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The Journal of Neuroscience, May 1994, f4(5): 2953-2965
&Adrenergic Receptor-mediated Regulation of Extracellular
Adenosine in Cerebral Cortex in Culture
Paul A. Rosenberg, Roger Knowles, Kelly P. Knowles, and Ya Li
Department of Neurology and Program in Neuroscience, Children’s Hospital and Harvard Medical School, Boston,
Massachusetts 02115
Adenosine is an important inhibitory neuromodulator in the
CNS, yet the sources of extracellular adenosine have yet to
be well characterized. In this study we show that /3-adre-
nergic stimulation of cortical cultures results in the extra-
cellular accumulation of CAMP as well as adenosine, and
that the extracellular adenosine derives from extracellular
CAMP. The concentration dependence of isoproterenol in
evoking CAMP secretion was determined by radioimmuno-
assay, and the EC,, for this effect was found to be approx-
imately 100 nM. In order to investigate the effect of &adre-
nergic sti’mulation on the regulation of extracellular
adenosine, the effect of isoproterenol in stimulating the ex-
tracellular accumulation of adenine-containing compounds
was examined by HPLC. lsoproterenol stimulated CAMP se-
cretion in both astrocyte cultures and astrocyte-rich mixed
cultures of astrocytes and neurons. However, no extracel-
lular CAMP was detectable in neuron-enriched astrocyte-
poor cultures. Extracellular adenosine increased in response
to isoproterenol in the astrocyte-rich mixed cultures, but not
in the neuron-enriched astrocyte-poor cultures. After 30 min
exposure to isoproterenol, the concentration of adenosine
in the extracellular medium increased by 47% in 56 exper-
iments in the mixed astrocyte-rich cultures.
In order to establish whether the adenosine that accu-
mulates in response to isoproterenol stimulation actually de-
rives from extracellular CAMP, phosphodiesterase inhibitors
were tested for their ability to block isoproterenol-stimulated
adenosine accumulation. lsobutylmethylxanthine (IBMX; 100
AM), RO 20-l 724 (180 AM), carbazeran (10 PM), dipyridamole
(10 @I), and trifluoperazine (10 AM) had no inhibitory effect
on the isoproterenol-stimulated accumulation of extracel-
lular adenosine. However 100 AM IBMX plus 180 @I RO 20-
1724 effectively blocked isoproterenol-stimulated adeno-
sine accumulation and, as expected, increased extracellular
CAMP.
As a further test of the origin of isoproterenol-stimulated
adenosine accumulation, we attempted to block this phe-
nomenon by blocking CAMP secretion itself. For this purpose
Received Aug. 2, 1993; revised Oct. 15, 1993; accepted Oct. 26, 1993.
We thank Can Zhong and Sara Vasquez for excellent technical assistance, and
Dr. Mohamad Mikati for heluful discussions earlv in this work. P.A.R. is suuoorted
by U.S. Public Health Se&ice Grants NS26SiO and NS31353, an Established
Investigatorship from the American Heart Association, and a Mental Retardation
Core Grant to Children’s Hospital.
Correspondence should be addressed to Dr. Paul A. Rosenberg, Enders Research
Building, Department of Neurology, Children’s Hospital, 300 Longwood Avenue,
Boston, MA 02 115.
Copyright 0 1994 Society for Neuroscience 0270-6474/94/ 142953-13$05.00/O
probenecid, a known inhibitor of CAMP secretion in many
different cell types, was used. We found that probenecid at
1 mM blocked isoproterenol-stimulated adenosine accu-
mulation.
These studies suggest that one potentially important
source of extracellular adenosine in the cerebral cortex is
endogenous extracellular CAMP, secreted from astrocytes
in response to @-adrenergic receptor stimulation. Since the
receptors of neuromodulators other than norepinephrine may
also be coupled to adenylyl cyclase in the cerebral cortex,
there may be several neuromodulatory systems that regulate
extracellular adenosine levels by this mechanism.
[Key words: norepinephrine, CAMP, phosphodiesterase,
astrocytes, secretion, transport, B-adrenergic, isoprotere-
no/]
Adenosine is an inhibitory modulator of central excitatory neu-
rotransmission. Adenosine receptors in the CNS have been well
characterized in ligand binding studies and have been mapped
by autoradiography (Bruns et al., 1980; Goodman et al., 1983).
Biochemical experiments have shown that adenosine blocks re-
lease of glutamate and aspartate from a number of preparations
(Dolphin and Archer, 1983; Corradetti et al., 1984; Fastbom
and Fredholm, 1985; Burke and Nadler, 1988), while electro-
physiological experiments have shown that adenosine blocks
excitatory postsynaptic events (Schubert and Mitzdorf, 1979;
Dunwiddie, 1984; Okada and Ozawa, 199 1; Scholz and Miller,
199 1; Yoon and Rothman, 199 1). Adenosine appears to block
transmitter release by a presynaptic action limiting calcium in-
flux (Wu et al., 1982; Proctor and Dunwiddie, 1983; Madison
et al., 1987; Fredholm and Dunwiddie, 1988), and considerable
evidence has been gathered showing that adenosine blocks cal-
cium currents in peripheral (Dolphin et al., 1986; MacDonald
et al., 1986) and central neurons (Proctor and Dunwiddie, 1983).
Recently a strong case has been put forward that the effect of
adenosine on excitatory transmitter release is mediated by an
effect on calcium currents observable postsynaptically and me-
diated by the adenosine A, receptor (Scholz and Miller, 199 1).
Interestingly, both biochemical and physiological studies have
shown that the effect of adenosine on synaptic neurotransmis-
sion is restricted to excitatory synapses (Dolphin and Archer,
1983; Yoon and Rothman, 1991). In addition, adenosine has
been shown to activate a potassium conductance mechanism in
hippocampal and striatal neurons (Trussel and Jackson, 1985;
Scholz and Miller, 1991; Yoon and Rothman, 199 1).
The sources of extracellular adenosine under physiological
conditions in the CNS have not been established. Extracellular
adenosine accumulation as a consequence of depolarization of
2954 Rosenberg et al. - Regulation of Extracellular Adenosine in Cortex
neurons has been demonstrated many times in a variety of
preparations (for review, see Dunwiddie, 1985). However, there
still is considerable uncertainty about which cell types this ex-
tracellular adenosine derives from in each stimulation para-
digm, whether adenosine itself is actually the species trans-
ported, and what the mechanisms oftransport are. A substantial
part of the difficulty arises from the fact that adenosine is a
ubiquitous substance in cells. Therefore, it is likely that, for
every cell type, conditions exist that result in the efflux of aden-
osine, for which a transport system is present in many cell
membranes (Plagemann and Wohlhueter, 1980). The problem
is determining how adenosine is transported under specific phys-
iologic and pathologic conditions, and from what cell types
under these conditions. For this purpose simple systems need
to be studied using stimulus paradigms with simple rather than
complicated effects on the tissue being studied. For an example
of the problem, veratridine is often used as a depolarizing agent
in studies of adenosine release, and unlike potassium depolar-
ization, veratridine depolarization results in adenosine release
that is not calcium dependent (MacDonald and White, 1985).
However, veratridine causes not only depolarization but rapid
depletion of cellular ATP stores through indirect activation of
the Na/K ATPase (Erecinska and Dagani, 1990), as well as
neuronal swelling and death (Ramnath et al., 1992). Therefore,
it is expected that there would be confusion concerning injury-
dependent release of adenosine, as opposed to physiological
release, especially since both depolarization-associated toxicity
and depolarization-evoked exocytotic release would be expected
to be calcium dependent (Douglas and Rubin, 1963; Choi, 1987).
It is doubtful whether veratridine stimulation experiments will
tell us much about the physiological release of adenosine; how-
ever, they might be a model for what happens in pathological
states of oxygen or glucose deprivation in which tissue metabolic
needs exceed the supply of energy, resulting in depletion of ATP
and accumulation of intracellular adenosine.
In addition to these problems, many of the earlier studies
looked at release of radiolabeled adenosine. Typically, tissues
are preincubated with radioactive adenosine, which accumu-
lates intracellularly as the nucleotides; the tissue is stimulated
in some way, and efflux of radioactivity is measured. It is crucial
to know what is the transported species (ATP or adenosine),
and yet many studies have not addressed this issue. Further-
more, quantitation is impossible with radiolabel techniques
because the specific activities of the released purines are un-
known. Finally, there is always uncertainty whether radiolabel-
ing techniques adequately label the pools that are most impor-
tant for physiological activity. These problems have been
addressed with the adoption of HPLC techniques for the mea-
surement of adenine-containing compounds down to a subpico-
molar level.
At this point it appears that we can identify several different
possible mechanisms underlying adenosine release: (1) a calci-
um-dependent mechanism demonstrable in potassium-stimu-
lated cortical slices (Hoehn and White, 1990a), synaptosomes
(MacDonald and White, 1985) cerebellar neuronal cultures
(Philibert and Dutton, 1989) and embryonic chick retinal neu-
ronal cultures (Paes de Carvalho et al., 1990); (2) a calcium-
independent mechanism that is demonstrable using veratridine
stimulation (MacDonald and White, 1985) metabolic poisoning
(Meghji et al., 1989) as well as glutamate receptor stimulation
(both NMDA and non-NMDA receptors) (Hoehn and White,
1990~) and that probably utilizes the bidirectional nucleoside
transporter (Meghji et al., 1989); (3) a glutamate uptake-acti-
vated mechanism demonstrable in synaptosomes (Hoehn and
White, 1990b); and (4) a mechanism dependent upon the hy-
drolysis of extracellular ATP that is itself released as a conse-
quence of calcium-dependent (potassium stimulation) and cal-
cium-independent (veratridine stimulation) processes (White,
1978).
It is striking that for all the attention that adenosine release
has received, so little has been made of the possibility that
norepinephrine-stimulated CAMP secretion and extracellular
hydrolysis to adenosine might be important in determining the
extracellular concentration of adenosine. There is a great deal
of evidence showing that astrocytes are an important target for
the actions of norepinephrine (Stone and Ariano, 1989). It has
been shown previously that norepinephrine stimulates intra-
cellular CAMP accumulation in cortical cultures, and that this
accumulation of CAMP occurs primarily in astrocytes. In ad-
dition, it was demonstrated that stimulation with isoproterenol
caused the efflux of relatively large quantities of CAMP into the
culture medium (Rosenberg and Dichter, 1989). This raised the
question whether the secretion of CAMP might have a role in
modulating the activity of nearby neurons by a paracrine mech-
anism. Since exogenous CAMP added to the medium was hy-
drolyzed to adenosine, one possible mechanism by which se-
cretion of CAMP might influence nearby neurons would be if
this CAMP were a significant source of adenosine (Rosenberg
and Dichter, 1989). However, the demonstration of CAMP se-
cretion and of metabolism of exogenous CAMP to adenosine
does not establish that endogenously derived extracellular CAMP
is a source of adenosine, and so the present investigations were
undertaken to determine whether P-adrenergic stimulation of
cultures could be shown to cause an increase in extracellular
adenosine, and if so, whether such extracellular adenosine ac-
cumulation could be shown to be derived from extracellular
CAMP.
Materials and Methods
Tissue culture.
Astrocyte-rich, astrocyte-poor, and astrocyte cultures
were prepared according to methods previously published (Rosenberg
and Aizenman, 1989; Rosenberg, 199 1). Astrocyte-rich and astrocyte-
poor mixed (neurons plus glia) cultures were derived from embryonic
day 16 CD
rat embryos (Charles River). Astrocyte-rich cultures were
subjected to mitotic inhibition using cytosine arabinoside when glial
cells became confluent, at 15 d in vitro (DIV). Astrocyte-poor cultures
were subjected to mitotic inhibition at 4 DIV. Astrocyte-rich cultures
contained 90-95% astrocytes, whereas astrocyte-poor cultures contained
approximately 30% non-neuronal cells, mostly astrocytes. Astrocyte-
poor cultures were used from 18 to 40 DIV. Astrocyte-rich cultures
were used from 24 to 56 DIV. Astrocyte cultures were used from 24 to
56 DIV. Astrocyte cultures were prepared from postnatal day 1 animals
according to methods previously published (Rosenberg and Dichter,
1989).
Isoproterenol stimulation. Cultures were washed in physiological sa-
line and then placed in Earle’s salt solution without phenol red (ESS).
1.2 ml/35 mm dish, with slow rotary agitation at 36°C in 95% air, 5%
CO, for 4 hr. We found that medium change itself produces a significant
release of adenosine into the medium, which then declines and reaches
a plateau by approximately 2 hr; therefore, we used a 24 hr preincu-
bation period for our stimulation experiments. At zero time, isopro-
terenol was added from a 100 x stock in 1 mM HCI. Controls received
1 mM HCl only. Buffering was adequate in ESS to prevent a significant
pH change (less than 0.1 unit). Catecholamines are relatively stable in
I mM HCI, and stock solutions can be kept frozen for weeks without
significant oxidation. At selected times, 1 ml of medium was removed
from the culture dish, made 10 mM in EDTA, and then placed in a
boiling water bath for 3 min.
Derivatization of samples to form the etheno derivatives. We used the
method of Perrett (1987) for the determination of adenine-containing
The Journal of Neuroscience, May 1994, 74(5) 2955
Table 1. The elation times of selected adenine-containing compounds
Comoound
k
(Per-
Retention time rett,
(min) k 1987)
2’,5’-Dideoxyadenosine
Adenine
NADP
AMP
NAD
ADP
Adenosine
DeoxyAMP
Deoxyadenosine
ATP
2,3-CAMP
Cyclic deoxyadenosine
monophosphate
Cyclic adenosine
monophosphate
4.49 f 0.18 2.73 k 0.06
4.57 f 0.17 2.55 + 0.23
4.7 + 0.04 2.9 + 0.06
4.84 f 0.04 3.07 t 0.09
5.45 + 0.06 3.51 + 0.09
5.81 + 0.03 3.91 t 0.12
6.85 + 0.06 4.16 t 0.13
8.33 zk 0.31 5.92 + 0.26
8.73 f 0.19
6.25 + 0.16
8.92 f 0.12 6.65 f 0.07
9.21 + 0.08 6.6 * 0.14
16.54 + 0.12
22.58 k 0.79
12.9 f 0.3
17.98 k 0.9
2.99
3.40
3.14
4.09
4.84
5.08
6.43
7.09
18.56
The values represent the mean and
SD from at least
three separate determinations.
k’ is the capacity factor [k’ = @, - t,J/f,., where t,” is the retention time of the
mobile phase and t, is the retention time of the compound of interest].
compounds by reversed-phase ion-pair high-performance liquid chro-
matography. This method achieves the separation of the 1 -N6-etheno
derivatives of many adenine-containing compounds. Derivatization to
form the etheno derivatives was accomplished by a simple precolumn
step in which the samples were heated with chloracetaldehyde (Secrist
et al., 1972). Samples were derivatized immediately or after 24 hr storage
at 4°C. In initial experiments, 100 ~1 of 2
M
potakium phosphate (pk
6.0). and 50 ~1 of 55% chloracetaldehvde @luka) were added. mixed.
and’placed in a boiling water bath for a 10 min pkriod, after which thd
reaction was stopped by removing the tubes to an ice bath. Samples
were then spun at 14,000 rpm in an Eppendorf microcentrifuge. Deri-
vatized samples were stored at 4°C until assay, which was within 72 hr.
In later experiments, the pH of the sample was lowered using potassium
phosphate at pH 5.6 and adding 50 ~1 of 1 M HCl to the sample. Final
pH was 5.6. Fifty microliters of a 10% solution of chloracetaldehyde
were used, and samples were boiled for 45 min. These modifications of
the procedure considerably decreased the presence of background flu-
orescence in the samples and increased the yield of formation of the
etheno derivatives.
Table 1 shows the retention times and capacity factors obtained in
at least three chromatographic analyses for various adenine-containing
compounds using this procedure. Based on this data, we determined
that cyclic deoxyadenosine monophosphate would be a useful com-
pound as an internal standard in the HPLC assay, given the chemical
similarity between it and the compounds of interest in these studies,
and its elution time, which is significantly different fi-om the other com-
pounds of interest. The use of an internal standard controls for several
possible sources of measurement error: pipetting error, variation in
derivatization yield, variation in lamp intensity, variation in photo-
multiplier sensitivity, and variation in recorder sensitivity. Cyclic deox-
yadenosine monophosphate was therefore used as an internal standard
in some experiments, added to each sample to achieve a final concen-
tration of 8.3 pmol/50 ~1.
Yield. In six experiments the following yields of the etheno com-
pounds were obtained starting with the indicated adenine-containing
compounds: adenosine, 77.1 f 2.5%; AMP, 82.4 & 4.9%; CAMP, 89.3
+ 9.3%.
Chromatography. Standards of adenosine, AMP, and CAMP were
derivatized and run with samples in each experiment, Complete stan-
dard curves for each substance were run with the samples-from each
experiment, with standards bracketing the range in which the com-
pounds appeared in the samples. Absolute quantities of adenine-con-
taining compounds were determined by comparing peak heights of sam-
ples with those of standards. In samples run with the internal standard,
the ratios ofpeak heights in the samples to the peak height ofthe internal
standard were compared with the same for standards. 1 -N6-etheno stan-
dards (Sigma) for each compound were also run in each set of samples
in order to calculate yield for the derivatization procedure for each
compound in each experiment.
Samples were analyzed using a 50 ~1 loop. An isocratic elution system
was used with 10% methanol, 50 mM ammonium acetate, 1 mM EDTA,
0.2 mM tetrabutylammonium hydrogen sulfate, pH 5.6 as the running
buffer: flow rate = 1 ml/min. A Cl 8 silica column was used 13 urn ODS
Hypeisil, Keystone Scientific, Inc., Bellefonte, PA). Fluores&ce of the
eluate was monitored using a fluorescence detector (Schoeffel model FS
970) equipped with a xenon lamp, a monochromator (Schoeffel GM
970), and a >360 emission filter. Excitation wavelength was 212 nm.
In some experiments, a McPherson fluorescence detector with a 200 W
xenon-mercury lamp was used with excitation at the mercury emission
band at 265 nm. Data was acquired on chart recorders, and peak heights
were measured manually in initial experiments. Subsequently, data were
acquired and analyzed using System Gold software from Beckman In-
struments and a Beckman model 406 analog-to-digital interface.
Verification of identity of peaks. Verification of the identification of
peaks was accomplished by three criteria: (1) identity of elution times,
(2) coelution of added compound with the putatively identified com-
pound, and (3) elimination of the peak with incubation of sample with
adenosine deaminase (for adenosine) and cyclic nucleotide phospho-
diesterase (for CAMP).
For identification of the CAMP peak, 3’:5’-cyclic nucleotide phos-
phodiesterase from beef heart was used (Sigma P-0134); 0.012 U was
added to a 300 ~1 sample (without EDTA) and incubated for 3 hr at
37°C. This procedure specifically eliminated the putative CAMP peak.
For identification of the adenosine peak, adenosine deaminase from
calf intestine was used (Boehringer-Mannheim 102 091); 2.0 U was
added to a 500 ~1 sample, which was then incubated for 3 hr at 37°C.
This procedure specifically eliminated the putative adenosine peak.
The identification of AMP was more problematic. There was a large
peak that eluted with a retention time very close if not identical to that
of AMP. When authentic AMP was added to the sample, in some runs
it coeluted with this peak, suggesting that it might be AMP. However,
we found that tryptophan, which is a fluorescent amino acid that is
present in the growth medium, and which is apparently secreted by cells
in the cultures, also coeluted with this peak. Because of the difficulty in
resolving AMP from tryptophan, in this study we have not attempted
to quantitate AMP.
Radioimmunoassay of CAMP. Cyclic AMP was assayed by radio-
immunoassay kit from New England Nuclear, using methods previously
published (Rosenberg and Dichter, 1989). These experiments were con-
ducted in 24-well plates, using one glass coverslip per well (transferred
to the wells at the start of the experiment), and 0.5 ml of ESS per well.
In these experiments (data shown in Figs. 1, 2), the quantity of tissue
to medium volume (100 mg/500 ~1) was considerably less than in the
later experiments (Figs. 3-lo), which were assayed by HPLC, and in
which the tissue to medium volume was 850 mg/l200 ~1.
At the appropriate times, 400 ~1 samples were removed from wells.
Zero time point samples were treated the same as other samples but
were not exposed to cells. Samples were placed in 400 ~1 of ice-cold 0.6
M HClO, and placed on ice for 15 min. Eighty microliters of 3
M
KHCO,
were added for 30 min. This procedure resulted in a dilution of the
medium CAMP to 0.45 times the original concentration. Tubes were
centrifuged for 1 min and 700 ~1 was decanted and stored at -20°C
until CAMP concentration of each sample was assayed.
The difference in tissue mass/medium volume in the experiments
assayed by radioimmunoassay and by HPLC, together with the dilution
of the sample during preparation in the experiments assayed by radio-
immunoassay, would account for an eightfold difference between the
concentrations of CAMP actually measured in the two sets of experi-
ments. In fact, differences of this order of magnitude in medium CAMP
concentration in the two sets of experiments were observed, as seen in
the medium CAMP concentrations presented in Figures 1 and 2 com-
pared with those presented in Figures 3-10.
Chemicals. Adenine-containing compounds and their etheno adducts
as well as all other compounds were obtained from Sigma. HPLC-grade
methanol and chloracetaldehyde (55% solution) were obtained from
Fluka. Rolipram was obtained from Schering AG, Berlin, West Ger-
many. RO 20-l 724 was obtained from Calbiochem. Carbazeran citrate
was obtained from Pfizer Central Research, Kent, UK.
Statistics. Tests of significance were performed using the
INSTAT
pro-
gram From GraphPad So&are. Data were subjected to an analysis of
variance (ANOVA) followed by a Tukey-Kramer multiple comparisons
2956 Rosenberg et al. - Regulation of Extracellular Adenosine in Cortex
35 T
20
?
f 15
E
2 10
$5 /’
‘\
e----f 1
0’
I
0 30 60 80 120 150 150
Time (min)
25
I
I
300nM
20
-i
0 30 60 80 120 150 160
Time (anin)
25
20
I
Time (min)
25
Time (min)
Of
0 30 60 90 120 150 150
Time (min)
-8
-7
-6 -5
LOG [ISOPROTERENOL]
Figure
2. Dos+response relationship for isoproterenol-stimulated se-
cretion of CAMP. The results from five experiments ofthe type displayed
in Figure 1 were pooled, and the peak concentrations of CAMP attained
at each concentration of isoproterenol were plotted. Means plus SD are
shown. A given point represents two to five individual values because
the selected concentrations were not the same in all experiments. The
EC,, for this pooled data was 100 nM.
test. The parametric assumption was tested by Bartlett’s test for ho-
mogeneity of variances. When a nonparametric test of significance was
required, the Kruskal-Wallis nonparametric ANOVA was used, fol-
lowed by Dunn’s multiple comparisons test.
Results
Concentration dependence
of eflect of
isoproterenol on CAMP
accumulation
Previously, we showed that 10
FM
isoproterenol produced in-
tracellular and extracellular
CAMP accumulation in cortical cul-
tures (Rosenberg and Dichter, 1989). Figure 1 shows a dose-
response experiment in which at each of several concentrations
of isoproterenol the secretion of CAMP from astrocyte-rich cul-
tures was measured by radioimmunoassay at selected time points
during continuous exposure to the drug. The radioimmunoassay
results were expressed as concentrations, and the units given in
Figures 1 and 2 for the y-axis are picomoles CAMP per milliliter
of culture medium treated with perchloric acid and potassium
bicarbonate as described in Materials and Methods (resulting
Figure 1.
Time course of CAMP secretion at varying concentrations
ofisoproterenol. Astrocyte-rich cortical cultures were exposed to varying
concentrations of isoproterenol from 10 nM to 1
PM
and the appearance
of CAMP in the extracellular medium was measured by radioimmu-
noassay. An effect of isoproterenol was detectable at 10 nM isoproter-
enol. Maximal effect was attained at 1
PM.
The time to the peak response
(maximal extracellular concentration ofcAMP) was different at different
concentrations. Coverslip cultures of rat cerebral cortex at 34 DIV were
exposed to selected concentrations of isoproterenol, prepared by serial
dilutions into ESS. Isoproterenol was prepared in a 10 mM stock in 1
mM HCl and stored at -20°C. Coverslip cultures were placed in ESS
in a 24-well plate for 20 min to preincubate, and then medium was
replaced with 0.5 ml of medium plus isoproterenol. All the coverslips
from one dish were exposed to one concentration of isoproterenol. Plates
were placed on a rotary shaker at 36°C in a 5% CO, incubator. The
resulting data were plotted (means f SD) without further manipulation
and are shown in Figures 1 and 2. Samples were run in duplicate. Each
point shown represents data from a single coverslip. The figure repre-
sents data obtained from a single experiment using coverslips from five
dishes.
The Journal of Neuroscience, May 1994, 14(5) 2957
in a 0.45 dilution of the actual concentration of
CAMP). CAMP
secretion appears to be maximal at 1
PM,
but an effect is de-
tectable at concentrations as low as 10 nM isoproterenol. The
peak concentrations
ofcAMP attained in a set of five time course
experiments using concentrations of isoproterenol from 10 nM
to
10
FM
were chosen and plotted [the time course of the effect
of 10
FM
isoproterenol on extracellular
CAMP accumulation has
been shown previously (Rosenberg and Dichter, 1989); in the
particular experiment shown in Fig. 1, a 10
PM
category was
not included], and the results of these experiments were pooled,
generating the dose-response curve shown in Figure 2. The EC,,
for the effect of isoproterenol on CAMP secretion in these ex-
periments was approximately 100 nM. In all subsequent exper-
iments, 1
PM
isoproterenol was used to stimulate cortical cul-
tures.
Assay of adenine nucleotides
The specific experimental question that we addressed was whether
fl-adrenergic receptor stimulation of cortical cultures would re-
sult in a detectable increase in extracellular adenosine. For this
experiment it was necessary to assay not only CAMP but also
adenosine. To accomplish this, we used chloracetaldehyde de-
rivatization of adenine compounds to form their fluorescent
etheno derivatives (Secrist et al., 1972; Perrett, 1987), with sep-
aration by ion-pair reverse-phase HPLC on a C 18 column, using
fluorescence detection (Perrett, 1987). Using this method, we
determined the effect of stimulation of astrocyte-rich cortical
cultures with 1
PM
isoproterenol on the appearance of CAMP
and adenosine in the medium. Figure 3A shows a chromatogram
ofa sample containing known concentrations ofadenosine, AMP,
and CAMP (2.5,2.5, and 5 pmol/SO ~1, respectively), which were
derivatized and assayed by HPLC together with the test samples
shown in Figure 3, B and C. Figure 3A is shown in order to
indicate the elution times of the peaks of adenosine, AMP, and
CAMP. Figure
3B
shows the chromatogram of a media sample
taken from a control culture that was not exposed to drug. In
this sample, adenosine but not CAMP was present. Figure 3C
shows a chromatogram of a medium sample taken from cultures
that were exposed to 1
KM
isoproterenol for 30 min. Following
exposure to 1
FM
isoproterenol, CAMP appeared in the medium,
and adenosine was increased, in this experiment to 2 14% of the
control value.
In Figure 3, B and C, the adenosine and CAMP peaks have
been tentatively identified by the similarity of their elution time
to the elution time of the authentic standards shown in Figure
3A. Two additional criteria were used in order to make these
identifications with certainty: coelution of authentic compound
with the putatively identified compound in the sample and elim-
ination of the peak by enzymatic digestion (see Materials and
Methods).
Reproducibility of phenomenon
The experiments described in this report extended over a period
of 29
months. In this time, a total of 56 experiments were
performed in cultures 24-56 DIV in which the adenosine con-
centration was measured in the extracellular medium of cortical
cultures exposed to 1
I.LM
isoproterenol for 30 min and compared
to
control cultures. In 49 of these experiments, the mean of the
isoproterenol-treated group was greater than the mean of the
control group. The mean adenosine concentration in the control
cultures was 1.5 + 1 pm01150 ~1 (30 ? 20 nq
n
= 155 deter-
minations). The mean adenosine concentration in the isopro-
c
Adenosine \4
cqMP
Adenosine
\
200 set
3
Inject
3-
IAject
4
r”
Inject
Figure 3. Elution profile of adenine-containing compounds present
in
medium of cortical cultures exposed to isoproterenol: sample chro-
matograms of a standard solution containing 2.5 pmol adenosine, 2.5
pmol AMP, and 5 pmol CAMP per 50 ~1, derivatized prior to assay (A);
a sample of control medium from astrocyte-rich cultures incubated for
30 min with vehicle only, derivatized prior to assay (B); and a sample
of medium from astrocyte-rich cultures incubated with 1
NM
isoproter-
enol for 30 min, derivatized prior to assay (C). Adenine-containing
compounds in the samples were assayed by HPLC with fluorescence
detection. Isoproterenol causes the appearance of a large peak that mi-
grates with the same retention time as CAMP, and causes an increase
in the peak that has the same elution time as adenosine. In addition,
there is a peak that migrates with the retention time of AMP, and that
does not seem to be affected by the presence of isoproterenol. Fluores-
cence of the eluate was monitored using a Schoeffel fluorometer with
monochromator at 2 12 nm. Fluorescence intensity is given in arbitrary
units.
Arrows
display time of injection of samples. Data were digitized
directly from the chart paper; recorder was run at 0.2 mm/set with
sensitivity of 5 fiV/mm.
2958 Rosenberg et al. - Regulation of Extracellular Adenosine in Cortex
10
9
8
7
T
Adenosine
0 control
0 isoproterenol
80
T
CAMP
6o t
30
20
10
0
0
60 120
180
Time (minutes)
Figure 4.
Time course of appearance of adenosine and CAMP in the
extracellular medium of astrocyte-rich mixed cultures of astrocytes and
neurons following stimulation by 1
FM
isoproterenol. Astrocyte-rich
cultures (39 DIV) were exposed to 1
PM
isoproterenol and media samples
were taken at selected time points and assayed by HPLC. CAMP attains
a maximum between 60 and 120 min and then declines. At the maxi-
mum, CAMP in the extracellular medium was 640 nM. Adenosine dem-
onstrated a time-dependent and continuous increase in the presence of
isoproterenol. The baseline concentration was 17 nM, and this increased
over 120 min to 52 nM.
terenol-treated cultures was 2.2 + 1.3 pmol/50 pl(44 f 26 nM;
n = 159 determinations; p < 0.000 1 ), a 47% increase.
We investigated the time course of isoproterenol-stimulated
extracellular adenosine and CAMP accumulation in order to
characterize these phenomena further, including the relation-
ship of the adenosine accumulation to CAMP transport. Our
previous work suggested that P-adrenergic receptors in cortical
cultures are localized primarily on astrocytes (Rosenberg and
Dichter, 1989). Furthermore, preliminary studies suggested to
us that an extracellular cyclic nucleotide phosphodiesterase is
present in cortical cultures (Rosenberg and Dichter, 1989; Vas-
quez and Rosenberg, 1989), and that it appeared to be localized
primarily to neurons (Vasquez and Rosenberg, 1989). In order
to understand the role of astrocytes and neurons in these phe-
nomena, time course experiments were conducted on astrocyte-
rich cultures, composed of 90-95% neurons; astrocyte-poor cul-
tures, which are 20-30% astrocytes [containing approximately
l/50 the number of astrocytes as in the astrocyte-rich cultures
(Rosenberg, 199 l)]; and astrocyte cultures.
Assay of adenine nucleotides in astrocyte-rich cultures
stimulated with isoproterenol
Figure 4 shows a time course experiment plotting CAMP and
adenosine appearing in the medium of astrocyte-rich cultures
(39 DIV, containing 740 f 90 mg of protein) stimulated with
1
WM
isoproterenol. CAMP secretion achieved a peak concen-
tration of 32 pmol/50 ~1 or 640 nM, at 60-l 20 min, subsequently
declining, as has been noted before (Rosenberg and Dichter,
1989). Adenosine continuously increased in response to isopro-
terenol stimulation, in this experiment from a basal concentra-
tion of 17-52 nM after 120 min exposure to isoproterenol. In
seven time course experiments the extracellular adenosine con-
centration rose with isoproterenol from a basal level of 10 f 6
nM to 41 f 19 nM at 120 min (p < 0.002).
Assay of adenine nucleotides in astrocyte cultures stimulated
with isoproterenol
For comparison with the response to isoproterenol in astrocyte-
rich cultures, Figure 5 shows a similar time course experiment
but using astrocyte cultures (4 1 DIV), with a protein content of
440 f 60 mg/dish. Here extracellular CAMP accumulation was
also seen, and was in fact greater than that observed with the
cultures containing neurons. In this experiment, basal extracel-
lular adenosine was 5 nM and rose to 9 nM after 120 min ex-
posure to isoproterenol. In six experiments with astrocyte cul-
tures, extracellular adenosine after exposure to isoproterenol for
120 min (11 -t 7 nM) was not significantly different from basal
levels (7 f 4 nM).
Extracellular CAMP concentrations found in the media of
astrocyte-rich and astrocyte cultures exposed to isoproterenol
were compared. No CAMP was detected in the absence of iso-
proterenol stimulation. In astrocyte-rich cultures, following 60
min stimulation, the extracellular CAMP concentration was 482
? 208 nM (n = 7). In astrocyte cultures, following 60 min stim-
ulation, the extracellular CAMP concentration was 904 ? 468
nM (n = 6).
Assay of adenine nucleotides in neuron-enriched, astrocyte-
poor cultures stimulated with isoproterenol
Figure 6 shows an experiment using neuron-enriched astrocyte-
poor cultures (19 DIV), with a protein content of 140 * 10 mg/
dish. Here we saw no CAMP accumulation, consistent with pre-
vious work (Rosenberg and Dichter, 1989) and with the fact
that relatively few astrocytes are present. In addition, no sig-
nificant change in adenosine was observed from the basal level,
which in this experiment was 36 nM. In five experiments, we
found a basal concentration for adenosine in astrocyte-poor
cultures of 55 f 22 nM. After exposure to isoproterenol for 120
min, the adenosine concentration was 61 f 27 nM.
The demonstration that isoproterenol stimulated the secre-
tion of CAMP and also caused an increase in the extracellular
concentration of adenosine suggested that the increase in aden-
osine had extracellular CAMP as its source. The relationship
between CAMP secretion and extracellular adenosine is impor-
The Journal of Neuroscience, May 1994, 74(5) 2959
Adenosine
0 control
0 isoproterenol
80
70
60
-3 50
20
10
0
Time (minutes)
Time (minutes)
Figure 5. Time course of appearance of adenosine and CAMP in the
extracellular medium of astrocyte cultures following stimulation by 1
PM isoproterenol. Astrocyte cultures (41 DIV) were exposed to 1 PM
isoproterenol, and medium was sampled at selected time points. No
significant effect of isoproterenol
on
adenosine accumulation was ob-
served. CAMP progressively accumulates over the time course of this
experiment, and reaches 1.4 PM.
Figure 6. Time course of appearance of adenosine and CAMP in the
extracellular medium of neuron-enriched astrocyte-poor cultures fol-
lowing stimulation by 1 PM isoproterenol. Neuron-enriched astrocyte-
poor cultures (19 DIV) were exposed to 1
PM
isoproterenol, and medium
was sampled at selected time points. No effect on adenosine or CAMP
accumulation was observed.
tam to our understanding of the regulation of extracellular aden-
osine concentration, and so we undertook to establish whether
the increase in adenosine that appears to be a consequence of
p-adrenergic stimulation in fact derives from secreted CAMP.
Two approaches to establishing the relationship between extra-
cellular CAMP and extracellular adenosine that we have taken
rely on inhibition of cyclic nucleotide phosphodiesterase and
inhibition of CAMP secretion.
Effect of cyclic nucleotide phosphodiesterase inhibitors on
isoproterenol-stimulated adenosine accumulation
We tried to block the isoproterenol-stimulated increase in ex-
traceliular adenosine using phosphodiesterase inhibitors. If for-
mation of adenosine were dependent upon hydrolysis of CAMP
10
9
8
i
Adenosine
0 control
0 isoproterenol
80
?-
70
i
60 +
0 60 120 180
by phosphodiesterase, then inhibition of phosphodiesterase
should prevent an increase in adenosine. In order to pursue this
test of the hypothesis that isoproterenol-stimulated extracellular
adenosine accumulation derives from extracellular CAMP, we
tested a variety of cyclic nucleotide phosphodiesterase inhibi-
tors: isobutylmethylxanthine (IBMX), a nonspecific cyclic nu-
cleotide phosphodiesterase inhibitor (Beavo, 1988); RO 20-l 724,
an inhibitor of calcium-independent CAMP-specific phospho-
diesterase prevalent in the brain (Beavo, 1988; Beavo and Re-
ifsnyder, 1990; Challiss and Nicholson, 1990; Nicholson et al.,
199 1); carbazeran, an inhibitor of cGMP-inhibited phospho-
diesterase (Weishaar et al., 1986); dipyridamole, an inhibitor of
cGMP-specific phosphodiesterase (Weishaar et al., 1986); and
trifluoperazine, an inhibitor of calcium/calmodulin-dependent
phosphodiesterase (Levin and Weiss, 1977). However, we found
2960 Rosenberg et al. * Regulation of Extracellular Adenosine in Cortex
8r
0 IBMX
0 IBMX+ISO
V IBMX+RoZO
. IBMX+RoZO+lSO
120 r
B
7r
[<---------IBMX-------->I
[<R020- 1724->]
IS0 IS0
Figure 7. Effect of IBMX plus RO 20-1724
on isoproterenol-stimu-
lated adenosine accumulation. RO
20- 1724 plus IBMX
block isopro-
terenol-stimulated adenosine accumulation. Cultures were incubated
with or without 1
PM
isoproterenol for 30 min in the presence of IBMX
100
PM
and with or without 180
PM
RO 20-1724. The results of 15
experiments were pooled. Error bars show SEM. In the presence of
IBMX,
isoproterenol produced a significant (p < 0.05) increase in ex-
tracellular adenosine
(asterisks).
This effect was eliminated by the in-
clusion of RO 20-
1724.
that several of these inhibitors by themselves, in the absence of
isoproterenol, caused an increase in extracellular adenosine.
Therefore, we restricted ourselves to working with concentra-
tions of phosphodiesterase inhibitors that produced a doubling
or less of the baseline concentrations of adenosine. We found
that IBMX at 100
PM
(n = 30 experiments; see, e.g., Fig. 7)
RO 20-l 724 (180
PM,
n = 9) dipyridamole (10
PM,
IZ = 2)
carbazeran citrate (10
KM,
n = 3) and trifluoperazine (10
MM,
n
= 2) failed to block the effect of isoproterenol on extracellular
adenosine.
In 15 more experiments we combined the use of 100
FM
IBMX
with 180
PM
RO 20- 1724. We found that when cortical cultures
were exposed to RO 20- 1724 together with IBMX, isoproterenol
no longer produced a significant increase in extracellular aden-
osine accumulation (Fig. 7). In these experiments, in the pres-
ence of IBMX alone, isoproterenol produced a 56% increase in
extracellular adenosine accumulation (p < 0.05). In contrast,
with the addition of 180
FM
RO 20- 1724, there was no signif-
icant change in extracellular adenosine concentration with iso-
proterenol.
We also demonstrated the effect of the combination of IBMX
with RO 20- 1724 on the time course of extracellular adenosine
as well as CAMP accumulation (seven experiments performed).
As expected from the data presented in the previous figure,
isoproterenol-stimulated adenosine accumulation was signifi-
cantly inhibited by RO 20-l 724 plus IBMX at 30 min (p <
0.01) (Fig. 8A). Although there have been reports of phospho-
diesterase inhibitors blocking CAMP secretion (King and Mayer,
1974; Wu et al., 1978; Nemecek et al., 1980) we found that
IBMX plus RO 20- 1724, at the doses used, did not block CAMP
0 30 60 90
120 150
O-
120 150
Time (minutes)
Time (minutes)
Figure
8. Effect of IBMX plus RO 20-1724 on the time course of
isoproterenol-stimulated adenosine and CAMP accumulation. Astro-
cyte-rich cultures were preincubated with 100
PM
IBMX alone, or IBMX
plus 180
PM
RO 20- 1724. At zero time, 1
PM
isoproterenol or vehicle
was added to cultures in each of these groups, and medium was collected
at selected intervals for assay of adenosine and CAMP. Isoproterenol
evoked a significant increase in extracellular adenosine in this experi-
ment, in the presence of 100
PM
IBMX alone, which peaked at 30 min.
RO 20- 1724 plus IBMX inhibited the accumulation of adenosine (p <
0.01 at 30 min). In B, the effect of isoproterenol on extracellular CAMP
accumulation in the presence of IBMX and in the presence of IBMX
plus RO 20- 1724 is shown. CAMP measured in the medium was sig-
nificantlv increased bv the addition of RO 20- 1724 to IBMX at 60 min
(p < 0.65) and 120 &in (p < 0.001). Data shown are from a single
experiment of seven that were performed.
secretion and, in fact, that the extracellular CAMP was equal to
or greater than control levels stimulated by isoproterenol, as
seen in this representative single experiment (Fig. 8B). Isopro-
terenol-stimulated extracellular CAMP accumulation was sig-
nificantly greater in the presence of RO 20-l 724 than in its
absence at 60 (min p < 0.05) and at 120 min (p < 0.001).
We have tried using representatives of other classes of phos-
phodiesterase inhibitors in conjunction with 100
FM
IBMX,
such as dipyridamole, carbazeran, and trifluoperazine (all at 10
PM),
but found no blockade of isoproterenol-stimulated extra-
cellular adenosine accumulation. Higher concentrations of in-
hibitors were not used in these experiments because they were
found to have large effects (greater than a doubling) on adenosine
levels themselves.
Efect of probenecid on isoproterenol-stimulated extracellular
CAMP and adenosine accumulation
The pharmacology of inhibition of CAMP efflux has been well
characterized in a number of cell types, including astrocytoma-
derived cell lines (Doore et al., 1975; Henderson and Strauss,
199 1). If extracellular adenosine that appears as a result of p-ad-
renergic receptor stimulation is derived from extracellular CAMP,
then drugs such as probenecid, which block CAMP efflux, should
eliminate this effect of isoproterenol. The effect of probenecid
on isoproterenol-stimulated adenosine accumulation is shown
in Figure 9, which represents data pooled from 10 experiments.
In these experiments, the adenosine concentration in medium
from isoproterenol-treated cultures was 177 & 66% of control
values, and this effect was blocked by 1000
PM
probenecid (p
< 0.05). [In these experiments the baseline adenosine concen-
tration was 1.8 ? 1.2 pmoV50 ~1 without probenecid and 2.0
3.5
3.0
.---. 2.5
::
.-
-4
$ 2.0
.-
+
.E 1.5
:
6
2 1.0
0.5
0.0
[--*--------------------*--,
0
100 1000
[Probenecid] (PM)
Figure 9. Effect of probenecid on isoproterenol-stimulated adenosine
accumulation. Probenecid blocks isoproterenol-stimulated adenosine
accumulation in astrocyte-rich cultures. For each concentration of pro-
benecid the ratio of the adenosine concentration with isoproterenol
stimulation over the adenosine concentration without isoproterenol
stimulation was obtained. The results from 10 separate experiments
were pooled. There was a significant difference (p < 0.05) between the
values obtained in the absence of probenecid and in the presence of
1000 PM probenecid (populations being compared indicated by asterisks
embedded in dashed line). Astrocyte-rich cultures were exposed to 0,
100, and 1000
PM
probenecid in the presence or absence of 1
PM
iso-
proterenol for 30 min. Media samples were then taken and assayed for
adenosine, shown here, and CAMP (shown in Fig. 10). These data show
the pooled results from 10 similar experiments. In four of these exper-
iments, 100
PM
IBMX was present in the medium, but there were no
significant differences between the results from these experiments with
IBMX and experiments performed without IBMX, and therefore they
were pooled.
? 1.4 pmol/50 ~1 with probenecid (1000
FM).]
This concentra-
tion of probenecid also decreased extracellular CAMP by 78%
in these experiments (p < 0.01) (Fig. 10).
Discussion
We have shown that cortical cultures respond to stimulation by
the /3-adrenergic agonist isoproterenol by secreting CAMP into
the extracellular medium. This response can be detected at con-
centrations as low as 10 nM, and saturates by 1
FM.
We noticed
in some experiments what appeared to be a concentration de-
pendence of the time for the extracellular concentration of CAMP
to attain a maximum, as seen in Figure 1, and assume that this
is due to a change in the relative contributions of the source of
extracellular CAMP, CAMP transport, and the sink for CAMP,
extracellular cyclic nucleotide phosphodiesterase activity, at dif-
ferent isoproterenol concentrations.
Using the peak concentrations of extracellular CAMP at dif-
ferent concentrations of isoproterenol, we obtained a value for
the EC,, for the stimulation of CAMP transport by isoproterenol
of approximately 100 nM (Fig. 2). By comparison, in other work
using glial cultures and investigating the activation by isopro-
terenol of adenylyl cyclase by measuring intracellular CAMP,
24
22
20
18
16
14
12
10
8
6
4
2
0
The Journal of Neuroscience, May 1994, 14(5) 2961
* vehicle, no CAMP detected
m isoproterenol
* * *
0 0.1
1.0
ir
0.1 1.0
[Probenecid] (mM)
Figure 10. Effect of probenecid on isoproterenol-stimulated CAMP
accumulation. Probenecid blocks isoproterenol-stimulated CAMP se-
cretion. This figure shows pooled data from six experiments, in which
probenecid is shown to have a dose-dependent effect on CAMP secretion.
The IC,, for the inhibition of CAMP secretion by probenecid in these
experiments was 89 & 6
PM.
No CAMP was detectable in the medium
without isoproterenol stimulation (shown by asterisks).
an EC,, of 43 nM was determined (Ebersolt et al., 1981). A
number of factors might account for the difference between the
value we have determined and this value, including the fact that
we performed our experiments on mixed cultures of neurons
and glial cells, whereas the previous experiments were per-
formed on glial cultures, or that in our experiments we measured
extracellular CAMP whereas in the previous experiments intra-
cellular CAMP was assayed.
The principal question that we have examined in this study
is whether P-adrenergic receptor stimulation might cause an
increase in extracellular adenosine derived from extracellular
CAMP. In fact, we were able to demonstrate, using an HPLC
method, that stimulation of astrocyte-rich cultures with isopro-
terenol resulted in an increase in extracellular adenosine. This
effect was not observed in every experiment, but in a large series
of experiments, we found a highly significant increase in extra-
cellular adenosine in cultures stimulated with isoproterenol. Our
experiments comparing astrocyte-rich cultures, neuron-en-
riched astrocyte-poor cultures, and astrocyte cultures suggest
one explanation why this might be the case. The fact that iso-
proterenol produces an increase in CAMP in both astrocyte
cultures as well as astrocyte-rich cultures, but an increase in
adenosine only in mixed cultures of neurons and astrocytes
(astrocyte-rich cultures), implies that one or both ofthe enzymes
required for the breakdown of CAMP occurs in neurons. Since
we know that astrocytes (in addition to neurons) (Nagy et al.,
1986; Trapido-Rosenthal et al., 1990) possess abundant 5’-nu-
cleotidase (Kreutzberg et al., 1978) it seems likely that extra-
cellular cyclic nucleotide phosphodiesterase activity is localized
to neurons in the cultures. We do not know whether this enzyme
is present on all neurons or only on a subpopulation of neurons,
2962 Rosenberg et al. - Regulation of Extracellular Adenosine in Cortex
or what is the subcellular localization of this enzyme on neurons
that possess it. In any case, this apparent requirement for neu-
rons in order to detect isoproterenol-stimulated adenosine ac-
cumulation suggests that there might be a minimum density of
neurons below which an insufficient amount of enzyme would
be present to produce a detectable amount of adenosine derived
from CAMP. Because it is possible that extracellular cyclic nu-
cleotide phosphodiesterase is present not on all cortical neurons
but on a subpopulation, it is conceivable that that subpopulation
is variably represented in different cultures. This might also
account for the variability in the response to isoproterenol. Fi-
nally, the fact that no effect of probenecid is seen below 100
pM
suggests that the phosphodiesterase is probably saturated at low
levels of CAMP and, as a result, no effect of suppressing CAMP
transport is seen until extracellular CAMP concentrations are
markedly reduced. This observation suggests that the absolute
amount of enzyme might be limiting, and therefore a major
factor in determining whether an effect of isoproterenol is ob-
servable in these cultures. Another issue that might bear on the
reproducibility of this phenomenon is that these cultures are
not a monolayer. They consist instead of a sandwich of neuropil
with an astrocyte layer both underneath, in contact with the
substrate, and on top, sequestering the neuropil from the extra-
cellular medium (Harris and Rosenberg, 1993). Therefore, CAMP
secreted from the surface of astrocytes may go into the medium
without ever coming into contact with cyclic nucleotide phos-
phodiesterase associated with neurons. CAMP secreted by as-
trocytes that are in contact with the neuropil, on the other hand,
may come in contact with a neuronal extracellular enzyme, but
because of the tortuous route to the surface, which might be
occluded by tight junctions (Harris and Rosenberg, 1993), may
never gain access to the extracellular medium where it could be
detected. Therefore, the fact that our sampling is restricted to
the extracellular medium may limit our ability to detect the
conversion of CAMP to a metabolite if this conversion were to
depend on an interaction with dendrites. Nonetheless, we have
been able to demonstrate an effect of isoproterenol in vitro, and
this provides strong motivation to pursue these studies in vivo,
using microdialysis techniques and sampling directly in the ex-
tracellular space. Using these techniques it has already been
demonstrated that P-adrenergic stimulation produces secretion
of CAMP in viva, in the frontal cortex of living rats (Stone and
John, 1990).
The demonstration that /3-adrenergic stimulation of cortical
cultures increases extracellular adenosine still leaves open the
question of whether the adenosine that accumulates in the ex-
tracellular space actually derived from extracellular CAMP. If
this were true, then it should be possible to block this effect of
isoproterenol both with an inhibitor of cyclic nucleotide phos-
phodiesterase and with an inhibitor of CAMP transport. The
usefulness of a number of phosphodiesterase inhibitors tested
was limited by their tendency to increase baseline levels of aden-
osine, in the absence of P-adrenergic receptor stimulation. We
do not understand the basis for this effect, but it may be due to
activity of these agents as adenosine receptor antagonists (Bruns
and et al., 1986; Parsons et al., 1988; Williams and Jarvis, 1988;
Nicholson et al., 1989). We used concentrations of these drugs
that did not on their own increase adenosine concentrations by
more than lOO%, and with these constraints, found no effect of
a variety of phosphodiesterase inhibitors, including the non-
specific inhibitor IBMX (Fig. 7). This apparent resistance to the
effect of IBMX is unusual, though not unprecedented @van et
al., 1989). However, we found that the combination of RO 20-
1724 with IBMX did block isoproterenol-stimulated adenosine
accumulation (Figs. 7, 8) and, as expected, increased CAMP
accumulation (Fig. 8). The exact relationship between extracel-
lular adenosine levels and extracellular CAMP levels in these
experiments is complicated by a number of factors, including
the effects of the phosphodiesterase inhibitors on the intracel-
lular level of CAMP (which determines the rate of CAMP trans-
port) (Barber and Butcher, 1983) and adenosine uptake. For this
reason, one would not necessarily expect a stoichiometric re-
lationship between the decrease in the adenosine and the in-
crease in the CAMP detected in the medium. Nonetheless, the
data support the hypothesis that CAMP is a source of the aden-
osine, since a decrease in adenosine is associated with an in-
crease in CAMP. In the experiment shown in Figure 8, a decrease
in adenosine content of 3.2 pmol/50 ~1 was associated with an
increase in CAMP content of 8.9 pmoV50 ~1.
The significance of the requirement for two different phos-
phodiesterase inhibitors is unclear. Previous studies have dem-
onstrated a subtype of cyclic nucleotide phosphodiesterase that
has both regulatory and catalytic cyclic nucleotide binding sites
that interact (Francis et al., 1990; Thomas et al., 1992). There-
fore, it is conceivable that the interaction of IBMX with the
enzyme we are studying changes the affinity of the catalytic site
for RO 20-l 724, or vice versa. Clearly, more detailed phar-
macological studies need to be performed in order to charac-
terize the extracellular phosphodiesterase activity that we ob-
serve in cortical cultures. These studies would best be performed
in a simple assay of phosphodiesterase activity, rather than in
the experimental paradigm we have employed here, which dem-
onstrates phosphodiesterase activity indirectly.
As another test of the hypothesis that the adenosine that
accumulates in response to isoproterenol stimulation derives
from extracellular CAMP, we attempted to block the effect of
isoproterenol by using probenecid, an inhibitor of CAMP trans-
port. We found an EC,, for the inhibition by probenecid of
CAMP secretion of 89 f 6
PM
(Fig. 10). This is similar to the
value obtained previously by others for the effect of probenecid
on CAMP extrusion from C6 rat glioma cells (83 ? 17
1~)
(Henderson and Strauss, 199 1). In contrast, with respect to the
potency of probenecid as an inhibitor of isoproterenol-stimu-
lated adenosine accumulation, we found little effect below 100
PM.
In the experiments shown in Figure 9, the EC,, appears to
be between 200 and 300
FM.
These data are consistent with the
hypothesis that the origin of the adenosine that accumulates in
response to isoproterenol is extracellular CAMP, but also suggest
that CAMP secretion itself is not the rate-limiting step.
Until recently, no studies have examined the possibility that
hydrolysis of extracellular CAMP might provide a significant
source of extracellular adenosine in cerebral cortex. In spinal
cord, serotonin (5-HT) has been shown to release a nucleotide
from dorsal horn spinal cord synaptosomes that is degraded
extracellularly to adenosine, leading to the hypothesis that this
mechanism might in part underlie the antinociception produced
by 5-HT (DeLander and Hopkins, 1987; Sweeney et al., 1988;
Sawynok and Sweeney, 1989). Sweeney et al. (1990) present
evidence that this nucleotide is, in fact, CAMP. Interestingly, in
the dorsal horn synaptosomes, purine release is Ca2+ dependent,
originates in capsaicin-sensitive small-diameter primary affer-
ent nerve terminals (Sweeney et al., 1988), and is not elicited
by noradrenergic stimulation. Therefore, the source of extra-
cellular CAMP in the spinal cord appears to be neurons, and
The Journal of Neuroscience, May 1994, 14(5) 2963
CAMP in this case appears to be transported by a calcium-
dependent process-possibly exocytosis. In contrast, in the pro-
cess we are suggesting as a source of adenosine in cortex, extra-
cellular CAMP derives from astrocytes (Rosenberg and Dichter,
1989) secreted in response to norepinephrine via a transport
process that is ATP dependent (Barber and Butcher, 1983; Hen-
derson and Strauss, 199 1) and calcium independent (Barber and
Butcher, 198 1).
CAMP efflux occurs in all cells that accumulate CAMP intra-
cellularly in response to @-adrenergic stimulation (Barber and
Butcher, 1983) and was originally discovered by Davoren and
Sutherland (1963) in the pigeon erythrocyte. This transport pro-
cess has been studied in several different preparations, and has
been demonstrated in the frontal cortex of living rats (Egawa et
al., 1988) in the striatal slice preparation (Schoffelmeer et al.,
1985; Headley and O’Shaughnessy, 1986; O’Shaughnessy et al.,
1987) in glioma-derived cell lines (Doore et al., 1975; Hen-
derson and Strauss, 199 l), and in rat cerebra1 cortex in culture
(Rosenberg and Dichter, 1989). The transport mechanism is
characteristically inhibited by several structurally diverse com-
pounds including probenecid, verapamil, dipyridamole, and
prostaglandin Al, and this pharmacology is similar to that of
the multidrug resistance transporter, with which it appears to
be closely related (Henderson and Strauss, 199 1). It is note-
worthy that the structure of adenylyl cyclase itself is similar to
that of the multidrug resistance transporter, leading to the sug-
gestion that the adenylyl cyclase molecule provides an efflux
route for CAMP (Krupinski et al., 1989). The function of CAMP
efflux has been a subject of speculation since its discovery, but,
in any case, this transport route appears to provide a pathway
of efflux for large organic ions from cells in which it appears,
as is the case for the multidrug resistance transporter or P-gly-
coprotein. The possible functional significance of CAMP efflux
has been previously reviewed (Barber and Butcher, 1983; Ro-
senberg, 1993). Recently, a new addition has been made to the
list of possibilities with the demonstration that P-glycoprotein
is itself or is capable of regulating a volume-sensitive chloride
conductance (Valverde et al., 1992). In addition, Sorbera and
Morad (199 1) have shown that CAMP diminishes sodium cur-
rents in myocytes, suggesting the existence of an extracellular
CAMP receptor on this cell type. This phenomenon has not yet
been described in neurons.
Although there is ample evidence for the presence of p-ad-
renergic receptors on neurons in the adult cerebral cortex and
hippocampus (Madison and Nicoll, 1986a,b; Aoki et al., 1987;
Schwindt et al., 1988; Aoki and Pickel, 1990, 1992), there is
very strong evidence as well that astrocytes are a major target
for the P-adrenergic actions of norepinephrine (Stone and Ari-
ano, 1989; McCarthy et al., 199 1). In fact, in embryonic cultures
of cerebral cortex there is compelling evidence that cortical neu-
rons do not have P-adrenergic receptors (Rosenberg and Dichter,
1989; McCarthy et al., 1991). What role might p-adrenergic
stimulation of adenosine accumulation play in the physiology
of norepinephrine? Since Woodward and colleagues proposed
that the function of the norepinephrine projection to cortex is
to increase the signal-to-noise relationship for significant stim-
uli, this hypothesis has become widely accepted as useful in
understanding the function of norepinephrine (Woodward et al.,
1979; Foote et al., 1983). It has received considerable support
in studies (Haas and Konnerth, 1982; Madison and Nicoll, 1982)
that provided a cellular basis for the strengthening of strong
inputs that is one role for norepinephrine required by the Wood-
ward hypothesis. However, another function for norepinephrine
posited by the Woodward hypothesis is the suppression of weak
inputs. Madison and Nicoll showed that norepinephrine causes
a small hyperpolarization associated with an increase in mem-
brane conductance in approximately 70% of pyramidal neurons
in the CA1 region of hippocampus, and demonstrated that this
was sufficient to inhibit action potential generation in response
to a near-threshold stimulus (Madison and Nicoll, 1986a). An-
other, possibly additional, mechanism for the suppression of
weak inputs suggested by the present work is that by increasing
extracellular adenosine concentration, excitatory synaptic in-
puts might be effectively weakened by decreasing excitatory
transmitter release.
An unresolved issue is determining in what time frame this
effect might occur. The phenomenon we have demonstrated in
culture is slow, occurring over many minutes, whereas one would
expect an effective physiological mechanism to occur over sec-
onds or fractions of a second. However, our sampling of the
extracellular medium is at best an imperfect window onto events
that are taking place next to synapses that are surrounded by
other cells, including astrocytes, from which they are separated
by very small volumes. In these small volumes, rapid changes
in the concentration of solutes is possible with the transport of
small quantities of substrates across cell membranes of either
neurons or adjacent glial cells. Although tissue culture provides
an extremely useful model system in which it is possible to apply
drugs at known concentrations, to measure their effects on cells,
and to study the role of cellular interactions in generating these
effects, ultimately it will be necessary to investigate the phe-
nomenon we have described in vivo using microdialysis tech-
niques. In addition, once the components of the system have
been elucidated, as well as how to manipulate them pharma-
cologically, it would be of great interest to use this information
to study the role ofthe mechanism we have described in a variety
of electrophysiological model systems.
In addition to monoamines, the cortex is richly endowed with
neuropeptides, some of which are coupled to adenylyl cyclase.
Any hormone coupled to adenylyl cyclase (positively or nega-
tively) might be expected to modulate CAMP efflux. Therefore,
a mechanism deriving adenosine from CAMP might be a final
common pathway by which multiple neuromodulators could
converge to control extracellular adenosine, and thereby inhib-
itory tone. Such a mechanism might have important effects in
modulating synaptic transmission, both under normal circum-
stances and under the abnormal circumstances of hypoxia/isch-
emia or seizures (Dragunow et al., 1985; Dragunow and Rob-
ertson, 1987; Dragunow, 1988; Dragunow and Faull, 1988).
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