587
Pharmacological Research, Vol. 22, No. 5, 1990
STIMULATORY EFFECT OF S E R U M O N 86Rb WASHOUT F R O M VASCULAR S M O O T H MUSCLE CELLS IN CULTURE KOJI N A K A M U R A , SATORU KUR1YAMA, H A R U O TOMONARI, YOSHIHIKO KAGUCHI, TAKAO HASHIMOTO and OSAMU SAKAI
Second Department of Internal Medicine, Jikei UniversitySchool of Medicine, Tokyo, Japan Received in final form 8 February 1990
SUMMARY In order to elucidate the effect of serum on passive K permeability of vascular smooth muscle cells (VSMC) membrane, outward passive K permeability yielded as washout rate constant (Ke) of 86Rb washout, was measured in the presence and in the absence of cation transport modulators using VSMC in culture. The overall K e of S6Rb washout subjected to 1% serum was significantly larger than that in controls. This stimulated K~ was substantially blunted in the presence of 10-4M amiloride and was partially inhibited with 5 x 10-4 M bumetanide. Angiotensin II of 10-5M exerted to a lesser extent, a similar significant stimulatory effect on Ke of S6Rb washout, which effect was inhibited with application of amiloride. Additionally, Ca-antagonist, 1 0-5 M nifedipine reduced serum-stimulated Ke to the basal level. It is concluded that both serum and angiotensin lI increase K permeability in cultured VSMC. A part of this effect of serum may be attributable to angiotensin II in the serum. Furthermore, it is suggested that the stimulatory effect of serum on membrane permeability may be exerted, at least in part, via activation of both NaIl antiport and Na-K co-transport, possibly through mechanisms in conjunction with intracellular Ca. KEY WORDS"serum, angiotensin I1, amiloride, bumetaifide, nifedipine, K permeability.
INTRODUCTION Recent investigations on cellular physiology have unravelled to a great extent a specific relationship between the effect of serum and the alteration in membrane permeability to cations [1-4]. It is widely known that cells initiate proliferation in the presence of serum in in vitro VSMC culture systems, although little is known of the precise mechanisms which account for this phenomenon in association with Na Address for correspondence and proofs: Satoru Kuriyama M.D., Second Department of Internal Medicine, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105, Japan. 1043-6618/90/050587-09/S03.00/0
© 1990 The Italian Pharmacological Society
588
Pharmacological Research, Vol. 22, No. 5, 1990
and K transport. The possible involvement of Na-H antiport along with intracellular Ca in the process of cell division has been postulated [5]. This is a ubiquitous Na transport system driven by Na and is activated by many agonists and blocked by amiloride. Angiotensin lI is known to exert its effect on membrane permeability through enhancement of Na-H antiport in relation to intracellular Ca mobilization [1, 3, 4]. It is also postulated that angiotensin II may stimulate cellular proliferation of vascular smooth muscle cells, suggesting the involvement of this hormone in hypertrophy or hyperproliferation of the cells seen in the vascular walls of the patients with hypertension [6-8]. Regardless of abundant studies regarding serum and angiotensin II on Na transport, much less is known as to the effect of this hormone on K permeability across the cell membrane of VSMCs. Outward K current comprises mainly passive diffiasional movement of this ion which depends on a concentration gradient across the cell membrane. It includes passive outward K movement, outward Na-K co-transport, and the transport systems associated secondary with the activation of other transport systems including Na-H antiport. Utilizing 86Rb washout, a refined analytical method developed recently in our laboratory renders it possible to investigate a subtle change in membrane permeability of K [9, 10]. In this communication, we have made a specific attempt to study the effect of serum and angiotensin II on passive K permeability by measuring the washout rate constant of 86Rb washout. Several pharmacological tools bearing inhibitory action on cation transport systems enabled us to gain further insight into these mechanisms.
METHODS Cultured vascular smooth muscle cells (VSMC) were derived from thoracic arteries of rats (American Cell Type Collection; A7r5, passage approximately 15-20). For S6Rb washout experiments, a total of 1 × 105 cells/ml were inoculated into 12 well Nunc cluster, 3 or 4 days prior to the experiment. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (5% C O 2 and 95% 02) with 10% FCS. After reaching confluence, on the day of the experiment, cells were washed with 1.0 ml aliquot of D M E M three times, and cells were preloaded with 15/~Ci/ ml 86Rb for approximately 120 min in an incubator. Thereafter, loaded cells were rapidly washed with ice-cold DMEM three times to remove the tracer. The washout experiment was then initiated by periodic addition and suction of the medium for as long as 60 min. Indicated probes (angiotensin II 10 -5 M, amiloride 10 .4 M, bumetanide 5 x 10 4 M, and nifedipine 10 -5 M) were included only in the washout medium. During the experimental period, 10% FCS was not included in the wells and temperature was kept at 37°C throughout the process. At the end of the experiment, cells were extracted by addition of 5% trypsin. Suctioned medium was counted in a scintillation counter and each count was added in a reverse manner., As we described previously [10], the data were fitted to a monoexponential function. The fraction of the activity in the cells at a given time of the experiment (A~) can be expressed as:
589
Pharmacological Research, Vol. 22, No. 5, 1990
where Y A~ is the total activity in the cells at initiation of the washout experiment and Z A t is the activity that left the cells at a given time t. An approximation of the instantaneous rate constant (Ke) for the 86Rb washout in a time interval (h - t2) was calculated as: Ke - Ac,2 - Ac,1 t2- h
A slope (initial washout rate constant: Ke) and an intersection (size of the pool) of the curve were therefore obtained. Each experiment was performed individually in parallel to a control. For the experiment using serum, whole blood from the ventral aorta bifurcation of male Sprague-Dawley rats, was drawn by a syringe under light pentobarbitone anaesthesia. The blood samples were immediately transferred to tubes and were centrifuged to remove red blood and white blood cells at 4°C and stored in a deep freezer until use. The serum of several rats was pooled for the study. Preliminary experiments showed that apparent stimulatory effect of serum on 86Rb washout occurred at a concentration above 0.l%, thus a 1% concentration of serum was used for the experiments. Data consist of the average of duplicate observations from several washout experiments in cultured VSMC. For intracellular Na and K concentration determination, cells were washed three times with ice-cold 0.11 M MgCI2. The wells were then left at room temperature until dry. Deionized distilled water was added and cells were frozen in a freezer. On the day of the experiment, cells were thawed in a shaking incubator at 37°C and Na and K concentration were measured by atomic absorption spectrometry. Amiloride and angiotensin lI were purchased from Sigma Co Ltd. Bumetanide and nifedipine were gifts from Sankyo Pharmaceutical Co. Ltd and Bayer pharmaceutical Co. Ltd respectively. All probes were originally dissolved in DMEM without using organic material as a vehicle. Statistical analyses employed were paired and unpaired t-test, as appropriate. Data are expressed as mean + SD, unless otherwise indicated.
RESULTS The determination of intracellular Na and K concentration after an exposure to l% serum for 180 min and depicted in Table I. Intracellular Na and K concentration were unchanged compared with the control, for this manoeuvre throughout the experimental period. Therefore, the washout experiments performed in the present study clearly reflect the results under steady state conditions with respect to Na and K concentration, in which Na and K efflux equals their uptake. Figures 1, 2, 3 and 4 are a representative experiment of S6Rb washout curve, all of which were described as monoexponential functions. The slope of the line,
590
Pharmacological Research, Vol. 22, No. 5, 1990
Table I I n t r a c e l l u l a r N a and K c o n c e n t r a t i o n in t h e p r e s e n c e o f 1% s e r u m d e t e r m i n e d by a t o m i c a b s o r p t i o n s p e c t r o m e t r y Na (nmol/lO 5 cells)
K (nmol/lO 5 cells)
1.8 + 0.37 2.0 _+0.42
8.9 _+0.57 9.4 + 0.62
Control (n = 6) 1% Serum (n = 6)
,01i,q 8
0.1
0
I
I
1
I
15
30
45
60
Time (rain) Fig. 1. A representative experiment of 8~Rb washout. Symbols denote control (open circle), 1% serum (closed circle), amiloride 10-4M (open triangle), and serum plus amiloride (open square).
f.oL
I,.,1 8
O.I
0
I
I
I
I
15
30
45
80
Time (rain) Fig. 2. A representative experiment of 8~Rb washout. Symbols denote control (open circle), angiotensin II 10- s M (closed circle), amiloride 10-4 M (open triangle), and angiotensin II plus amiloride (open square).
591
Pharmacological Research, VoL 22, No. 5, 1990
J.oL I,q
0.1 0
[ 15
I 50
I 45
,I 60
Time (rain)
Fig. 3. A representative experiment of S6Rb washout. Symbols denote control (open circle), 1% serum (closed circle), nifedipine 10 -5 M (open triangle), and serum plus nifedipine (open square).
t.0
O.
0.1
_
0
I 15
I 50
I 45
I
60
Time (rain)
Fig. 4. The effect of bumetanide on serum-stimulated S6Rb washout. Symbols represent control (open circle), 1% serum (closed circle), and serum plus bumetanide 5 x 10 -4 M (open square). washout rate constant (Ke) reflects mainly passive K permeability and the intersection of the line indicates the size of the K pool inside the cells. Note that the y-axis is expressed as a logarithmic function and the x-axis is expressed as a function of time after starting the washout experiment. Figure 1 depicts the results of 86Rb washout in the presence of 1% serum. As shown here, serum (S) exerted a significant stimulating effect on K e of S6Rb washout compared to that in the control (C) by approximately 60% (143.2 _+ 17.8 for C versus 227.3_+21.5 ( x 10-4/min)
592
Pharmacological Research, Vol. 22, No. 5, 1990
for S, P < 0.001, n = 18). With the application of 10 -4 M amiloride (Am), the serumstimulated steep slope of the washout returned to its basal level (227.3 + 21.5 for S versus 128.3 + 20.7 ( x 10- 4/rain) for S + Am, P < 0.001, n = 18). Amiloride alone exerted a significant reductive effect on the Ke (143.2+-17.8 for C versus 94.8 + 14.6 (10-4/min) for Am, P < 0.001, n = 18). Figure 2 depicts the effect of angiotensin II (AII) on K e of 86Rb washout. Angiotensin II stimulates K e by approximately 10% (129.5 _+ 15.4 for C versus 138.5 + 25.9 ( x 10-4/min) for AII, P< 0.05, n = 18), although this effect was substantially inhibited in the presence of amiloride (138.5 _+25.9 for AII versus 109.6 + 17.6 ( x 10-4/min) for A I I + A m , P < 0.001, n = 18). Figure 3 demonstrates the effect of the Ca antagonist, nifedipine (Nif) on the serum-stimulated Ke of 86Rb washout. As was previously reported [10], nifedipine alone decreases K e of 86Rb washout (138.3+16.0 for C versus 121.4 + 33.3 ( x 10-4/min) for Nif, P < 0.001, n = 4). Subsequently, an additional set of experiments shows that nifedipine exerts its ameliorating effects on the serum stimulated 86Rb washout (165.1 + 21.6 for S versus 142.2 _+26.3 ( x 10-4/min) for S+Nif, P
Pharmacological Research, Vol. 22, No. 5, 1990
STIMULATORY EFFECT OF S E R U M O N 86Rb WASHOUT F R O M VASCULAR S M O O T H MUSCLE CELLS IN CULTURE KOJI N A K A M U R A , SATORU KUR1YAMA, H A R U O TOMONARI, YOSHIHIKO KAGUCHI, TAKAO HASHIMOTO and OSAMU SAKAI
Second Department of Internal Medicine, Jikei UniversitySchool of Medicine, Tokyo, Japan Received in final form 8 February 1990
SUMMARY In order to elucidate the effect of serum on passive K permeability of vascular smooth muscle cells (VSMC) membrane, outward passive K permeability yielded as washout rate constant (Ke) of 86Rb washout, was measured in the presence and in the absence of cation transport modulators using VSMC in culture. The overall K e of S6Rb washout subjected to 1% serum was significantly larger than that in controls. This stimulated K~ was substantially blunted in the presence of 10-4M amiloride and was partially inhibited with 5 x 10-4 M bumetanide. Angiotensin II of 10-5M exerted to a lesser extent, a similar significant stimulatory effect on Ke of S6Rb washout, which effect was inhibited with application of amiloride. Additionally, Ca-antagonist, 1 0-5 M nifedipine reduced serum-stimulated Ke to the basal level. It is concluded that both serum and angiotensin lI increase K permeability in cultured VSMC. A part of this effect of serum may be attributable to angiotensin II in the serum. Furthermore, it is suggested that the stimulatory effect of serum on membrane permeability may be exerted, at least in part, via activation of both NaIl antiport and Na-K co-transport, possibly through mechanisms in conjunction with intracellular Ca. KEY WORDS"serum, angiotensin I1, amiloride, bumetaifide, nifedipine, K permeability.
INTRODUCTION Recent investigations on cellular physiology have unravelled to a great extent a specific relationship between the effect of serum and the alteration in membrane permeability to cations [1-4]. It is widely known that cells initiate proliferation in the presence of serum in in vitro VSMC culture systems, although little is known of the precise mechanisms which account for this phenomenon in association with Na Address for correspondence and proofs: Satoru Kuriyama M.D., Second Department of Internal Medicine, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105, Japan. 1043-6618/90/050587-09/S03.00/0
© 1990 The Italian Pharmacological Society
588
Pharmacological Research, Vol. 22, No. 5, 1990
and K transport. The possible involvement of Na-H antiport along with intracellular Ca in the process of cell division has been postulated [5]. This is a ubiquitous Na transport system driven by Na and is activated by many agonists and blocked by amiloride. Angiotensin lI is known to exert its effect on membrane permeability through enhancement of Na-H antiport in relation to intracellular Ca mobilization [1, 3, 4]. It is also postulated that angiotensin II may stimulate cellular proliferation of vascular smooth muscle cells, suggesting the involvement of this hormone in hypertrophy or hyperproliferation of the cells seen in the vascular walls of the patients with hypertension [6-8]. Regardless of abundant studies regarding serum and angiotensin II on Na transport, much less is known as to the effect of this hormone on K permeability across the cell membrane of VSMCs. Outward K current comprises mainly passive diffiasional movement of this ion which depends on a concentration gradient across the cell membrane. It includes passive outward K movement, outward Na-K co-transport, and the transport systems associated secondary with the activation of other transport systems including Na-H antiport. Utilizing 86Rb washout, a refined analytical method developed recently in our laboratory renders it possible to investigate a subtle change in membrane permeability of K [9, 10]. In this communication, we have made a specific attempt to study the effect of serum and angiotensin II on passive K permeability by measuring the washout rate constant of 86Rb washout. Several pharmacological tools bearing inhibitory action on cation transport systems enabled us to gain further insight into these mechanisms.
METHODS Cultured vascular smooth muscle cells (VSMC) were derived from thoracic arteries of rats (American Cell Type Collection; A7r5, passage approximately 15-20). For S6Rb washout experiments, a total of 1 × 105 cells/ml were inoculated into 12 well Nunc cluster, 3 or 4 days prior to the experiment. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (5% C O 2 and 95% 02) with 10% FCS. After reaching confluence, on the day of the experiment, cells were washed with 1.0 ml aliquot of D M E M three times, and cells were preloaded with 15/~Ci/ ml 86Rb for approximately 120 min in an incubator. Thereafter, loaded cells were rapidly washed with ice-cold DMEM three times to remove the tracer. The washout experiment was then initiated by periodic addition and suction of the medium for as long as 60 min. Indicated probes (angiotensin II 10 -5 M, amiloride 10 .4 M, bumetanide 5 x 10 4 M, and nifedipine 10 -5 M) were included only in the washout medium. During the experimental period, 10% FCS was not included in the wells and temperature was kept at 37°C throughout the process. At the end of the experiment, cells were extracted by addition of 5% trypsin. Suctioned medium was counted in a scintillation counter and each count was added in a reverse manner., As we described previously [10], the data were fitted to a monoexponential function. The fraction of the activity in the cells at a given time of the experiment (A~) can be expressed as:
589
Pharmacological Research, Vol. 22, No. 5, 1990
where Y A~ is the total activity in the cells at initiation of the washout experiment and Z A t is the activity that left the cells at a given time t. An approximation of the instantaneous rate constant (Ke) for the 86Rb washout in a time interval (h - t2) was calculated as: Ke - Ac,2 - Ac,1 t2- h
A slope (initial washout rate constant: Ke) and an intersection (size of the pool) of the curve were therefore obtained. Each experiment was performed individually in parallel to a control. For the experiment using serum, whole blood from the ventral aorta bifurcation of male Sprague-Dawley rats, was drawn by a syringe under light pentobarbitone anaesthesia. The blood samples were immediately transferred to tubes and were centrifuged to remove red blood and white blood cells at 4°C and stored in a deep freezer until use. The serum of several rats was pooled for the study. Preliminary experiments showed that apparent stimulatory effect of serum on 86Rb washout occurred at a concentration above 0.l%, thus a 1% concentration of serum was used for the experiments. Data consist of the average of duplicate observations from several washout experiments in cultured VSMC. For intracellular Na and K concentration determination, cells were washed three times with ice-cold 0.11 M MgCI2. The wells were then left at room temperature until dry. Deionized distilled water was added and cells were frozen in a freezer. On the day of the experiment, cells were thawed in a shaking incubator at 37°C and Na and K concentration were measured by atomic absorption spectrometry. Amiloride and angiotensin lI were purchased from Sigma Co Ltd. Bumetanide and nifedipine were gifts from Sankyo Pharmaceutical Co. Ltd and Bayer pharmaceutical Co. Ltd respectively. All probes were originally dissolved in DMEM without using organic material as a vehicle. Statistical analyses employed were paired and unpaired t-test, as appropriate. Data are expressed as mean + SD, unless otherwise indicated.
RESULTS The determination of intracellular Na and K concentration after an exposure to l% serum for 180 min and depicted in Table I. Intracellular Na and K concentration were unchanged compared with the control, for this manoeuvre throughout the experimental period. Therefore, the washout experiments performed in the present study clearly reflect the results under steady state conditions with respect to Na and K concentration, in which Na and K efflux equals their uptake. Figures 1, 2, 3 and 4 are a representative experiment of S6Rb washout curve, all of which were described as monoexponential functions. The slope of the line,
590
Pharmacological Research, Vol. 22, No. 5, 1990
Table I I n t r a c e l l u l a r N a and K c o n c e n t r a t i o n in t h e p r e s e n c e o f 1% s e r u m d e t e r m i n e d by a t o m i c a b s o r p t i o n s p e c t r o m e t r y Na (nmol/lO 5 cells)
K (nmol/lO 5 cells)
1.8 + 0.37 2.0 _+0.42
8.9 _+0.57 9.4 + 0.62
Control (n = 6) 1% Serum (n = 6)
,01i,q 8
0.1
0
I
I
1
I
15
30
45
60
Time (rain) Fig. 1. A representative experiment of 8~Rb washout. Symbols denote control (open circle), 1% serum (closed circle), amiloride 10-4M (open triangle), and serum plus amiloride (open square).
f.oL
I,.,1 8
O.I
0
I
I
I
I
15
30
45
80
Time (rain) Fig. 2. A representative experiment of 8~Rb washout. Symbols denote control (open circle), angiotensin II 10- s M (closed circle), amiloride 10-4 M (open triangle), and angiotensin II plus amiloride (open square).
591
Pharmacological Research, VoL 22, No. 5, 1990
J.oL I,q
0.1 0
[ 15
I 50
I 45
,I 60
Time (rain)
Fig. 3. A representative experiment of S6Rb washout. Symbols denote control (open circle), 1% serum (closed circle), nifedipine 10 -5 M (open triangle), and serum plus nifedipine (open square).
t.0
O.
0.1
_
0
I 15
I 50
I 45
I
60
Time (rain)
Fig. 4. The effect of bumetanide on serum-stimulated S6Rb washout. Symbols represent control (open circle), 1% serum (closed circle), and serum plus bumetanide 5 x 10 -4 M (open square). washout rate constant (Ke) reflects mainly passive K permeability and the intersection of the line indicates the size of the K pool inside the cells. Note that the y-axis is expressed as a logarithmic function and the x-axis is expressed as a function of time after starting the washout experiment. Figure 1 depicts the results of 86Rb washout in the presence of 1% serum. As shown here, serum (S) exerted a significant stimulating effect on K e of S6Rb washout compared to that in the control (C) by approximately 60% (143.2 _+ 17.8 for C versus 227.3_+21.5 ( x 10-4/min)
592
Pharmacological Research, Vol. 22, No. 5, 1990
for S, P < 0.001, n = 18). With the application of 10 -4 M amiloride (Am), the serumstimulated steep slope of the washout returned to its basal level (227.3 + 21.5 for S versus 128.3 + 20.7 ( x 10- 4/rain) for S + Am, P < 0.001, n = 18). Amiloride alone exerted a significant reductive effect on the Ke (143.2+-17.8 for C versus 94.8 + 14.6 (10-4/min) for Am, P < 0.001, n = 18). Figure 2 depicts the effect of angiotensin II (AII) on K e of 86Rb washout. Angiotensin II stimulates K e by approximately 10% (129.5 _+ 15.4 for C versus 138.5 + 25.9 ( x 10-4/min) for AII, P< 0.05, n = 18), although this effect was substantially inhibited in the presence of amiloride (138.5 _+25.9 for AII versus 109.6 + 17.6 ( x 10-4/min) for A I I + A m , P < 0.001, n = 18). Figure 3 demonstrates the effect of the Ca antagonist, nifedipine (Nif) on the serum-stimulated Ke of 86Rb washout. As was previously reported [10], nifedipine alone decreases K e of 86Rb washout (138.3+16.0 for C versus 121.4 + 33.3 ( x 10-4/min) for Nif, P < 0.001, n = 4). Subsequently, an additional set of experiments shows that nifedipine exerts its ameliorating effects on the serum stimulated 86Rb washout (165.1 + 21.6 for S versus 142.2 _+26.3 ( x 10-4/min) for S+Nif, P
