Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6632-6636, December 1979
Medical Sciences
a- and f3-adrenergic stimulation of arachidonic acid metabolism in cells in culture (phospholipase A2/prostaglandins/adrenergic antagonists/norepinephrine/adrenergic receptors)
LAWRENCE LEVINE* AND MICHAEL A. MOSKOWITZtt *Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154; tLaboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and *Section of Neurology, Peter Bent Brigham Hospital, Harvard Medical School, Boston, Massachusetts 02115
Communicated by H. N. Munro, September 12,1979
We now report that the regulation of PG biosynthesis is controlled, in part, by a- or f3-adrenergic receptors which, when stimulated, promote the deacylation of phospholipids and subsequent metabolism of arachidonic acid. MATERIALS AND METHODS Cell Cultures. Exponentially growing dog kidney (MDCK) cells were treated with 0.25% trypsin and seeded at 2 X 105 cells per 60-mm Falcon tissue culture dish in 4 ml of Eagle's minimal essential medium containing 2 mM i-glutamate and supplemented with 10% (vol/vol) fetal bovine serum, 250 units of penicillin per ml, and 250 jg of streptomycin per ml; they were incubated for 24 hr. In the experiments described below, the cells were washed twice with 2 ml of the medium lacking fetal bovine serum and incubated with 4 ml of the medium lacking the fetal bovine serum but containing the experimental reagents. Assay of Arachidonic Acid Metabolites. Thromboxane A2 (TBXA2) (measured as TBXB2), PGE2, PGF2., and PGD2 were measured in culture fluids by radioimmunoassay using antisera whose serologic properties have been described (13, 14). Prostacyclin, measured as 6-keto-PGF1a, was also measured by radioimmunoassay. In this system, 10 pg of 6-keto-PGFia inhibited the binding of 6-keto-[3H]PGFia to anti-6-keto-PGFia by 50%; PGE2, PGF2a, and PGA2 crossreacted less than 1%. In some experiments, separation and quantitation of arachidonic acid metabolites were performed as reported (15). Briefly, conditioned medium from MDCK cells was first acidified to pH 3.5 and the arachidonic acid metabolites were adsorbed onto XAD-2 resin (ISOLAB, Akron, OH). The resin was washed with 20 ml of H20, and arachidonic acid metabolites were eluted with 100% ethanol. The eluates were dried under nitrogen at room temperature and resuspended in ethanol. Samples were then clarified by centrifugation, concentrated by drying under nitrogen, and subjected to high-pressure liquid chromatography using a reversed-phase system (15). The eluted fractions were assayed by radioimmunoassay. In other experiments, the conditioned media were analyzed directly by radioimmunoassay. Chemicals. Drugs used in this study were purchased from Sigma except as noted. The tritiated arachidonic acid metabolites were purchased from New England Nuclear. Stock solutions of a-adrenergic antagonists in dimethyl sulfoxide were stored in the dark at -20°C. Dilutions were made in medium lacking fetal bovine serum just prior to the experiment, and the appropriate amount was added to the
Madin-Darby canine kidney cells (MDCK) synthesize prostaglandin (PG) F2, PGI2 (measured as 6-ketoPGEia), PGE2, PGD2, and thromboxane A2 (measured as thromboxane B2). When incubated in the presence of norepinephrine (6 p&M), the syntheses of these arachidonic acid metabolites are stimulated -fold. Norepinephrine's effect can be antagonized by the addition of a-adrenergic receptor blocking agents (phenoxybenzamine>phentolamine>yohimbine>dibenamine>tolazoline) but not by the 0-adrenergic blocking drug propranolol. Norepinephrine's stimulation is also inhibited by low concentrations of dihydroergotamine, bromocryptine, ergocryptine, and ergotamine. The stimulation of PG synthesis by norepinephrine is reversible, continues during the 24 hr of incubation, and requires the presence of norepinephrine at the receptor site but it is not blocked by the addition of colchicine, cytochalasin B, or cycloheximide. Neither phenoxybenzamine nor ergotamine at concentrations that block norepinephrine's stimulation of PG biosynthesis suppresses the increase in PG synthesis induced by exogenous arachidonic acid, suggesting that the a-adrenergic regulation is not occurring primarily at the cyclooxygenase step in the metabolism of arachidonic acid. In mouse lymphoma cells (WEHI-5), low concentrations of isoproterenol or norepinephrine stimulate the synthesis of thromboxane, an effect that can be blocked by the addition of propranolol but not by relatively high concentrations of phenoxybenzamine or ergotamine. Taken together, these results suggest that a-adrenergic receptor stimulation promotes the deacylation of phospholipids by MDCK cells whereas 0adrenergic mechanisms lead to activation of similar pathways in WEHI-5 cells. The mechanism by which catecholamines stimulate the biosynthesis of prostaglandin-like substances in adipose tissue (1, 2), spleen (3-6), lungs (7), phrenic diaphragm (8,9), brain (10), kidney (11), and skin (2) is poorly understood. It is possible that these compounds stimulate via receptor-mediated mechanisms; for example, treatment with the a-adrenergic receptor blocking agent phenoxybenzamine inhibits the appearance of prostaglandin-like material from dog spleen (3, 4, 6) and rabbit kidney (11) after the administration of norepinephrine (NE). It is also possible that prostaglandin-like substances are released as a result of tissue contraction (e.g., splenic capsule) induced by the catecholamines (6). On the other hand, stimulation of prostaglandin (PG) synthesis by the catecholami-nes may simply reflect their properties as cofactors for the cyclooxygenation of arachidonic acid, as shown by studies using microsomes prepared from seminal vesicles (12). In the present study, we examined the relationship between catecholamine receptors and PG synthesis by cells in culture.
ABSTRACT
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: NE, norepinephrine; PG, prostaglandin; TBX, thromboxane. 6632
Proc. Natl. Acad. Sci. USA 76 (1979)
Medical Sciences: Levine and Moskowitz culture dish. The highest level of dimethyl sulfoxide used (0.1%) had no effect on MDCK cells or production of PGs. Solutions of the a- and f3-adrenergic agonists as well as propranolol were freshly made for each experiment in the medium lacking fetal bovine serum. None of the reagents at the concentrations used interfered with the radioimmunoassays. RESULTS The MDCK cells synthesized PGF2a, PGI2 (measured as 6keto-PGFIa), PGE2, PGD2, and' TBXA2 (measured as TBXB2) (Fig. 1). In order to maximize arachidonic acid metabolism so that the metabolic profile could be clearly demonstrated, in the experiment shown in Fig. 1 the cells were stimulated to metabolize polyenoic acids by incubation with a tumor-promoting phorbol diester [the phorbol diester stimulates deacylation of cellular lipids (16, 17) and provides the polyenoic substrates required for the synthesizing enzymes of PG, TBX, and prostacyclin]. Such treatment stimulates PG production 5- to 10-fold (16,'17). Radioimmunoassay of arachidonic acid metabolites before and after separation by high-pressure liquid chromatography gave similar values. [Radioimmunoassay of conditioned media from cells established from mouse lymphoma, bovine aorta, rabbit aorta smooth muscle, normal human foreskin, normal human embryonic lung, and a rat adult type II alveolar cell before and after separation also gave comparable 1000
K
PGF20f
6KFia
10o
1C
values (unpublished data).] Hence, all subsequent analyses were performed by radioimmunoassay of the conditioned media without separation, unless otherwise indicated. NE in doses as low as 2-10 ,tM stimulated the production of PGE2, PGF2., PGI2, TBXA2, and PGD2 without affecting the number or viability of cells. When measured after 24 hr of incubation, 6 AM norepinephrine stimulated synthesis of all of these products 3-fold (Fig. 2). The NE effect continued for at least 24 hr but was not inhibited by addition of cycloheximide (0.2 ,ug/ml), colchicine (1 Mug/ml), or cytochalasin B (0.1 ug/ml) and therefore does not depend upon the synthesis of protein or integrity of microtubules or microfilaments. The possibility that NE was inhibiting catabolism of PGE2, PGF2a, TBXA2, PGI2, and PGD2 by 15-hydroxydehydrogenase and A13-reductase was unlikely because the metabolites of PGE2 and PGF2a, the 15-keto- and 13,14-dihydro-15-keto derivatives, are not found in MDCK cells or their culture fluids (15). Epinephrine and dopamine also stimulate PG production. At 10 ,uM, epinephrine was the most potent of the agonists tested; isoproterenol did not stimulate PG production at any of the concentrations tested (2-20 MM). The effects of NE (6 ,M) could be blocked by addition of low concentrations of the a-adrenergic receptor blocking agents phenoxybenzamine or ergotamine (Fig. 3). The blockade (like the stimulation by NE) seems to occur at either the cyclooxygenase or phospholipase reaction because all of the arachidonic acid metabolites were inhibited to a similar extent. To distinguish between these two possible loci of activity, PG synthesis was stimulated by incubating the cells in the presence of arachidonic acid. The arachidonic acid-induced stimulation was not blocked by high concentrations of phenoxybenzamine (3 MM) or ergotamine (0.7 MM) (Table 1). Because it has been shown (17) that little, if any, free arachidonic acid exists in MDCK cells, it seems most likely that NE promotes the deac0.07
0.6 -0.5
_ 0.06
-
-E0, 0.05
-
c'
._r
TBXB2
1
c
0.4
OA O/
u 0.3
PGD2
'
R
0
0.7
0
M 4(U
6633
0.2
', 0.04
X 0.03 H- 0.02 -5 -
0.01
0.1
)
5
10
0
15 20 25
5
10 15 20 25
6.0
0.1 I
_ 5.0
E
c
0~~~
, 4.0
O 2.0
0.01 0
10
20
30 Fraction
40
50
60
FIG. 1. High-pressure liquid chromatogram of cyclooxygenase products of arachidonic acid metabolism by MDCK cells. MDCK cells (2 X 105 cells per 60-mm tissue culture dish) were incubated in minimal essential medium (4.0 ml) containing 12-O-tetradecanoylphorbol 13-acetate (1 ng/ml) for 24 hr. The media from 20 dishes were pooled and the arachidonic acid metabolites were adsorbed on and eluted from XAD-2 resin. The concentrated eluate was subjected to highpressure liquid chromatography and the fractions were assayed by radioimmunoassay. After similar analysis, essentially the same metabolic profile was obtained in a second experiment. Also, in at least 50 experiments with MDCK cells, similar metabolic profiles have been obtained after radioimmunoassay of the culture fluids before chromatographic separation.
6 U-
6 3.0
0
a.
01'
1.0
6 CD
10 15 20 25 Time, hr Time, hr FIG. 2. Effect of NE on PGE2, PGF2a, PGI2 (6-keto-PGFia"), and 0
5
TBXB2 synthesis by MDCK cells. Multiple dishes of MDCK cells (2 X 105 cells per 60-mm tissue culture dish) were incubated in minimal essential medium lacking 10% fetal bovine serum (-) or in minimal essential medium lacking 10% fetal bovine serum but containing 6 AM NE (0). At various periods of time the medium from three dishes was removed and assayed with antisera of the appropriate serologic specificity. Each point gives the mean for the three dishes (±SD). This experiment was done three times. The times of incubation varied among the experiments, but in each experiment the degree of stimulation of each arachidonic acid metabolite was the same.
Proc. Natl. Acad. Sci. USA 76 (1979)
Medical Sciences: Levine and Moskowitz
6634 100[
loor B
A
6
.!uw
80F
80
t-
vo
60
c
lk
60k
0 c
0
,, 40
401
._
c II
20
20 0
10O9
10-8
10-
10-6
Phenoxybenzamine, M
10i9
108
10-'
106
Ergotamine, M
FIG. 3. Effect of phenoxybenzamine (A) and ergotamine (B) on NE stimulation of PG synthesis by MDCK cells. Multiple dishes of MDCK cells (2 X 105 cells per dish) were incubated for 24 hr with minimal essential medium alone, with 6,uM NE, or with 6,MM NE plus increments of phenoxybenzamine or ergotamine. The medium was removed and assayed for PGF2q (0), 6-keto-PGFIa (0), PGE2 (A), and TBXB2 (a). The data are expressed as the percentage of inhibition of NE stimulation. Duplicate dishes were used and the values obtained were within 20% of the mean value. This experiment with increments of each drug was done twice. Concentration-dependent inhibition was found. Experiments with the two drugs at a single concentration have been done at least five times.
ylation of phospholipids (and the availability of arachidonic acid) by perhaps stimulating a phospholipase pathway rather than by affecting the cyclooxygenase enzyme. Additional studies are needed, however, to confirm experimentally the site of NE action. Other a-adrenergic antagonists suppressed the ability of NE to stimulate the entire spectrum of arachidonate metabolites with the following order of potency: phenoxybenzamine>phentolamine>yohimbine>dibenamine>tolazoline (Table 2). Among the ergot alkaloids, bromocryptine, ergocryptine, dihydroergotamine, and ergotamine were effective inhibitors. L-Ergothioneine and ergonovine were not effective blockers Table 1. Effect of phenoxybenzamine and ergotamine on stimulation of PG production by exogenous arachidonic acid Mean + SD, ng/ml culture fluid 6-KetoDishes, PGFia PGF2a no. Treatment 0.87 0.11 0.31 i 0.06 18 Minimal essential medium 6 3.86 0.32 1.56 + 0.14 Arachidonic acid (2 M^g/ml) 3 0.92 : 0.13 0.33 + 0.09 Phenoxybenzamine (3.2 IiM) 3 0.91 ± 0.08 0.30 + 0.04 Phenoxybenzamine (0.64,gM) Phenoxybenzamine (3.2,uM) 3.47 ± 0.33 1.44 + 0.08 3 + arachidonic acid (2 Mg/ml) Phenoxybenzamine (0.64 MM) 4.33 0.47 1.27 : 0.17 3 + arachidonic acid (2,ug/ml) 0.83 0.08 0.34 + 0.09 3 Ergotamine (0.76 MM) Ergotamine (0.76 MM) 3.72 i 0.22 1.59 + 0.11 3 + arachidonic acid (2 Mg/mi) MDCK cells (2 X 105 cells per 60-mm dish) were incubated for 24 hr in minimal essential medium lacking 10%/ fetal bovine serum but containing the indicated reagents. The medium was collected and subjected to radioimmunoassay. This experiment was done twice. The normalized mean values obtained agreed within 20% of the mean values shown.
Table 2. Inhibition of NE stimulation of arachidonic acid metabolism by a-adrenergic receptor antagonists Antagonist Ic50, M 1.8 X 10-8 Dihydroergotamine 2.1 X 10-8 Bromocryptine 3.0 X 10-8 Ergotamine 3.8 X 10-8 Ergocryptine 7.9 X 10-8 Phenoxybenzamine 1.2 X 10-7 Phentolamine 2.7 X 10-7 Yohimbine 1.4 X 10-6 Dibenamine 7.6 X 10`6 Tolazoline >1 X 106 Ergothioneine >1 X 1o-6 Ergonovine The inhibition of stimulation of PGF2,, PGE2, 6-keto-PGFia, and TBXB2 synthesis by 6 MM NE was determined as described in Fig. 3. The concentrations inhibiting stimulation 50%6 were determined by interpolation of such inhibition curves. Experiments were done at least twice with each drug and more than five times with some drugs. The Ic5o values agreed within 20% of the mean values shown.
even at concentrations of 4.4 and 2.3 AM, respectively. The ability of NE to enhance PG synthesis appears to depend upon its occupation of the receptor site. When MDCK cells were incubated for 3 hr with 6 iM NE and then washed and reincubated for 21 hr in the absence of the agonist, the levels of synthesized products did not increase much beyond those observed in the unstimulated state (Fig. 4). Addition of 1 ,LM dihydroergotamine to the culture after incubation for 3 hr with 6 IAM NE suppressed the ability of NE to stimulate PG synthesis over the next 21 hr. By contrast, the addition of 1 MiM propranolol, a f3-adrenergic receptor blocker, did not affect the subsequent stimulation by NE. Dopamine appears to stimulate PG synthesis by a different mechanism of action, because the addition of phenoxybenzamine, ergotamine, or propranolol (5 MM) did not alter dopamine's ability to increase PG synthesis by these cells. Addition of an equivalent amount of other amines (serotonin, histamine) was without effect. To determine the ability of another cell line to respond to the addition of adrenergic compounds, cells established from a mouse lymphoma (WEHI-5) were incubated with NE, dopamine, or isoproterenol. In the presence of 6 ,MM NE or isoproterenol but not dopamine, the synthesis of TBX was stimulated 3- to 4-fold. Propranolol at 1 MiM suppressed this stimulation whereas 3 ,M phenoxybenzamine or 0.7 ,uM ergotamine did not inhibit this effect (Table 3). Thus, in contrast to the MDCK cells, PG biosynthesis in WEHI-5 cells may result from a direct action of NE on a f3-adrenergic receptor that controls PG synthesis, most likely by stimulating the deacylation of cellular lipids. DISCUSSION PGs, prostacyclin, and TBX are synthesized in most mammalian cells after the deacylation of membrane phospholipids by the enzyme phospholipase A2. This first step provides the cell with arachidonic acid, the PG precursor that normally does not exist free in cells but is bound to the glycerol moiety of phosphoglycerides. Once cleaved, arachidonic acid serves as substrate for the cyclooxygenase to form cyclic endoperoxides and lipoxygenase to form hydroxy fatty acids. The short-lived endoperoxides then become rapidly converted to TBX, PGs, and prostacyclin by specific enzymes and nonenzymatic degradation. A number of hormones and other metabolically active compounds have been shown to release PGs from organs and
Medical Sciences: Levine and Moskowitz 8
LL
0~
10
15
Time, hr
FIG. 4. Requirement for the presence of NE to stimulate the synthesis of PGs during incubation with MDCK cells, and the effect of 1 MM dihydroergotamine or 1 uM propranolol on NE-induced stimulation when added at 3 hr. Multiple dishes of MDCK cells (2 X 105 cells per dish) were incubated in minimal essential medium alone (0) or containing 6 ,M NE (@). At 3 hr of incubation, the media containing NE were removed and replaced with 4.0 ml of minimal essential medium. The cells in these dishes were incubated for 21 more hr (N). Also at 3 hr, propranolol (o) or dihydroergotamine (A) was added to the cells incubating with NE (final concentrations of the propranolol and dihydroergotamine, 1 MM). These cells were incubated for 21 additional hr. Propranolol and dihydroergotamine also were added at 3 hr to dishes containing only minimal essential medium and these dishes were incubated another 21 hr; in addition, at 3 hr, the cells incubating in minimal essential media were given another 4.0 ml of fresh medium to measure the effects of these variables in the absence of NE. There was no significant change in PGF2a production. Each point gives the mean (±SD) for three culture dishes. This experiment was done twice. Propranolol had no effect; the effective inhibition of stimulation by dihydroergotamine or removal of NE was found. In addition, in two experiments in which dihydroergotamine was added or NE was removed after 1 hr, propranolol had no effect but in the presence of dihydroergotamine or after removal of NE, PG production was not stimulated.
tissues (18). Most "releasing compounds" probably act by stimulating PG biosynthesis because PGs, TBXs, and prostacyclins are not stored in cells or tissues to any extent. One such "releasing" group is the catecholamines (1-11). For example, the addition of NE to synaptosomes stimulates the production of the PG precursor arachidonic acid and other fatty acids (19). Sympathetic nerve stimulation releases large amounts of PGs in the effluent of isolated perfused organs such as the spleen (3, 4) and kidney (20). NE, epinephrine, and methoxamine stimulate the release of PGE or PGF from contracted splenic capsular tissue; pretreatment with phentolamine blocks these effects (6). NE and epinephrine also stimulate the release of PGs from isolated perfused rabbit kidney; phenoxybenzamine, but not propranolol, blocks this release (11). Although suggestive of an a-adrenergic receptor-mediated response, these latter experiments do not exclude the possibility that PG release is evoked by the drug-induced mechanical stimulation and not primarily by the action of the drug itself. Other studies also provide suggestive evidence of a cause-and-effect relationship between the catecholamines and the biosynthesis of PGs
(21). Our data establish a clear relationship between NE and the stimulation of PG synthesis by cells in culture. Whereas the MDCK cells are stimulated by a-adrenergic receptor mechanisms, the WEHI-5 cells appear to become activated by occu-
Proc. Natl. Acad. Sci. USA 76 (1979)
6635
Table 3. Stimulation by NE and isoproterenol of TBX synthesis by WEHI-5 cells and the effects of a- and f-adrenergic antagonists on NE stimulation TBX, ng/ml culture fluid Treatment 0.028 + 0.004 Minimal essential medium 0.088 h 0.009 Norepinephrine (6 tM) 0.114 I 0.013 Isoproterenol (6,MM) 0.031 I 0.002 Dopamine (6 MM) 0.031 + 0.001 Phenoxybenzamine (3MM) 0.036 + 0.002 Phenoxybenzamine (0.6 MM) 0.087 + 0.012 Phenoxybenzamine (3MM) + NE (6 MM) 0.081 + 0.011 Phenoxybenzamine (0.6 MM) + NE (6 ,uM) 0.030 + 0.003 Ergotamine (0.8 MM) 0.036 + 0.006 Ergotamine (0.15 MM) 0.087 + 0.005 Ergotamine (0.8 ,M) + NE (6MuM) 0.081 + 0.005 Ergotamine (0.15,MM) + NE (6AM) 0.031 + 0.006 Propranolol (3.9 AM) 0.044 I 0.002 Propranolol (3.9 MAM) + NE (6 AM) WEHI-5 cells (5 X 105 cells per 60-mm dish) were incubated for 24 hr in 4 ml of minimal essential medium lacking 10% fetal bovine serum but containing the indicated reagents. The media were collected and assayed for TBX with an antiserum to TBXB2. The results given are the mean (4SD) of three dishes. This experiment has been done three times. In each experiment, NE and isoproterenol stimulated PG production and dopamine did not; propranolol inhibited NE stimulation and phenoxybenzamine and ergotamine did not.
pation of the f3-adrenergic receptor by NE. NE stimulation of PG production by MDCK cells is dose dependent, reversible, and not dependent upon protein synthesis or microtubular or microfilament integrity, but it requires the presence of NE at the receptor site. The ability of a-adrenergic receptor blocking agents to inhibit NE-induced stimulation correlates with the potency of these drugs in vvo and their ability to inhibit radioligand binding in vitro (22). MDCK cells respond to NE by increasing, to the same extent, the synthesis of all arachidonate metabolites made by the cell. This suggests that receptormediated stimulation occurs at the cyclooxygenation of arachidonic acid or deacylation of phospholipids; Bradykinin, thrombin, tumor-promoting phorbol diesters, adriamycin, aromatic polycyclic hydrocarbons, retinoids, epidermal growth factor, and polypeptide component of bee venom (melittin) have been found to stimulate PG production in vitro by increasing the deacylation of cellular lipids (23). This was not surprising because it had already been found that virtually all available arachidonic acid is incorporated by MDCK cells into phospholipids (23). Thus it seems likely, but not certain, that NE, like the compounds listed above, activates phospholipase A2 to liberate arachidonic acid. Further indirect evidence for such a mechanism is provided by experiments which showed that the aadrenergic receptor blocking agents were unable to block the stimulation of PG synthesis induced by arachidonic acid, although they were effective inhibitors of NE activity. The ability of NE to stimulate the deacylation of phospholipids and biosynthesis of PGs may or may not be related to drug-induced changes in membrane permeability reported for MDCK cells. These cells retain in culture many features characteristic of distal tubular epithelial cells such as tight junctions, transport of ions, permeability to water, and sensitivity to several hormones (24). The addition of pharmacological doses of PGE1 or PGE2 causes a marked increase in the activity of adenylate cyclase and formation of cyclic AMP by MDCK cells,
6636 a
Proc. Nati. Acad. Sci. USA 76 (1979)
Medical Sciences: Levine and Moskowitz
property shared with other hormones such
as
vasopressin,
oxytocin, and glucagon and with cholera toxin. PGs have pre-
viously been thought to modulate cellular permeability to water and ionic fluxes induced by hormones such as vasopressin (25). In cortical collecting tubules, NE has been shown to affect adenylate cyclase activity and to modify water and electrolyte transport in various epithelia (26). It is possible that the PGs themselves alter membrane properties and account for NEinduced changes in permeability or ion transport; alternatively, receptor activation and PG biosynthesis, or the resulting generation of lysophosphatides within the membrane, may be coupled to methylation of membrane phospholipids to modify membrane structure and function (27). Recently, 3-adrenergic receptor-mediated mechanisms have been reported to increase phospholipid methylation of erythrocyte membranes, thereby altering membrane viscosity and enhancing membrane fluidity (28). The extent that a-adrenergic receptor-mediated PG synthesis modifies membrane function in transport epithelium remains to be determined. L.L. is a Research Professor of Biochemistry of the American Cancer Society (Award PRP-21). M.A.M. is a recipient of Teacher-Investigator Award 11081 from the National Institute of Neurologic and Communicative Diseases and Stroke. This is publication no. 1279 from the Department of Biochemistry, Brandeis University, Waltham, MA 02254. 1. Shaw, J. E. & Ramwell, P. W. (1968) J. Biol. Chem. 243, 1498-1503. 2. Ramwell, P. W. & Shaw, J. E. (1970) Recent Prog. Horm. Res. 26, 139-187. 3. Davies, B. N., Horton, E. W. & Wirthington, P. G. (1968) Br. J. Pharmacol. 32, 127-135. 4. Ferreira, S. H. & Vane, J. R. (1967) Nature (London) 216, 868-873. 5. Gilmore, N., Vane, J. R. & Wyllie, J. H. (1968) Nature (London) 218, 1135-1140. 6. Jobke, A., Peskar, B. A. & Hertting, G. (1976) NaunynSchmiedebergs Arch. Pharmakol. 292,35-42. 7. Liebig, R., Bernauer, W. & Peskar, B. A. (1974) NaunynSchmiedebergs Arch. Pharmakol. 284, 279-293.
8. Ramwell, P. W., Shaw, J. E. & Kucharski, J. (1965) Science 149, 1390-1391. 9. Laity, J. L. H. (1969) Br. J. Pharmacol. 37,698-704. 10. Leslie, C. A. (1976) Res. Commun. Chem. Pathol. Pharmacol. 14,455-469. 11. Needleman, P., Douglas, J. R., Jr., Jakschik, B., Stoecklein, P. B. & Johnson, E. M., Jr. (1974) J. Pharmacol. Exp. Ther. 188,
453-460. 12. Takaguchi, C., Kohno, E. & Sih, C. J. (1971) Biochemistry 10, 2372-2376. 13. Pong, S. S. & Levine, L. (1977) in The Prostaglandins, ed. Ramwell, P. W. (Plenum, New York), Vol. 3, pp. 41-76. 14. Levine, L., Alam, I. & Langone, J. L. (1979) Prostaglandins Med. 2, 177-189. 15. Alam, I., Ohuchi, K. & Levine, L. (1979) Anal. Biochem. 93, 339-345. 16. Levine, L. & Hassid, A. (1977) Biochem. Blophys. Res. Commun.
792477-484.
17. Ohuchi, K. & Levine, L. (1978) J. Biol. Chem. 253, 47834790. 18. Horton, E. W. (1973) Br. Med. Bull. 29, 148-151. 19. Price, C. J. & Rowe, C. E. (1972) Biochem. J. 126,575-585. 20. Dunham, E. W. & Zimmerman, B. G. (1970) Am. J. Physiol. 219, 1279-1285. 21. Hedqvist, P. (1973) in The Prostaglandins, ed. Ramwell, P. W. (Plenum, New York), Vol. 1, pp. 101-131. 22. Williams, L. T. & Lefkowitz, R. J. (1978) Receptor Binding Studies in Adrenergic Pharmacology (Raven, New York). 23. Levine, L. (1979) in Hormones and Cell Culture, eds. Sato, G. & Ross, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 623-640. 24. Rindler, M. J., Chuman, L. M., Shaffer, L. & Saier, M. H., Jr. (1979) J. Cell Biol. 81, 635-648. 25. Grantham, J. J. & Orloff, J. (1968) J. Clin. Invest. 47, 11541161. 26. liono, Y., Flier, S. R. & Brenner, B. M. (1979) Clin. Res. 27, 418a. 27. Hirata, F., Corcoran, B. A., Venkatasubramanian, K., Schiffman, E. & Axelrod, J. (1979) Proc. Natl. Acad. Sca. USA 76, 26402643. 28. Hirata, F. & Axelrod, J. (1978) Proc. Natl. Acad. Sci. USA 75, 2348-2352.
Medical Sciences
a- and f3-adrenergic stimulation of arachidonic acid metabolism in cells in culture (phospholipase A2/prostaglandins/adrenergic antagonists/norepinephrine/adrenergic receptors)
LAWRENCE LEVINE* AND MICHAEL A. MOSKOWITZtt *Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154; tLaboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and *Section of Neurology, Peter Bent Brigham Hospital, Harvard Medical School, Boston, Massachusetts 02115
Communicated by H. N. Munro, September 12,1979
We now report that the regulation of PG biosynthesis is controlled, in part, by a- or f3-adrenergic receptors which, when stimulated, promote the deacylation of phospholipids and subsequent metabolism of arachidonic acid. MATERIALS AND METHODS Cell Cultures. Exponentially growing dog kidney (MDCK) cells were treated with 0.25% trypsin and seeded at 2 X 105 cells per 60-mm Falcon tissue culture dish in 4 ml of Eagle's minimal essential medium containing 2 mM i-glutamate and supplemented with 10% (vol/vol) fetal bovine serum, 250 units of penicillin per ml, and 250 jg of streptomycin per ml; they were incubated for 24 hr. In the experiments described below, the cells were washed twice with 2 ml of the medium lacking fetal bovine serum and incubated with 4 ml of the medium lacking the fetal bovine serum but containing the experimental reagents. Assay of Arachidonic Acid Metabolites. Thromboxane A2 (TBXA2) (measured as TBXB2), PGE2, PGF2., and PGD2 were measured in culture fluids by radioimmunoassay using antisera whose serologic properties have been described (13, 14). Prostacyclin, measured as 6-keto-PGF1a, was also measured by radioimmunoassay. In this system, 10 pg of 6-keto-PGFia inhibited the binding of 6-keto-[3H]PGFia to anti-6-keto-PGFia by 50%; PGE2, PGF2a, and PGA2 crossreacted less than 1%. In some experiments, separation and quantitation of arachidonic acid metabolites were performed as reported (15). Briefly, conditioned medium from MDCK cells was first acidified to pH 3.5 and the arachidonic acid metabolites were adsorbed onto XAD-2 resin (ISOLAB, Akron, OH). The resin was washed with 20 ml of H20, and arachidonic acid metabolites were eluted with 100% ethanol. The eluates were dried under nitrogen at room temperature and resuspended in ethanol. Samples were then clarified by centrifugation, concentrated by drying under nitrogen, and subjected to high-pressure liquid chromatography using a reversed-phase system (15). The eluted fractions were assayed by radioimmunoassay. In other experiments, the conditioned media were analyzed directly by radioimmunoassay. Chemicals. Drugs used in this study were purchased from Sigma except as noted. The tritiated arachidonic acid metabolites were purchased from New England Nuclear. Stock solutions of a-adrenergic antagonists in dimethyl sulfoxide were stored in the dark at -20°C. Dilutions were made in medium lacking fetal bovine serum just prior to the experiment, and the appropriate amount was added to the
Madin-Darby canine kidney cells (MDCK) synthesize prostaglandin (PG) F2, PGI2 (measured as 6-ketoPGEia), PGE2, PGD2, and thromboxane A2 (measured as thromboxane B2). When incubated in the presence of norepinephrine (6 p&M), the syntheses of these arachidonic acid metabolites are stimulated -fold. Norepinephrine's effect can be antagonized by the addition of a-adrenergic receptor blocking agents (phenoxybenzamine>phentolamine>yohimbine>dibenamine>tolazoline) but not by the 0-adrenergic blocking drug propranolol. Norepinephrine's stimulation is also inhibited by low concentrations of dihydroergotamine, bromocryptine, ergocryptine, and ergotamine. The stimulation of PG synthesis by norepinephrine is reversible, continues during the 24 hr of incubation, and requires the presence of norepinephrine at the receptor site but it is not blocked by the addition of colchicine, cytochalasin B, or cycloheximide. Neither phenoxybenzamine nor ergotamine at concentrations that block norepinephrine's stimulation of PG biosynthesis suppresses the increase in PG synthesis induced by exogenous arachidonic acid, suggesting that the a-adrenergic regulation is not occurring primarily at the cyclooxygenase step in the metabolism of arachidonic acid. In mouse lymphoma cells (WEHI-5), low concentrations of isoproterenol or norepinephrine stimulate the synthesis of thromboxane, an effect that can be blocked by the addition of propranolol but not by relatively high concentrations of phenoxybenzamine or ergotamine. Taken together, these results suggest that a-adrenergic receptor stimulation promotes the deacylation of phospholipids by MDCK cells whereas 0adrenergic mechanisms lead to activation of similar pathways in WEHI-5 cells. The mechanism by which catecholamines stimulate the biosynthesis of prostaglandin-like substances in adipose tissue (1, 2), spleen (3-6), lungs (7), phrenic diaphragm (8,9), brain (10), kidney (11), and skin (2) is poorly understood. It is possible that these compounds stimulate via receptor-mediated mechanisms; for example, treatment with the a-adrenergic receptor blocking agent phenoxybenzamine inhibits the appearance of prostaglandin-like material from dog spleen (3, 4, 6) and rabbit kidney (11) after the administration of norepinephrine (NE). It is also possible that prostaglandin-like substances are released as a result of tissue contraction (e.g., splenic capsule) induced by the catecholamines (6). On the other hand, stimulation of prostaglandin (PG) synthesis by the catecholami-nes may simply reflect their properties as cofactors for the cyclooxygenation of arachidonic acid, as shown by studies using microsomes prepared from seminal vesicles (12). In the present study, we examined the relationship between catecholamine receptors and PG synthesis by cells in culture.
ABSTRACT
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: NE, norepinephrine; PG, prostaglandin; TBX, thromboxane. 6632
Proc. Natl. Acad. Sci. USA 76 (1979)
Medical Sciences: Levine and Moskowitz culture dish. The highest level of dimethyl sulfoxide used (0.1%) had no effect on MDCK cells or production of PGs. Solutions of the a- and f3-adrenergic agonists as well as propranolol were freshly made for each experiment in the medium lacking fetal bovine serum. None of the reagents at the concentrations used interfered with the radioimmunoassays. RESULTS The MDCK cells synthesized PGF2a, PGI2 (measured as 6keto-PGFIa), PGE2, PGD2, and' TBXA2 (measured as TBXB2) (Fig. 1). In order to maximize arachidonic acid metabolism so that the metabolic profile could be clearly demonstrated, in the experiment shown in Fig. 1 the cells were stimulated to metabolize polyenoic acids by incubation with a tumor-promoting phorbol diester [the phorbol diester stimulates deacylation of cellular lipids (16, 17) and provides the polyenoic substrates required for the synthesizing enzymes of PG, TBX, and prostacyclin]. Such treatment stimulates PG production 5- to 10-fold (16,'17). Radioimmunoassay of arachidonic acid metabolites before and after separation by high-pressure liquid chromatography gave similar values. [Radioimmunoassay of conditioned media from cells established from mouse lymphoma, bovine aorta, rabbit aorta smooth muscle, normal human foreskin, normal human embryonic lung, and a rat adult type II alveolar cell before and after separation also gave comparable 1000
K
PGF20f
6KFia
10o
1C
values (unpublished data).] Hence, all subsequent analyses were performed by radioimmunoassay of the conditioned media without separation, unless otherwise indicated. NE in doses as low as 2-10 ,tM stimulated the production of PGE2, PGF2., PGI2, TBXA2, and PGD2 without affecting the number or viability of cells. When measured after 24 hr of incubation, 6 AM norepinephrine stimulated synthesis of all of these products 3-fold (Fig. 2). The NE effect continued for at least 24 hr but was not inhibited by addition of cycloheximide (0.2 ,ug/ml), colchicine (1 Mug/ml), or cytochalasin B (0.1 ug/ml) and therefore does not depend upon the synthesis of protein or integrity of microtubules or microfilaments. The possibility that NE was inhibiting catabolism of PGE2, PGF2a, TBXA2, PGI2, and PGD2 by 15-hydroxydehydrogenase and A13-reductase was unlikely because the metabolites of PGE2 and PGF2a, the 15-keto- and 13,14-dihydro-15-keto derivatives, are not found in MDCK cells or their culture fluids (15). Epinephrine and dopamine also stimulate PG production. At 10 ,uM, epinephrine was the most potent of the agonists tested; isoproterenol did not stimulate PG production at any of the concentrations tested (2-20 MM). The effects of NE (6 ,M) could be blocked by addition of low concentrations of the a-adrenergic receptor blocking agents phenoxybenzamine or ergotamine (Fig. 3). The blockade (like the stimulation by NE) seems to occur at either the cyclooxygenase or phospholipase reaction because all of the arachidonic acid metabolites were inhibited to a similar extent. To distinguish between these two possible loci of activity, PG synthesis was stimulated by incubating the cells in the presence of arachidonic acid. The arachidonic acid-induced stimulation was not blocked by high concentrations of phenoxybenzamine (3 MM) or ergotamine (0.7 MM) (Table 1). Because it has been shown (17) that little, if any, free arachidonic acid exists in MDCK cells, it seems most likely that NE promotes the deac0.07
0.6 -0.5
_ 0.06
-
-E0, 0.05
-
c'
._r
TBXB2
1
c
0.4
OA O/
u 0.3
PGD2
'
R
0
0.7
0
M 4(U
6633
0.2
', 0.04
X 0.03 H- 0.02 -5 -
0.01
0.1
)
5
10
0
15 20 25
5
10 15 20 25
6.0
0.1 I
_ 5.0
E
c
0~~~
, 4.0
O 2.0
0.01 0
10
20
30 Fraction
40
50
60
FIG. 1. High-pressure liquid chromatogram of cyclooxygenase products of arachidonic acid metabolism by MDCK cells. MDCK cells (2 X 105 cells per 60-mm tissue culture dish) were incubated in minimal essential medium (4.0 ml) containing 12-O-tetradecanoylphorbol 13-acetate (1 ng/ml) for 24 hr. The media from 20 dishes were pooled and the arachidonic acid metabolites were adsorbed on and eluted from XAD-2 resin. The concentrated eluate was subjected to highpressure liquid chromatography and the fractions were assayed by radioimmunoassay. After similar analysis, essentially the same metabolic profile was obtained in a second experiment. Also, in at least 50 experiments with MDCK cells, similar metabolic profiles have been obtained after radioimmunoassay of the culture fluids before chromatographic separation.
6 U-
6 3.0
0
a.
01'
1.0
6 CD
10 15 20 25 Time, hr Time, hr FIG. 2. Effect of NE on PGE2, PGF2a, PGI2 (6-keto-PGFia"), and 0
5
TBXB2 synthesis by MDCK cells. Multiple dishes of MDCK cells (2 X 105 cells per 60-mm tissue culture dish) were incubated in minimal essential medium lacking 10% fetal bovine serum (-) or in minimal essential medium lacking 10% fetal bovine serum but containing 6 AM NE (0). At various periods of time the medium from three dishes was removed and assayed with antisera of the appropriate serologic specificity. Each point gives the mean for the three dishes (±SD). This experiment was done three times. The times of incubation varied among the experiments, but in each experiment the degree of stimulation of each arachidonic acid metabolite was the same.
Proc. Natl. Acad. Sci. USA 76 (1979)
Medical Sciences: Levine and Moskowitz
6634 100[
loor B
A
6
.!uw
80F
80
t-
vo
60
c
lk
60k
0 c
0
,, 40
401
._
c II
20
20 0
10O9
10-8
10-
10-6
Phenoxybenzamine, M
10i9
108
10-'
106
Ergotamine, M
FIG. 3. Effect of phenoxybenzamine (A) and ergotamine (B) on NE stimulation of PG synthesis by MDCK cells. Multiple dishes of MDCK cells (2 X 105 cells per dish) were incubated for 24 hr with minimal essential medium alone, with 6,uM NE, or with 6,MM NE plus increments of phenoxybenzamine or ergotamine. The medium was removed and assayed for PGF2q (0), 6-keto-PGFIa (0), PGE2 (A), and TBXB2 (a). The data are expressed as the percentage of inhibition of NE stimulation. Duplicate dishes were used and the values obtained were within 20% of the mean value. This experiment with increments of each drug was done twice. Concentration-dependent inhibition was found. Experiments with the two drugs at a single concentration have been done at least five times.
ylation of phospholipids (and the availability of arachidonic acid) by perhaps stimulating a phospholipase pathway rather than by affecting the cyclooxygenase enzyme. Additional studies are needed, however, to confirm experimentally the site of NE action. Other a-adrenergic antagonists suppressed the ability of NE to stimulate the entire spectrum of arachidonate metabolites with the following order of potency: phenoxybenzamine>phentolamine>yohimbine>dibenamine>tolazoline (Table 2). Among the ergot alkaloids, bromocryptine, ergocryptine, dihydroergotamine, and ergotamine were effective inhibitors. L-Ergothioneine and ergonovine were not effective blockers Table 1. Effect of phenoxybenzamine and ergotamine on stimulation of PG production by exogenous arachidonic acid Mean + SD, ng/ml culture fluid 6-KetoDishes, PGFia PGF2a no. Treatment 0.87 0.11 0.31 i 0.06 18 Minimal essential medium 6 3.86 0.32 1.56 + 0.14 Arachidonic acid (2 M^g/ml) 3 0.92 : 0.13 0.33 + 0.09 Phenoxybenzamine (3.2 IiM) 3 0.91 ± 0.08 0.30 + 0.04 Phenoxybenzamine (0.64,gM) Phenoxybenzamine (3.2,uM) 3.47 ± 0.33 1.44 + 0.08 3 + arachidonic acid (2 Mg/ml) Phenoxybenzamine (0.64 MM) 4.33 0.47 1.27 : 0.17 3 + arachidonic acid (2,ug/ml) 0.83 0.08 0.34 + 0.09 3 Ergotamine (0.76 MM) Ergotamine (0.76 MM) 3.72 i 0.22 1.59 + 0.11 3 + arachidonic acid (2 Mg/mi) MDCK cells (2 X 105 cells per 60-mm dish) were incubated for 24 hr in minimal essential medium lacking 10%/ fetal bovine serum but containing the indicated reagents. The medium was collected and subjected to radioimmunoassay. This experiment was done twice. The normalized mean values obtained agreed within 20% of the mean values shown.
Table 2. Inhibition of NE stimulation of arachidonic acid metabolism by a-adrenergic receptor antagonists Antagonist Ic50, M 1.8 X 10-8 Dihydroergotamine 2.1 X 10-8 Bromocryptine 3.0 X 10-8 Ergotamine 3.8 X 10-8 Ergocryptine 7.9 X 10-8 Phenoxybenzamine 1.2 X 10-7 Phentolamine 2.7 X 10-7 Yohimbine 1.4 X 10-6 Dibenamine 7.6 X 10`6 Tolazoline >1 X 106 Ergothioneine >1 X 1o-6 Ergonovine The inhibition of stimulation of PGF2,, PGE2, 6-keto-PGFia, and TBXB2 synthesis by 6 MM NE was determined as described in Fig. 3. The concentrations inhibiting stimulation 50%6 were determined by interpolation of such inhibition curves. Experiments were done at least twice with each drug and more than five times with some drugs. The Ic5o values agreed within 20% of the mean values shown.
even at concentrations of 4.4 and 2.3 AM, respectively. The ability of NE to enhance PG synthesis appears to depend upon its occupation of the receptor site. When MDCK cells were incubated for 3 hr with 6 iM NE and then washed and reincubated for 21 hr in the absence of the agonist, the levels of synthesized products did not increase much beyond those observed in the unstimulated state (Fig. 4). Addition of 1 ,LM dihydroergotamine to the culture after incubation for 3 hr with 6 IAM NE suppressed the ability of NE to stimulate PG synthesis over the next 21 hr. By contrast, the addition of 1 MiM propranolol, a f3-adrenergic receptor blocker, did not affect the subsequent stimulation by NE. Dopamine appears to stimulate PG synthesis by a different mechanism of action, because the addition of phenoxybenzamine, ergotamine, or propranolol (5 MM) did not alter dopamine's ability to increase PG synthesis by these cells. Addition of an equivalent amount of other amines (serotonin, histamine) was without effect. To determine the ability of another cell line to respond to the addition of adrenergic compounds, cells established from a mouse lymphoma (WEHI-5) were incubated with NE, dopamine, or isoproterenol. In the presence of 6 ,MM NE or isoproterenol but not dopamine, the synthesis of TBX was stimulated 3- to 4-fold. Propranolol at 1 MiM suppressed this stimulation whereas 3 ,M phenoxybenzamine or 0.7 ,uM ergotamine did not inhibit this effect (Table 3). Thus, in contrast to the MDCK cells, PG biosynthesis in WEHI-5 cells may result from a direct action of NE on a f3-adrenergic receptor that controls PG synthesis, most likely by stimulating the deacylation of cellular lipids. DISCUSSION PGs, prostacyclin, and TBX are synthesized in most mammalian cells after the deacylation of membrane phospholipids by the enzyme phospholipase A2. This first step provides the cell with arachidonic acid, the PG precursor that normally does not exist free in cells but is bound to the glycerol moiety of phosphoglycerides. Once cleaved, arachidonic acid serves as substrate for the cyclooxygenase to form cyclic endoperoxides and lipoxygenase to form hydroxy fatty acids. The short-lived endoperoxides then become rapidly converted to TBX, PGs, and prostacyclin by specific enzymes and nonenzymatic degradation. A number of hormones and other metabolically active compounds have been shown to release PGs from organs and
Medical Sciences: Levine and Moskowitz 8
LL
0~
10
15
Time, hr
FIG. 4. Requirement for the presence of NE to stimulate the synthesis of PGs during incubation with MDCK cells, and the effect of 1 MM dihydroergotamine or 1 uM propranolol on NE-induced stimulation when added at 3 hr. Multiple dishes of MDCK cells (2 X 105 cells per dish) were incubated in minimal essential medium alone (0) or containing 6 ,M NE (@). At 3 hr of incubation, the media containing NE were removed and replaced with 4.0 ml of minimal essential medium. The cells in these dishes were incubated for 21 more hr (N). Also at 3 hr, propranolol (o) or dihydroergotamine (A) was added to the cells incubating with NE (final concentrations of the propranolol and dihydroergotamine, 1 MM). These cells were incubated for 21 additional hr. Propranolol and dihydroergotamine also were added at 3 hr to dishes containing only minimal essential medium and these dishes were incubated another 21 hr; in addition, at 3 hr, the cells incubating in minimal essential media were given another 4.0 ml of fresh medium to measure the effects of these variables in the absence of NE. There was no significant change in PGF2a production. Each point gives the mean (±SD) for three culture dishes. This experiment was done twice. Propranolol had no effect; the effective inhibition of stimulation by dihydroergotamine or removal of NE was found. In addition, in two experiments in which dihydroergotamine was added or NE was removed after 1 hr, propranolol had no effect but in the presence of dihydroergotamine or after removal of NE, PG production was not stimulated.
tissues (18). Most "releasing compounds" probably act by stimulating PG biosynthesis because PGs, TBXs, and prostacyclins are not stored in cells or tissues to any extent. One such "releasing" group is the catecholamines (1-11). For example, the addition of NE to synaptosomes stimulates the production of the PG precursor arachidonic acid and other fatty acids (19). Sympathetic nerve stimulation releases large amounts of PGs in the effluent of isolated perfused organs such as the spleen (3, 4) and kidney (20). NE, epinephrine, and methoxamine stimulate the release of PGE or PGF from contracted splenic capsular tissue; pretreatment with phentolamine blocks these effects (6). NE and epinephrine also stimulate the release of PGs from isolated perfused rabbit kidney; phenoxybenzamine, but not propranolol, blocks this release (11). Although suggestive of an a-adrenergic receptor-mediated response, these latter experiments do not exclude the possibility that PG release is evoked by the drug-induced mechanical stimulation and not primarily by the action of the drug itself. Other studies also provide suggestive evidence of a cause-and-effect relationship between the catecholamines and the biosynthesis of PGs
(21). Our data establish a clear relationship between NE and the stimulation of PG synthesis by cells in culture. Whereas the MDCK cells are stimulated by a-adrenergic receptor mechanisms, the WEHI-5 cells appear to become activated by occu-
Proc. Natl. Acad. Sci. USA 76 (1979)
6635
Table 3. Stimulation by NE and isoproterenol of TBX synthesis by WEHI-5 cells and the effects of a- and f-adrenergic antagonists on NE stimulation TBX, ng/ml culture fluid Treatment 0.028 + 0.004 Minimal essential medium 0.088 h 0.009 Norepinephrine (6 tM) 0.114 I 0.013 Isoproterenol (6,MM) 0.031 I 0.002 Dopamine (6 MM) 0.031 + 0.001 Phenoxybenzamine (3MM) 0.036 + 0.002 Phenoxybenzamine (0.6 MM) 0.087 + 0.012 Phenoxybenzamine (3MM) + NE (6 MM) 0.081 + 0.011 Phenoxybenzamine (0.6 MM) + NE (6 ,uM) 0.030 + 0.003 Ergotamine (0.8 MM) 0.036 + 0.006 Ergotamine (0.15 MM) 0.087 + 0.005 Ergotamine (0.8 ,M) + NE (6MuM) 0.081 + 0.005 Ergotamine (0.15,MM) + NE (6AM) 0.031 + 0.006 Propranolol (3.9 AM) 0.044 I 0.002 Propranolol (3.9 MAM) + NE (6 AM) WEHI-5 cells (5 X 105 cells per 60-mm dish) were incubated for 24 hr in 4 ml of minimal essential medium lacking 10% fetal bovine serum but containing the indicated reagents. The media were collected and assayed for TBX with an antiserum to TBXB2. The results given are the mean (4SD) of three dishes. This experiment has been done three times. In each experiment, NE and isoproterenol stimulated PG production and dopamine did not; propranolol inhibited NE stimulation and phenoxybenzamine and ergotamine did not.
pation of the f3-adrenergic receptor by NE. NE stimulation of PG production by MDCK cells is dose dependent, reversible, and not dependent upon protein synthesis or microtubular or microfilament integrity, but it requires the presence of NE at the receptor site. The ability of a-adrenergic receptor blocking agents to inhibit NE-induced stimulation correlates with the potency of these drugs in vvo and their ability to inhibit radioligand binding in vitro (22). MDCK cells respond to NE by increasing, to the same extent, the synthesis of all arachidonate metabolites made by the cell. This suggests that receptormediated stimulation occurs at the cyclooxygenation of arachidonic acid or deacylation of phospholipids; Bradykinin, thrombin, tumor-promoting phorbol diesters, adriamycin, aromatic polycyclic hydrocarbons, retinoids, epidermal growth factor, and polypeptide component of bee venom (melittin) have been found to stimulate PG production in vitro by increasing the deacylation of cellular lipids (23). This was not surprising because it had already been found that virtually all available arachidonic acid is incorporated by MDCK cells into phospholipids (23). Thus it seems likely, but not certain, that NE, like the compounds listed above, activates phospholipase A2 to liberate arachidonic acid. Further indirect evidence for such a mechanism is provided by experiments which showed that the aadrenergic receptor blocking agents were unable to block the stimulation of PG synthesis induced by arachidonic acid, although they were effective inhibitors of NE activity. The ability of NE to stimulate the deacylation of phospholipids and biosynthesis of PGs may or may not be related to drug-induced changes in membrane permeability reported for MDCK cells. These cells retain in culture many features characteristic of distal tubular epithelial cells such as tight junctions, transport of ions, permeability to water, and sensitivity to several hormones (24). The addition of pharmacological doses of PGE1 or PGE2 causes a marked increase in the activity of adenylate cyclase and formation of cyclic AMP by MDCK cells,
6636 a
Proc. Nati. Acad. Sci. USA 76 (1979)
Medical Sciences: Levine and Moskowitz
property shared with other hormones such
as
vasopressin,
oxytocin, and glucagon and with cholera toxin. PGs have pre-
viously been thought to modulate cellular permeability to water and ionic fluxes induced by hormones such as vasopressin (25). In cortical collecting tubules, NE has been shown to affect adenylate cyclase activity and to modify water and electrolyte transport in various epithelia (26). It is possible that the PGs themselves alter membrane properties and account for NEinduced changes in permeability or ion transport; alternatively, receptor activation and PG biosynthesis, or the resulting generation of lysophosphatides within the membrane, may be coupled to methylation of membrane phospholipids to modify membrane structure and function (27). Recently, 3-adrenergic receptor-mediated mechanisms have been reported to increase phospholipid methylation of erythrocyte membranes, thereby altering membrane viscosity and enhancing membrane fluidity (28). The extent that a-adrenergic receptor-mediated PG synthesis modifies membrane function in transport epithelium remains to be determined. L.L. is a Research Professor of Biochemistry of the American Cancer Society (Award PRP-21). M.A.M. is a recipient of Teacher-Investigator Award 11081 from the National Institute of Neurologic and Communicative Diseases and Stroke. This is publication no. 1279 from the Department of Biochemistry, Brandeis University, Waltham, MA 02254. 1. Shaw, J. E. & Ramwell, P. W. (1968) J. Biol. Chem. 243, 1498-1503. 2. Ramwell, P. W. & Shaw, J. E. (1970) Recent Prog. Horm. Res. 26, 139-187. 3. Davies, B. N., Horton, E. W. & Wirthington, P. G. (1968) Br. J. Pharmacol. 32, 127-135. 4. Ferreira, S. H. & Vane, J. R. (1967) Nature (London) 216, 868-873. 5. Gilmore, N., Vane, J. R. & Wyllie, J. H. (1968) Nature (London) 218, 1135-1140. 6. Jobke, A., Peskar, B. A. & Hertting, G. (1976) NaunynSchmiedebergs Arch. Pharmakol. 292,35-42. 7. Liebig, R., Bernauer, W. & Peskar, B. A. (1974) NaunynSchmiedebergs Arch. Pharmakol. 284, 279-293.
8. Ramwell, P. W., Shaw, J. E. & Kucharski, J. (1965) Science 149, 1390-1391. 9. Laity, J. L. H. (1969) Br. J. Pharmacol. 37,698-704. 10. Leslie, C. A. (1976) Res. Commun. Chem. Pathol. Pharmacol. 14,455-469. 11. Needleman, P., Douglas, J. R., Jr., Jakschik, B., Stoecklein, P. B. & Johnson, E. M., Jr. (1974) J. Pharmacol. Exp. Ther. 188,
453-460. 12. Takaguchi, C., Kohno, E. & Sih, C. J. (1971) Biochemistry 10, 2372-2376. 13. Pong, S. S. & Levine, L. (1977) in The Prostaglandins, ed. Ramwell, P. W. (Plenum, New York), Vol. 3, pp. 41-76. 14. Levine, L., Alam, I. & Langone, J. L. (1979) Prostaglandins Med. 2, 177-189. 15. Alam, I., Ohuchi, K. & Levine, L. (1979) Anal. Biochem. 93, 339-345. 16. Levine, L. & Hassid, A. (1977) Biochem. Blophys. Res. Commun.
792477-484.
17. Ohuchi, K. & Levine, L. (1978) J. Biol. Chem. 253, 47834790. 18. Horton, E. W. (1973) Br. Med. Bull. 29, 148-151. 19. Price, C. J. & Rowe, C. E. (1972) Biochem. J. 126,575-585. 20. Dunham, E. W. & Zimmerman, B. G. (1970) Am. J. Physiol. 219, 1279-1285. 21. Hedqvist, P. (1973) in The Prostaglandins, ed. Ramwell, P. W. (Plenum, New York), Vol. 1, pp. 101-131. 22. Williams, L. T. & Lefkowitz, R. J. (1978) Receptor Binding Studies in Adrenergic Pharmacology (Raven, New York). 23. Levine, L. (1979) in Hormones and Cell Culture, eds. Sato, G. & Ross, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 623-640. 24. Rindler, M. J., Chuman, L. M., Shaffer, L. & Saier, M. H., Jr. (1979) J. Cell Biol. 81, 635-648. 25. Grantham, J. J. & Orloff, J. (1968) J. Clin. Invest. 47, 11541161. 26. liono, Y., Flier, S. R. & Brenner, B. M. (1979) Clin. Res. 27, 418a. 27. Hirata, F., Corcoran, B. A., Venkatasubramanian, K., Schiffman, E. & Axelrod, J. (1979) Proc. Natl. Acad. Sca. USA 76, 26402643. 28. Hirata, F. & Axelrod, J. (1978) Proc. Natl. Acad. Sci. USA 75, 2348-2352.
