1 Department of Pediatrics, The source of
early production of
sn-1,2-diacylglycerol (DAG) has for a
long time been exclusively linked to hydrolysis of phosphatidylinositol
4,5-diphosphate, which on receptor activation is hydrolyzed into DAG
and inositol 1,4,5-trisphosphate. We have investigated the
origin of lipid sources of DAG production in smooth muscle cells, in
response to contraction induced by peptide agonists. We have performed
a quantitative analysis of the molecular species of DAG formed in
relation to the known molecular composition of parent phospholipids.
The molecular species of phospholipids are sufficiently unique that the
phospholipid origin of DAGs and its quantitative contribution to their
formation can be measured by HPLC. Cell suspensions (10-15 × 106 cells/ml) from the circular
muscle of rabbit rectosigmoid were incubated in the presence of the
contractile peptide agonist bombesin (BB) at
10
diacylglycerol analysis; phosphatidylcholine; phosphatidylinositol
THE SIGNAL-TRANSDUCTION events that stimulate
functional responses in many cell systems are associated with
activation of phospholipase C (PLC) and the hydrolysis of
phosphatidylinositol 4,5-diphosphate
(PIP2), resulting in the
production of inositol 1,4,5-trisphosphate
(IP3) and
sn-1,2-diacylglycerol (DAG).
IP3 causes the release of
Ca2+ from intracellular stores,
producing a Ca2+-calmodulin
complex, which (in smooth muscle) activates myosin light-chain kinase
and causes phosphorylation of the 20,000-Da myosin light chain,
resulting in contraction. DAG causes activation of protein kinase C
(PKC), a family of isoenzymes implicated in signal transduction, which
induces sustained contraction of smooth muscle cells (4).
Early studies had focused on PLC-mediated phosphoinositide hydrolysis
as an intracellular signaling system for DAG generation. The
establishment of DAG as a second messenger was confirmed by the
measurement of the increase in the mass of DAG after stimulation, and
the enhancement of stearic and arachidonic acids in the incremental fraction of DAG supported the involvement of phosphoinositide hydrolysis (27). More recently, it has become increasingly apparent that nonphosphoinositide sources of DAG, especially arising from agonist-stimulated PLC- and PLD-activated phosphatidylcholine (PC)
breakdown, exist (11, 27). DAG coproduced from hydrolysis of other
phospholipid sources, such as PC, is important in the PKC-mediated
pathway for contraction (3, 9, 11). The activation of PKC by DAG
appears to play a pivotal role in signal- transduction events involved
in such processes as growth, differentiation, and contraction (25).
Evidence has been cited in support of a PKC-mediated pathway as an
alternative signaling mechanism for regulating excitation-contraction
coupling in smooth muscle and explains phorbol-induced contraction of
smooth muscle without increases in cytosolic
Ca2+ or in myosin light- chain
phosphorylation (17). It was of interest to us, therefore, to examine
the contribution of nonphosphoinositide DAG sources in
isolated smooth muscle cells of the rabbit rectosigmoid. For this
purpose, the use of benzoylated DAG ( We focused on the time course of formation of the molecular species of
newly generated DAG to 1) deduce the
phospholipid origin of the new DAG formed during bombesin-induced
contraction and 2) measure the
approximate relative contributions of these phospholipid DAG
precursors. The data indicate that PC appears to be the major phospholipid source for newly generated DAG during both short- and
long-term stimulation with bombesin. In addition, our data support the
hypothesis that PKC plays an important role as a mediator of this
contractile response in rabbit rectosigmoid smooth muscle cells. This
in turn not only supports similar findings made by others but also
reaffirms our knowledge of the importance of nonphosphoinositide phospholipids as participants in second-messenger generation during the
early stages of upstream signaling at the cell membrane.
Materials
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
6 M. Reactions were
stopped at different time intervals from 30 s to 4 min. DAGs were
extracted, purified by TLC, and benzoylated with benzoic anhydride. The
benzoylated DAGs were first purified by TLC and then by normal phase
HPLC before they were injected onto a reverse-phase column and eluted
isocratically. Furthermore, phospholipids in the lipid extract
[phosphatidylinositol (PI), phosphatidylcholine (PC),
phosphatidylserine (PS), and phosphatidylethanolamine (PE)] were
purified by TLC and similarly analyzed after hydrolysis to DAGs with
phospholipase C (PLC). The DAG molecular species profiles for PI, PC,
PS, and PE were all unique. Contraction of cells with BB gave
noticeable increases (17-55%) in newly formed DAGs. The major
phospholipid source of the newly formed DAGs at 30 s was only ~30%
from PI, and the remainder was from PC. In contrast, after 4 min of BB
stimulation, a decrease was seen in newly formed DAGs in the peak
specific for PI hydrolysis. The data suggest that BB-induced
contraction by activation of PLCs results in hydrolysis of different
phospholipids. The DAGs formed as a result are qualitatively and
quantitatively distinct. This could be the basis for the kinetically
different pattern of sustained contraction observed with BB.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-DAG) derivatives and their
analysis by HPLC, known to be both sensitive and reliable (18, 19), was
adopted as the preferred method of analysis. This technique allows the
quantitative measurement of the actual new DAG mass formed, the
determination of which may be somewhat complicated with the use of
radiolabeling approaches. Another significant advantage of using this
chromatographic method over a popular radiometric assay, which relies
on enzymatically phosphorylating DAG by DAG kinase with
[
-32P]ATP and
quantitating the resulting
[32P]phosphatidic
acid, is that it allows a profiling, or fingerprinting, of the
different molecular species of DAG (18).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-D-glucopyranoside (O
DG), diethylenetriamine-pentaacetic acid
,
-bis(biocytinamide)
(DETAPAC), and ATP were from Sigma Chemical (St. Louis, MO). Micelles
were prepared by dissolving 25 mg of cardiolipin (CL; Avanti Polar Lipids) and 250 mg of recrystallized O
DG simultaneously with 3.33 ml
of 1 mM DETAPAC. The mixture was then warmed to 37°C for 30 min and
gently vortexed. Aliquots were then frozen and stored at
70°C. DAG kinase was purchased from Lipidex (Westfield, NJ), and dithiothreitol (DTT) was from Boehringer-Mannheim (Indianapolis, IN). [
-32P]ATP (10 µCi/µl) and enhanced chemiluminescence detection reagents were
obtained from Amersham (Arlington Heights, IL).
Collagenase type 2 (CLS type II) was from Worthington Biochemical
(Freehold, NJ). Calphostin C was from Kamiya Biomedical (Thousand Oaks,
CA) and chelerythrine from Alomone Labs (Jerusalem, Israel). All other reagents were purchased from Sigma Chemical.
HPLC assay. HEPES, Tris, phospholipid standards, Triton X-100, 1,2-distearoyl-rac-glycerol (DSG), 14% (wt/vol) boron trifluoride-methanol, 4-pyrrolidinopyridine, benzoic anhydride, B. cereus PLC, bombesin, 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine, and cyclohexane (redistilled) were from Sigma Chemical. Ham's F-12 medium, DMEM, and N-2 supplement were obtained from GIBCO (Grand Island, NY). PUFA-2 mix fatty acid methyl ester standard for gas liquid chromatography was from Matreya (Pleasant Gap, PA). HPLC grade solvents were from Mallinckrodt Specialty Chemicals (Paris, KY) or Burdick & Jackson (Baxter Healthcare, Muskegon, MI). An HPLC solvent-delivery system consisted of two Waters M-45 pumps (Milford, MA) coupled to a Rheodyne model 7125 syringe-loading rotary sample-injector valve equipped with a 50-µl sample loop (Rheodyne Cotati). Analytic Silica gel 60-precoated glass TLC plates were from Merck (Darmstadt, Germany). All other reagents were purchased from Sigma Chemical.
Methods
Isolation of smooth muscle cells from rabbit rectosigmoid. The internal anal sphincter, consisting of the most distal 3 mm of the circular muscle layer, ending at the junction of skin and mucosa, was removed by sharp dissection. A 5-cm length of the rectosigmoid orad to the junction was dissected and digested to yield isolated smooth muscle cells. Cells were isolated as previously described (4, 5). The tissue was incubated for two successive 60-min periods at 31°C in 15 ml of HEPES buffer (pH 7.4) containing 0.1% (wt/vol) collagenase (150 U/mg, Worthington CLS type II) and 0.01% (wt/vol) soybean trypsin inhibitor. At the end of the second enzymatic incubation period, the medium was filtered through 500-µm Nitex mesh. The partially digested tissue left on the filter was washed four times with 50 ml of collagenase-free buffer solution. The tissue was then transferred into 15 ml of fresh buffer solution and incubated for 30 min to allow the cells to disperse spontaneously. After a hemocytometric cell count, the harvested cells were resuspended in maintenance medium, pH 7.4, containing Ham's F-12 medium, DMEM, and N-2 supplement (50:50:1, vol/vol) and aliquoted into glass scintillation vials in 1-ml portions. These cells were allowed to equilibrate for 90 min at 37°C in a humidified 95% air-5% CO2 (vol/vol) incubator atmosphere before either control, inhibitor, or agonist-stimulated treatments were initiated. Each rectosigmoid yielded 15-20 × 106 cells.
Biosynthesis and extraction of DAG. Smooth muscle cells at a concentration of 1.5 × 106 cells/ml were dispensed in 200-µl aliquots in 16 × 100 mm glass tubes. The tubes were incubated in a humidified CO2 incubator at 37°C and allowed to equilibrate for 2 h. An aliquot of cells was examined under the microscope, and cell measurement was performed at this stage. We then added 22-µl aliquots of DMEM or agonists at 10 times the desired concentration, and the reaction was stopped at the appropriate times by adding 0.6 ml ice-cold 1% perchloric acid and placing the tubes in a melting ice bath. We then added 3 ml ice-cold chloroform-methanol (1:1, vol/vol), and the lipids were extracted for 15 min. To separate the phases, 1 ml of 1 M NaCl and 1 ml of CHCl3 were added to each tube and vortexed. The tubes were then centrifuged at 1,500-2,000 g for 5 min. Taking care not to disturb the interface, we then aspirated and discarded part of the aqueous top layer. A 1.5-ml aliquot of the organic lower layer was then removed from each tube, evaporated to dryness under N2, and capped immediately under N2. The extracted lipids were assayed within 24 h.
DAG (phosphatidic acid) assay.
To each cell extract and/or diolein standard, 20 µl of
micelles prewarmed to 37°C were added, and the mixture was vortexed and sonicated for 10 min at room temperature. An assay buffer was
prepared for each sample (50 µl of 2× assay buffer, 10 µl of
20 mM DTT, 10 µl DAG kinase, and 5 µl of 20 mM fresh ATP), and the
mixture was allowed to incubate at room temperature for 20 min. Then 1 µCi
[-32P]ATP per
sample was added, and 80 µl of this mixture were added to each sample
and allowed to incubate for 30 min at 25°C. The reaction was
stopped with 0.7 ml of cold 1%
HClO4. The lipids were then
extracted by adding 3 ml
CHCl3/MeOH (1:2, vol/vol) and vortexing. Phases were then separated by adding 1 ml of 1 M NaCl and 1 ml CHCl3, followed by vortexing
and centrifugation. We then evaporated 1 ml of the organic layer to
dryness under N2. The dried lipid
was reconstituted in 5% MetOH
CH3OH/MeOH in
CHCl3 and applied to preactivated
TLC plates (Silica gel 60). The plates were placed in a preequilibrated
chromatography chamber containing a mixture of
CHCl3, MeOH, acetone, acetic acid,
and water (30:9.8:11.3:9:5.3, vol/vol) for 30 min and then exposed to
X-ray film (X-OMat, XAR-2). The positions of phosphatidic
acid spots were determined on the film, and then they were scraped from
the TLC plate and quantified by liquid scintillation counting in 5 ml
Ecolume (ICN Radiochemicals, Costa Mesa, CA).
Preparation of whole lipid extracts.
After an appropriate incubation period with or without agonist at
37°C, 3.4 ml of 1:2 (vol/vol) chloroform-methanol quenching solution was added to each 1-ml cell suspension (10-20 × 106 cells), and the mixture was
shaken vigorously. We then added 2-3 nmol of DSG internal standard
to each sample and mixed them well. The samples were sonicated for 20 s
in an ultrasonic bath and then centrifuged at 10,000 g for 10 min. The supernatants were
collected, and the pellets were reextracted with 2 ml of 1:1 (vol/vol)
chloroform-methanol, sonicated, and centrifuged as described above.
With the combined supernatants from each sample, 1 ml of chloroform was
mixed in well and then 1.4 ml of 0.9% (wt/vol) NaCl was added and
mixed by vortexing. The aqueous and organic layers were resolved by
centrifuging at 10,000 g for 5 min.
After the upper layer was removed, the lower layer was dried under
blowing N2 and redissolved with
200-500 µl of 2:1 (vol/vol) chloroform-methanol. The DAGs were
immediately purified from this extract by TLC and then quickly
benzoylated as described below. Otherwise, the extract was stored under
N2 at 20°C for
isolation and analysis of phospholipids.
Purification and quantitation of phospholipids. Individual major phospholipid bands [i.e., PC, phosphatidylserine (PS), phosphatidylinositol (PI), CL, phosphatidylethanolamine (PE), and sphingomyelin (SM)] were purified from neutral lipid extracts by applying ~50-100 nmol of the phospholipids as 2-cm bands on 20 × 20-cm Silica gel 60 glass TLC plates (0.25-mm layer thickness) and doubly developing up to 1 cm from the top using the chloroform-methanol-0.25% (wt/vol) KCl-ethyl acetate-isopropanol (150:45:30:90:125, vol/vol) solvent system of Hedegaard and Jensen (14). The bands were visualized by spraying them with 0.001% (wt/vol) primulin in 4:1 (vol/vol) acetone-water and circling them with a pencil under long-wave ultraviolet (365 nm) light. Then we scraped each band and equivalent blank areas as blanks. The amount of each phospholipid was determined by washing the bands with Mg(NO3)2 over a flame and measuring the liberated total Pi with acid-molybdate reagent by the method of Ames (2). Using a standard curve prepared simultaneously with potassium dihydrogen phosphate as a standard, we determined the amount of phospholipid from the absorbance measured at 660 nm after removal of silica by centrifuging at 1,000 g for 5 min.
PLC treatment of phospholipids or whole lipid extracts. Individual phospholipid bands purified by TLC [chloroform-methanol-0.25% (wt/vol) KCl-ethyl acetate-isopropanol (150:45:30:90:125, vol/vol)] as described above were extracted with 2 × 3 ml of 2:1 (vol/vol) chloroform-methanol. For PI, 2 × 3 ml of 2:1 (vol/vol) chloroform-methanol containing 0.25% (wt/vol) HCl were used instead, and the HCl was removed by shaking with 1.5 ml of water, centrifuging at 1,000 g for 5 min to separate the phases, and collecting the lower layer. After the extracts were pooled from several bands and dried under N2, they were treated as described below for the preparation of DAGs derived from whole lipid extracts. Dried whole lipid extracts (20 × 106 cells) or purified phospholipid extracts (as described above) were sonicated for 1 min with 0.3 ml of 0.25% (wt/vol) Triton X-100 in 50 mM Tris · HCl buffer (pH 7.5) containing 10 U of B. cereus PLC and incubated overnight at 37°C under N2 in sealed glass test tubes. The liberated DAGs were extracted with 5 ml of 2:1 (vol/vol) chloroform-methanol and 1 ml of 0.9% (wt/vol) NaCl; the lower layer collected after centrifuging at 1,000 g for 5 min was dried under N2, redissolved with 200 µl (600 µl for whole lipid extracts) of 2:1 (vol/vol) chloroform-methanol, and then purified by TLC and benzoylated as described below.
DAG purification and benzoylation protocol. Samples of 200 µl of whole lipid extract or phospholipid from PLC treatment were applied as 2-cm bands on a Silica gel 60 20 × 20 cm glass plate (0.25-mm layer thickness) and developed in a mixture of toluene, ether, and methanol (80:10:10, vol/vol) up to 10 cm from the origin. The DAG band [retention factor (Rf) = 0.58], corresponding with the internal standard, DSG (Rf = 0.58), was visualized by spraying with 0.001% (wt/vol) primulin in acetone-water (4:1, vol/vol) and then circling the bands with a pencil under long-wave ultraviolet (365 nm) light. The DAG bands were scraped into test tubes, extracted once with 2 ml ether (distilled), and centrifuged at 1,000 g for 5 min. The pellet was then reextracted twice with 2 ml ether each time. The combined extracts were dried under blowing N2. To each tube of dried DAGs were added 100 µl of dry benzene, 10 µl (6.8 µmol) of 0.68 M 4-pyrrolidinopyridine in dry benzene, and 10 µl (5.5 µmol) of 0.55 M benzoic anhydride in dry benzene. The contents were mixed well and the tubes were gassed with N2 and incubated overnight at room temperature. The next morning, the reaction was stopped by adding 100 µl of methanol to each tube. The tubes were allowed to stand for 5 min after methanol was added. (A similar control reaction mixture was run in a separate tube containing no DAG.)
Purification and reverse-phase HPLC analysis of
-DAGs.
The benzoylation reactions were applied in toto to a 20 × 20 cm
glass plate of Silica gel 60 (0.25-mm layer) as a 2-cm band along with
the control reaction. After development of the plate in a mixture of
benzene, hexane, ether, and 30% (wt/wt) ammonia (50:45:5:1, vol/vol)
up to 1 cm from the top, the
-DAG
(Rf = 0.34) band and corresponding
benzoylated internal standard,
-DSG (Rf = 0.34), were visualized with
primulin after 10-15 min air-drying and scraped into clean glass
tubes. The
-DAGs were extracted with 2 ml of ether, centrifuged at
1,000 g for 5 min, and reextracted from the pellets twice more with 2 ml ether each time. The combined ether extracts were dried under blowing
N2 and taken up with 200 µl of
distilled cyclohexane-ether (95.5:4.5, vol/vol) after filtering through
a 0.45-µm Teflon syringe filter. The sample was stored under
N2 at
20°C for further
purification by normal-phase HPLC before analysis on a reverse-phase
HPLC column (Ultrasphere ODS, 5 µm, 4.6 mm × 25 cm; Beckman,
San Ramon, CA). For cleanup on a normal-phase column (Microsorb Si, 5 µm, 4.6 mm × 25 cm; Rainin Instrument, Woburn, MA), the sample, in
normal-phase solvent, was concentrated under blowing
N2 to ~50-100 µl before
injecting ~50 µl onto the column and then equilibrated with
cyclohexane-ether (95.5:4.5, vol/vol) at 1.0 ml/min.
-DAG (retention
time = ~10 min) was monitored at 228 nm with a Spectroflow 773 absorbance detector (Kratos Analytical Instruments, Westwood, NJ). Then
the effluent was collected and stored under
N2 at
20°C or dried
under blowing N2 and redissolved
with ~200 µl of reverse-phase solvent and either stored at
20°C under N2 or
concentrated to ~50-100 µl with blowing
N2 before injection of ~50 µl
for reverse-phase analysis on a column equilibrated at 1.0 ml/min with
acetonitrile-isopropanol (70:30, vol/vol).
-DAG peaks detected
on-line at 228 nm were quantitated by electronic integration of peak
areas with a Macintosh SE personal computer interfaced with an on-line
data acquisition system (MacIntegrator II, Rainin Instrument). To
calculate actual amounts present in the original sample, we multiplied
areas relative to the
-DSG internal standard by the fixed amount of
internal standard added at the beginning of sample workup, assuming
equal and linear detector responses for all
-DAG.
Formation and purification of fatty acid methyl esters from
phospholipids and DAGs.
-DAGs isolated from reverse-phase HPLC or individual phospholipid
bands (~50-100 nmol) purified by Silica gel TLC
[chloroform-methanol-0.25% (wt/vol) KCl-ethyl
acetate-isopropanol (150:45:30:90:125, vol/vol)] from neutral
lipid extracts (i.e., PC, PS, PI, CL, PE, and SM) were placed in 16 × 100 mm Teflon-lined screw cap glass test tubes. A known
quantity of triheptadecanoyl glycerol (5-10 nmol) was added to
each tube, and the contents were dried under blowing N2. The glycerolipids were then
methanolyzed in 0.5 ml of 14% (wt/vol)
BF3-methanol (Sigma Chemical) in
sealed tubes for 1 h at 60°C after flushing the tubes with
N2. SM was methanolyzed under the
same conditions, except that 0.5 ml chloroform (for solubilization) and
0.5 ml of methanol-concentrated HCl (5:1, vol/vol) were used for 20 h.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phospholipid Composition of Isolated Rabbit Rectosigmoid Smooth Muscle Cells
Table 1 shows the relative amounts of the major phospholipid components found in rabbit colon smooth muscle cells after their separation from whole lipid extracts by TLC. In order of increasing polarity, the relative amounts were (in mol/100 mol) 24 PE, 2 CL, 6 PI, 11 PS, 38 PC, and 20 SM. These values are comparable to those seen in other tissues, such as rat mesenteric and aortic vascular smooth muscle cells [(in mol/100 mol) 25 PE, 8 PI, 10 PS, 40 PC, and 15 SM] (10).
|
Molecular Species Composition of DAGs from Whole Lipid Extracts of Isolated Smooth Muscle Cells
As shown in Fig. 1, reverse-phase HPLC analysis after purification and benzoylation of smooth muscle diglycerides present in whole lipid extracts revealed the presence of 11 major DAG peaks. These peaks were subsequently collected and individually analyzed for fatty acid content by gas chromatography of the fatty acid methyl esters formed by transesterification with boron trifluoride-methanol. Table 2 lists the identity of the major fatty acids found by gas chromatographic analysis for each major DAG peak. The species identification of the DAGs in our extracts was facilitated by comparison of our retention time data with those previously published by Lee and Hajra (19). After the relative retention times are normalized, the data indicate a good correspondence in retention between the molecular species of DAG as identified in smooth muscle and in other tissues.
|
|
Phospholipid Fingerprinting: Molecular Species Composition of DAGs of Phospholipids Purified from Whole Lipid Extracts of Isolated Rabbit Smooth Muscle Cells
Figure 2 shows the reverse-phase HPLC DAG profiles of the individual phospholipids purified from isolated smooth muscle cells of the rabbit rectosigmoid. Distinctive patterns of DAGs are evident for each individual phospholipid. Because these unique profiles can serve as phospholipid "fingerprints," they are useful in tracing the origin of new DAGs formed on agonist stimulation. For example, if DAG production after a contractile stimulus is due solely to phosphoinositide hydrolysis, then the pattern of newly formed DAGs, i.e., above the control values, should mirror those that were obtained for PI. However, more complex patterns can arise if there are a multiplicity of phospholipids contributing to the production of new agonist-induced DAGs, and one would then require a calculation of the expected distribution of DAGs that would match the observed profile by assuming various values for the fractional contributions made by each contributing phospholipid. In this manner, we were able to give an approximation of the relative contributions of PI, PS, PC, and PE that is most consistent with the profile of newly formed DAGs, using the values shown in Table 3 for the various major DAG peaks obtained from each phospholipid HPLC profile.
|
|
Effect of Bombesin Stimulation on DAG Levels in Isolated Rabbit Colon Smooth Muscle Cells
Stimulation of isolated smooth muscle cells with the contractile agonist bombesin causes a substantial elevation of newly formed DAG levels at 30 s and 4 min. Newly formed DAG increased significantly (0.01 > P > 0.001) by 29.2 ± 4.1% (n = 3) at 30 s and by 81.5 ± 9.6% (n = 3) at 4 min above the control levels. The results indicate that bombesin-induced sustained contraction is associated with an increase in newly formed DAG resulting from hydrolysis of phospholipids and that DAG production is associated with both early transient (phasic) and sustained (tonic) smooth muscle contraction induced by bombesin.
|
Effect of TPA Stimulation on DAG Levels and Inhibition of TPA-Induced DAG Production by PKC Inhibitor Calphostin C in Isolated Rabbit Colon Smooth Muscle Cells
We have previously shown that bombesin-induced contraction is inhibited by the PKC antagonists (28). We have investigated whether direct activation of PKC results in the formation of newly formed DAG. Because many signal-transduction processes are mediated by PKC activation, to further elucidate the early steps of signal transduction in the contractile response we next investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA), a DAG analog known both as an effector/activator of PKC and as a contractile agent. Figure 3 shows that in response to TPA ester, a known regulator/activator of PKC, smooth muscle-induced contraction was associated with an increase in newly formed DAG. This stimulation is significantly blocked after preincubation with calphostin C, an inhibitor of the regulatory site of PKC (12). The data suggest that activation of PKC by TPA results in DAG production, and this stimulation is blocked by calphostin C.Origin of DAG Production Stimulated by Bombesin
The distinctive patterns of DAGs that are evident for each individual phospholipid (Fig. 2) are useful in tracking the origin of new DAGs formed on agonist stimulation. For example, if DAG production after stimulation is due to merely a random fluctuation in our experimental measurement of DAG levels or, possibly, an identical release of more old, "preexisting DAGs" from the same pool of DAG precursors existing before stimulation, then the pattern of elicited DAGs should mirror those that were obtained for the control (unstimulated) samples. A comparison of the DAG profile in response to bombesin (Table 4) with the corresponding profile in unstimulated controls (Table 2) reveals that there is a significant change in the contribution of certain DAG molecular species after stimulation. The largest of these changes, shown graphically in Fig. 4, are marked by a relative increase in the molar fraction (mol/100 mol) for peaks 2, 4, and 6 but a relative decrease in the molar fraction of peaks 9 and 11. These changes indicate that the new DAG must be coming from a new source or process, presumably via glycerophospholipid hydrolysis stimulated by bombesin.
|
|
If one were to consider the possibility that the new DAG produced in response to bombesin might be due solely to phosphoinositide hydrolysis, then the pattern of elicited DAGs should reflect those that were obtained for PI (Fig. 2). Figure 5 compares the profile of new DAG in response to bombesin stimulation with that of PI (mol/100 mol). These new DAG and PI profiles do not match, and therefore we must consider alternatives to account for the experimentally observed composition of new DAG molecules in response to bombesin stimulation. Comparisons made with other phospholipid DAG profiles (Fig. 5) also indicate that no single phospholipid can uniquely determine the experimentally observed new DAG pattern formed in response to bombesin stimulation. The remaining consideration is that matching DAG patterns can arise if many phospholipids contribute to the production of new agonist-induced DAGs. This requires a calculation of the expected distribution of DAGs that would match the observed profile by assuming various values for the fractional contributions made by each contributing phospholipid. In this manner, we are able to give an approximation of the relative contributions of PI, PC, PS, and PE consistent with the profile of newly formed DAGs, using the data from Table 3 for the various major DAG peaks obtained from each phospholipid HPLC profile.
|
Approximation of Relative Contributions of PI, PC, PS, and PE Most Consistent with Profile of Newly Formed DAGs
Figures 6-9 show the comparison between observed and calculated new DAG values (mol/100 mol) using the original calculated fractional values that were supposed to give a good fit to the experimental data. The calculated fractional values for the major phospholipids (PC, PE, PS, and PI) that were assumed to contribute to DAG production were arrived at by calculations involving solutions for three unknown variables, i.e., the DAG values (mol/100 mol) for three phospholipids, in two simultaneous equations (Table 5). One of the variables was set to a fixed value (e.g., setting the fraction of PS or PE at 0.1, or 0.2, or 0.3,...etc.) and solving for the other two (e.g., PI and PC) according to the equation
![]() |
(1) |
|
After the PI fraction values from several (n = 3 and and n = 5) experiments were calculated for each peak and then averaged, the major peaks having the lowest SE for those values were found to also yield values that were consistent, or close to each other. It was also noted that these peaks corresponded to peaks where there was a large difference in the DAG values (mol/100 mol) for PI and PC. Peaks 2, 4, and 6 gave the most consistent values, because the differences in the PI and PC values (mol/100 mol) were greatest for these peaks, and were deemed to be the most reliable ones for determining which PI fraction values fit the data most closely.
For each value of the fixed variable, these PI fraction values were then averaged and a standard deviation was calculated to determine their closeness. The calculation for a given fixed variable value showing the greatest consistency in PI fraction values, i.e., having the lowest standard deviation, was selected as the one yielding the best fractional values. Thus the calculated values of DAG (mol/100 mol) would most closely match the experimental data for DAG (mol/100 mol) in response to bombesin stimulation.
The logic in these calculations is that if our calculated mean PI fraction value is a "best-fit" value for the PI fraction value and is actually a good approximation to some true hypothetical mean PI fraction value for the observed data, then there should be a minimum spread, measured by the standard deviation, in the computed PI fraction values among peaks. In other words, two calculated PI fraction values that are close together (low standard deviation) will probably give a better approximation to the mean value than two that are further apart. Because each peak should ideally give the same hypothetical mean PI fraction value, then deviations in calculated PI fraction value among peaks at a given value of fixed variable reflect the variation in the observed new DAG value (mol/100 mol) from some true hypothetical mean new DAG value (mol/100 mol) for those peaks.
The validity of using this approach was tested by using hypothetical data, which showed that the calculated PI fraction values varied more from their true value as the new DAG value (mol/100 mol) varied more from its true value, for a given value of fixed variable. Also, from this hypothetical data, the spread of calculated PI fraction values was found to increase as the fixed variable (either the PS fraction value or the PC fraction value) was allowed to vary from its true value, and the approximation to the true mean PI fraction value was poorer as well.
Having found our approximate solution to the fractional values for PI, PC, PS, and PE, their goodness of fit to the experimentally observed data was then verified by substituting those values back into the equation
![]() |
(2) |
Figure 6 shows the comparison between observed and calculated new DAG values (in mol/100 mol, at 30 s poststimulation with bombesin) using the original calculated fractional values that were supposed to give a good fit to the experimental data. There was good agreement for peak 6, but for the other two major peaks, peaks 4 and 8, there was a rather large relative difference between calculated and observed values. However, by increasing the relative contributions from PC we found a better fit for peaks 4 and 8, i.e., the relative error, or deviation, between calculated and observed DAG values (mol/100 mol) was now more uniform.
|
At 30 s after stimulation with bombesin, it appears that a good approximation for the fractional value from PI is somewhere in the range of 0.1-0.4, with the remainder of the new DAG produced apparently coming from either PC (0.66-0.70) and PS (0-0.10) or a combination of PC (0.45-0.50) and PE (0.1-0.45). A similar analysis suggested that PI = 0.3 ± 0.1 and PC = 0.7 ± 0.1 would be a best estimate for the data at 4 min (Fig. 7). As shown in Fig. 8, preincubation of the cells with the PC-specific PLC inhibitor D609 resulted in inhibition of DAG production in response to bombesin, confirming that the main source of DAG is from hydrolysis of PC. Comparable results were also obtained for the new DAG elicited by stimulation with TPA, which revealed that, at both 30 s and 4 min (Fig. 9), there are contributions of ~20 ± 10% from PI and 80 ± 10% from PC. The data clearly indicate that PI cannot account solely for the newly formed DAG, during both short-term phasic (30-s) and long-term sustained (4-min) contraction, and that PC appears to be the major contributor for DAG production in both of these stages.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In many types of smooth muscle cells, agonist-induced contraction has been associated with a rise in intracellular Ca2+ concentration (6, 21, 24). The rise in intracellular Ca2+ concentration activates the calmodulin-dependent enzyme, myosin light-chain kinase (1, 13, 29), which results in phosphorylation of the 20-kDa myosin light chain. This leads to interaction between myosin and actin, which, in turn, initiates the contractile response.
Agonist-induced production of IP3 and DAG is not always equivalent. In Swiss 3T3 mouse fibroblasts (7), bombesin stimulates the release of both IP3 and DAG. The increase in IP3 is rapid and transient, returning to basal levels by 30 s. In contrast, the increase in DAG is biphasic: the first phase (0-30 s) mirrors the transient IP3-mediated response, whereas the second phase is sustained (up to 60 min) and occurs in the absence of elevated IP3, at a time when IP3 generation is not occurring. The possibility that PC serves as an alternative source for DAG production is suggested by a strong temporal correlation between bombesin- and PMA-stimulated [3H]choline release (8) and by a correlation between PMA-stimulated DAG production (30) and the second phase of bombesin-stimulated DAG production.
It is, therefore, possible that PIP2 hydrolysis may not be the sole source of bombesin-stimulated DAG generation, but that a second source may be present, possibly PC. In addition to bombesin, other peptides such as endothelin, a contractile peptide of vascular smooth muscle, activate with unique selectivity and potency the DAG/PKC pathway (20). Thus the differential activation of IP3 and PKC may depend on differential activation of distinct phospholipases, resulting in different mixes of second messengers such as phosphoinositides and/or DAG, depending on the agonist employed.
Phosphoinositides represent ~6% of the phospholipids present in the
plasmalemma of total cellular phospholipid of rectosigmoid smooth
muscle cells. Ligand binding causes PLC-induced PI hydrolysis and
results in equimolar production of
IP3 and DAG. As suggested by
others (8), there is much evidence that PC hydrolysis is a major source
for sustained DAG production in cellular control mechanisms requiring
prolonged activity of PKC and that this role is well suited to
PC. PC is the most abundant cellular phospholipid, accounting for ~50%, and thereby can ensure an ample source of DAG
without dangerously altering the structural/functional integrity of the
membrane (3, 11). We have investigated the relevance of this hypothesis
in smooth muscle cells and the source of DAG and its relationship to
sustained contraction. It was crucial to have a reliable quantitative
measurement of DAG. This is often done radiometrically by an enzymatic
phosphorylation method that, using commercially obtained DAG kinase
contained in a crude membrane preparation from
Escherichia coli, phosphorylates DAG
with [-32P]ATP and
then quantitates the resulting
[32P]phosphatidic acid
after TLC. Lee and Hajra (19) noted difficulty in using this commercial
preparation, as the presence of other hydrolytic enzymes (e.g.,
phosphatases or phospholipases) in this crude preparation could result
in artifactual formation of higher amounts of DAG from phospholipids
present in the cellular lipid extracts being analyzed, or possibly even
those present in the enzyme/phospholipid reagent mixture. It is
possible that even the lipid extracts being analyzed might have
contained some of these contaminating enzymes. The radiometric
technique does not provide a qualitative distinction of the source of
the new DAG produced. Available evidence indicates that the rapid
increase in DAG at 30 s and 4 min after stimulation with bombesin is
all due to hydrolysis. The biosynthetic rate of DAG is too slow to contribute a significant amount to the DAG pool during this time period. For these reasons, the established techniques (18, 19) using
-DAG derivatives and their analysis by HPLC were adopted as the
preferred method of analysis.
Examination of the different phospholipid classes present in smooth muscle cells from rabbit colon confirmed that PC is the most prominent phospholipid, accounting for ~40% of the total cellular phospholipid, whereas PI contributes only 6% (Table 1). Other prominent phospholipids in our system are PE and SM, each constituting ~20% of the total cellular phospholipid pool. The finding of a large amount of SM is rather striking and may be related to our recent discovery of its role as a precursor source for ceramide, an important second messenger and PKC activator in PKC-mediated MAP kinase activation in smooth muscle contraction (28). Reverse-phase analysis of DAGs liberated after individual treatment of the major glycerophospholipids (PC, PE, PS, and PI) with PLC revealed characteristic molecular species differences (Table 3, Fig. 2), which allowed us to determine the source of DAG induced in bombesin-stimulated cells.
Stimulation of contraction in isolated rabbit colon smooth muscle cells with 1 µM bombesin led to a significant (0.01 > P > 0.001) 29.2 ± 4.1% (n = 3) increase in DAG production above the control levels within 30 s of agonist addition (Fig. 3), this period being associated with the transient phase of contraction. In the later stages of the response beyond 30 s, representing sustained tonic contraction, the DAG remained significantly elevated (0.01 > P > 0.001) above the control after 4 min, at a level 81.5 ± 9.6% (n = 3) above control. We have compared the profile of endogenous DAGs present in controls before stimulation with that of DAGs elicited by bombesin. A comparison (Fig. 4) of the newly formed DAG profile in response to stimulation with bombesin (Table 4) with the corresponding profile in unstimulated controls (Table 2) reveals that there was a significant change in the relative moles per 100 mol contribution of certain DAG molecular species after stimulation. The pattern of elicited DAGs in response to bombesin stimulation is different and does not mirror those that were obtained from the unstimulated control samples. The profile indicates the presence of DAG, in specific peaks, where it is not present in the control (unstimulated) cells. These changes are important and indicate that the new profile represents an increase in total DAG.
By comparing the molecular species profiles for newly formed DAGs, arising from bombesin-stimulated contraction (Table 4), with those profiles obtained for the major cellular glycerophospholipids (PC, PE, PS, and PI), we were able to calculate that ~70% of the DAGs formed at both 30 s (Fig. 6) and 4 min (Fig. 4) are due to PC breakdown and that a PLC/PLD-mediated hydrolysis of PC plays a major role in both short- and long-term bombesin-stimulated smooth muscle contraction. The data reaffirm previous reports (3, 11) that in many cellular systems phosphoinositide hydrolysis is not the only source for DAG production and that PC breakdown plays a more prominent role in generating DAG.
It has been reported that stimulation of many cell types by tumor-promoting phorbol esters promotes PC hydrolysis, which may or may not be mediated by PKC, as shown by the use of inactive phorbol ester analogs, PKC inhibitors, or the phenomenon of phorbol ester-induced downregulation of PKC under prolonged exposure (3, 11). This hydrolysis exclusively produces DAG from PC via both PLC- and PLD-mediated pathways and occurs without provoking phosphoinositide hydrolysis. Therefore, stimulation with a phorbol ester such as TPA would be expected to give rise to DAG molecular species that have arisen predominantly, if not exclusively, from PC hydrolysis and provides us with a reference response with which to compare and assess the reliability of our conclusion. Our analysis of the new DAG species induced in TPA-stimulated smooth muscle cells confirmed this expectation. The distribution of new TPA-induced DAG species at both 30 s and 4 min (Fig. 9) was very similar to that obtained with bombesin, except that in the TPA-induced DAG there was a smaller contribution from peak 6, which represents an 18:0-20:4 DAG molecular species (Table 2) containing stearic and arachidonic acids and is the single most prominent characteristic of phosphoinositides.
Using calculations similar to those explained above for bombesin-stimulated new DAG, our data were consistent with ~80% of the TPA-generated DAG being derived from PC hydrolysis, with the remainder coming from phosphoinositides. PC hydrolysis was almost completely blocked by a specific PKC inhibitor, calphostin C. This is also consistent with the knowledge that phorbol esters induce contraction in smooth muscle by a process dependent on PKC, because it can be blocked by PKC inhibitors (17). Despite the shortcomings of PKC inhibitors (31), the ability to mimic the action of a physiological ligand (bombesin) with an exogenous activator of PKC (TPA) and to block its action with calphostin C suggests that the natural contractile response to bombesin may be a PKC-mediated process and validates our conclusion that DAG production indeed arises primarily from increased PC breakdown.
Our findings are consistent with proposed models for receptor-mediated
PC hydrolysis (3, 11). Bombesin appears to induce a sustained
contraction by stimulating PC hydrolysis through a PKC-mediated
activation of either PLC- or PLD-mediated pathways or both. In these
models, it is reported that G proteins are involved in mediating PC
hydrolysis by activating PLC and/or PLD in some cell systems,
as shown by the use of nonhydrolyzable GTP analogs such as GTPS,
which in addition to potentiating an agonist-induced response, by
itself can activate both phospholipase activities. In some systems,
activation of both phospholipases appears to be operative and is not
dependent on G proteins. For example, in rat vascular smooth muscle
cells the platelet-derived growth factor (PDGF)-activated formation of
phosphatidic acid, an important mitogen in these cells, occurs both
indirectly by the combined but sequential action of PC-PLC and DAG
kinase and also directly by activation of PLD (15). Furthermore,
PDGF-stimulated phosphatidic acid synthesis could occur independently
of receptor tyrosine kinase or PKC activity through activation of a DAG
kinase pathway, suggesting an important independent role for DAG kinase
in signal transduction (16). However, in endothelin-induced vascular
smooth muscle contraction, which occurs through a G protein-coupled
pathway, activation of PLD appears to be more important for the
continued activation of PKC and maintenance of sustained entry of
Ca2+ through PKC-dependent
activation of L-type Ca2+
channels, implying that phosphorylation of
Ca2+ channels may have an
important modulatory function, although evidence for regulation of
these channels by PKC phosphorylation was not reported (32).
These results add to the growing body of evidence that DAG production
can occur in more than one phase and simultaneously from more than one
source. In the case of smooth muscle, it could be derived from a dual
source, PI and PC. The activation of distinct PLC- isozymes by
bombesin has been shown in pancreatic acinar cells (26), and in smooth
muscle cells receptor activation of distinct G proteins (23) and
isozymes of PLC (22) has been demonstrated. Our results also support
our earlier findings of the important role of PKC in mediating
calmodulin-independent sustained contraction of GI smooth muscle.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-42876.
![]() |
FOOTNOTES |
---|
Address for reprint requests: K. N. Bitar, Univ. of Michigan Medical School, 1150 W. Medical Center Dr. A520D, MSRB I, Ann Arbor, MI 48109-0656.
Received 11 March 1997; accepted in final form 11 March 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adelstein, R. S.,
and
E. Eisenberg.
Regulation and kinetics of the actin-myosin-ATP interaction.
Annu. Rev. Biochem.
49:
921-956,
1980[Medline].
2.
Ames, B. N.
Assay of inorganic phosphate, total phosphate and phosphatases.
Methods Enzymol.
8:
115-118,
1966.
3.
Billah, M. M.,
and
J. C. Anthes.
Regulation and cellular functions of phosphatidylcholine hydrolysis.
Biochem. J.
269:
281-291,
1990[Medline].
4.
Bitar, K. N.,
C. Hillemeier,
P. Biancani,
and
K. J. Balazovich.
Regulation of smooth muscle contraction in rabbit internal anal sphincter by protein kinase C and Ins(1,4,5)P3.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G537-G542,
1991
5.
Bitar, K. N.,
and
G. M. Makhlouf.
Measurement of function in isolated single smooth muscle cells.
Am. J. Physiol.
250 (Gastrointest. Liver Physiol. 13):
G357-G360,
1986[Medline].
6.
Capponi, A. M.,
P. D. Lew,
and
M. B. Vallotton.
Cytosolic free calcium levels in monolayers of cultured rat aortic smooth muscle cells. Effects of angiotensin II and vasopressin.
J. Biol. Chem.
260:
7836-7842,
1985
7.
Cook, S. J.,
S. Palmer,
R. Plevin,
and
M. J. Wakelam.
Mass measurement of inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 mouse fibroblasts.
Biochem. J.
265:
617-620,
1990[Medline].
8.
Cook, S. J.,
and
M. J. Wakelam.
Analysis of the water-soluble products of phosphatidylcholine breakdown by ion-exchange chromatography. Bombesin and TPA (12-O-tetradecanoylphorbol 13-acetate) stimulate choline generation in Swiss 3T3 cells by a common mechanism.
Biochem. J.
263:
581-587,
1989[Medline].
9.
Dennis, E. A.,
S. G. Rhee,
M. M. Billah,
and
Y. A. Hannun.
Role of phospholipases in generating lipid second messengers in signal transduction.
FASEB J.
5:
2068-2077,
1991
10.
Dominiczak, A. F.,
D. F. Lazar,
A. Das,
and
D. F. Bohr.
Lipid bilayer in genetic hypertension.
Hypertension
18:
748-757,
1991[Abstract].
11.
Exton, J. H.
Signaling through phosphatidylcholine breakdown.
J. Biol. Chem.
265:
1-4,
1990
12.
Gschwendt, M.,
W. Kittstein,
and
F. Marks.
Protein kinase C activation by phorbol esters: do cysteine-rich regions and pseudosubstrate motifs play a role?
Trends Biochem. Sci.
16:
167-169,
1991[Medline].
13.
Hartshorne, D. J.,
and
R. F. Siemankowski.
Regulation of smooth muscle actomyosin.
Annu. Rev. Physiol.
43:
519-530,
1981[Medline].
14.
Hedegaard, E.,
and
B. Jensen.
Nano-scale densitometric quantitation of phospholipids.
J. Chromatogr. Sci.
225:
450-454,
1981.
15.
Inui, H.,
T. Kondo,
F. Konishi,
Y. Kitami,
and
T. Inagami.
Participation of diacylglycerol kinase in mitogenic signal transduction induced by platelet-derived growth factor in vascular smooth muscle cells.
Biochem. Biophys. Res. Commun.
205:
1338-1344,
1994[Medline].
16.
Kanoh, H.,
K. Yamada,
and
F. Sakane.
Diacylglycerol kinase: a key modulator of signal transduction?
Trends Biochem. Sci.
15:
47-50,
1990[Medline].
17.
Khalil, R. A.,
and
K. G. Morgan.
Protein kinase C: a second E-C coupling pathway in vascular smooth muscle?
News Physiol. Sci.
7:
10-15,
1992.
18.
Lee, C.,
and
A. K. Hajra.
Quantitative analysis of changes in the molecular species of glycerolipids in cultured cells during signal transduction.
Neuroprotocols
3:
83-90,
1993.
19.
Lee, C. H.,
and
A. K. Hajra.
Molecular species of diacylglycerols and phosphoglycerides and the postmortem changes in the molecular species of diacylglycerols in rat brains.
J. Neurochem.
56:
370-379,
1991[Medline].
20.
Lee, T. S.,
T. Chao,
K. Q. Hu,
and
G. L. King.
Endothelin stimulates a sustained 1,2-diacylglycerol increase and protein kinase C activation in bovine aortic smooth muscle cells.
Biochem. Biophys. Res. Commun.
162:
381-386,
1989[Medline].
21.
Morgan, J. P.,
and
K. G. Morgan.
Vascular smooth muscle: the first recorded Ca2+ transients.
Pflügers Arch.
395:
75-77,
1982[Medline].
22.
Murthy, K. S.,
J. F. Kuemmerle,
and
G. M. Makhlouf.
Agonist-mediated activation of PLA2 initiates Ca2+ mobilization in intestinal longitudinal smooth muscle.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G93-G102,
1995
23.
Murthy, K. S.,
and
G. M. Makhlouf.
Adenosine A1 receptor-mediated activation of phospholipase C-3 in intestinal muscle: dual requirement for
and
subunits of Gi3.
J. Pharmacol. Exp. Ther.
47:
1172-1179,
1995.
24.
Nabika, T.,
P. A. Velletri,
W. Lovenberg,
and
M. A. Beaven.
Increase in cytosolic calcium and phosphoinositide metabolism induced by angiotensin II and [Arg]vasopressin in vascular smooth muscle cells.
J. Biol. Chem.
260:
4661-4670,
1985[Abstract].
25.
Nishizuka, Y.
Studies and prospectives of the protein kinase C family for cellular regulation.
Cancer
10:
1892-1903,
1989.
26.
Piiper, A.,
D. Stryjek-Kaminska,
R. Klengel,
and
S. Zeuzem.
CCK, carbachol, and bombesin activate distinct PLC- isoenzymes via Gq/11 in rat pancreatic acinar membranes.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G135-G140,
1997
27.
Rana, R. S.,
and
L. E. Hokin.
Role of phosphoinositides in transmembrane signaling.
Physiol. Rev.
70:
115-164,
1990
28.
Sbrissa, D. D. L.,
H. Yamada,
and
K. N. Bitar.
Bombesin-stimulated ceramide production and MAP kinase activation in isolated smooth muscle cells from rabbit colon.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G1615-G1625,
1997
29.
Stull, J. T.
Phosphorylation of contractile proteins in relation to muscle function.
Adv. Cyclic Nucleotide Res.
13:
39-93,
1980[Medline].
30.
Takuwa, N.,
Y. Takuwa,
and
H. Rasmussen.
A tumour promoter, 12-O-tetradecanoylphorbol 13-acetate, increases cellular 1,2-diacylglycerol content through a mechanism other than phosphoinositide hydrolysis in Swiss-mouse 3T3 fibroblasts.
Biochem. J.
243:
647-653,
1987[Medline].
31.
Wilkinson, S. E.,
and
T. J. Hallam.
Protein kinase C: is its pivotal role in cellular activation over-stated?
Trends Pharmacol. Sci.
15:
53-57,
1994[Medline].
32.
Xuan, Y.-T.,
O. L. Wang,
and
A. R. Whorton.
Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1560-C1567,
1994
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |