European Journal of Pharmacology, 187 (1990) 183-191
183
Elsevier EJP 51482
Angiotensin stimulates Ca2+-dependent action potentials in cultured smooth muscle cells D e a r i n g W . J o h n s i a n d N i c h o l a s Sperelakis 2 Departments of lnternal Medicine and Physiology, University of Virginia, Charlottesville, VA, U.S.A. Received 27 April 1990, accepted 26 June 1990
The steady-state angiotensin II response was measured in primary cultures of reaggregated vascular smooth muscle cells derived from rat aorta by use of intracellular microelectrode recording of membrane potentials. Angiotensin II ( 1 0 - 9 - 1 0 - 6 M) produced a depolarization which triggered a single action potential, consisting of a spike plus plateau. In addition, angiotensin II prolonged the action potential plateau and lowered input resistance. The angiotensin II-induced action potentials and the action potential plateau prolongation were inhibited by verapamil. Saralasin blocked the occurrence of angiotensin II-induced action potentials and reversed the increase in action potential duration provoked by angiotensin II. Saralasin, in the absence of angiotensin II, exhibited agonistic activity which was manifest by plateau prolongation. Therefore, angiotensin II, through interaction of the peptide with its receptor, depolarizes cultured vascular smooth muscle cells and prolongs the calcium-dependent action potentials. These effects could be mediated by the known ability of angiotensin II to stimulate production of inositol trisphosphate and diacylglycerol, and activation of protein kinase C. Angiotensin I I; Smooth muscle (vascular); Action potentials; Membrane depolarization; Ion conductance; Ca 2 + currents
1. Introduction
Angiotensin II is an octapeptide pressor hormone with potent vasoconstrictive activity. In addition, angiotensin II stimulates aldosterone biosynthesis in the adrenal cortex (Fraser et al., 1983), induces the release of catecholamines from the adrenal medulla and facilitates norepinephrine (NE) release from sympathetic nerve terminals
1 Supported by an NIH Clinical Investigator Award K08-HL01365 and in part by HL-31942, T32-HL-07355 and P01HL-19242. 2 Current address: University of Cincinnati, Department of Physiology and Biophysics, Cincinnati, OH, U.S.A. Correspondence to: D.W. Johns, Box 146, Division of Cardiology, University of Virginia School of Medicine, Charlottesville, VA 22908, U.S.A.
(Peach, 1977). Except for studies in the heart (Freer et al., 1976; Dosemeci et al., 1988), there have been few reports describing the electrophysiological properties of angiotensin II. Recent study indicates angiotensin II action in cardiocytes is mediated by diacylglycerol and phosphokinase-C stimulation of calcium current in heart cells (Allen et al., 1988). In order to isolate the direct vascular action of angiotensin II from its multiple physiological effects, we have chosen to study cultured vascular smooth muscle cells from rat aorta. Such cultured cells are devoid of endothelium, nerve terminals, and adrenal gland influence. It was previously demonstrated that the action potentials in these cultured cells are dependent o n C a 2+ ions (Harder and Sperelakis, 1979a). An earlier report from this laboratory (Zelcer and Sperelakis, 1981) noted a
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184 transient depolarization produced by exposure to angiotensin II, administered as a bolus, on similar cultured vascular smooth muscle cells (reaggregates). The present study has characterized the steady-state angiotensin II response and established the presence of functional angiotensin II receptors on the primary cultures of vascular smooth muscle cells.
2. Materials and methods
Reaggregates of cultured vascular smooth muscle cells were prepared from rat aortas (Harder and Sperelakis, 1979a). In brief, using sterile technique, adventitia-free aortas from Sprague-Dawley rats (250 g) were incubated at 37°C in enzyme solution containing 1 m g / m l collagenase (Worthington, CLS II) and 0.5 m g / m l elastase (Type I, Sigma) in Hank's balanced salt solution with 1.8 mM Ca 2÷ and 0.76 mM Mg ÷. Dispersed cells were then filtered through gauze, centrifuged (75 × g for 8 rain), and the pellet washed twice in sterile culture medium (Medium-199, North American Biologicals). The final suspension containing 10% fetal calf serum (Gibco) and penicillin (100/~/ml)/streptomycin (100 ~tg/ml) was placed in Carrel flasks having dialysis tubing on the bottom. The cells did not adhere tightly to the cellophane and therefore spontaneously detached from this substrate and formed reaggregates of cells after approximately 36 h in culture. Reaggregates used in this study were maintained in culture (37°C) for 5-12 days before use. For electrophysiological recording, reaggregates were transferred by Pasteur pipette to a heated bath (1.5 ml, 37°C) and superfused at a rate of 3 m l / m i n with Ringer solution containing (mM): 146 Na +, 5.0 K ÷, 1.0 Mg 2+, 1.5 Ca 2+, 130 CI-, 1.0 H 2 P O 4 , 25 H C O 3 , 11 glucose. The solution was equilibrated with 95% 02-5% CO 2 (pH 7.4). Transmembrane potentials were recorded with conventional glass microelectrodes filled with 3 M KC1 and having resistances of 30-100 M~2, usually about 80 M g . The microelectrodes were attached to Zeiss sliding micromanipulators and Ag-AgC1 half cells were used. The recording preamplifier (Dagan model No. 8500) possessed capacitance
neutralization and an internal bridge circuit. Responses were visualized on a Tektronix oscilloscope and photographed with a Grass C-4 camera. A successful impalement was defined as one associated with a sharp drop in voltage on entry of the microelectrode into the cell and a sharp return to baseline (zero) upon exit. In many cases the microelectrode could be maintained inside a cell with stable membrane potential, for several hours. No reaggregate was seen to move or change shape in association with an action potential. Reaggregates were stimulated electrically either intracellularly through the bridge circuit or extracellularly by rectangular current pulses (1-4 ms in duration) applied through platinum plate electrodes (field stimulation). Input resistance (Rin) was determined by applying rectangular current pulses (Io) of varying intensities through the microelectrode while in the cell and recording the associated voltage changes (AEm). Upon withdrawal of the microelectrode from the cell, the bridge balance was checked again. If the bridge was significantly out of balance (e.g. more than 5 mV) that measurement of Rin was discarded. Drugs were added to the superfusion solution at the following final concentrations: tetraethylammonium-chloride 10 mM (TEA, Eastman Kodak Co., Rochester, NY); angiotensin II 10 -9 t o 10 - 6 M (Sigma Chemical Co., St. Louis, MO); verapamil-HC1 10 -5 M (Knoll Pharmaceuticals, Whippany, N J); Saralasin 10 -6 to 10 -5 M (Sarl,ValS,AlaS-angiotensin II; United States Biochemical Corp., Cleveland, OH); norepinephrine 10 -5 M (NE, Sigma Chemical Co., St, Louis, MO). Duration of exposure to various drugs was varied as described in the results and figures. The dead time in the perfusion system was about 30 s. Statistical comparisons were performed using Student's t-test or analysis of variance. A P-value less than 0.05 was considered significant. Results are reported as mean _+ S.E.M.
3. Results
The range of resting membrane potentials recorded from reaggregates cultured for 5-12 days was between - 3 0 and - 7 5 mV in Ringer saline
185 TABLE 1
TABLE 2
Steady-state depolarization induced by continuous exposure to angiotensin II.
Effect of 1 0 - 6 M angiotensin II on input resistance. Input resistance was measured after equilibration for 20 rain in each solution. N = number of cells/number of preparations.
Solution
RMP (mV) No. No. prepmean + S.E. cells arations
Solution
Ringer +Ang II (10 -6 M)
-41.8+0.7 -33.5-t-9
183 95
20 9
Ringer Ang II
10 mM TEA TEA+Ang II ( 1 0 - 9 M ) TEA+Ang II ( 1 0 - 7 M ) TEA+Ang II (10 -6 M) TEA + Ang II (10- 5 M)
- 39.0+0.5 -38.8+1.1 - 35.4+3 a -31.0+1 a - 34.6 + 3 a
380 84 17 154 28
38 13 1 15 4
Ringer TEA (10 raM) TEA + Ang II
Rin (M$2)
N
9.4 + 0.7 7.8 + 1.1 a
45/3 32/3
10.9 + 1.9 11.3 + 1.4 a 9.2 -l-1.4 b
119/15 164/13 134/13
a p < 0.05 compared with Ringer alone, b p < 0.05 compared with 10 mM TEA.
a p < 0.05 compared with 10 mM TEA alone. Note: resting membrane potentials obtained between 10-90 min in solution.
solution with a m e a n of - 4 1 . 8 ___0.7 m V (183 cells i n 20 reaggregates) as can b e seen from table 1. T h e m e a n resting m e m b r a n e p o t e n t i a l after addition of 10 m M T E A was - 39.0 _ 0.5 m V (range - 1 5 to - 8 0 mV). There was n o difference i n the shape or time course of the action potential evoked b y electrical field s t i m u l a t i o n in T E A solution a n d R i n g e r solution (fig. 1) a n d n o significant change in resting m e m b r a n e potential. A l t h o u g h electrical field s t i m u l a t i o n sometimes elicited action p o t e n -
Normal Ringer
10
mM TEA
A
OmVI
25 .
.
.
.
° ° • . . . . . . .
I • IO-'~M
°
NE
Fig. 1. Two representative examples of TEA-induced excitability. (A) Electrical stimulation failed to elicit an action potential in normal Ringer solution. (B) After exposure to 10 mM TEA, 1 h electrical stimulation evoked action potentials (same vascular smooth muscle reaggregate as panel A). (C) A second vascular smooth muscle preparation exposed to 10 mM TEA × 45 rain exhibited action potentials following electrical stimulation. (D) Norepinephrine (NE, 10 -5 M) shortened the action potential plateau (same preparation as panel C). Solid line above action potential tracing represents 0 voltage line.
tials in R i n g e r solution alone, in the presence of 10 m M T E A action potentials always could be elicited b y electrical field stimulation. F o r this reason, d a t a reported here have b e e n o b t a i n e d in the presence of 10 m M T E A unless otherwise stated.
3.1. Effects of angiotensin H A n g i o t e n s i n II depolarized the cultured smooth muscle cells in a d o s e - d e p e n d e n t fashion (table 1). T h e threshold c o n c e n t r a t i o n of a n g i o t e n s i n II for steady-state d e p o l a r i z a t i o n was above 10 - 9 M a n d the m a x i m u m effect occurred at a c o n c e n t r a t i o n of 10 - 6 M a n g i o t e n s i n II. U s u a l l y the steady-state d e p o l a r i z a t i o n was stable t h r o u g h o u t exposure to a n g i o t e n s i n II (up to 5 h), b u t sometimes the m e m b r a n e p o t e n t i a l w o u l d r e t u r n slowly to baseline level. As c a n be seen from table 2, a n g i o t e n s i n II decreased i n p u t resistance from 9.4 + 0.7 to 7.8 + 1.1 M~2 ( P < 0 . 0 5 ) . I n the presence of T E A , a n g i o t e n s i n II also lowered i n p u t resistance from 11.3 + 1.4 to 9.2 + 1.4 MI2 (P < 0.05). As might be expected, the a d d i t i o n of 10 m M T E A increased i n p u t resistance from 10.9 + 1.9 to 11.3 + 1.4 MI2
(P < 0.05). W i t h i n 1-2 min, a n g i o t e n s i n II (10 -6 M) always provoked a s p o n t a n e o u s action p o t e n t i a l (fig. 2B). A s p o n t a n e o u s action p o t e n t i a l sometimes was observed at low c o n c e n t r a t i o n s (10 -9 M) of a n g i o t e n s i n II, b u t rarely before 10 m i n of exposure to the peptide. A t all c o n c e n t r a t i o n s of a n g i o t e n s i n II tested, the p l a t e a u of the electrically
186 Control tn 10 m M
ANG-I~ 10 -6 M
TEA
4OmVI ls
ANG-I[ 10 -9 M
>
H .~E~~~ F ~ 2s
2=
--
- -
m= 211
2s
. . . . . . .
G~.~
20 rain
.
_
.
.
H
60
rnin
5s
Fig. 2. Representative examples of angiotensin-induced action potential facilitation in three separate experiments (preparation 1 = panels A and B; preparation 2 = panels C and D; preparation 3 = panels E - H ) . (A) Electrically evoked action potential of 4.5 s duration. (B) Induction of spontaneous action potential 1 min after exposure to angiotensin II (10 - 6 M) (note change in time scale between A and B). (C) In a second vascular smooth muscle preparation control action potential duration is 4.5 s. (D) Exposure to angiotensin II for 17 rain prolonged the action potential duration to 9.8 s. (E) In a third vascular smooth muscle preparation (E-H) control action potential duration was 5 s (E 1 expanded time scale reveals shape and action potential overshoot). (F) Prolongation of action potential duration from 5 to 8 s after exposure to angtiotensin II for 5 min. (G) Angiotensin II exposure for 20 min prolongs action potential to 13 s. (H) Angiotensin II exposure for 60 min prolongs action potential to 55 s. Solid line above action potential represents 0 voltage line.
TABLE 3 Effect of angiotensin II on resting potential and electrically evoked action potentials. S u m m a r y of experiments in which the microelectrode remained in a single cell while 5-10 action potentials were evoked in control (10 m M TEA) solution and then again after addition of angiotensin II. Data given as means + S.E. Solution (in 10 m M TEA)
N
RMP (mV)
Spike amp. (mV)
AP duration (APD9o) (s)
Vma~ (V/s)
Control
9
- 50.8 + 2.0
54.2 + 3.0
4.5 ± 0.6
0.87 4- 0.1
9
-- 48.4 + 2.0
57.6 + 3.0
5.9 ± 0.6 a
1.26 + 0.1 ~
A n g It (10-
9 M)
a p < 0.05 when compared with control (minus angiotensin II).
187 (10 mm TEA present) Control A
40 mV
10-SANG-II pre-trut with saralasin (10-5M)
B
2s • .J.I
. . . . . . .
2s
C
~ntml
D
3 x 10-6M Saralasln (x 10 rain)
Ill
E .j..lx?O?rANGII . . . . . .
F
+ 6 x 10-6 M Saralasln
Fig. 3. Saralasin effect on angiotensin II induced excitability in three separate vascular smooth muscle preparations. (A) Action potential in 10 mM TEA (action potential duration approximately 7 s). (B) Pretreatment with saralasin prevents spontaneous action potential induced by exposure to angiotensin II (10 -6 M). (C) Action potential in 10 mM TEA in another vascular smooth muscle preparation (action potential duration approximately 5 s). (D) Action potential prolongation by saralasin (3 x 10 -6 M x 10 rain) to approximately 8 s. (E) In another vascular smooth muscle preparation, action potential duration approximately 9 s after exposure to angiotensin II (10-6 M×15 rain). (F) Inhibition by saralasin (6x10-6 M x5 min) of angiotensin II-induced plateau prolongation (action potential duration approximately 6 s).
induced action potential was prolonged markedly (fig. 2D-H). Table 3 depicts results from single-ceU experiments which compare the shape of the electrically induced action potential in control solution (TEA present) to those elicited in the same cell after exposure t o 10 - 9 M angiotensin II (TEA present). Angiotensin II prolonged the action potential plateau from 4.5 + 0.6 to 5.9 + 0.6 s (P < 0.05) and increased the action potential rate of rise (Vmax) from 0.87 +_ 0.1 to 1.26 +__0.1 V / s (P