Tohoku

J. Exp.

Med., 1992,

166, 107-122

Phosphoinositide Mobilization

Induced

Angiotensin Muscle

Hydrolysis

and

Calcium

by Vasopressin

II in Cultured

and

Vascular

Smooth

Cells

KAZUHISATAKEUCHI,KEISHI ABE*, MINORUYASUJIMA, MAKITOSATO, M., KAZUTAKAMAEYAMA '~, TAKEHIKO WATANABEt, SHUNICHISATO, HUMIDINABA4and KAORU YOSHINAGA The SecondDepartment of Internal Medicine,*Department of Clinical Biology and Hormonal Regulation, and 'the First Department of Pharmacology, Tohoku University School of Medicine and $ Research Institute of Electrical Communication, Tohoku University,Sendai 980 TAKEUCHI,K., ABE, K., YASUJIMA, M., SATO,M., MAEYAMA, K., WATANABE, T., SATO,S., INABA,H. and YOSHINAGA, K. Phosphoinositide Hydrolysis and Calcium Mobilization Induced by Vasopressin and Angiotensin II in Cultured Vascular Smooth Muscle Cells. Tohoku J. Exp. Med., 1992, 166 (1), 107-122 The cellular action of vasoconstrictive hormones, angiotensin II (All) and Arg8vasopressin (AVP), on vascular smooth muscle (VSM) in cultured VSM cells from rat mesenteric artery was studied. Both All and AVP specifically induce a transient increases in cytosolic free calcium independent of entracellular calcium or calcium channels activated by high potassium depolarization in VSM cells loaded with Fura-2. Vasoconstrictive hormones induce a dose-dependency with formation of inositolphosphates. Analysis using high pressure liquid chromatography has shown that AVP stimulates rapid and transient increases in inositol 1, 3, 4-trisphosphate, inositol 1, 4, 5-trisphosphate and inositol 1, 3, 4, 5tetrakisphosphate within 1 minute. Moreover, a laser-excitation fluorescence system reveals high calcium concentration sites in subsarcolemmal region. These results indicate that, unlike voltage-dependent calcium influx across the cell membrane, All and AVP induce receptor-mediated increases in cytosolic free calcium via phosphoinositide hydrolysis creating an intracellular messenger for calcium release from intracellular calcium stores. cytosolic calcium ; fura-2 ; Indo-1; inositol phosphate ; laser-excitation fluorescence microscopy ; angiotensin II receptor

sion",

This paper was Sendai, Japan

presented 1988.

at the international

meeting,

"The

Kidney

and Hyperten-

Address for correspondence and reprints : Kazuhisa Takeuchi, M.D. Ph. D., The Second Department of Internal Medicine, Tohoku University School of Medicine, l-1 Seiryo-machi, Aoba-ku

Sendai

980, Japan. 107

108

K. Takeuchi

et al.

Angiotensin II (All) and Arg8-Vasopressin (AVP) are known to play a homeostatic role in the regulation of the vascular tone, contributing to blood pressure regulation. It has been suggested that the contraction of vascular smooth muscle (VSM) is due to an increase in cytosolic free calcium concentration ( [Ca2]+i), which activates calcium-calmodulin-dependent myosin light chain kinase which, in turn, initiates the phosphorylations resulting in smooth muscle contraction (Kamm and Stull 1985). VSM has been shown to have specific receptors for the vasoconstrictive hormone, AVP and All (Gunther et al. 1982; Pinet et al. 1983), and the hormones trigger the VSM contraction by binding to their specific receptors which may cause calcium mobilization. However, the post-receptor mechanism of the hormones action has not been fully elucidated. Berridge (1983) has hypothesized that an agonist-induced water soluble metabolite of membrane phosphoinositide is involved in intracellular calcium mobilization, and Streb et al. (1983) have shown that inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) can indeed release calcium from intracellular calcium storage sites, presumably a component of endoplasmic reticulum. Since the findings occurred, a link between phosphoinositide metabolism and [Ca2+] i has been investigated, in various types of cells (Berridge 1987). Recently, a method for the analysis of inositol phosphates has been developed by high pressure liquid chromatography (HPLC) (Heslop et al. 1985; Tilly et al. 1987; Cunha-Melo 1987), making it accessible to separate isomers of InsP3, inositol tetrakisphosphate (InsP4 ), inositol pentakisphosphate (InsP5) and inositol hexahisphosphate (InsP6 ), showing the kinetics of these metabolites. In VSM cells, however, the metabolism of inositol phosphates has not been fully studied. In the present study, to evaluate the relationship between inositol phosphates and calcium mobilization in the cultured VSM cells, we have examined the effects of AVP and All on intracellular inositol phosphate formation and [Ca2+] i. Moreover, we have developed a laser-excitation fluorescence microscope system to examine the possible existence of intracellular calcium storage sites where calcium may be released, presumably by the inositol phosphate. MATERIALS AND METHODS The sources of materials used in the present study were as follows ; type III collagenase, deoxyribonuclease I, elastese, soybean trypsin inhibitor, digitonin, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP) from Sigma Chemical, St, Louis, MO, USA ; medium 199, minimal essential medium (MEM), Lglutamine solution, and trypsin-EDTA from Gibco, Grand Island, NY, USA ; fetal bovine serum from Flow Laboratory, North Ryde, Australia ; 12-well sterile culture dishes from Coaster, Cambridge, MA, USA, and 6-well dishes from Nunc, Copenhagen, Demmark ; Arg8-vasopressin, angiotensin II and Sarl-Ilea-angiotensin II from Protein Research Institute, Osaka, Japan ; [ 1-/3-mercapto-R, fl-cyclopentamethyl-enepropionic acid), 2-0methyltryrosine, Arg8-vasopressin from Peninsula Laboratories, Belmont, CA, USA ; fura-2 acetoxy methylester (fura-2/AM) from Dojin, Kumamoto ; ionomycin from Calbiochem, La Jolla, CA, USA, Nicardipine was a gift from Yamanouchi Pharmaceutical Company, Tokyo.

Phosphoinositide

Hydrolysis

and Calcium

Mobilization

109

Tritiated myo-[2-3H(N)]-inositol, D-[inositol-2-3H(N)]-inositol 1-monophosphate, D[inositol-2-3H(N)]-inositol 4-monophosphate, D- [inositol-2-3H(N)] -inositol( 1, 4)bisphosphate, D- [inositol-2-3H(N)] -inositol( 1, 4, 5)-trisphosphate and D-[inositol-23H(N)] -inositol(1 , 3, 4, 5)-tetrakisphosphate were obtained from Dupont-New England Nuclear, Boston, NA, USA. Culture of vascular smooth muscle cells Cultured VSM cells were prepared by enzymatic dispersion from rat mesenteric artery as previously reported (Sato, M, et al. 1986), based on the methods by Ives et al. (1978) and Gunther et al. (1982) modified by Hassid and Williams (1978). Briefly, male SpragueDawley rats weighing between 200-400 g were anesthetized by nembutal®, and the superior mesenteric artery was removed. These arteries were freed of surrounding tissues and incubated for 15-20 min at 3TC, being shaken in a modified Krebs-Ringer solution composed of the following gradients (in mM) : NaC1,105 ; KCI, 5 ; KH2PO4, 1; HEPES, 25; MgSO4,1; glucose, 14 ; CaC12,0.2 ; NaHCO3, 25. The solution was further supplemented with collagenase (340 U/ml), elastase (96 U/ml), DNase (56 U/ml) and soybean trypsin inhibitor (1 mg/ml). Cells which had dissociated during the first 15 to 20 min of incubation were discarded because they were considered to be rich in adventitial fibroblasts as well as endothelial cells. The remaining portion of the vessels was incubated for another 30 to 90 min. Cells dissociated during the second incubation were centrifuged and filtered through a 150pm Nytex filter before they were allowed to adhere to culture dishes in the medium 199 lacking fetal bovine serum. After 2 hr, most cells adhered to the bottom of the wells and were further cultured in medium 199 supplemented with 10% fetal bovine serum. Cells were subcultured by treatment with 0.08% trysin-EDTA. A morphological examination revealed characteristic features of smooth muscle cells with a criss-cross or a hills and valleys pattern under phase contrast microscopy. Ultrastructural examination by electronmicroscopy, also revealed the presence of abundant filaments resembling myofilaments and dense bodies. For the experiments, the cells in subcultures 4 to 15 were used. Measurement of cytosolicfree calcium in monolayers of cultured vascular smooth muscle cells Cytosolic free calcium concentration ( [Ca2+]i) was measured in monolayers loaded with fura-2. The cells were cultured on 2.5 X 1.3 cm cover glasses in medium 199 supplemented with 10% fetal bovine serum. One day before the experiments, the medium was replaced with serum free medium. Confluent cells on cover glasses were incubated with fura-2/AM 4 ~cMin balanced salt solution ((in mM) : NaC1130, KC15, CaC121.5, MgC121.0,glucose 10, HEPES 20, buffered to pH 7.4) for 30-40 min at 3TC. After washing the monolayer on cover glasses three times, it was incubated with the balanced salt solution in the cuvette. The cover glass was set diagonally to the light axis in the holder of the fluorometer, Hitachi MPF4. In the fluorometer, a temperature of 3TC was maintained. The excitation wavelength was set at 340 nm and the fluorescence was measured at the emission wavelength of 505 nm. Calibration was performed at the end of each experiment. [Ca2+]i was calculated by the following equation proposed by Tsien et al. (1982) with Ka taken to be 224 nM, which has been estimated by Grynkiewicz et al. (1985) : [Ca2+] i = (F - Fmin) / (Finax F) x Kd. Finax or Fmin was obtained by the addition of ionomycin 2,aM or EGTA 2.5 mM, respectively. In the experiment, agents were cumulatively added to the medium in the cuvett. In a portion of this study, the fluorescence intensities at the excitation wavelength of 340 nm and 380 nm were monitored and [Ca2+]i was estimated by the ratio of the intensities by a dual excitation wavelength fluorescence analyzer (CAF100 ; Nihon-Bunko, Tokyo). Measurement of [Ca2+] i by laser-excitation fluorescence Microscope system This fluorescence system is based on the method described by Sato, S. et al. (1986), and the block diagram is shown in Fig. 1. An argon laser (excitation wavelength= 355 nm) is

K. Takeuchi

110

Fig

et al.

.1. Block diagram of the laser-excitation fluorescence microscope system. The microscope (Nikon XF) is commercially available. SIT camera Hamamatsu C1000-18. Image Intensifier : Hamamatsu 02100.

:

used in the experiment. The cells adherent to the cover glass are loaded with indo-1 through incubation with 4,uM indo-1/AM for 30 min, 37°C. The emission wavelengths (Ems.) of 405 nm and 485 nm are measured, and [Ca2+]i is estimated by the ratio between the fluorescence intensities (Em. 450/Em. 485) (Grynkiewicz et al. 1985). Measurement of inositol phosphates by column chromatography The measurement of inositol phosphates was based on reports described by Maeyama et al. (1986) and Nabika et al. (1985). Briefly, the confluent cultures in 24-well dished were prelabeled by incubation with serum free M199 containing 4pCi/ml myo-[2-3H(N)]-inositol for 24 hr. In the experiments, the cells were stimulated by agonists (300,1 of incubation volume) in the presence of 10 mM LiCI (which inhibits inositol 1-phosphatase) for 30 min and the incubations were terminated by the addition of a chloroform/methanol mixture (1: 2), 750 gal. The chloroform/methanol extracts from the labeled cultures were separated into aqueous and organic phases in order to assay phospholipids in the organic phase and different water-soluble phosphates in the aqueous phase. The aqueous extracts were then transferred to polypropylene tubes, and the phases were separated by addition of 250,1 of chloroform and 250,1 of water followed by mixing and centrifugation. The upper aqueous phase was collected and the chloroform layer was washed twice with 200 u 1 of a mixture of chloroform, methanol, 100 mM sodium cyclohexane-1, 2-diaminetetraacetate (16: 8 : 5, v/v). The upper aqueous phase and washing aqueous fraction were pooled and diluted to 3 ml with water and then transferred to a Dowex 1 formate column. Inositol, glycerophosphoinositol, inositol monophosphate, inositol bisphosphate, inositol trisphosphate were sequentially eluted with water, 5 mM sodium tetraborate in 60 mM sodium formate, 200 mM amonium formate in 100 mM formic acid 400 mM amonium formate, and finally 1 M ammonium formate in 100 mM formic acid. Aliquots were taken from each batch elution for the determination of the content of 3H. Total phosphoinositides were eluted with 1 M sodium formate after eluting inositol and glycerophosphoinositol with water and then with 5 mM sodium. The organic phase was washed twice with a methanol/aqueous (1 M KC1,10

Phosphoinositid

e Hydroly

mM inositol) solution (1:1), and the solvent liquid scintillation counting.

sis and Cal cium was evaporated.

Mo bilization The 3H was determined

111

by

Separation of inositol phosphates by HPLC Inositol phosphates were separated by a modification of the method described by Cunha-Melo et al. (1987). Briefly, the prelabeled cultures in 6-well dished was prepared as described above. The incubation was terminated, after the removal of the medium, by the addition of 1 ml (15%, w/v) trichloroacetic acid. Insoluble debris were removed by centrifugation and the supernatant fraction was washed three times by diethylether. The ether was removed by evaporation under a stream of N2. The extracts were then lyophylized and the residue was dissolved in 200 u l of 10 mM ammonium phosphate for application to a Whatman Partisil 10 SAX Anion exchange column (25 X 0.46 cm). In the present study, a Gilson model two pump system was used and the size of sample injected into the column was 100 ,al. Adenine nucleotides (AMP, ADP and ATP) were added to each sample and the absorbance readings (254 nm) were monitored. The validity of this procedure was verified by calibration using authentic 3H-inositol phosphates standards (Ins(1)P1, Ins(4)P1, Ins(1, 4)P2, Ins(1, 3, 4)P3, Ins(1, 4, 5)P3 and Ins(1, 3, 4, 5)P4). The solvents for elution consist of 10 mM and 1.75M ammonium phosphate (pH 3.8). The gradients of ammonium phosphate used were (flow rate 1 ml) : 0.01-0.09 M for 30 min (to separate the 3H-labeled inositol, glycerophosphoinositol, and isomers InSPI) ; 0.2-0.29 M for 30 min (to separate isomers of InsP2) ; 0.48-0.53 for 30 min (to separate isomers of InsP3 ), and 0.53-1.75 M for

Fig

. 2. Effects of Arg8-vasopressin (AVP) and angiotensin II (All) on cytosolic free calcium ([Ca2~]i) in cultured vascular smooth muscle (VSM) cells loaded with Fura-2(a). [Sarl, IIee] angiotensin II(b) and 1-f3-Mercapto-/l,,8cyclopentamethyienepropionic acid(c) was used as an All antagonist and AVP antagonist, respectively.

112

K. Takeuchi et al.

10 min (to separate InsP4) followed by an isocratic elution (1.75 M) for 30 minutes. The elutes were collected in 0.5 ml fractions, and radioactivity was determined by liquid scintillation counting, using Atomlight (Dupont-New England Nuclear, Boston, MA, USA)/ water (10: 1) as counting fluid.

RESULTS Calcium mobilization by vasoconstrictive hormones and high potassium depolarization AVP and All immediately increased the [Ca2+] i, as measured by fura-2 fluorescence (Fig. 2a); No cross tachyphylaxis was observed between them. According to the calibration, the resting [Ca2]+i was estimated to be 152 nM. AVP 10-' M elicited a peak increase within 20 sec, reaching a 3-4 fold increase in [Ca2]+i. As shown in Fig. 2b and 2c., the hormone-induced increase in [Ca2+] i were abolished by their respective receptor antagonists. AVP-induced increases in the peak value of [Ca2]+i were dose-dependent (Fig. 3). AVP and All induced-increases in [Ca2+] i were not attenuated by the treatment with the

Fig.

3. Dose-response of AVP-induced increase in cytosolic free calcium ([Ca2+] i) in cultured VSM cells. Each point (•) represents the value measured in a single experiment. Group means (a) ±s.E. of the fluorescence intensities are shown adjacent to sets of data points.

Phosphoinositide

Hydrolysis

and Calcium

Mobilization

113

Fig. 4. Effect of a calcium antagonist, nicardipine, on increases in cytosolic free calcium ( [Ca2+]i induced by AVP and All (a), (b) or by high potassium depolarization (c).

calcium antagonist, nicardipine

(Fig. 4a, b).

On the other hand, [Ca2]i +was

gradually increased by high potassium depolarization was inhibited by nicardipine, in a dose-dependent absence of extracellular calcium, AVP still induced (Fig. 5), while high potassium depolarization did not shown).

and the increase in [Ca2]+i manner (Fig. 4c). In the a transient rise in [Ca2+] i increase [Ca2]+i (results not

Localization of intracellular calcium The cytosolic fluorescence of the cells loaded with fura-2 is nonhomogeneous with a perinuclear-reticular or a filamentous structure, and appears to be compartimentalized in some subcellular organelles (Takeuchi et al. 1989). On the other hand, the cytosolic Tndo-1 fluorescence is rather homogeneous. A three dimensional display of [Ca2]+i demonstrates the high calcium concentration sites on the

114

K. Takeuchi

et al.

Fig. 5. Effect of extracellular calcium depletion (b) or AVP-induced increase in [Caz+]i (a). Extracellular zero calcium was brought about through the addition of EGTA (final concentration 2.5 mM) to calcium free Hanks' balanced salt solution. Changes in [Caz+]i were monitored by a dual excitation wavelength fluorescence analyzer (CAF 100). [Caz+]i is expressed by the ratio between the fluorescence intensities at the excitation wavelength of 340 nm and 380 nm.

inner surface of the cell membrane (Fig. 6). AVP-Induced inositol phosphates formation measured by column chromatography Fig. 7 shows the elution profiles of inositol phosphates (Ins Ps) in the basal condition and after incubation with AVP for 30 minutes. The amount of 3H-Ins P1, 3H-Ins P2 or 3H-Ins P3 increased two-fold in response to AVP 10-' M, whereas the amount of 3H-inositol decreased. The addition of All or AVP to confluent cultures of VSM cells (in the presence of 10 mM LiCI) caused a dose-dependent accumulation of inositol phosphates (Fig. 8a, b). A saturation response was observed at approx. 10-6 M AVP, where the Ins Ps level was approx. 2-fold of the basal level. Fig. 9 shows the effect of nicardipine on All-induced Ins Ps formation. Nicardipine did not affected the Ins Ps formation. Separation of inositol phosphates by HPLC The separation of inositol phosphates from prelabeled, unstimulated VSM cells yielded twelve peaks of radioactivity (Fig. 10). By means of calibration with the 3H-labelled inositol phosphate standards we identified Ins(1)P1, Ins(4)

Phosphoinositide

Hydrolysis

and Calcium

Mobilization

Fig

. 6. Three dimensional display of distribution of [Ca2+ ] i in a single VSM cell in unstimulated condition. The cell was loaded with indo-1 and the fluorescence was analyzed by the laser-excitation fluorescence microscope system. a) and b) shows the fluorescence at the emission wavelength of 405 nm and 485 nm, respectively. [Ca2+]i is estimated by the ratio between the two fluorescence intensities (c).

Fig

7. Elution profiles of inositol phosphates in basal condition (open circle) and after incubation with AVP 10-' M for 30 min (closed circle) in the presence of 10 mM LiCI and 1.5 mM Ca2t

115

K. Takeuchi

116

Fig

et al.

. 8. Dose-response relationships of either AVP- or All-induced total inositol phosphate accumulation. Effects of AVP (a) and All (b) are shown respectively. Asterisk indicates statistical significance at p