Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECTS OF SOME INHIBITORS OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ON PROTEIN PHOSPHORYLATION IN I S O L A T E D N E U R O N S AND GLIA FROM RAT BRAIN William J. Kinnier and John Eric Wilson Department of Biochemistry and Nutrition, University of North Carolina School of Medicine, Chapel Hill, NC 27514 Received
July
1,1977
SUMMARY: The effects of the cyclic nucleotide phosphodiesterase inhibitors theophylline, l-methyl-3-isobutylxanthine, and R0-20-1724 on protein phosphorylation in homogenates of isolated neurons and glia were examined. Theophylline (10mM) and l-methyl-3-isobutylxanthine (imM) inhibit protein phosphorylation by more than 40%. In both neuronal and glial preparations these agents lowered the basal rate of protein phosphorylation and, to a slightly lesser extent, the rate in the presence of added cyclic AMP. It also appears that the predominant form of cyclic nucleotide phosphodiesterase in isolated neurons is calcium-dependent since it is markedly inhibited by EGTA, while that in isolated glia is predominantly independent of calcium ions. Since the discovery two decades ago of cyclic adenosine 3', 5' -monophosphate by Sutherland and Rall
(i), cyclic AMP has been found
to be produced by adenylate cyclase phosphodiesterase
(i).
(2) and hydrolyzed by cyclic nucleotide
It appears to affect "cyclic AMP mediated hormone-
triggered events" by activating a cyclic AMP-dependent protein kinase
(3).
In order to study these enzymes individually or their interrelationships, cyclic nucleotide phosphodiesterase
inhibitors are routinely utilized to
maintain levels of cyclic AMP endogenously produced or exogenously added. Theophylline
has been the most widely used of these inhibitors and its
action has been generally attributed to inhibition of the phosphodiesterase; however, other anomolous effects of theophylline have been described. phylline has been reported to affect calcium efflux secretion
(4) and endocrine
(5), to inhibit intrinsic protein kinase activity of brain membrane
preparations homogenates
Theo-
(6), and to lower basal protein phosphorylation
in rat caudate
(7).
We now report that theophylline,
l-methyl-3-isobutylxanthine
*Abbreviations: cyclic adenosine -3', 5'-monophosphate, isobutyl-l-methyl xanthine, IBMX.
Copyright © 1977 by Academic Press, Inc. All rights o/reproduction in any form reserved.
and RO-20-
cyclic AMP; 3-
1369 1SSN 0006-291X
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1724, in concentrations commonly used for the inhibition of cyclic nucleotide phosphodiesterase activity, suppress both basal and cyclic AMP-stimulated protein kinase activity in homogenates of isolated neurons and glia. METHODS Materials: Theophylline, cyclic AMP, and beef heart cyclic AMP-dependent protein kinase were from Sigma Chemical Company. A cyclic AMP assay kit TRK .432 was from Amershara-Searle. EGTA was from Calbiochem. The l-methyl-3isobutylxanthine was a gift from Dr. Richard Miller of Burroughs Wellcome. The RO-20-1724 was a gift from Hoffmann La Roche Inc. All other chemicals were of reagent grade. I s o l a t i 0 n o f neurons and glia: Neurons and glia were isolated from the cerebral cortices of 100 g, male, Wistar rats by a modification of the method of Sellinger et al. ( 8 ) . Bovine serum albumin was omitted from all media and MgCl 2 was substituted for CaCl 2 in the 7.5% polyvinylpyrrolidone solution. The gradients were centrifuged at 3,200 g in a Sorvall RC-3 centrifuge using an HL-8 rotor. Under a phase contrast microscope the neuronal and glial fractions prepared in this way appeared to be indentical to those prepared by the unmodified procedure. The caleulated RNA/DNA ratios were 1.92 for neuronal perikarya and 1.86 for the glia. Determinations of protein, RNA, and DNA were done as described by Sellinger et al. (8). Assay for protein phosphor~lation: Neurons or glia were homogenized by hand in 1.4 ml of homogenization medium (2mM EGTA, 10mM K2HP04, pH 7.4), using i0 strokes with a Teflon and glass Potter-Elvehjem homogenizer (clearance 0.i - 0.15 mm). Of this suspension 25~i (approximately 35~g protein) was added to 75~i of incubation medium (2mM MgS04, 0.2mM K2HP04, 0.2mM EGTA, pH 7.4, with or without phosphodiesterase inhibitor of cyclic AMP) which had been preincubated for 1 min at 37°C. Incubation was continued for 1 min at 37°C, followed by the addition of 0.5-1~Ci/150mM [y_32p] ATP (prepared as previously described (7)) to a final concentration of 5~M and carried out at 37°C for 1 min. Kinase activity was terminated with 2ml of 10% trichloroacetic acid at 4°C. The tubes were chilled in ice for 30 min, heated to 90°C for 20 min, and again chilled for 30 min. With two washes of 2ml of 10% trichloroacetic acid, the contents of each tube were vacuum filtered onto 2.3cm Whatman 3MM filter discs, that had been prewashed with 2ml of 10% trichloroacetic acid. The discs were then washed with 15ml of 5% trichloroacetic acid followed by 5ml of ethanol: ether (i:i) and placed in scintillation vials to dry. Radioactivity was assayed as previously described (7). Assay of cyclic AMP: Cyclic AMP was assayed by the method of Cooper et al. (9) by means of an Amersham-Searle cyclic AMP assay kit. RESULTS Effects of ph0s~hodiesterase inhibitors on ~rotein phosphorylation:
Although
absolute levels of protein phosphorylation and the magnitudes of responses to activators and inhibitors varied from one cell preparation to another, each preparation gave results that were internally consistent, and results were qualitatively consistent from one batch of cells to another.
1370
Table 1
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Theophylline effects on protein phosphorylation of homogenized neurons and ~lia
Cell type
Theophylline concentration (mM)
cyclic AMP
% control ± s.d.* (n=5)
% basalt
(uM) Neurons
Glia
0 0
0 i0
i00.0 ± 4.7 i16.9 ± 2.6
100.0 116.9
1 1
0 l0
i00.0 ± 6.4 124.5 ± 6.9
71.3 88.8
i0 l0
0 l0
100.0 ± 5.5 138.1 ± 7.0
46.3 63.9
0 0
0 i0
i00.0 ± 3.6 130.2 ± 0.4
100.0 130.2
1
0
i00.0 ± 6,0
1
l0
i17.8 + 0.6
92.0 108.4
i0 i0
0 i0
i00.0 + 3.4 163.1 +- 3.6
49.4 63.9
* % control = epm32p incorporated/m ~ protein cpm32p incorporated/mg protein in absence of cyclic AMP
X i00
T % basal
X 100
= c~m32P inc°r~°ratgd/mg pr°tein cpm32p incorporated/mg protein in absence of cyclic and phosphodiesterase inhibitor
gives typical results obtained from one preparation of isolated neurons and glia.
It shows the effects of various concentrations of theophylline on
protein phosphorylation in homogenates of these cells. Other preparations gave results that were in agreement with those presented here except that the stimulation caused by cyclic AMP in the absence of phosphodiesterase inhibitors was variable.
The results suggest
a K i of approximately 10mM for the action of theophylline on basal protein kinase activity in both neurons and glia, and somewhat higher than 10mM in the presence of added cyclic AMP.
Similar, but less marked, effects
were seen in the presence of imM theophylline.
1371
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 2 I BMX and RO-20-1724
effects on ~rotein phosphorylation
of homogenized neurons
and glia
Cell type
Phosphodiesterase (PDE) inhibitor
Inhibitor concentration
cyclic AMP
% control ± s.d. (n=5)
% basal
(uM)
Neurons
None None
0 0
0 i0
i00.0 ± 4.4 144.3 ± 0.4
i00.0 144.3
i0.0 i0.0
0 i0
i00.0 ± 4.4 169.4 ± 1.9
58.1 98.4
IBMX IBMX
0.i 0.i
0 10
100.0 ± 4.8 155.5 ± 3.0
89.4 139.0
IBMX IBMX
1.0 1.0
0 i0
i00.0 ± 7.3 151.9 ± 5.5
60.2 91.4
RO 20-1724 RO 20-1724
0.I 0.i
0 i0
i00.0 ± 8.3 136.8 ± 5.8
97.5 133.4
RO 20-1724 RO 20-1724
1.0 1.0
0 i0
i00.0 ± 7.1 125.0 ± 3.1
93.2 116.5
None None
0 0
0 i0
i00.0 ± 5.6 118.3 ± 6.6
i00.0 118.3
i0.0 i0.0
0 i0
i00.0 ± 4.6 180.4 ± 2.5
52.9 95.4
IBMX IBMX
0.i 0.i
0 i0
i00.0 ± 5.9 130.7 ± 4.6
92.7 121.1
IBMX IBMX
1.0 1.0
0 i0
i00.0 ± 8.1 156.6 ± 2.9
56.2 88.0
RO 20-1724 RO 20-1724
0.i 0.i
0 i0
i00.0 ± 3.4 138.7 ± 2.3
84.6 117.3
RO 20-1724 RO 20-1724
1.0 1.0
0 i0
I00.0 ± 6.2 131.4 ± 6.8
82.6 108.8
Theophylline Theophylline
Glia
Theophylline Theophylline
Results expressed
as in Table i.
The effects of IBMX and of RO-20-1724 compared with those of 10mM theophylline.
on protein phosphorylation IBMX and RO-20-1724
reported to be more potent than theophylline
1372
as inhibitors
were
have been
of the
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 3 Phosphodiesterase activity of homo$enized isolated neurons and $1ia
Cell type
EGTA (650~M)
Phosphodiesterase inhibitor
Neurons +
Inhibitor concentration (mM)
Phosphodiesterase activity*
0
2.72
0
0.80
Structure
O +
Theophylline
i0
0.72 O~I"-.N/--N ~H 3
O I! +
IBMX
0.i
0.88
CN3-NA~--,N~
o t.
!
C4H 9
+
RO-20-1724
0.i
0.72
C 4 H 9 0 ~ H > : o CH 3 0 ~
--N H
GI ia +
0
1.56
0
1.31
i0
0.40
Same as above
+
Theophylline
+
IBMX
0.I
0.84
Same as above
+
RO-20-1724
0.I
0.84
Same as above
*nanomoles cyclic AMP hydrolyzed/mg protein/minute
phosphodiesterase
(i0, ii), and Table 2 shows that imM IBMX inhibits basal
protein phosphorylation in neurons and glia to about the same extent that 10mM theophylline does.
The inhibition of cyclic AMP-stimulated phos-
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Table 4 Theophylline inhibition of beef heart cAMP dependent protein kinase
cAMP 10~M
Theophylline 10mM
% control ± s.d. (n=3)
i00.0 ± 4.1
I00.0
210.6 ± 9.3
210.6
+
i00.0 ± 5.9
59.0
+
330.6 ± 8.6
195.0
+
-
+
% basal
Beef heart cyclic AMP-dependent protein kinase activity was determined using 200~g of casein in homogenization medium and 10~g of the enzyme in incubation medium in the presence of 5~M [y_32p] ATP as described in methods. Results expressed as in Table i.
phorylation by the isobutyl derivative, on the other hand, is slightly greater than the theophylline inhibition.
Thus phosphorylation responses to
cyclic AMP appear to be less when IBMX is used to inhibit phosphodiesterase than when theophylline is used. R0-20-1724 is of particular interest because it is widely used as a phosphodiesterase inhibitor and it is an imidazolidinone derivative rather than a xanthine.
It has relatively little effect on basal or cyclic AMP-
stimulated phosphorylation,
although glial preparations show greater
responses to cyclic AMP in the presence of RO-20-1724 than in its absence, while neuronal preparations show the opposite effect. Effect of theophylline on the activity of a protein kinase, of non-neural origin: Table 4 shows that 10mM theophylline suppresses the activity of the cyclic AMP-dependent protein kinase of beef heart. Comparative effects of the inhibitors on phos~hodiesterase activities To gain more information about the effects of these compounds on
1374
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
protein phosphorylation, we compared their effectiveness as inhibitors of phosphodiesterase activities in homogenates of neurons and glia, in the presence and absence of EGTA (Table 3).
EGTA itself proved to be a rather
effective inhibitor of the neuronal phosphodiesterase activity.
None of the
three specific inhibitors significantly increased the EGTA inhibition of neuronal activity when used at concentrations that were about 1-3 times the K i values reported in the literature. Glial phosphodiesterase activity, on the other hand, was only slightly (16%) inhibited by EGTA, and all three inhibitors gave additional inhibition when added to the incubation media containing the chelator.
At the
concentrations used in this experiment, theophylline was the most effective inhibitor of glial phosphodiesterase activity. DISCUSSION Theophylline, a phosphodiesterase inhibitor with a K i close to imM (12), has been widely utilized to inhibit cyclic nucleotide degradation in various preparations while other more potent phosphodiesterase inhibitors such as IBMX and RO-20-1724 have been introduced more recently.
Since
these compounds are so widely used in the study of cyclic nucleotidemediated events, their effects on other enzymes of the cyclic nucleotide system other than the cyclic nucleotide phosphodiesterase deserve consideration. In the homogenates of isolated neurons and glia the xanthines appear to inhibit protein kinase activity, lowering both basal and cyclic AMPstimulated protein phosphorylation.
The latter, however, is inhibited less
than basal activity by these compounds.
Hence, the percentage increase in
phosphorylation in response to cyclic AMP is greater in the presence of the methyl xanthines than in their absence, even though absolute increases are diminished.
This phenomenon is not specific to brain since it is also seen
with purified cyclic AMP-dependent protein kinase from beef heart. This supports similar observations by Weller and Rodnight on crude
1375
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cerebral membrane preparations of Hullihan et al.
(6) and is in harmony with the observations
(7).
The potencies of the methyl xanthines, theophylline and IBMX, as protein kinase inhibitors seem
to parallel their phosphodiesterase inhibitory
activity with Ki's for the kinase of 10mM and imM respectively. Although no detailed study of structure-activity relationship was done, the methyl xanthines inhibitors inhibit protein phosphorylation more than R0-20-1724, at concentrations commonly used.
This suggests that the
xanthine structure is important in lowering the activity of the kinase. In addition, it also appears that the calcium-dependent form of the phosphodiesterase
(13) is the predominant form in isolated neurons while
that in isolated glia is predominantly independent of calcium. In conclusion, if phosphodiesterase inhibitors must be used to maintain levels of cyclic AMP, concentrations of 0.1mM IBMX or imM RO-20-1724 do not inhibit protein kinase activity more seriously than about i0 percent. ACKNOWLEDGEMENTS The authors would like to thank Robert Berman and John P. Hullihan for their helpful discussions. This work was supported in part by NIH grant NS-07457. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii.
12. 13.
Sutherland, E. W. and Rall, T. W. (1958) J. Biol. Chem. 232, 1077-1091. Sutherland, E. W., Rall, T. W. and Nemon, T. (1962) J. Biol. Chem. 237, 1220-1227. Langan, T. (1972) Advances in Cyclic Nucleotide Research vol. 3 pp. 99153, Raven Press, New York. Brisson, G. R., Malaisse-Lagae, F. and Malaisse, W. J. (1972) J. Clinical Invest. 5_~i, 232-241. Fleisher, N. and Donald, R. A. (1969) Amer. J. Physiology 217, 1287-1291. Weller, M. and Rodnight, R. (1971) Biochem. J. 124, 393-406. Hullihan, J. P., Wilson, J. E. and Williams, M. (1977) Biochem. Biophys. Acta in press. Sellinger, O., Azcurra, J., Johnson, D., Ahlsson, W. and Lodin, Z. (1971) Nature New Biol. 230, 253-256. Cooper R. H., McPherson, M. and Schofield, J. G. (1972) Biochem. J. 127, 143-154. Fredholm, B. B., Fuxe, K. and Agnati, L. (1976) E. J. Pharm. 38, 31-38. Weinryb, I., Chasin, M., Free, C. A., Harris, D. N., Goldberg, H., Mitchell, I. M., Paik, V. S., Phillips, M., Samaniego, S. and Hess, S. M. (1972) J. Pharm. Sci. 61, 1556-1567. Appleman, M. M., Thompson, W. J. and Russell, T. R. (1973) Advances in cyclic Nucleotide Research vol. 3 pp. 65-98, Raven Press, New York. Weiss, B. (1975) Advances in Cyclic Nucleotide Research Vol. 5, 195211, Raven Press, New York.
1376
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EFFECTS OF SOME INHIBITORS OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ON PROTEIN PHOSPHORYLATION IN I S O L A T E D N E U R O N S AND GLIA FROM RAT BRAIN William J. Kinnier and John Eric Wilson Department of Biochemistry and Nutrition, University of North Carolina School of Medicine, Chapel Hill, NC 27514 Received
July
1,1977
SUMMARY: The effects of the cyclic nucleotide phosphodiesterase inhibitors theophylline, l-methyl-3-isobutylxanthine, and R0-20-1724 on protein phosphorylation in homogenates of isolated neurons and glia were examined. Theophylline (10mM) and l-methyl-3-isobutylxanthine (imM) inhibit protein phosphorylation by more than 40%. In both neuronal and glial preparations these agents lowered the basal rate of protein phosphorylation and, to a slightly lesser extent, the rate in the presence of added cyclic AMP. It also appears that the predominant form of cyclic nucleotide phosphodiesterase in isolated neurons is calcium-dependent since it is markedly inhibited by EGTA, while that in isolated glia is predominantly independent of calcium ions. Since the discovery two decades ago of cyclic adenosine 3', 5' -monophosphate by Sutherland and Rall
(i), cyclic AMP has been found
to be produced by adenylate cyclase phosphodiesterase
(i).
(2) and hydrolyzed by cyclic nucleotide
It appears to affect "cyclic AMP mediated hormone-
triggered events" by activating a cyclic AMP-dependent protein kinase
(3).
In order to study these enzymes individually or their interrelationships, cyclic nucleotide phosphodiesterase
inhibitors are routinely utilized to
maintain levels of cyclic AMP endogenously produced or exogenously added. Theophylline
has been the most widely used of these inhibitors and its
action has been generally attributed to inhibition of the phosphodiesterase; however, other anomolous effects of theophylline have been described. phylline has been reported to affect calcium efflux secretion
(4) and endocrine
(5), to inhibit intrinsic protein kinase activity of brain membrane
preparations homogenates
Theo-
(6), and to lower basal protein phosphorylation
in rat caudate
(7).
We now report that theophylline,
l-methyl-3-isobutylxanthine
*Abbreviations: cyclic adenosine -3', 5'-monophosphate, isobutyl-l-methyl xanthine, IBMX.
Copyright © 1977 by Academic Press, Inc. All rights o/reproduction in any form reserved.
and RO-20-
cyclic AMP; 3-
1369 1SSN 0006-291X
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1724, in concentrations commonly used for the inhibition of cyclic nucleotide phosphodiesterase activity, suppress both basal and cyclic AMP-stimulated protein kinase activity in homogenates of isolated neurons and glia. METHODS Materials: Theophylline, cyclic AMP, and beef heart cyclic AMP-dependent protein kinase were from Sigma Chemical Company. A cyclic AMP assay kit TRK .432 was from Amershara-Searle. EGTA was from Calbiochem. The l-methyl-3isobutylxanthine was a gift from Dr. Richard Miller of Burroughs Wellcome. The RO-20-1724 was a gift from Hoffmann La Roche Inc. All other chemicals were of reagent grade. I s o l a t i 0 n o f neurons and glia: Neurons and glia were isolated from the cerebral cortices of 100 g, male, Wistar rats by a modification of the method of Sellinger et al. ( 8 ) . Bovine serum albumin was omitted from all media and MgCl 2 was substituted for CaCl 2 in the 7.5% polyvinylpyrrolidone solution. The gradients were centrifuged at 3,200 g in a Sorvall RC-3 centrifuge using an HL-8 rotor. Under a phase contrast microscope the neuronal and glial fractions prepared in this way appeared to be indentical to those prepared by the unmodified procedure. The caleulated RNA/DNA ratios were 1.92 for neuronal perikarya and 1.86 for the glia. Determinations of protein, RNA, and DNA were done as described by Sellinger et al. (8). Assay for protein phosphor~lation: Neurons or glia were homogenized by hand in 1.4 ml of homogenization medium (2mM EGTA, 10mM K2HP04, pH 7.4), using i0 strokes with a Teflon and glass Potter-Elvehjem homogenizer (clearance 0.i - 0.15 mm). Of this suspension 25~i (approximately 35~g protein) was added to 75~i of incubation medium (2mM MgS04, 0.2mM K2HP04, 0.2mM EGTA, pH 7.4, with or without phosphodiesterase inhibitor of cyclic AMP) which had been preincubated for 1 min at 37°C. Incubation was continued for 1 min at 37°C, followed by the addition of 0.5-1~Ci/150mM [y_32p] ATP (prepared as previously described (7)) to a final concentration of 5~M and carried out at 37°C for 1 min. Kinase activity was terminated with 2ml of 10% trichloroacetic acid at 4°C. The tubes were chilled in ice for 30 min, heated to 90°C for 20 min, and again chilled for 30 min. With two washes of 2ml of 10% trichloroacetic acid, the contents of each tube were vacuum filtered onto 2.3cm Whatman 3MM filter discs, that had been prewashed with 2ml of 10% trichloroacetic acid. The discs were then washed with 15ml of 5% trichloroacetic acid followed by 5ml of ethanol: ether (i:i) and placed in scintillation vials to dry. Radioactivity was assayed as previously described (7). Assay of cyclic AMP: Cyclic AMP was assayed by the method of Cooper et al. (9) by means of an Amersham-Searle cyclic AMP assay kit. RESULTS Effects of ph0s~hodiesterase inhibitors on ~rotein phosphorylation:
Although
absolute levels of protein phosphorylation and the magnitudes of responses to activators and inhibitors varied from one cell preparation to another, each preparation gave results that were internally consistent, and results were qualitatively consistent from one batch of cells to another.
1370
Table 1
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Theophylline effects on protein phosphorylation of homogenized neurons and ~lia
Cell type
Theophylline concentration (mM)
cyclic AMP
% control ± s.d.* (n=5)
% basalt
(uM) Neurons
Glia
0 0
0 i0
i00.0 ± 4.7 i16.9 ± 2.6
100.0 116.9
1 1
0 l0
i00.0 ± 6.4 124.5 ± 6.9
71.3 88.8
i0 l0
0 l0
100.0 ± 5.5 138.1 ± 7.0
46.3 63.9
0 0
0 i0
i00.0 ± 3.6 130.2 ± 0.4
100.0 130.2
1
0
i00.0 ± 6,0
1
l0
i17.8 + 0.6
92.0 108.4
i0 i0
0 i0
i00.0 + 3.4 163.1 +- 3.6
49.4 63.9
* % control = epm32p incorporated/m ~ protein cpm32p incorporated/mg protein in absence of cyclic AMP
X i00
T % basal
X 100
= c~m32P inc°r~°ratgd/mg pr°tein cpm32p incorporated/mg protein in absence of cyclic and phosphodiesterase inhibitor
gives typical results obtained from one preparation of isolated neurons and glia.
It shows the effects of various concentrations of theophylline on
protein phosphorylation in homogenates of these cells. Other preparations gave results that were in agreement with those presented here except that the stimulation caused by cyclic AMP in the absence of phosphodiesterase inhibitors was variable.
The results suggest
a K i of approximately 10mM for the action of theophylline on basal protein kinase activity in both neurons and glia, and somewhat higher than 10mM in the presence of added cyclic AMP.
Similar, but less marked, effects
were seen in the presence of imM theophylline.
1371
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Table 2 I BMX and RO-20-1724
effects on ~rotein phosphorylation
of homogenized neurons
and glia
Cell type
Phosphodiesterase (PDE) inhibitor
Inhibitor concentration
cyclic AMP
% control ± s.d. (n=5)
% basal
(uM)
Neurons
None None
0 0
0 i0
i00.0 ± 4.4 144.3 ± 0.4
i00.0 144.3
i0.0 i0.0
0 i0
i00.0 ± 4.4 169.4 ± 1.9
58.1 98.4
IBMX IBMX
0.i 0.i
0 10
100.0 ± 4.8 155.5 ± 3.0
89.4 139.0
IBMX IBMX
1.0 1.0
0 i0
i00.0 ± 7.3 151.9 ± 5.5
60.2 91.4
RO 20-1724 RO 20-1724
0.I 0.i
0 i0
i00.0 ± 8.3 136.8 ± 5.8
97.5 133.4
RO 20-1724 RO 20-1724
1.0 1.0
0 i0
i00.0 ± 7.1 125.0 ± 3.1
93.2 116.5
None None
0 0
0 i0
i00.0 ± 5.6 118.3 ± 6.6
i00.0 118.3
i0.0 i0.0
0 i0
i00.0 ± 4.6 180.4 ± 2.5
52.9 95.4
IBMX IBMX
0.i 0.i
0 i0
i00.0 ± 5.9 130.7 ± 4.6
92.7 121.1
IBMX IBMX
1.0 1.0
0 i0
i00.0 ± 8.1 156.6 ± 2.9
56.2 88.0
RO 20-1724 RO 20-1724
0.i 0.i
0 i0
i00.0 ± 3.4 138.7 ± 2.3
84.6 117.3
RO 20-1724 RO 20-1724
1.0 1.0
0 i0
I00.0 ± 6.2 131.4 ± 6.8
82.6 108.8
Theophylline Theophylline
Glia
Theophylline Theophylline
Results expressed
as in Table i.
The effects of IBMX and of RO-20-1724 compared with those of 10mM theophylline.
on protein phosphorylation IBMX and RO-20-1724
reported to be more potent than theophylline
1372
as inhibitors
were
have been
of the
Vol. 77, No. 4, 1977
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 3 Phosphodiesterase activity of homo$enized isolated neurons and $1ia
Cell type
EGTA (650~M)
Phosphodiesterase inhibitor
Neurons +
Inhibitor concentration (mM)
Phosphodiesterase activity*
0
2.72
0
0.80
Structure
O +
Theophylline
i0
0.72 O~I"-.N/--N ~H 3
O I! +
IBMX
0.i
0.88
CN3-NA~--,N~
o t.
!
C4H 9
+
RO-20-1724
0.i
0.72
C 4 H 9 0 ~ H > : o CH 3 0 ~
--N H
GI ia +
0
1.56
0
1.31
i0
0.40
Same as above
+
Theophylline
+
IBMX
0.I
0.84
Same as above
+
RO-20-1724
0.I
0.84
Same as above
*nanomoles cyclic AMP hydrolyzed/mg protein/minute
phosphodiesterase
(i0, ii), and Table 2 shows that imM IBMX inhibits basal
protein phosphorylation in neurons and glia to about the same extent that 10mM theophylline does.
The inhibition of cyclic AMP-stimulated phos-
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Table 4 Theophylline inhibition of beef heart cAMP dependent protein kinase
cAMP 10~M
Theophylline 10mM
% control ± s.d. (n=3)
i00.0 ± 4.1
I00.0
210.6 ± 9.3
210.6
+
i00.0 ± 5.9
59.0
+
330.6 ± 8.6
195.0
+
-
+
% basal
Beef heart cyclic AMP-dependent protein kinase activity was determined using 200~g of casein in homogenization medium and 10~g of the enzyme in incubation medium in the presence of 5~M [y_32p] ATP as described in methods. Results expressed as in Table i.
phorylation by the isobutyl derivative, on the other hand, is slightly greater than the theophylline inhibition.
Thus phosphorylation responses to
cyclic AMP appear to be less when IBMX is used to inhibit phosphodiesterase than when theophylline is used. R0-20-1724 is of particular interest because it is widely used as a phosphodiesterase inhibitor and it is an imidazolidinone derivative rather than a xanthine.
It has relatively little effect on basal or cyclic AMP-
stimulated phosphorylation,
although glial preparations show greater
responses to cyclic AMP in the presence of RO-20-1724 than in its absence, while neuronal preparations show the opposite effect. Effect of theophylline on the activity of a protein kinase, of non-neural origin: Table 4 shows that 10mM theophylline suppresses the activity of the cyclic AMP-dependent protein kinase of beef heart. Comparative effects of the inhibitors on phos~hodiesterase activities To gain more information about the effects of these compounds on
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
protein phosphorylation, we compared their effectiveness as inhibitors of phosphodiesterase activities in homogenates of neurons and glia, in the presence and absence of EGTA (Table 3).
EGTA itself proved to be a rather
effective inhibitor of the neuronal phosphodiesterase activity.
None of the
three specific inhibitors significantly increased the EGTA inhibition of neuronal activity when used at concentrations that were about 1-3 times the K i values reported in the literature. Glial phosphodiesterase activity, on the other hand, was only slightly (16%) inhibited by EGTA, and all three inhibitors gave additional inhibition when added to the incubation media containing the chelator.
At the
concentrations used in this experiment, theophylline was the most effective inhibitor of glial phosphodiesterase activity. DISCUSSION Theophylline, a phosphodiesterase inhibitor with a K i close to imM (12), has been widely utilized to inhibit cyclic nucleotide degradation in various preparations while other more potent phosphodiesterase inhibitors such as IBMX and RO-20-1724 have been introduced more recently.
Since
these compounds are so widely used in the study of cyclic nucleotidemediated events, their effects on other enzymes of the cyclic nucleotide system other than the cyclic nucleotide phosphodiesterase deserve consideration. In the homogenates of isolated neurons and glia the xanthines appear to inhibit protein kinase activity, lowering both basal and cyclic AMPstimulated protein phosphorylation.
The latter, however, is inhibited less
than basal activity by these compounds.
Hence, the percentage increase in
phosphorylation in response to cyclic AMP is greater in the presence of the methyl xanthines than in their absence, even though absolute increases are diminished.
This phenomenon is not specific to brain since it is also seen
with purified cyclic AMP-dependent protein kinase from beef heart. This supports similar observations by Weller and Rodnight on crude
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cerebral membrane preparations of Hullihan et al.
(6) and is in harmony with the observations
(7).
The potencies of the methyl xanthines, theophylline and IBMX, as protein kinase inhibitors seem
to parallel their phosphodiesterase inhibitory
activity with Ki's for the kinase of 10mM and imM respectively. Although no detailed study of structure-activity relationship was done, the methyl xanthines inhibitors inhibit protein phosphorylation more than R0-20-1724, at concentrations commonly used.
This suggests that the
xanthine structure is important in lowering the activity of the kinase. In addition, it also appears that the calcium-dependent form of the phosphodiesterase
(13) is the predominant form in isolated neurons while
that in isolated glia is predominantly independent of calcium. In conclusion, if phosphodiesterase inhibitors must be used to maintain levels of cyclic AMP, concentrations of 0.1mM IBMX or imM RO-20-1724 do not inhibit protein kinase activity more seriously than about i0 percent. ACKNOWLEDGEMENTS The authors would like to thank Robert Berman and John P. Hullihan for their helpful discussions. This work was supported in part by NIH grant NS-07457. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii.
12. 13.
Sutherland, E. W. and Rall, T. W. (1958) J. Biol. Chem. 232, 1077-1091. Sutherland, E. W., Rall, T. W. and Nemon, T. (1962) J. Biol. Chem. 237, 1220-1227. Langan, T. (1972) Advances in Cyclic Nucleotide Research vol. 3 pp. 99153, Raven Press, New York. Brisson, G. R., Malaisse-Lagae, F. and Malaisse, W. J. (1972) J. Clinical Invest. 5_~i, 232-241. Fleisher, N. and Donald, R. A. (1969) Amer. J. Physiology 217, 1287-1291. Weller, M. and Rodnight, R. (1971) Biochem. J. 124, 393-406. Hullihan, J. P., Wilson, J. E. and Williams, M. (1977) Biochem. Biophys. Acta in press. Sellinger, O., Azcurra, J., Johnson, D., Ahlsson, W. and Lodin, Z. (1971) Nature New Biol. 230, 253-256. Cooper R. H., McPherson, M. and Schofield, J. G. (1972) Biochem. J. 127, 143-154. Fredholm, B. B., Fuxe, K. and Agnati, L. (1976) E. J. Pharm. 38, 31-38. Weinryb, I., Chasin, M., Free, C. A., Harris, D. N., Goldberg, H., Mitchell, I. M., Paik, V. S., Phillips, M., Samaniego, S. and Hess, S. M. (1972) J. Pharm. Sci. 61, 1556-1567. Appleman, M. M., Thompson, W. J. and Russell, T. R. (1973) Advances in cyclic Nucleotide Research vol. 3 pp. 65-98, Raven Press, New York. Weiss, B. (1975) Advances in Cyclic Nucleotide Research Vol. 5, 195211, Raven Press, New York.
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