Increase in (Tytosolic Calcium and Phosphoinositide Metabolism

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 260, No. 8, Issue of April 25, pp. 46614670,1985 Printed in U.S.A.

Increase in (Tytosolic Calcium and Phosphoinositide Metabolism Induced by Angiotensin I1 and [ArglVasopressin in Vascular Smooth Muscle Cells*

(Received for publication, October 23, 1984)

Toru NabikaS, Paul A. VelletriS, Walter LovenbergS, and Michael A. Beavens From the $Section on Biochemical Pharmacology and the $Laboratory of Chemical Pharmacology, National Institutes of Health, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20205

Effects of angiotensin I1 and [Arglvasopressin on cytosolic free Ca” concentration ([Ca”’],) and phosphoinositide metabolism were studied in cultured aortic smooth muscle cells obtained from Wistar-Kyoto rats and their spontaneously hypertensive substrain. [Ca2+], was measured using the fluorescent Ca2+ indicator quina. No clear differences in basal [Ca2+Ii were detected between cells derived from the two strains. High concentrations of angiotensin I1 (210 nM) and [Arglvasopressin (2100 nM) elicited large and rapid increases in [Ca2+Ji, followed by a rapid return to control values. Low concentrations of these peptides (51.0 nM) elicited small and slow increases in [CaZ+li that persisted for minutes. These responses were blocked by specific antagonists for each of these peptides. Only high concentrations of angiotensin I1 caused [Ca2+Ii increases in “Ca2+-free” medium, which suggested that high concentrations of angiotensin I1 could release Ca2+ from intracellular pools. A high concentration of angiotensin 11 and [Arglvasopressin elicited progressive accumulations of inositol phosphates. Only high concentrations of angiotensin I1 caused inositol phosphate accumulation in Cas+-free medium. Maximal accumulation of inositol phosphate elicited by angiotensin I1 and [Arglvasopressin was found to be additive. A desensitization to the effects of both peptides on Ca2+ mobilization occurred despite the continued accumulation of inositol phosphates. These observations indicated that angiotensin I1 and [Arglvasopressin inter- acted with independent receptors, both of which are linked to phosphoinositide breakdown and Ca” mobilization.

Intracellular mobilization of Ca2+ is an essential event in the regulation of vascular smooth muscle tone (1). Alterna- tions in Ca2+ mobilization in vascular smooth muscle are thought to play an important role in the development of hypertension (1). Many studies have suggested that various neuronal and humoral factors, such as catecholamines, angiotensin 11, [Arglvasopressin, and certain prostaglandins, lead to the contraction of vascular smooth muscle through Ca2+ mobilization (2). It has been postulated that these agents bind to specific receptors on cell membranes to induce accumulation in cytoplasm of Ca2+, which originates either from extracellular or intracellular Ca2+ pools. These Ca2+ fluxes are

*This research was done as part of the agreement on United States-Japan Cooperation in Cardiovascular Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

thought to increase the cytosolic free Ca2+ concentration ( [CaZ+li)’ and then activate Ca2+-calmodulin-dependent myosin light chain kinase, which initiates the cascade of phosphorylations resulting in smooth muscle contraction (3).

Angiotensin I1 and [Arglvasopressin are believed to be among the most potent vasoconstrictors yet identified (4). Little is known about the mechanisms by which [Ca2+]i is regulated following exposure of smooth muscle cells to angiotensin I1 or [Arglvasopressin. Some studies have described specific binding sites for these peptides on vascular smooth muscle cells (5, 6) and Ca2+-dependent phosphorylation of myosin light chain subsequent to treatment with angiotensin I1 (7).

The vascular smooth muscle cell culture system originally described by Ross (8) provides a convenient model to study the cellular mechanisms by which angiotensin I1 and [Arg] vasopressin regulate [Ca2+Ii in smooth muscle. This cell culture system is homogeneous, free of contamination by fibro- blasts and endothelial cells, and maintains specific receptors for these vasoactive peptides (6), many of which are thought to transduce messages to the cells by elevating [CaZ+li. The fluorescent Ca2+ indicator quin2 developed by Tsien et al. (9) can be readily employed for the measurement of free intracellular Ca2+ in cell culture systems (9-15). quin2, which is hydrolyzed intracellularly from the lipophilic derivative quin2/acetoxymethyl ester by cellular esterases, is believed to remain in the cytoplasm due to its hydrophilicity.

Of interest to this laboratory are reports that phosphoinositide hydrolysis by a phospholipase C may play a key role in Ca2+ mobilization following the interaction of certain ligands with their surface receptors (16). Angiotensin I1 and [Arg] vasopressin have been postulated to cause the turnover of membrane phosphoinositides and the mobilization of Ca2+ in hepatocytes (15, 25) and adrenal glomerulosa cells (17). In- deed, many reports suggest that phosphoinositide breakdown is closely related to Ca2+ mobilization, although it remains unclear whether phosphoinositide breakdown is a primary phenomenon causing Caz+ mobilization or is an event that occurs in parallel with changes in intracellular Ca2+ levels (18).

In this study, we describe characteristic changes in [CaZ+li and their relationship to phosphoinositide turnover that occur in vascular smooth muscle cell cultures following exposure to varying concentrations of angiotensin I1 and [Arglvasopres- sin. We note that the persistency of phosphotidylinositol breakdown after levels of intracellular Ca2+ have returned to

The abbreviations used are: [Ca*+],, free cytosolic calcium concentration; [Ca2+],, free extracellular calcium concentration; EGTA, eth- ylene glycol bis(8-aminoethyl ester)-N,N,N’,N’-tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

4661

4662 ea2+ and Phosphoinositides in Smooth Muscle Cells

prestimulated values, an additive degree of phosphatidylinositol breakdown that occurs with optimal concentrations of angiotensin I1 and [Arglvasopressin, and the occurrence of phosphatidylinositol breakdown under conditions where [Ca2+Ii can be mobilized from either external or intracellular Ca2+ pools.

EXPERIMENTAL PROCEDURES

Materials-Angiotensin 11, [Arglvasopressin, and [Sar',Ala']angiotensin I1 were from Sigma. [ 1-(P-mercapto-P,@-cyclopentamethyl- enepropionic acid),2-O-methyltyrosine, [ArglVasopressin and A23187 were purchased from Peninsula Laboratories (Belmont, CA) and Calbiochem-Behring, respectively. [3H]quin2/acetoxymethyl ester was synthesized by Dr. G. A. Smith, Department of Biochemistry, University of Cambridge, England, or purchased from Amersham Corp. Nifedipine, diltiazem, and verapamil were gifts from Pfizer Inc. (New York, NY), Marion Laboratories, Inc. (Kansas City, MO), and Knoll Pharmaceutical Co. (Whippany, NJ), respectively. My0[2-~Hl binositol(15.6 Ci/mmol) was obtained from Amersham Corp. Culture medium and fetal bovine serum were purchased from Gibco Labora- tories (Grand Island, NY). The ATP assay kit was obtained from DuPont Instruments (Wilmington, DE). Collagenase type I1 (186 units/mg) and trypsin (1:250) were from Worthington and Difco, respectively.

Preparation of Vascular Smooth Muscle Cell Cultures-Wistar- Kyoto rats and their spontaneously hypertensive strains were from an inbred colony maintained by the Division of Research Services at National Institutes of Health, Bethesda, MD. They are descendants of original strains developed by Okamoto and Aoki (19). Primary cultures of vascular smooth muscle cells were obtained by a modification of the explanation method of Ross (8), as described previously (20). Briefly, thoracic aortas of 5-6-week-old Wistar-Kyoto rats and spontaneously hypertensive rats were dissected free of surrounding tissue and transferred to Petri dishes. The aortas were cut longitu- dinally to expose the tunica intima and were then incubated for 20 min at 37 "C in serum-free modified Medium 199 (21) in the presence of 1.0 mg/ml collagenase type 11. Intimal-medial tissue was dissected away from the tunica adventitia and cut into 1-2-mm2 sections, which were placed in culture flasks and kept in modified Medium 199 containing 10% fetal bovine serum, as described by Lewis et al. (21). After 10-14 days of incubation at 37 "C in a humidified atmosphere of 5% C02, 95% air, cultured cells that had migrated from the explanted tissue were trypsinized with 0.2% (w/v) trypsin for 5 min at 37 "C and transferred into new flasks. Cell cultures were studied under a phase-contrast microscope and were characterized as a homogeneous population of smooth muscle cells if cell morphologies corresponded to typical characteristics described by Chamley-Camp- bell et al. (22). Stock cultures were trypsinized and passaged at a 1:4 or 1:8 split every week. Cells of the 3rd to 15th passage were employed in experiments. No differences in the response of smooth muscle cells to the experimental conditions described in this paper were observed when cells of the 3rd to 15th passage were employed.

quin2 Loading and Measurement o i Ca2+-quin2 Fluorescence-The use of the fluorescent Ca2+ indicator quin2 for the assay of free cytoplasmic Ca2+ was as previously described for rat leukemic baso- phil cultures (11). Smooth muscle cells were grown to reach confluency in 10-cm cultures dishes and were incubated in serum-free medium for 24-48 h before experiments. Cells were trypsinized (0.05% (w/v) trypsin, 5 min, 37 "C), and cell suspensions were washed once with medium A (133 mM NaCI, 3.6 mM KCl, 1.0 mM CaC12, 0.4 mM MgC12, 16.0 mM D-glucose, 0.1% (w/v) bovine serum albumin, 3.0 mM HEPES buffer, pH 7.4). Cells were counted in a Coulter counter and were resuspended in medium A so that a final cell concentration of 1 x lo6 cells/ml was obtained. Cell viability was checked by the dye- exclusion method and found to be over 95%.

[3H]quin2/acetoxymethyl ester (10 p ~ ) was added to cell suspensions, and these suspensions were incubated at 37 'C with continuous shaking. Hydrolysis of quin2/acetoxymethyl ester to quin2 by intracellular esterases was routinely determined by monitoring the shift in the emission wavelength from 430 to 492 mm (9). Rates of hydrolysis varied with different batches of cells. Usually 20-50 min were needed to allow for optimal loading of the cell with quin2. At the end of the loading period, cell suspensions were centrifuged (200 X g, 5 min), washed, and resuspended in medium A to a concentration of 1 x lo6 cells/ml. Aliquots of the cell suspension were counted in a @- scintillation counter to determine the extent of [3H]quin2 loading.

Intracellular quin2 concentrations were calculated from an estimated cell volume of 1.7 X &ell as determined in a Celloscope cell counter (Particle Data, Inc.) with ancillary equipment for cell size determination. Intracellular quin2 concentrations varied from 2.4 to 6.0 mM. After loading the cells with quin2, cell suspensions were kept on ice until they were employed in measurements of Ca2+-quin2 fluorescence. This treatment minimized cell aggregation and leakage of quin2 into the extracellular medium. Leakage was less than 15% in 60 min under these conditions. Cell suspensions were maintained on ice for no longer than 1 h.

of Ca2+-quin2 and [Ca2+li were performed as described by Hesketh et Fluorescent measurements and calculations of per cent saturation

al. (10). Two-ml aliquots of quin2-loaded cell suspensions were transferred to quartz cuvettes and were stirred with a magnetic stir bar continuously, and temperature was maintained at 36 "C. quin2 fluorescence was detected with a Perkin-Elmer LS-5 spectrofluorometer at an excitation wavelength of 339 nm and an emission wavelength of 492 nm. After each experiment, [Ca2+]; was estimated by lysing cells with Triton X-100 to give 100% saturation of quin2. This treatment was followed by the addition of MnC12 to quench quin2 fluorescence. The [Ca2+]; was calculated from the formula in Ref. 10 after correction for extracellular quin2 as determined by the addition of Mn2+ (10). In some cases, after the addition of Triton X-100, we observed a rapid decay of maximal fluorescence that could be prevented by addition of 1.0 mM EGTA to scavenge heavy metal ions. In such cases, free Ca2+ concentration was calculated to be 25 mM, which was sufficient to obtain 100% saturation of quin2 with Ca2+, Nevertheless, 1.0 mM MnC12 was required to obtain complete quench- ing of quin2 fluorescence.

Phosphoinositide Breakdown-Phosphoinositide breakdown was measured by a modification of the procedures of Berridge et al. (23) as described by Beaven et al. (18) and Moore et al. (24). Smooth muscle cells in 10% (v/v) fetal bovine serum and modified Medium 199 were seeded into microplate wells (24 X 1.6 cm wells) so that a final concentration of 0.2 X lo6 cells/well was achieved. Cells were grown for 24 h in 5% C02,95% air at 37 "C. Cells were then incubated for an additional 24 h under identical conditions, but in the absence of fetal bovine serum. Cells were then washed and exposed to 6 or 10 pCi/ml my0[2-~H]inositol for approximately 16 h in serum-free medium. The my0[2-~H]inositol was treated with Dowex (formate form) prior to use to remove decomposition products (24).

For the determination of phosphoinositide breakdown, cells labeled with [3H]inositol were washed twice with medium B (medium A minus bovine serum albumin). Cells were then preincubated for 10 min in medium B in the presence of 10 mM LiCI, which inhibits inositol-1-phosphatase (23). In the experiments summarized in Fig. 10, 1.2 mM EGTA was added 2 min prior to the completion of the preincubation period. The measurement of phosphoinositide breakdown was commenced by the addition of 20 pl of various test reagents or medium B alone, which acted as a control. Reactions were terminated after 0-20 min by the addition of 750 pl of chloroform/methanol (1:2) to the microplate wells. Extracts were then transferred to polypropylene tubes, and the phases were separated by the addition of 250 pl of chloroform and 250 pl of water followed by Vortex mixing and centrifugation (200 X g, 3 rnin). An aliquot of the upper aqueous phase was transferred to Dowex 1 formate column and washed three times with 5.0 mM inositol. [3H]Inositol phosphates were eluted with 1.5 ml of 1 M sodium formate, 0.1 M formic acid. The 3H content in the eluate was counted by a liquid scintillation counter. The remaining 3H-labeled lipids in the lower chloroform phase, which were predominantly composed of phosphoinositides (see "Results"), were washed twice with a methanol/aqueous (1 M KCl, 10 mM inositol) solution (1:1), and the solvent was evaporated. The 3H content was determined by a liquid scintillation counter. Data on [3H]inositol phosphates and 3H-labeled lipids were calculated as a per cent of control (no agonist) values of total 3H-labeled lipids. In the experiments shown in Table I, various fractions of the [3H]inositol phosphates were separated as described previously (18). After addition of chloroform/methanol to tissue samples, the upper aqueous phase was collected and the chloroform layer was washed twice with 200 pl of the upper phase from a mixture of chloroform, methanol, 100 mM sodium cyclohexane-1,2-diaminetetraacetate (16:85, by volume). The upper aqueous phase and washings were pooled and diluted to 3 ml with water. Inositol, glycerophosphorylinositol, inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate were se- quentially eluted with 1) water, 2) 5 mM sodium tetraborate and 60 mM sodium formate, 3) 100 mM formic acid and 200 mM ammonium

ea2+ and Phosphoinositides in Smooth Muscle Cells 4663

formate, 4) 100 mM formic acid and 400 mM ammonium formate, and 5) 100 mM formic acid and 1 M ammonium formate. Aliquots were taken from each batch elution for the determination of the content of 3H.

ATP Measurements-To determine whether quin2 had detrimental effects on the intermediary metabolism of smooth muscle cells, ATP levels in cells following exposure to quina were determined as described previously (11). Briefly, aliquots (2.0-4.0 X 106 cells) of quin2- loaded cell suspensions were centrifuged (18,000 X g, 60 s), and cell pellets were suspended in 200 pl of ice-cold 2% perchloric acid. After neutralization with KHCOa solution and sonication, the supernatants were analyzed for ATP content using the DuPont 760 luminescence biometer and luciferin-luciferase reagent kit.

Presentation of Results-Because no apparent differences were observed when smooth muscle cell values from Wistar-Kyoto or spontaneously hypertensive rats were compared, results of these two strains were at times mixed, unless stated otherwise. The Student's t test was employed in all statistical analyses.

RESULTS

Determination of quin2 Fluorescence following Exposure of Vascular Smooth Muscle Cells to Vasoactive Agents-A variety of biologial compounds with known actions on intact vascular preparations were studied to determine their effects on [Ca2+Ii in cultured vascular smooth muscle cells, as indicated by changes in quin2 fluorescence. Among these reagents, only angiotensin 11, [Arglvasopressin, 5-hydroxytryptamine, and prostaglandin F2a had notable effects on the free intracellular Ca2+ levels (Fig. 1). Angiotensin I1 (100 nM) and [Arglvaso- pressin (1 FM), the two most potent agents studied, caused rapid and pronounced increases in quin2 fluorescence, which indicated an increase in the free cytoplasmic concentration of Caz+. In contrast, 5-hydroxytryptamine (10 FM) and prostaglandin F2, (10 FM) induced smaller and less rapid increases in quin2 fluorescence. Other agents with known vasoactive properties, such as phenylephrine, isoproterenol, and car- bachol, failed to exhibit any effect on quin2 fluorescence at concentrations up to 10 p~ (data not shown). Additionally, 60 mM KC1, a concentration of KC1 known to depolarize vascular smooth muscle cells (26), did not result in a change in quin2 fluorescence (data not shown).

The [Ca2+Ii in unstimulated vascular smooth muscle cells was calculated to be 114 f 2.9 nM (mean +. S.E., n = 17). quin2, at concentrations up to 6.0 mM, had little effect either on the apparent [Ca"+Ii or on intracellular ATP levels (Fig. 2).

Angiotensin ZZ-induced Changes in [CaZ+li of Vascular Smooth Muscle Cells-Angiotensin I1 caused increases in [Ca2+Ii in a concentration-dependent manner (Fig. 3A). Changes were detectable when as little as 0.1 nM angiotensin I1 was employed. On exposure to higher concentrations of angiotensin I1 (10 or 100 nM), a characteristic temporal pattern of immediate and rapid increases in quin2 fluorescence was observed, These increases reached peak levels within 20- 30 s and were followed by a rapid decay in fluorescence. In contrast, lower concentrations of angiotensin 11 (1.0 or 0.1 nM) caused slower increases and slower rates of decay in quin2 fluorescence. Typically, maximal levels of fluorescence were reached after 1 min, and levels were maintained for up to 4 min after the addition of the peptide.

A specific inhibitor of angiotensin 11, [Sar',Alas]angiotensin 11, blocked increases in quin2 fluorescence elicited by angiotensin 11, but not those elicited by [Arglvasopressin (Fig. 3B), which suggested that [Arglvasopressin acted on a different receptor than did angiotensin 11. Furthermore, [Sar',Alas] angiotensin I1 enhanced the return to base-line of quin2 fluorescence that was induced by angiotensin 11.

In order to establish whether the angiotensin-induced in-

L P I 1-

10 )rM

FIG. 1. Caa*-quin2 fluorescence in vascular smooth muscle cells induced by selected vasoactive agents. Cell suspensions were loaded with quin2 as described under "Experimental Proce- dures" and transferred into cuvettes and stirred at 36 "C. quin2- loaded cell suspensions were challenged with angiotensin I1 (AZO, [Arglvasopressin ( A VP), 5-hydroxytryptamine (5HT), or prostaglandin FP. (PGF,) at concentrations indicated in the appropriate panels. After maximal responses were obtained, Triton X-100 (Tx) was added to lyse the cells and saturate quin2. This treatment was followed by the addition of Mnz+ (Mn) to quench quin2 fluorescence as described under "Experimental Procedures." [Ca2+]i in unstimulated cells and maximal values of [Ca2+Ii after stimulatlons were calculated as described under "Experimental Procedures." Each panel shows a typical trace from a representative experiment.

creases in free cytoplasmic Ca" required the presence of extracellular Ca", cell suspensions were treated with 1.2 mM EGTA to chelate Ca" prior to treatment with angiotensin 11. Under these conditions, 10 nM ( d a t a not shown) and 100 nM (Fig. 3C) angiotensin 11 elicited a rise in quin2 fluorescence, while a concentration of 1.0 nM did not. In addition, the Ca2+ channel blocker, La3+, in concentrations up to 1 mM, partially attenuated the response to 1 nM but not 100 nM angiotensin I1 (data not shown). These results indicated that changes in [Caz+]i following exposure of cells to higher concentrations (greater than 10 nM) of angiotensin I1 were independent of [Caz+]o, whereas the elevations in [Ca2+Ii following exposure of cells to lower concentrations (1.0 nM or less) required the presence of [CaZ+],,.

Previous studies have demonstrated that vascular strips exposed briefly to angiotensin I1 diminish in their ability to contract further following additional treatment with the peptide (27). In the present study, cultured smooth muscle cells that were exposed to an initial pulse of high (100 nM), but

4664 Ca2+ and Phosphoinositides in Smooth Muscle Cells

following additional exposure of cells to the peptide (Fig. 4D),

However, the dependency of the cellular Ca2+ response on

markedly different than that observed with angiotensin 11.

m quired the presence of extracellular Ca2+ to elicit a response m- 100 (Fig. 4C). EGTA at a concentration of 1.2 mM, which was

calculated to decrease [Ca2+]o to 2.0 p ~ , obliterated the cel-

[Arglvasopressin employed, unlike the effect noted with angiotensin I1 (Fig. 3C).

tion of angiotensin I1 or [Arglvasopressin is summarized in Fig. 5. The EC, of angiotensin I1 and [Arglvasopressin ex- pressed as per cent saturation of quinf with Ca2+ was calculated to be approximately 4 and 20 nM, respectively. The

sin I1 was calculated to be greater, being 88.2 2.6% (mean

A - 200 as had been observed with angiotensin 11.

0

+- Both high and low concentrations of [Arglvasopressin re- 0

2 extracellular Ca2+ following exposure to [Arglvasopressin was 1 0 0 8 .

+ o o .= . N

m a

0 lular increase in quin2 fluorescence at all concentrations of I I I

0 8 B The relationship of [Ca2+ji accumulation to the concentra-

0

m

-t S.E.) for angiotensin I1 as compared to 74.1 f 1.0% for O rn

maximal response (per cent saturation of quinf) to angioten-

0 0 [Arglvasopressin. The calculated values for maximal [Ca2+Ii m rn after angiotensin I1 or [Arglvasopressin were 678 * 136 and

257 f 14 nM, respectively. 1 1 I 4 5 6 nifedipine, or diltiazem, which are known to block voltage-

Of further interest was the observation that verapamil,

[Quin 2Ii, mM dependent Ca2+ channels and therefore the influx of Ca2+ into cells (29), had no apparent effect on the increase in [Ca"']i

FIG. 2. Effects of quin2 loading on [Caa'l, and intracellular ATP levels in unstimulated vascular smooth cells. Vascular induced by either angiotensin 'I Or [Arglvasopressin at drug smooth muscle cells obtained from either spontaneously hypertensive concentrations up to 50 nM (data not shown)- rats (.) or Wistar-Kyoto normotensive controls (0) were grown in Effects of Angiotensin II a d IArglVasopressin on culture dishes until confluency was reached. After trypsinization, cell phoinositide Turnover-In cultures previously incubated with

During fluorescence measurements, aliquots were taken from cuvettes trations of angiotensin 11 (100 nM) induced hy&olysis of the to determine the amounts of ATP or intracellular quin2 concentra-

statistical differences were observed when [Ca2+], or ATP levels in phosphates for up to 2o min (Fig* 6B)* In contrast* the either strain of rats were compared. [Caz+]; was calculated to be 114 accumulation of inositol phosphates induced by lower concen- f 2.9 nM (mean f one standard error, n = 17), and ATP was 5.21 f trations of angiotensin 11 (1.0 nM) seemed to plateau after 5 0.73 fmol/cell (n = 12). min (Fig. 6B). [ArglVasopressin (100 nM) elicited a slower

rate of accumulation of inositol phosphates than did 100 nM angiotensin I1 (data not shown).

not low (1.0 nM), angiotensin 11 lost the ability to increase The accumulation of inositol Phosphates was not accom- their free intracellular Ca2+ concentration following further panied by a Progressive decrease in 3H-labeled lipids- After exposure to angiotensin 11 (e.g. responses in left and an initial decrease in the amount of 3H associated with lipids, right paneh, ~ i ~ . 30) . The loss of respons~veness to further the level of 3H in the lipid fraction increased rapidly to levels

exposure of cells to the peptide (data not shown). The appar- '1. [ArglVasopressin (loo n') and A23187 (loo n') ent c6tachyphylaxis~~ did not appear to be due to a loss of cell elicited small increases in 3H-labeled lipids after 20 min (data

without an initial exposure to angiotensin did not lose their 98% phosphatidylinositol, with the remaining 3H associated ability to respond to the peptide (data not shown).

rAr~Vasopressin-induced changes in rca~+li of Vascular Table I summarizes accumulation of various fractions of

Smooth Muscle Cel~--Responses in the quin2 fluorescence of inositol phosphates after the additions of angiotensin I1 and

vascular smooth muscle cells exposed to [Arg]vasopressin [Arglvasopressin in the presence of 10 mM LiCl. Progressive

displayed qualitatively similar characteristics as those in- accumulations of inositol 1-phosphate were observed when

duced by angiotensin 11. First, the responses to [Arglvaso- cells were stimulated by 100 nM angiotensin I1 and [Arg]

pressin were dose-dependent. The temporal pattern of vasopressin. The accumulation of inositol 1,4-bisphosphate

suspensions were loaded with [3H]quin2 as described previously. [3H]inositol to label the phosphoinositide pool, high concen-

tions ([Quin2]i) as described under "Experimental Procedures." No phospholipids with a progressive accumulation Of

exposure to peptide was still apparent 20 min after the initial greater than those Cherved at time (Fig- GA and Table

viability, as cells incubated under identical conditions. but not shown). The 3H label in the lipid Pool was approximately

with polyphosphoinositides.2


FIG. 3. Increases in Caa+-quin2 fluorescence of smooth muscle cells induced by exposure to angiotensin 11. quin2-loaded cell suspensions were prepared as described in the legend to Fig. 1. Angiotensin I1 (AZO, [Sar',Alaa] angiotensin I1 (ISar',Ala81AZI), [Arg] vasopressin (AVP), and EGTA were added to quin2-loaded cell suspensions at concentrations indicated. Each panel shows a typical trace of a representative experiment. A, concentration-response relationship of angiotensin 11-induced Ca2+-quin2 fluorescence; B, effect of [Sarl,Alas]angiotensin on angiotensin I1 response (experiments have shown consistently a more rapid return to base-line upon addition of antagonist); C, effect of EGTA on angiotensin I1 response; D, effect of pretreatment of cells with angiotensin I1 on subsequent cellular response to angiotensin I1 treatment.

100

% t IPP 100 All nM 10 t nM 4 1 .O $. nM

N c .- d N +' rn

[Sar,Ala]All All AVP 103 nM 10 nM 1.0 nM All [Sar,AlalA

1.0 nM 10 nM

E G ~ A Ail A'II 1.2 mM 1.0 nM 100 nM

seemed to reach a plateau or to decrease after 1 min. Only small changes were detected in the accumulation of inositol 1,4,5-trisphosphate. Increases in radiolabeled glycerophosphorylinositol were apparent at 20 min (but not 1 min) and may have been a consequence of increases in [Ca2+Ii.

In the absence of Li+, smaller accumulations of inositol phosphates were observed at 1 min, while no accumulations were detected at 20 min (data not shown). Li+, at concentrations of 10 mM, had no effects on the inositol phosphate levels in unstimulated cells (data not shown).

Accumulations of inositol phosphates induced by angiotensin I1 and [Arglvasopressin were concentration-dependent (Fig. 7), and good correlations were observed between inositol phosphate accumulation and the increase in [Ca"]; (Fig. 8). The effect of these two peptides on the accumulation of inositol phosphates was inhibited by specific antagonists (Fig. 9). The observations correlate well with the results obtained in the quin2 fluorescence studies discussed earlier (see Figs. 3B and 4B). Although the Ca2+ ionophore, A23187 (100 nM) caused a large and sustained increase in quin2 fluorescence (data not shown), this compound failed to induce an accumulation of inositol phosphates (Fig. 9). This observation confirmed previous results, which indicated that phosphatidylinositol breakdown was not a secondary phenomenon to

t t t t 1.O'nM 1OO'nM 100'nM 100'nM

I 1 MIN

an increase in [Ca2+]i ( l l ) , and that increases in [CaZ+li by non-receptor-mediated processes do not necessarily require phosphatidylinositol breakdown.

Fig. 9 also shows that angiotensin I1 and [Arglvasopressin had an additive effect on the accumulation of inositol phosphates. Both of these two peptides caused maximal inositol phosphate accumulation at concentrations of 100 nM (Fig. 7). When 100 nM angiotensin I1 and [Arglvasopressin was added simultaneously to cell cultures, accumulation of inositol phosphates was purely additive when compared to values achieved with angiotensin I1 or [Arglvasopressin alone (Fig. 9).

A comparison of the [Ca2+]; response and of the accumulation of inositol phosphates elicited by angiotensin 11 or [Arg] vasopressin in the presence or absence of extracellular Ca2+ is depicted in Fig. 10. High concentrations of angiotensin I1 (100 nM) resulted in the accumulation of [Ca2+Ii and inositol phosphates even in the presence of 1.2 mM EGTA. However, this concentration of EGTA abolished the cellular accumulation of both [CaZ+li and inositol phosphates following exposure to low dose angiotensin 11 (1.0 nM) and to 100 nM [Arglvasopressin. Hence, receptor-mediated increases in [Ca"']i appeared to correlate well with the accumulation of inositol phosphates, a phenomenon in contrast to the increase in [Ca2+]; elicited by the Ca2+ ionophore A23187, which did


A

FIG. 4. Increases in quin2 fluorescence induced by [Arglvasopres- sin. quin2-loaded cell suspensions were prepared as described. [ArglVasopressin ( A V P ) , angiotensin I1 (AII), [l-(D-mercapto-j3,@-cyclopentamethylenepro- pionic acid), 2-0-methyltyrosine,Arg] vasopressin (AVP antag.), and EGTA were added to quin2-loaded cell suspensions at concentrations indicated. Each panel shows a typical trace of a representative experiment. A, concentration- response relationship of [Arglvasopres- sin-induced Ca2+-quin2 fluorescence; E , effect of [Arglvasopressin antagonist on [Arglvasopressin response (experiments have shown consistently a more rapid return to base-line upon addition of antagonist); C, effect of EGTA on [Arg] vasopressin response; D, effect of pretreatment of cells with [Arglvasopressin on subsequent cellular response to [Arg] vasopressin treatment.

+ 4 4 AVP 1 .O pM 100 nM 10 nM

5 o k I I t- AVP h a g .

1.0 pM AGP All

100nM 100nM

I C t 100 nM 1.0 pM 1.0 nM

AVP AVP antag All

d

t t t t 1.2mM 10nM 100nM EGTA AVP AVP All 100 nM

C

not affect inositol phosphate turnover. Of importance is the observation that 1.2 mM EGTA had little effect on the incorporation of my~[~H]inositol into the total 3H lipid pool or on basal incorporation of 3H label into inositol phosphates (data not shown).

DISCUSSION

The experiments reported in this study demonstrate the ability of smooth muscle cells from the aortas of Wistar- Kyoto and spontaneously hypertensive rats to accumulate intracellular Ca2+ in response to the vasopressor peptides angiotensin I1 and [Arglvasopressin. As has been reported in other cell systems, increases in [Caz+Ii induced by specific hormones were associated with enhanced phosphoinositide turnover, and close correlations between the two events were noted with respect to the concentration of hormones required to elicit responses. Of particular interest was that high levels of angiotensin I1 do not require the presence of extracellular Ca2+ to induce an elevation in [Ca2+Ii and phosphoinositide turnover, a result that suggests that accumulation of [Ca2+Ii can result solely from mobilization of intracellular stores of

D

+ I I I AVP AVP AVP All 100nM

1.0nM 100nM 100nM - 1 MIN

Ca2+. It was also apparent that phosphoinositide breakdown persisted after [Ca"+li had returned to prestimulated values, a finding that may be associated with the cellular mechanism resulting in desensitization to continued exposure to the vasopressor peptides. As no differences in the responses of cells derived from either Wistar-Kyoto or spontaneously hypertensive rats were noted in any of the experiments con- ducted in this study, no attempt has been made to relate findings in this study to the hypertensive disease process.

Numerous reports have demonstrated that events that stimulate Ca2+ mobilization are coupled to phosphoinositide breakdown in many cell systems and have proposed that phosphoinositide breakdown might play a primary role in initiating Ca" mobilization (16). For example, angiotensin I1 and [Arglvasopressin displayed good correlations between Ca2+ accumulation and phosphoinositide turnover in hepatocytes (15) and adrenal glomerulosa cells (17). The data pre- sented in the present study show that these two peptides induced Ca2+ mobilization and phosphoinositide turnover in a parallel manner in vascular smooth muscle cells. The data thus confirm studies in other cell systems.

Both angiotensin I1 and [Arglvasopressin were studied in


”L 0 ’ 0.1 1.0 I 10 I 1w I loo0 I

CONCENTRATION, nM

FIG. 5. Effect of various concentrations of angiotensin 11 and [Arglvasopressin on [Cas+], in smooth muscle cells. quin2- loaded cell suspensions were prepared as described in the legend to Fig. 1. quin2-loaded cell suspensions were challenged with various concentrations of angiotensin I1 (AZZ, 0) or [Arglvasopressin (AVP, A). After a maximal response was obtained, Triton X-100 and Mn2+ were added in a serial manner for the calibration of quin2 fluorescence as described under “Experimental Procedures.” Each point represent mean f one standard error of four or five experiments.

detail because, of all agents investigated in this study, they led to the most dramatic increases in [Ca”]i. Angiotensin I1 and [Arglvasopressin caused 2-6-fold increases in [Ca2+]i at concentrations of peptide that yielded a maximal response. These observed increases in [Ca2+Ii were similar to the proposed range of [Ca2+]; that regulates vascular smooth muscle tone (30). Angiotensin I1 and [Arglvasopressin increased [Ca2+Ii through the stimulation of different receptors, as experiments with specific receptor antagonists indicated. Fur- thermore, angiotensin I1 was five times more potent than [Arglvasopressin in enhancing [Ca2++li. These results were consistent with a previous study that reported KO values in rat aortic smooth muscle cells of 2.3 and 12 nM for angiotensin I1 and [Arglvasopressin, respectively (6).

The observation that Ca2+ channel blockers had no apparent effect on angiotensin 11- and [Arglvasopressin-induced increases in [Ca2+Ii suggested that these two peptides mobilized Ca2+ through mechanisms different from those requiring voltage-dependent Ca2+ channels. An absence of voltage-dependent Ca2+ channels on these smooth muscle cells was also suggested by the failure of KC1 to induce changes in [CaZ+li. In contrast to the present observation in smooth muscle cells, other studies indicate that high concentrations of KC1 can stimulate Ca2+ mobilization in adrenal glomerulosa cells (14), PC12 cells (12), and insulin-secreting RINmSF cells (13). However, Capponi et al. (14) have reported that angiotensin 11-induced mobilization of [Ca2+]; is not blocked by Ca2+- channel blockers in adrenal glomerulosa cells.

The stimulation of phosphoinositide breakdown by angiotensin I1 and [Arglvasopressin correlated well with increases in [Ca2+];, both in terms of the concentration of peptide required to initiate a response and in terms of the dependence on extracellular Ca2+. These observations were consistent with the data on antigen-stimulated leukemic basophils (18). Additionally, the observation that the Ca2+ ionophore A23187 failed to increase phosphoinositide breakdown confirmed previous findings that indicated that only the mobilization of [Ca2+Ii due to receptor-mediatedprocesses, but not Ca2+ influx per se, was coupled to phosphoinositide breakdown (18).

It was also apparent that when phosphoinositide breakdown was maximally stimulated with angiotensin 11, additional phosphoinositide breakdown could be evoked with [Arglvas- opressin. The data are thus consistent with the idea that separate receptor systems may activate a common mechanism

+1.0 nM

5 10 20 TIME, MIN

FIG. 6. Time course of phospholinositide turnover in smooth muscle cells following exposure to angiotensin 11. Vascular

and then labeled with 6 pCi of [3H]inositol as described under smooth muscle cells were grown in 24-well microplates to confluence

“Experimental Procedures.” Cells were then washed with medium B and preincubated for 10 min at 37 “C with 10 mM Li+. Experiments were initiated with the addition of either 100 nM (U) or 1.0 nM ( 0 - - 4) angiotenin I1 and terminated with the addition of chloro-

phosphates ( B ) and 3H-labeled lipids (A) were measured as described form/methanol(1:2) at various incubation times as indicated. Inositol

under “Experimental Procedures.” The release of [3H]inositol phosphates and the change in 3H-labeled lipid levels were represented as per cent of the amount of 3H-labeled lipids at zero time (60,900 f 8,100 dpm, mean & one standard error, n = 5). Each point represents mean .+ one standard error of three to five experiments. In each experiment, levels of 3H-labeled lipid at 20 min were greater than those at 0 min, although the averaged data did not show a statistically significant difference when compared to 0,min. *, p < 0.5 compared to 2 min (standard t test); t, p < 0.025 compared to 0 min (paired t test).

resulting in phosphoinositide breakdown. Others have reached similar conclusions from studies of 32P labeling of the phosphoinositide pool in parotid acinar cells. In these studies, however, 32P labeling in the presence of two agonists was not additive but intermediate of responses evoked by either agonist alone (31). The apparent discrepancy might be a consequence of different approaches used 32P labeling is a measure of phospholipid turnover rather than phospholipid hydrolysis as determined in our studies.

Certain characteristics of the [Ca2+Ii response of vascular smooth muscle cells to angiotensin I1 and [Arglvasopressin warrant discussion. First, different patterns of [Ca2+Ii accumulation were observed when cells were stimulated by either high or low doses of either peptide. The rapid decay in [Ca2+]; observed when cells were stimulated by high doses of angiotensin 11 (210 nM) and [Arglvasopressin (2100 nM) was not due to the metabolism of these peptides by cellular pro- teases, because subsequent treatment of cells with angiotensin I1 and [Arglvasopressin failed to increase [Ca2+Ii.

Second, the rapid decay of the Ca2+ signal did not appear to be a consequence of increased [Ca2+], as a similar decay


TABLE I Effects of angiotensin II and iArg/uasopressin on various fractions of inositol phosphates

Vascular smooth muscle cells were grown in 24-well microplates until confluency was reached. They were then labeled with 10 rCi/ml [3H]inositol as described under "Experimental Procedures." After washing the cells with medium B, cells were preincubated in the same medium with 10 mM Li+ for 10 min. Experiments were initiated at 37 'C by the addition of either 100 nM angiotensin I1 (AII) or 100 nM Arg-vasopressin (AVP) and terminated by the addition of chloroform/methanol(1:2) after a 1- or 20-min incubation. Various fractions of inositol phosphates and 3H-labeled lipids were measured as described under "Experimental Procedures." Each value represents the mean f one standard error of triplicate determinations from a typical experiment.

'H label in each fraction Condition Inositol Inositol Inositol Glycerophosphoryl-

1-phosphate 1,4-bisphosphate 1,4,5-trisphospbate inositol Inositol "H-lahe'ed Total lipids dpm X IO+

None 3.74 f 0.09 1.10 2 0.02 1.01 f 0.09 3.20 f 0.33 1050 f 16.2 214

AI1

1270 f30.8

1 min 11.7 f 2.35" 5.33 f 0.44' 1.30 f 0.04" 2.66 f 0.10 1080 f 11.3 187 1280

20 min 72.5 f 2.06 6.14 f 0.67' 1.49 +. 0.09" 10.5 f 0.61' 987 f 9.76 220 1290 f47.6

f17.2 AVP

1 min 9.36 f 1.63" 3.46 f 0.53" 1.20 f 0.25 2.91 f 0.21 1060 f 17.3 163 1240

20 min 28.1 f 1.20' 1.54 f 0.07' 1.30 f 0.31 5.02 f 0.56" 1060 f 19.7 238 1340 f20.8

f16.4 p < 0.05 compared with no additions.

' p < 0.005 compared with no additions.

CONCENTRATION, nM FIG. 7. Effect of various concentrations of angiotensin 11

and [Arglvasopressin on inositol phosphate accumulation in smooth muscle cells. Vascular smooth muscle cells were grown in 24-well microplates to confluence and then labeled with 6 pCi/ml [3H]inositol, followed by exposure to 10 mM Li+, as described in the legend to Fig. 6. Experiments were initiated by the addition of various concentrations of either angiotensin I1 (AZI, 0) or [Arglvasopressin ( A VP, A). After 20 min of incubation, chloroform/methanol(1:2) was added to cell cultures to terminate the reaction. [3H]Inositol phosphates were measured as described in the legend to Fig. 6. The release of [3H]inosit~l phosphates was represented as per cent of 3H]-labeled lipids in control cell cultures (58,400 f 6,200 dpm, mean f one standard error, n = 2). Each point represents mean f one standard error of two experiments performed in triplicate.

was not observed with A23187, which by itself caused a large increase in [Ca2+];. Nevertheless, Ca2+ efflux was involved in the regulation of [Ca2+]; in stimulated cells as indicated by the accelerated decay in Ca2+ signal upon displacement of

100 -

50-

0 - 4 1

I I I 1 0 l 0 . 1 1.0 10 l o o lo(

CONCENTRATION, nM FIG. 8. Correlation between [Ca'*Ii changes and inositol

phosphate accumulation in smooth muscle cells induced by angiotensin II and [Arglvasopressin. Values for the increase in [Ca2+Ii over unstimulated levels (0, A) and for the increase in inositol phosphate accumulation (0, A) were taken from Figs. 5 and 7, respectively. Maximal response was defined as the value generated in the presence of 100 nM angiotensin 11. Circles represent the response to angiotensin 11, while triangles represent the responses to [Arg] vasopressin.

angiotensin I1 or [Arglvasopressin with antagonists (Figs. 3B and 4B). Presumably, the extent of [CaZ+li increase represented the balance between influx and efflux rates. In previous studies with 2H3 cells, blockade of Ca2+ influx with La3+ ions or by removal of external Ca2+ ions with EGTA resulted in rapid loss of Ca2+ signal (11). From these experiments, it was inferred that Ca2+ efflux rates were increased to balance enhanced rates of influx and that such strategies permitted estimates to be made of efflux rates. The data did not permit conclusion as to whether the efflux pathway was altered kinetically (in K, or V,,,) or whether its kinetics efficiency was increased by, for example, the action of Ca2+-calmodulin on Ca2+-ATPase in the plasma membrane (11). The inability

Ca2' and Phosphoinositides in Smooth Muscle Celki 4669

" All AVP All All AVP A23187 + + + AVP [Sar,Ala] AVP

All antag FIG. 9. Effects of angiotensin 11, [Arg]vasopressin, and

A23187 on inositol phosphate accumulation in smooth muscle

to confluency and then labeled with 6 pCi/ml ['Hlinositol followed cells. Vascular smooth muscle cells were grown in 24-well microplates

by exposure to 10 mM Li+, as described. Experiments were initiated by the addition of 100 nM angiotensin I1 (AII), 100 nM [Arglvaso- pressin (AVP), 100 nM A23187, 1 p~ [Sar',Alaa]angiotensin 11 ([Sar',AlaalAII), or 1 p~ Arg-vasopressin antagonist (AVP antag.). Incubation was terminated by the addition of chloroform/methanol (1:2), and ['H]inositol phosphates were measured as described. The release of ['Hlinositol phosphates was represented as per cent of 'H- labeled in control cell cultures (62,400 -+ 760 dpm). Each bar indicates mean f one standard error of triplicate determinations from a representative experiment. * , p < 0.005 compared with angiotensin II; t, p c 0.005 compared with Arg-vasopressin.

to block the angiotensin 11-induced Caz+ signal in smooth muscle cells by reduction of [Caz+], with EGTA or by La3+ has prevented us from estimating changes in efflux rates. The two possible explanations for the rapid decay in signal, i.e. decreased rates of influx or increased rates of efflux, cannot, therefore, be assessed from the present series of experiments. Rapid loss of Caz+ mobilizing ability by inositol 1,4,5-trisphosphate, a putative second messenger in Ca2+-dependent responses (16), has been observed in permeabilized cell and endoplasmic reticulum membrane preparations (32-35). It was unlikely that loss of inositol, 1,4,5-trisphosphate's mobilizing ability was the explanation for the loss of the Ca" signal in smooth muscle cells. Additional phosphoinositide breakdown and increases in [Ca2+]; could be promoted by a second ligand (e.g. Fig. 40).

Third, the mobilization of [Ca2+]; and the enhanced turnover of phosphoinositides elicited by low concentrations of angiotensin I1 and all concentrations of [Arglvasopressin required the presence of extracellular Ca". In contrast, high doses of angiotensin I1 elicited changes in [Ca2+]i and phosphoinositide turnover in "Ca*+-free" medium ([Ca"'], 2.0 MM). These results suggested that high doses of angiotensin 11 could cause phosphoinositide breakdown and Ca" mobilization through the activation of different mechanisms than those initiated by [Arglvasopressin and low doses of angiotensin 11.

In the present study, angiotensin 11, but not [Arglvasopres- sin, caused statistically significant increases in inositol 1,4,5- trisphosphate, which has been implicated as the specific agent responsible for recruitment of Ca2+ from intracellular membrane Ca2+ pools (32-34). If so, its production might account for the induction of a Ca" signal in the absence of external Caz+ in response to high doses of angiotensin I1 (Fig. 3C). The responses to low doses of angiotensin I1 and [Arglvasopressin, however, appeared to be totally dependent on the supply of

m- 5 g 200- d

4

loo -

L

EGTA IS mM

All, nM

AVP, nM

- + - +

loo l o o 1.0 1.0

A

- +

" _ - l o o l o o

20

10

0 FIG. 10. Effects of EGTA on [Caa+]imobilization and inositol

phosphate accumulation in smooth muscle cells following exposure to angiotensin I1 or [Arglvasopressin. A, vascular smooth muscle cells were grown in culture dishes to confluency. Cells were trypsinized and loaded with quin2 as described. quin2-loaded cell suspensions were challenged with 100 nM angiotensin I1 (AZO or 100 nM [Arglvasopressin (AVP) in the presence or absence of 1.2 mM EGTA. Increase in [Ca"], (A[Ca2+],) was calculated using calibrations with Triton X-100 and MnZ+. Each bar indicates mean f one standard error of two to four experiments. t , p < 0.025. B, vascular smooth muscle cells were grown in 24-well microplates to confluency and then labeled with 6 pCi/ml [3H]inositol, followed by exposure to 10 mM Li', as described. Two min before preincubation with Li' was completed, 1.2 mM EGTA was added to the cells. Experiments were initiated by the addition of angiotensin I1 or [Arglvasopressin. Incu- bations were terminated after 20 min by the addition of chloroform/ methanol (1:2). [3H]Inositol phosphates were measured as described. The release of ['H]inositol phosphate were represented as per cent of 'H-labeled lipids in control cell cultures (63.,500 f 9,900 dpm, n = 6). The addition of EGTA did not alter the amount of 3H-labeled lipids (101 f 5.1% compared to control, n = 3). Each bar indicates mean f one standard error of two to five experiments. * , p < 0.05.

external Ca". As in other cell systems, the mechanism by which the plasma membrane is rendered permeable to Ca" upon receptor stimulation is uncertain (32), but hydrolysis of the phosphoinositides is induced in the smooth muscle cells irrespective of the source of Ca" drawn upon to generate the signal.

In summary, the present data extend earlier reports on the close relationship between phosphoinositide turnover and Ca2+ mobilization activated by receptor-mediated mechanisms. Furthermore, these data indicate that aortic smooth muscle cells respond to the low and high doses of angiotensin I1 and [Arglvasopressin in different ways. These observations suggest that cultured vascular smooth muscle cells might be a good model system by which to clarify the mechanisms of receptor-mediated Ca2+ mobilization, as well as by which to investigate the mechanisms of Ca2+ regulation in vascular smooth muscle.

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Increase in (Tytosolic Calcium and Phosphoinositide Metabolism