tone was mvestagated using the portal veto isolated from rat 2 Ammonmm chloride at 10-90 mM reduced a contractile response 3 Spontaneous twttch contracuon of portal strips was augmented by ammomum chlonde at 10-60 mM 4 Ammomum chlortde-mduced contracUon was abohshed m calcmm-free soluUon or m the presence of 1/a M mfed~pme 5 Methylamme (60 mM) also reduced a contractde response and augmented the spontaneous twitch contraction in rat portal vein 6 After w~thdrawal of ammonmm chloride or methylamme from the organ bath solutaon, the spontaneous twttch contracUon was strongly mlub~ted 7 These results suggest that ammomum compounds mcrease vascular tone by caustng mflux of extracellular calcmm through the voltage-dependent calcium channel and mtracellular alkalunzatmn Is revolved m this process
INTRODUCTION Intracellular a l k a h n t z a t l o n has been recently suggested as a signal for cellular functions such as c o n t r a c t i o n a n d p r o h f e r a u o n o f vascular s m o o t h muscle (Berk et al, 1988, Wray, 1988) ActavaUon o f p r o t e m kanase C sUmulates N a + - H + exchange a n d subsequent mtraceUular a l k a h m z a t m n m vascular s m o o t h muscle cells (Berk et al, 1987, D a n t h u l u n et al, 1987) However, the process o f signal transductlon after mtracellular a l k a h m z a t ] o n remains unk n o w n W e a k bases, such as a m m o m u m chloride a n d m e t h y l a n u n e , are c o m m o n l y used as pharmacological tools to directly elevate mtracellular p H In some arterial preparations, these bases are k n o w n to produce c o n t r a c t d e responses ( D a n t h u l u n a n d Deth, 1989, K r a m p e t z a n d Rhoades, 1991), the m e c h a m s m o f which has n o t been precasely deterrmned F u r t h e r more, the effect o f mtracellular a l k a h m z a U o n o n venous responsiveness ~s n o t yet k n o w n Thus, we dec~ded to mvesUgate w h e t h e r a m m o n m m chloride a n d m e t h y l a m m e c a n alter the tone o f portal veto isolated f r o m rat
10 glucose, pH 7 4) mamtmned at 37°C and aerated gath 95% 02 and 5% CO2
Contractwn study Each venous strip was attached to a force transducer connected to a polygraph under a load of 5 mN After 2 hr of stabd~zatmn, the changes m ~sometnc force were recorded and expressed m terms of mN of tension In the portal vein, mechamcal acUvRy ehc~ted by ammotuum compounds was analysed as the effects on two phases (I) a component of spontaneous tvatch contracUon and (2) a component of tomc contracUon The effects on the twitch contraOaon were expressed as the percentage of twitch contracule force at 15rmn after addmon of the agomst to the basal tmtch contractde force The concentratton of each drug was expressed as the final concentraUon in the organ bath The concentraUon-response relauonslup for the contractde effect of ammomum chlonde was obtained by single-dose addmons of ammomum chloride Expenments m calcmmfree soluUon were performed as follows the vessels were nnsed qutckly 3 tunes vath calcmm-free Krebs-lhnger solution contmmng 1 mM ethyleneglycol-bts-(.8-anunoethylether)-N,N'-tetraaceUc acid (EGTA) and allowed to eqmhbrate m tl~s solutmn for an addmonal 15mm Ammotuum chlonde was then added to the vessels
Drugs MATERIALS AND METHODS
Ttssue preparatwn Male WJstar rats (400-500 g) were lolled by a blow to the head The portal veto was &ssected free from surroundmg tzssues and extracted as a 6-mm long secuon Tlus stnp was suspended vertically m organ bath containing 10 ml of Krebs--Rmger soluUon (mM I18NaC1, 4 7 KC1, 25 NaHCO 3, 1 2 KH2PO4, 1 2 MgSO4, 2 5 CaC12 and *To whom all correspondence should be addressed
Ntfedlpme (Sigma Chermcal C o , St Lotus, Mo) was dtssolved m ethanol The final concentration of ethanol m bath soluUon contmnmg I # M mfechptue was 0 1%, wluch affected neither the spontaneous twRch contracUon nor contraction m response to 60 mM ammomum dalonde Other chemicals were obtmned from Wako Pure Cbenucal Industry (Osaka, Japan) and chssolved m chsttlled water
Data analyszs Data are expressed as means + SEM StaUsUcal analyses were performed w~th Student's t-test and a P value o f less than 0 05 was taken as stgmficant
1189
IcmRo WAKABAYASHIet al
1190 RESULTS
Effects o f ammonmm chiorMe and methylamme on spontaneous twttch contractton o f rat portal vem
Rat portal strips showed spontaneous twRch contraction under the basal condmon Figure IA shows the effects of various concentrations of ammomum chloride on the twitch contraction m portal veins Ammonium chloride (10-60raM) augmented the twitch contractaon Methylarmne (60 raM) also augmented it [384 0 + 60 6% (n = 6), P < 0 01, when the effect was compared with the basal spontaneous actmty (Fig 2B)] A bagh concentration of ammonmm chloride (90 raM), however, strongly inhibited the twitch contraction (Figs 1A, 2E) Basal spontaneous twitch contraction was abohshed m calcmm-free solution or m the presence of nlfedipme (1/tM) Ammonium chloride (60 raM) did not show any effect on the basal tone of the strips m calciumfree solution or m the presence of mfedipme (1/zM) (Figs 2C, D) After wRhdrawal of ammonium chloride (60 raM) or methylarmne (60 raM), the twitch contraction was strongly mlubRed temporartly (about 10-20 nun) (Figs 2A, B) Ammomum chlortde and methylamme-mduced contractde responses m rat portal veto Figure I B shows the concentration-response relationship for ammonium chloride-reduced contraction m the portal veins Ammomum chloride at 10-90raM produced a tonic contractile response
~
A
§00
400
oy lOO o
1o
30
8o
90
Ammonium Chloride (mM)
O.
10
$0 eO Ammonium Chloride (mM)
90
Fig 1 A~Effect ofammomum chloride on the spontaneous twitch contracUon m rat portal veins Data are expressed as the percentage of the twitch contractile force at 15 nun after addition of ammomum chlonde to the basal spontaneous twRch contractile force at the resting condmon Asterisks denote statistically slgmficant chfferences (P
