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J Biol Chem, Vol. 274, Issue 53, 37770-37780, December 31, 1999
From the Wenner-Gren Institute, The Arrhenius Laboratories F3,
Stockholm University, S-106 91 Stockholm, Sweden
The mechanism behind the distinctive
non-Michaelis-Menten, bell-shaped kinetics of cAMP accumulation in
brown adipocytes (which underlies the similar kinetics of UCP1 and
When stimulated with norepinephrine, brown adipocytes express the
gene for the uncoupling protein-1
(UCP1)1 (1-4). However, the
kinetics of the induction of the expression of this gene do not adhere
to simple, monophasic Michaelis-Menten kinetics. Rather, a distinctive
bell-shaped response is observed, with norepinephrine concentrations of
The observation of non-Michaelis-Menten kinetics such as these may
easily be considered an experimental artifact and/or interpreted as an
indication of "overstimulation" of the relevant receptor, leading
to an acute "desensitization" of the response. Because of
difficulties in determining kinetic parameters in
non-Michaelis-Menten-responding systems, the downward part of such
dose-response curves may often be ignored (or perhaps even omitted) in
the presentation of data.
However, in a theoretical analysis, Rovati and Nicosia (5) demonstrated
that one conceivable possibility for generation of bell-shaped
dose-response curves would be that these curves represent the resultant
of interacting stimulatory and inhibitory components. They also implied
that in such systems, large underlying stimulatory components, masked
by the inhibitory component, could be revealed.
Following this proposal, we have here attempted to resolve the
bell-shaped dose-response curve for norepinephrine into dual components. As the kinetics of UCP1 gene expression have been demonstrated to mirror those of norepinephrine-induced cAMP
accumulation in these cells (6), we have, de facto, examined whether
specific receptors and intracellular mediators could interact to create the unusual kinetics of cAMP accumulation.
We conclude that a Rovati/Nicosia model fully explains the kinetics of
the control of cAMP levels in brown adipocytes and that through the
elimination of the inherent inhibitory component, a strikingly high but
otherwise masked potential for stimulation of cAMP accumulation via
Cell Isolation--
Brown fat precursor cells were isolated from
3-4-week-old male mice of the NMRI strain, principally as described by
Néchad et al. (7). Tissue was combined from the
cervical, interscapular, and axillary depots and incubated in a
Hepes-buffered solution (7), containing 200 units/ml crude collagenase
type 2 (Sigma).
Cell Culture--
Cells were routinely cultivated in 12-well
plates (growth area 3.83 cm2/well) as described earlier (3,
6, 8) in 1 ml of a culture medium consisting of Dulbecco's modified
Eagle's medium ((Flow) 1 × liquid without glutamine, 4 mM glutamine (Flow) added) supplemented with 10% newborn
calf serum (Flow), 4 nM insulin (Actrapid Human, Novo), 10 mM Hepes (Flow), and 50 IU of penicillin, 50 µg of
streptomycin, and 25 µg of sodium ascorbate (Kebo) per ml, at
37 °C in a water-saturated atmosphere of 8% CO2 in air
in a Heraeus CO2-auto-zero B5061 incubator. The medium was
completely exchanged with fresh prewarmed medium on day 1 (when the
cultures were first washed with 2 ml of prewarmed Dulbecco's modified
Eagle's medium) and on days 3 and 6 (without wash).
cAMP Determinations--
On day 6 or 7, as indicated,
antagonists, PMA, or phosphodiesterase inhibitors were added 5-7 min,
and cirazoline or ionomycin 1 min, before (other) agonist addition.
After a further 20-25 min, the culture medium was aspirated, 0.8 ml of
95% ethanol added, the cells scraped off, and the suspension
transferred to Eppendorf tubes. The wells were washed with 0.5 ml of
70% ethanol, and the combined suspensions were dried in a Speedvac
centrifuge. The dried samples were dissolved in 0.3-1 ml of the Buffer
1 provided with the Cyclic AMP [3H]Assay System from
Amersham Pharmacia Biotech and centrifuged at 14,000 rpm, 10 min. Two
50-µl aliquots of the supernatants of every sample were analyzed
according to the description in the assay system. For every
concentration of any agent in each experiment, two wells were used.
Thus, in each experiment, each value used in later calculations is the
mean of the four measurements of cAMP. The results were routinely
normalized in each series of experiments by setting the value of the
cAMP response for 0.1 µM NE (or 0.1 µM
isoprenaline or 1 µM CGP-12177 where relevant) to
100%.
Measurement of Intracellular Ca2+--
The
concentration of cytosolic Ca2+ was measured with the
Fura-2 fluorescent dye technique (9), principally as earlier described (10), in a dual wavelength spectrophotometer (Sigma ZWS II) with
alternating excitation wavelengths of 355 and 395 nm, with an emission
cut-off filter (KV 470). The ratio of emission was sampled with
frequency of 2.5/s and smoothed by a running means technique (15 samples). Intracellular Ca2+ concentrations
([Ca2+]i) were calculated and graphs drawn
(KaleidaGraph) from these data using the Grynkiewicz et al.
(9) equation: [Ca2+]i = KD
·(R Analysis of Dose-Response Curves--
For analysis of
dose-response curves, the nonlinear regression analysis curve-fitting
option of the KaleidaGraph 3.0 application was used. Monophasic
dose-response data were analyzed with the rearranged Michaelis-Menten
equation,
Chemicals--
The following agents were used: norepinephrine
(( In the present experiments, precursors of brown adipocytes were
isolated from mice and cultured under conditions leading to confluence
on days 6 or 7. The cells are then fully differentiated, in that they
contain numerous fat droplets and are able to express high levels of
the brown fat-specific mitochondrial uncoupling protein (UCP1) in
response to norepinephrine stimulation (3, 4). It is in cells like this
that the unusual and distinctive kinetics for norepinephrine-induced
UCP1 expression (3, 4, 6) and An Unusual Norepinephrine Dose-Response Curve--
The
characteristic dose-response curve for cAMP generation induced by
norepinephrine (NE) is presented in Fig.
1A. As seen, in agreement with
Ref. 6, the response did not adhere to simple Michaelis-Menten
kinetics; rather, a very distinctive semi-bell-shaped dose-response
curve was obtained. In control experiments, it was confirmed that the
same relative relationships for the cAMP levels induced by 0.1, 1, and
10 µM NE were observed at any time point up to 90 min
after NE stimulation (when the cAMP levels were still markedly elevated
over unstimulated levels) and that the response peaked at
A possible explanation for a bell-shaped dose-response curve would be
the presence of an inhibitory component, activated by high
concentrations of NE. Following the theoretical suggestions of Rovati
and Nicosia (5) and Equation 2 derived therefrom (see "Experimental
Procedures"), we have analyzed in Fig. 1B the kinetics of
the NE dose-response curve and found that it could be resolved into two
hypothetical components: a stimulatory component with an
EC50 of 35 nM and an inherent ability to
increase the cAMP level to 250% of that maximally observed
experimentally, and an inhibitory component with a somewhat higher
IC50 value of 100 nM and an inherent ability to
reduce the cAMP level by 200%. Through the interaction of these
hypothetical components (the Resultant curve in Fig. 1B), a
very good description of the experimental results (Fig. 1A)
was obtained. Although it was thus possible with this theoretical
analysis to describe the observation by introducing two counteracting
components, the question to be addressed was whether the two
components postulated here had a true existence.
The Nature of the Inhibitory Component--
In mature brown
adipocytes, the stimulatory component of the cAMP response to NE is
mediated via
The hypothetical inhibitory component could be mediated via the same
The obvious candidate for such an inhibitory receptor is the
We therefore also examined whether the
In Fig. 1D, we have used the data from Fig. 1C to
characterize the components predicted by the theoretical analysis in
Fig. 1B. As seen, the experimentally obtained values,
indicating a stimulatory
To substantiate this interpretation, the dual nature of NE action was
mimicked by combining a The Response to Isoprenaline also Includes an Inhibitory
An Unabridged cAMP Response Is Only Observed with
Activation of Protein Kinase C Does Not Mediate the
Inhibition--
To understand the cellular mechanism of the
To investigate the possible participation of the diacylglycerol-PKC
pathway, we stimulated the cultured adipocytes by the phorbol ester PMA
(12-O-tetradecanoylphorbol-13-acetate); this had in itself
no detectable effect (Table II). If PKC
activation was responsible for the inhibitory effect of
Conversely, we attempted to inhibit the PKC pathway during adrenergic
stimulation. The addition of the PKC inhibitor chelerythrine (1 µM) before addition of NE or isoprenaline led to a
decrease in the responses to these agents by ~10%, i.e.
the opposite of what would be expected if PKC mediated the inhibitory
effect (not shown). Furthermore, long-term pretreatment with PMA, a
treatment that is expected to desensitize PKC (22) and earlier
indirectly shown to do so in these cells (10), did not lead to an
augmentation in the cAMP response to Ca2+ Is Necessary for the Inhibitory
NE stimulation of cultured brown adipocytes led to an elevated
intracellular Ca2+ level (Fig.
3) (10). To investigate whether this was
a necessary component in the inhibitory effect, we examined the effect
of incubation conditions intended to diminish the increase in
[Ca2+]i. Changing to a Ca2+-free
medium led to an approximate halving of the norepinephrine-induced [Ca2+]i response (Fig. 3); this was associated
with an increase in the level of norepinephrine-induced cAMP
accumulation (Table III). To further
diminish the [Ca2+]i levels, we added the
Ca2+ chelator BAPTA to the nominally Ca2+-free
medium. A further reduction in [Ca2+]i response
was observed (Fig. 3), and a higher cAMP accumulation (Table III). To
fully eliminate the increase in cytosolic Ca2+ levels, we
preincubated the cells with the permeable Ca2+ chelator
BAPTA/AM. As seen, no NE-induced increase in
[Ca2+]i was then observed (Fig. 3), and a further
increase in NE-induced cAMP accumulation was found. (The cell-permeable non-chelator "half-BAPTA/AM," which is not able to chelate
Ca2+, had only a limited effect on NE-induced
[Ca2+]i and did not influence the NE-induced cAMP
response (Fig. 3; Table III).) Thus, an increase in
[Ca2+]i was clearly a necessary step in the
mediation of the inhibitory Correspondence between Adrenergic Effects on
[Ca2+]i and on Inhibition of cAMP
Accumulation--
If an increase in [Ca2+]i was
fully responsible for the mediation of the inhibition, there should be
good correspondence between the relative ability of adrenergic agonists
to increase [Ca2+]i and their ability to inhibit
cAMP accumulation. In Fig. 4A,
we exemplify the effect of different adrenergic agonists on
[Ca2+]i levels in these cells. In Fig. 4,
B-D, dose-response curves for these effects are presented as
a means from a series of experiments.
As seen, NE increased [Ca2+]i levels markedly,
with an EC50 value of about 0.2 µM, and a
maximal induction of more than micromolar Ca2+
concentrations (Fig. 4B). At concentrations Effect of Increase in [Ca2+]i on cAMP
Accumulation--
A further, critical step in the demonstration that
[Ca2+]i mediates the inhibitory component in the
control of cAMP level in cultured brown adipocytes would be to
experimentally increase the intracellular levels of Ca2+.
Two methods were used for this. One was through activation of the
receptor-mediated pathway, i.e. through stimulation of
Cirazoline, just as NE, led to a rapid increase in
[Ca2+]i levels. The increase induced by both NE
and cirazoline displayed Michaelis-Menten kinetics (Fig. 4,
B and D), but the EC50 for cirazoline
was, as expected, lower than that for NE (35 and 200 nM,
respectively). The maximal level reached after NE stimulation was
consistently higher than that reached after selective stimulation of
the
The Ca2+ elevation initiated by ionomycin tended to be
stable with time. The effect of ionomycin was not saturable and may
consist of two components: a Michaelis-Menten part and a linear part
(Fig. 4D). This may relate to the Ca2+ coming
from two sources: intracellular and extracellular (cf. studies by Mason and Grinstein (23) in lymphocytes). Ionomycin concentrations of 1-10 µM induced increases in
[Ca2+]i within the physiological range induced by
NE.
We thereafter analyzed the effects of cirazoline- or ionomycin-induced
Ca2+ increases on the cAMP levels in the brown adipocytes.
The analysis was performed with CGP-12177 as the agonist since this
agent does not in itself alter [Ca2+]i (Fig.
4A). The effect of cirazoline on agonist-stimulated cAMP
accumulation is shown on Fig.
5A. The effect was
dose-dependent, and, in agreement with expectations and the
single-dose experiment in Table I, cirazoline strongly inhibited the
CGP-12177-induced response. When ionomycin was used to increase
[Ca2+]i levels, an even more pronounced effect
was observed. This agent drastically reduced (and at high levels fully
eliminated) cAMP accumulation (Fig. 5B). Thus, an increase
in [Ca2+]i, even when artificially induced, was
sufficient to fully mimic the inhibitory
The dose dependence for the effect of ionomycin on
The relationship between Is the Increase in [Ca2+]i Induced by
Norepinephrine Responsible for the Inhibition of cAMP
Accumulation?--
The relevant physiological question is really
whether the observed increase in [Ca2+]i during
adrenergic stimulation can quantitatively explain the inhibition
observed in response to norepinephrine addition. We therefore tested
whether the relationship between the increase in
[Ca2+]i and the level of cAMP can be considered
the same as that above, also when [Ca2+]i is
increased through the innate stimulation elicited by NE. As seen in
Fig. 6A, cirazoline, which may
further increase [Ca2+]i, further inhibited
NE-stimulated cAMP accumulation, although only to a fairly small
degree. Also when [Ca2+]i was increased by
ionomycin, cAMP accumulation could be practically fully inhibited (Fig.
6B). From this curve, it should be possible to relate the
physiologically (NE-) induced [Ca2+]i increase to
the degree of inhibition observed, provided that the initial functional
level of Mediation of the Ca2+ Effect on cAMP
Accumulation--
To identify the link between the increase in
[Ca2+]i and the inhibition of cAMP accumulation,
we used the fact that the level of cAMP is determined by the balance
between two processes, one synthetic (mediated by adenylyl cyclase(s))
and one degradative (mediated by cyclic nucleotide phosphodiesterase(s)
(PDE)). To allow for a distinction between these two sites of action,
experiments were performed in which adenylyl cyclase was stimulated
with the direct activator forskolin (in the brown adipocytes studied
here, forskolin does not affect the basal level of Ca2+ or
the NE-induced Ca2+ response (10)). These experiments were
first carried out in the presence of the general phosphodiesterase
inhibitor IBMX, i.e. under conditions under which all
observed effects of additions can be assumed to be due solely to
effects on adenylyl cyclase. As seen (Fig.
7A), the addition of forskolin
led to the expected increase in cAMP. When
[Ca2+]i was increased by the addition of 1 µM ionomycin, only a minor difference (~20%) in the
kinetic constants of cAMP accumulation was registered (Fig.
7A). This effect of Ca2+ on adenylyl cyclase
activity was thus too small to account for the large inhibition of cAMP
accumulation observed above (
We therefore investigated whether the effect could be mediated via
activation of a phosphodiesterase. Experiments were therefore performed
in the absence of IBMX, i.e. when the phosphodiesterases were active. A different pattern was then observed (Fig.
7B). The maximal cAMP level achieved by forskolin was now
only half that in the presence of phosphodiesterase inhibitor, implying the presence of a high basal phosphodiesterase activity in these cells
(principally in accordance with Ref. 24)). When
[Ca2+]i was increased by the addition of 1 µM ionomycin, the cAMP level was further decreased by a
factor of 2 (Fig. 7B). As the inhibitory effect of
Ca2+ was only evident when the phosphodiesterases were
active (compare Fig. 7, A and B), it was clear
that activation of a phosphodiesterase was responsible for the
inhibition of the cAMP accumulation by Ca2+ (and thus, by
NE).
Which Phosphodiesterase Mediates the Effect?--
Not all
phosphodiesterases are Ca2+ sensitive. Out of at least
seven distinct PDE gene families which generate more than 20 different
isozymes, only the subtypes II, III, and IV have so far been positively
identified in brown fat (25-27) but none of these PDEs are generally
accepted to be Ca2+ stimulated; this is only the case for
PDE I (CaM-PDE), which has not as yet been identified in brown adipose
tissue. However, if CaM-PDE is the PDE responsible for the bell shape
of the dose-response curve for NE, specific inhibition of this PDE
should transform the bell-shaped curve to a normal
Michaelis-Menten-type curve. The effect of several PDE inhibitors on
the cAMP dose-response curve for NE was therefore studied.
Three selective PDE inhibitors, 8-MM-IBMX, OPC-3911, and Ro 20-1724, i.e. the inhibitors of subtypes I, III, and IV,
respectively, were tested. At the concentrations used, the inhibitors
should inhibit only the indicated PDE subtypes (28-30). The inhibitors of PDE III and IV, i.e. OPC-3911 and Ro 20-1724, failed to
convert the bell shape of the dose-response curve into normal kinetics, despite the fact that they considerably increased the maximal cAMP-response (Fig. 8A). Only
8-MM-IBMX, the selective inhibitor of the CaM-PDE (PDE-I), converted
the dose-response curve to a normal Michaelis-Menten type (Fig.
8A). This thus indicated that the inhibition was mediated
via the PDE-I subtype.
Involvement of Calmodulin in the Mediation of the
Inhibition--
Mediation of the Ca2+ effect on PDE I
occurs through calmodulin (31). Thus, if it is directly through the
formation of a Ca2+-calmodulin complex and its interaction
with PDE-I that this phosphodiesterase is activated, antagonism of
calmodulin should remove the inhibitory component of norepinephrine
stimulation. In agreement with this, the potent antagonist of
calmodulin, calmidazolium (IC50 ~0.2 µM for
CaM-PDE-activity (32)) at a concentration of 3 µM
converted the bell-shaped curve to a Michaelis-Menten-type curve (Fig.
8B).
When stimulated with norepinephrine, brown adipocytes may exhibit
non-Michaelis-Menten response kinetics in their responses. This is,
e.g. evident for norepinephrine-induced UCP1 (3, 4) and
The goal of the present investigation was to elucidate the mechanism
behind this response pattern. The analysis was based on the theoretical
model of this type of response pattern as being the result of a
stimulatory and an inhibitory component, as presented in a generalized
theory by Rovati and Nicosia (5). Experimentally, it was possible in
the present study to substantiate the existence of these components.
The elimination of the inhibitory component revealed that the
stimulatory This dual component analysis thus fully explains the kinetics of
norepinephrine-stimulated UCP1 and The Nature of the Two Components--
In the dual component
analysis, the stimulatory component was undoubtedly a regular
The inhibitory component was mediated by Ca2+ stimulation
of a cAMP phosphodiesterase. The interaction is therefore principally an example of negative cross-talk between intracellular signaling pathways, a phenomenon amply described over the last decades (36, 37).
However, in its classical forms, cross-talk occurs as interaction between the signaling pathways of two different extracellular stimuli
(hormones). Norepinephrine may mediate the stimulatory or the
inhibitory component in different systems. Thus, cAMP levels induced
via
However, the present observations fall outside these types of classical
cross-talk, because NE activates both the stimulatory adenylyl cyclase
component and the inhibitory component, and thus the cross-talk occurs
between two pathways activated via the same activator. There is
apparently only one earlier case in which norepinephrine has been
implied to activate both pathways: heart cells (40). However, in those
cells, the inhibitory
Thus, the uniqueness of the mechanisms reported here is related to the
quantitative relationship between the two components: the inhibitory
The Large Effect of
The high sensitivity of the system to Physiological Relevance of the Inhibition--
An important
difference between brown adipocytes in situ and those
studied here in culture is that the cultured cells are naive with
respect to adrenergic stimulation, whereas the brown adipocytes
in situ are exposed to chronic adrenergic stimulation of
alternating intensity. However, this may even accentuate the situation
described here, because chronic adrenergic stimulation increases the
density of the
Functionally, all processes that are rate-limited by cAMP levels will
be expected to show bell-shaped dose-response curves, but this does not
mean that all responses mediated via an increase in cAMP levels will
show this pattern. Thus, processes saturated at fairly low cAMP levels
will display monophasic kinetics even if the underlying cAMP response
is biphasic. This may, e.g. be the case for thermogenesis
where saturation apparently occurs already at levels of cAMP only 50%
of those maximally induced by norepinephrine (24).
The
However, a series of observations indicate a positive or synergistic
interaction between
Thus, presently, it can only be concluded that cells of brown adipose
tissue have an elaborate system for interaction between We thank Eva Degerman for valuable discussions.
*
This work was supported by a grant from the Swedish Natural
Science Research Council (to B. C. and J. N.) and Russian
Foundation for Basic Research Grant 98-04-49214 (to G. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: The Wenner-Gren
Institute, The Arrhenius Laboratories F3, Stockholm University, S-106
91 Stockholm, Sweden. Tel.: 46-8-164128; Fax: 46-8-156756; E-mail:
jan@metabol.su.se.
2
G. E. Bronnikov, S-J. Zhang, B. Cannon, and
J. Nedergaard, unpublished observations.
The abbreviations used are:
UCP1, uncoupling
protein-1;
[Ca2+]i, intracellular
Ca2+;
PMA, phorbol 12-myristate 13-acetate;
BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
IBMX, isobutylmethylxanthine;
NE, norepinephrine;
PKC, protein
kinase C;
PDE, phosphodiesterase.
A Dual Component Analysis Explains the Distinctive Kinetics of
cAMP Accumulation in Brown Adipocytes*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-adrenoreceptor gene expression) was
investigated. A theoretical dual component analysis indicated that the
observed dose-response curves could be constructed as the resultant of
a stimulatory and an inhibitory component. Experimentally, inhibition
of the 
1-component of the norepinephrine response
revealed the underlying existence of a much larger stimulatory 3-component which displayed monophasic Michaelis-Menten
kinetics. The inhibitory 1-component (which was also
monophasic but had a 2-fold higher EC50) was mediated via
an increase in [Ca2+]i; the protein kinase C
pathway was not involved. The [Ca2+]i increase
which resulted in massive inhibition of cAMP accumulation was very low:
<100 nM. The [Ca2+]i signal
stimulated a calmodulin-controlled phosphodiesterase, possibly PDE-1. The acquirement of this specific
interaction pattern between - and 1-adrenergic
stimulation was thus part of the differentiation program of the brown
adipocytes. It was concluded that an array of synergistic or inhibitory
1/ interactions occur in the adrenergic regulation of
this cell type which is unique in its dependence upon adrenergic
stimulation for cellular proliferation, differentiation, and metabolic function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.1 µM being optimal for induction of expression, and
higher, as well as lower, concentrations being less efficient (3,
4).
3-receptors may be revealed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Rmin)/(Rmax R)·Fo/Fs, where R is
the running 355/395 fluoresence ratio, Rmin is
this ratio under Ca2+-free conditions,
Fo is the actual fluorescence at 395 nm under
Ca2+-free conditions, and Rmax and
Fs are these values under saturating
Ca2+ conditions. The KD value used was
that determined by Grynkiewicz et al. (9), i.e.
224 nM. Experimentally, each experimental trace was
finalized by adding to the cuvette 30 µM ionomycin
(yielding the Rmax and the Fs
values), followed by an addition of 20 µM digitonin with
2 mM MnCl2, yielding the
Rmin and the Fo values. Under
the conditions used here, the Fo/Fs value
was very close to 1.
where h is the Hill coefficient. If h was
estimated to be close to 1 in the initial analysis, the data were
recalculated with h = 1. For the analysis of the
biphasic ("semi-bell-shaped") dose-response data, a model (5) for
the interaction of a ligand with two different receptors, one
stimulatory (S) and one inhibitory (I), was used,
![V<SUB>A</SUB>=<IT>basal</IT>+V<SUB><UP>max</UP></SUB>/(1+(<UP>EC</UP><SUB>50</SUB>/[A])<SUP>h</SUP>)](/content/vol274/issue53/fulltext/37770/img001.gif)
(Eq. 1)
IC50 here denotes the EC50 of the
inhibitory component. In some calculations basal was set as
a constant to avoid a singular matrix.
![V<SUB>A</SUB>=<IT>basal</IT>+V<SUB><UP>max</UP></SUB>(S)/(1+(<UP>EC</UP><SUB>50</SUB>/[A])<SUP>h</SUP>)+V<SUB><UP>max</UP></SUB>(I)/(1+(<UP>IC</UP><SUB>50</SUB>/[A])<SUP>h</SUP>)](/content/vol274/issue53/fulltext/37770/img002.gif)
(Eq. 2)
)-arterenol bitartrate), isoprenaline (()-isoproterenol
(+)-bitartrate), ionomycin (calcium salt), forskolin, Fura-2/AM, BAPTA,
phorbol 12-myristate 13-acetate (PMA)
(12-O-tetradecanoylphorbol-13-acetate), prazosin, yohimbine
(hydrochloride), and dimethyl sulfoxide (for cell culture) all obtained
from Sigma; CGP-12177 (CGP-12177A, from Ciba-Geigy),
3-isobutyl-1-methylxanthine (IBMX),
8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MM-IBMX), BAPTA-AM (all
from Calbiochem), Ro 20-1724, cirazoline (hydrochloride) (from RBI,
Natick), half-BAPTA from Molecular Probes, and OPC-3911 was a gift from
Eva Degerman (Lund University). Stock solutions (10 mM) of
agents used were normally made in 0.05% ascorbic acid (norepinephrine
and isoprenaline) or in Dulbecco's modified Eagle's medium and stored
at 80 °C. Ionomycin, forskolin, Fura-2/AM, prazosin, PMA, IBMX,
8-MM-IBMX, Ro 20-1724, and OPC-3911 were dissolved (1 to 100 mM) and kept in dimethyl sulfoxide. Up to 3% of dimethyl
sulfoxide did not affect the cAMP accumulation. Routinely, the final
concentration of dimethyl sulfoxide was kept <1%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-adrenoreceptor
expression (11) have been observed, as well as the underlying similar
kinetics of cAMP accumulation (6). The aim of the present investigation
has been to obtain an explanation for these kinetics.
20 min for
all NE concentrations (not shown). Therefore, in the following
experiments, the cAMP levels were determined 20 min after NE
stimulation.
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Fig. 1.
Analysis of the dose-response curve for
NE-induced cAMP accumulation in cultured mature brown adipocytes.
A, dose-response curve for the effect of NE on cAMP
accumulation. Cultured brown adipocytes were stimulated on day 6 of
culture with the indicated NE concentrations and cAMP levels were
determined 20 min later. Points are means ± S.E. of eight
independent experiments. B, component analysis of the data
in A. The data in A were fitted to the
two-component model of Rovati and Nicosia (5) by Equation 2 (see
"Experimental Procedures"). The components revealed consisted as
indicated of a stimulatory component with Vmax = 250%, EC50 = 35 nM, h = 1.35, and an inhibitory component with Vmax = 200%,
IC50 = 100 nM, h = 1. C, effect of -antagonists on the dose-response curve for
NE-stimulated cAMP accumulation. 10 µM yohimbine
(Yoh) and/or prazosin (Praz) were added 5-7 min before the
indicated concentration of NE, and cAMP levels determined 20 min later.
Points are mean ± S.E. of three experiments; in each series the
effect of 0.1 µM NE alone was set to 100%, corresponding
to 25 ± 2 pmol of cAMP/well. D, component analysis of
the experimental data in C. The 
points are identical to
those for NE + prazosin in C but are here analyzed for
adherence to simple Michaelis-Menten kinetics, yielding the indicated
values. The 
points were calculated as the difference between the NE
points and the "NE + prazosin" points in C; they thus
represent the inhibitory component. The inhibitory component was also
analyzed for adherence to Michaelis-Menten kinetics, yielding the
indicated values.
3-adrenergic receptors (6), and undoubtedly
3-activation would be the stimulatory component in the
above theoretical analysis.
3-receptors that mediate the stimulatory component or via other adrenergic receptors. The activated
3-receptors in themselves could induce the inhibition,
since released 
-subunits of the Gs (or
Gi) protein can inhibit certain adenylyl cyclase subtypes
(12-14). If this effect were to occur at high intensity of
3-adrenoreceptor activation, it could explain the curve
shape but it would not be possible to block only the inhibitory
component by the use of a subtype-selective adrenergic antagonist.
However, if other adrenergic receptors were involved, the correct
subtype-selective adrenergic antagonist should be able to transform the
biphasic dose-response curve into a simple, monophasic curve with the
appearance of the hypothetical stimulatory component in Fig.
1B.
2-adrenergic receptor that mediates its inhibitory
effect directly on adenylyl cyclase via Gi proteins. We
therefore examined whether the 2-antagonist yohimbine
would be able to eliminate the inhibitory component of the NE response.
As seen in Fig. 1C, +Yoh, this was not the case:
although there was a clear augmenting effect of yohimbine on the
maximum response to NE, the inhibitory component was still very evident.
1-antagonist
prazosin could influence the dose-response curve. As seen in Fig.
1C, +Praz, this was indeed the case: prazosin not
only doubled the maximal response (as did yohimbine) but it also
eliminated the inhibitory component and converted the bell-shaped curve
into a typical Michaelis-Menten curve.
3-component with an
EC50 of 34 nM and a large increase in cAMP up
to 231% of that normally seen, plus an inhibitory
1-component with an IC50 of 74 nM and inducing an inhibition of 170%, were very close to
those predicted by the theoretical analysis. It was therefore concluded
that the inhibitory component was not inherent to the
3-receptors and was not mediated in the classical way
via 2-receptors. Instead, this inhibition was
unexpectedly mediated via 1-receptors.
3-selective agonist (BRL-37344 or CGP-12177) with an 1-selective agonist (cirazoline).
In the brown adipocytes studied here, 3-agonists induce
a monophasic cAMP response (6) and they were used here at saturating
concentrations. As expected, the 3-agonists increased
the cAMP level (Table I), and also as
expected, this response was not affected by the
1-antagonist prazosin. The 2-antagonist
yohimbine in itself exhibited a tendency to an unexpected inhibitory
effect. However, in accordance with the model presented above,
activation of 1-receptors with cirazoline led to a
significant suppression of the cAMP accumulation induced by either of
the 3-selective agonists, which could be prevented by
the 1-selective antagonist prazosin. Thus, this result
is in accordance with the implication that it is through
1-receptors that the inhibitory component of the
responses to NE is mediated.
Effect of the 1-selective agonist cirazoline on cAMP
accumulation induced by 3-selective agonists
3-agonists and, where indicated, 1 µM
cirazoline 1 min before the 3-agonists. The cultures were
harvested 20 min after 3-agonist addition and cAMP levels
were determined. Results are mean ± S.E. of five experiments
(three experiments with yohimbine). In each series, the effect of 0.1 µM BRL-37344 or 1 µM CGP-12177 alone was
set to 100% (which corresponded to 96 ± 11 pmol of cAMP per well
for BRL-37344 and 68 ± 17 for CGP-12177). The data were analyzed
for significant effect of further agents on the response to the
3-agonist alone.
1-Component--
The agonist isoprenaline is generally
considered to be a selective -agonist. Thus, isoprenaline is not
expected to stimulate 1-receptors, and its dose-response
curve would therefore not be expected to exhibit the
1-inhibitory component identified above. However (Fig.
2A), although the maximal
response to isoprenaline stimulation (66 pmol/well) was much higher
than that to NE (25 pmol/well), presumably because of a much lower
activation of 2-receptors, the dose-response curve for
isoprenaline was nonetheless bell-shaped, although the apparent
inhibitory component was smaller than that of norepinephrine (both
absolutely and relatively). A theoretical component analysis was
therefore also performed for isoprenaline (Fig. 2B),
indicating again that the response could be explained as the resultant
of counteracting components. Yohimbine was unable to transform the
bell-shaped curve to a simple Michaelis-Menten curve, whereas prazosin
was able to accomplish this (Fig. 2C). The response to
isoprenaline thus also contained an inhibitory 1-component. From the analysis of the experimental data
(Fig. 2D), it is evident that at experimental concentrations
where isoprenaline may be expected to be selective for -receptor
interaction (~1 µM), it displayed a prominent
1=effect in these cells.
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Fig. 2.
Analysis of the dose-response curve for
isoprenaline-induced cAMP accumulation in cultured mature brown
adipocytes. A, dose-response curve for
isoprenaline-induced cAMP accumulation. Cultured brown adipocytes were
stimulated on culture day 6 with the indicated isoprenaline
concentrations and cAMP levels were determined 20 min later. Points are
mean ± S.E. of seven independent experiments. B,
component analysis of the data in A. The data in
A were analyzed as described in the legend to Fig.
1B, yielding a stimulatory component
(Vmax = 110%, EC50 = 8 nM, h = 1.7) and an inhibitory component
(Vmax = 50%, IC50 = 500 nM, h = 1). C, effect of
-antagonists on the dose-response curve for isoprenaline-stimulated
cAMP accumulation. 10 µM yohimbine (Yoh)
and/or prazosin (Praz) were added 5-7 min before the indicated concentration of
isoprenaline, and cAMP levels determined 20 min later. In each series,
the effect of 0.1 µM isoprenaline alone was set to 100%,
corresponding to 66 ± 9 pmol of cAMP/well. Points are mean + S.E.
of five experiments. D, component analysis of the
experimental data in C. The points are identical to
those for isoprenaline + prazosin in Fig. 1C but are here
analyzed for adherence to simple Michaelis-Menten kinetics, yielding
the indicated values. The points were calculated as the difference
between the isoprenaline points and the "isoprenaline + prazosin"
points in Fig. 1C; they thus represent the inhibitory
component. The inhibitory component was also analyzed for adherence to
Michaelis-Menten kinetics, yielding the indicated values.
3-Selective Agonists--
The combined implication of
the above analyses of the responses to norepinephrine (Fig. 1) and to
isoprenaline (Fig. 2) is that these agonists to different degrees
interact with 1-receptors and that this can explain both
the differences in shape of their dose-response curves and most of
their apparently different efficacy. Based on the results presented in
Figs. 1B and 2B, and Table I it was calculated
that the inhibitory 1-component led to a 74% reduction
of the response to 1 µM NE and to a 39% reduction of the
response to 1 µM isoprenaline; no inhibitory
1-component was found in the response to CGP-12177 or
BRL-37344.
1-inhibition, we proceeded to identify the second
messenger responsible. As in other systems, stimulation of
1-adrenoreceptors in brown fat cells produces two second
messengers, inositol 1,4,5-trisphosphate (15, 16) and diacylglycerol;
the inositol 1,4,5-trisphosphate releases Ca2+ from
intracellular stores and [Ca2+]i is increased
(10, 17, 18), and the diacylglycerol activates protein kinase C (PKC)
(19).
1-stimulation, PMA would be expected to inhibit
selective 3-agonist-induced cAMP accumulation, just as
did 1-stimulation (Table I). However, PMA did not
inhibit, but rather enhanced the cAMP accumulation. The nature of this
PMA-elicited enhancement of 3-adrenoreceptor-induced cAMP accumulation was not further investigated but could conceivably be
related to reported stimulation of certain adenylyl cyclase subtypes by
PKC (20, 21). As a similar enhancing effect of PMA was observed also
when the cells were stimulated with isoprenaline or NE (Table II), it
is unlikely that this mechanism is actually activated when the cells
are responding to adrenergic stimulation.
Effect of phorbol ester treatment on cAMP accumulation induced by
adrenergic agonists
-agonists (Table II). Thus, the
PKC pathway was not involved in mediation of the inhibitory
1-component.
1-Response--
To examine whether the
1-induced inhibitory effect was mediated via an increase
in cytosolic Ca2+ levels, we manipulated the intracellular
Ca2+ response during adrenergic stimulation.
1-component.
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Fig. 3.
NE-induced increases in
[Ca2+]i levels: the effects of incubation
conditions. Measurements of [Ca2+]i levels
were performed as described under "Experimental Procedures," but
the Ca2+ concentrations in the preincubation buffer were
altered in parallel to the conditions described in the legend to Table
III. For each experiment, the time of addition of 1 µM NE
was set to 0.
Effect of Ca2+ elimination on NE-induced cAMP accumulation
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Fig. 4.
Effects of agents on cytosolic
Ca2+ levels in brown adipocytes. A,
kinetics of increases in cytosolic [Ca2+] levels in
response to different agents. Brown adipocytes were preincubated with
Fura/AM as described under "Experimental Procedures" before the
addition of 1 µM NE (0.1 µM where
indicated), 1 µM cirazoline, 10 µM
CGP-12177 or 0.1 µM ionomycin. For each experiment, the
time of addition (heavy arrowhead) was set to 0. B, dose-response curve for the effect of NE on
[Ca2+]i. The experiments were principally
performed as in A, except that different concentrations of
NE were added, in different incubations. For each concentration, the
maximal response to NE was measured, expressed as the increase over the
basal value, i.e. as 
[Ca2+]i. The
results are mean ± S.E. from five experiments. Data points were
analyzed for best fit to a Michaelis-Menten equation (Equation 1),
yielding an EC50 of 0.2 µM NE and a
Vmax of 1.1 µM Ca2+
(Hill coefficient h = 1.89). (Here and in subsequent
figures, where not visible, the S.E. is smaller than the size of the symbol.) C,
dose-response curve for the effect of isoprenaline on
[Ca2+]i. The experiments were principally
performed as in B, except that different concentrations of
isoprenaline were added. The results are mean ± S.E. from five
experiments. Data points were analyzed for best fit to a
Michaelis-Menten equation (Equation 1), yielding an EC50 of
36 µM isoprenaline and a Vmax of
0.89 µM Ca2+ (h = 1.14). D,
dose-response curves for the effects of cirazoline and ionomycin on
[Ca2+]i. The experiments were principally
performed as in A and B. The Michaelis-Menten
analysis yielded for cirazoline an EC50 of 0.034 µM cirazoline and a Vmax of 0.52 µM Ca2+ (h = 1.83). For estimation of
the parameters of the ionomycin dose-response curve, the sum of a
linear and a Michaelis-Menten function was used, i.e.
[Ca2+]i = m · [ionomycin] + Vmax/(1+(EC50/[ionomycin]). This
yielded an EC50 of 0.11 µM ionomycin, a
Vmax of 85 nM Ca2+, and
an m value of 0.08. The pure linear approximation of the
data is drawn on the figure as the curve indicated with an
arrow.
1
µM, isoprenaline had a clear, although modest, effect on
[Ca2+]i levels (Fig. 4C) (this effect
could be blocked by 1 µM prazosin but not by propranolol,
indicating that it was indeed mediated via 1-receptors;
not shown). CGP-12177, however, even at 10 µM, was fully
without effect on [Ca2+]i (Fig. 4A).
It is clear that this relationship is fully parallel to the inherent
ability of each of these compounds to self-inhibit cAMP accumulation
through 1-pathways: CGP-12177 had no such effect (Table
I), isoprenaline some (Fig. 2), and NE much greater (Fig. 1).
1-receptors by the 1-selective agonist
cirazoline. The other one was through the use of the
Ca2+-ionophore ionomycin.
1-receptors with cirazoline; as pure -stimulation
and an increase in cAMP with forskolin did not elevate
[Ca2+]i (not shown), a complex interaction is
indicated but this has not been further studied here. The induced level
was not stable but successively declined (Fig. 4A), although
it remained elevated over the basal level for more than 40 min. Further
analysis of the decline in [Ca2+]i indicated that
it could not be fitted to a single exponential function. However, it
could be analyzed as being composed of two exponential functions (not
shown) (or it could be described mathematically as a power function of
time, i.e. as basal + [Ca2+]i-max · tc, where
t is time and the constant c is 0.6-0.8).
1-component of
the adrenergic response.
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Fig. 5.
Effect of increases in cytosolic
Ca2+ levels on cAMP accumulation induced by CGP-12177.
A, brown adipocytes were incubated with 1 µM
CGP-12177 in the presence of the indicated levels of cirazoline. Points
are mean ± S.E. from five experiments; 100% corresponded to
45 ± 10 pmol of cAMP per well. The EC50 for
cirazoline was 28 nM and the maximal inhibition was 64%.
B, as in A, except that ionomycin was added.
Mean ± S.E. from five experiments; 100% corresponded to 59 ± 14 pmol of cAMP per well. The EC50 for ionomycin was 0.3 µM and the maximal inhibition was 90%. C,
inhibition of CGP-12177-induced cAMP accumulation as a function of
cytosolic Ca2+ levels. The curves were constructed by
combining the [Ca2+]i levels compiled in Fig.
4D with the corresponding cAMP levels compiled in
A and B (
refer to the cirazoline data and 
to the ionomycin data).
[Ca2+]i was then used to estimate the
quantitative relationship between [Ca2+]i and
cAMP accumulation. Using the dose-response curve for the effect of
ionomycin on [Ca2+]i levels (Fig.
4D), we replotted the inhibition of cAMP levels (Fig.
5B) as a function of the corresponding, maintained [Ca2+]i (Fig. 5C). As seen, very
low increases in [Ca2+]i, of only about 30 nM, induced an inhibitory effect on the cAMP level, and at
levels 1 µM, cAMP accumulation was totally inhibited.
[Ca2+]i and cAMP
accumulation was also plotted when cirazoline was the
[Ca2+]i-increasing agent (Fig. 5C).
Apparently, a Ca2+ elevation induced by the
1-agonist was only one-third as effective as a
Ca2+ elevation induced with ionomycin in bringing about
inhibition of cAMP accumulation (IC50 was about 300 and 80 nM Ca2+, respectively). This difference can,
however, be understood if the different kinetics of the
Ca2+ responses to these agents are taken into account (Fig.
4A). The dose-response curves for the increases in
Ca2+ (Fig. 4D) are based on the peak response.
As the [Ca2+]i for ionomycin is fairly stable
with time, the peak level is a good approximation of the mean level
during the 20-min incubation used for determination of cAMP levels.
However, for cirazoline, the maximal level is clearly an overestimate
of the mean elevation of [Ca2+]i. The mean level
of [Ca2+]i for the 20-min incubation may be
estimated through integration of the area under the curves in Fig.
4A and is about a factor 3 lower than the maximal values
(this proved to be valid for both cirazoline and NE). Thus, this
kinetic factor of 3 may fully explain the apparent 3-fold difference
between the cirazoline and the ionomycin curves in Fig. 5C,
and an increase in [Ca2+]i is equally effective
in inhibiting cAMP accumulation whether it originates via a
receptor-mediated pathway or is induced by a Ca2+ ionophore.
[Ca2+]i prior to ionomycin addition
was known. The effective level of [Ca2+]i was
estimated, as indicated above, from the area under the curve, to be
about one-third of the peak level, i.e. about 300 nM. From Fig. 1B and calculations therefrom, it
was clear that at this NE concentration, there was already a 74%
inhibition of the cAMP response. With these corresponding
[Ca2+]i and cAMP values as starting points, a
full dose-response curve for the effect of
[Ca2+]i on cAMP levels could be constructed
(Fig. 6C). As seen, the values originating from CGP-12177
and NE data could be superimposed. Thus, not only qualitatively but
also quantitatively, the inhibitory 1-component could be
described as a simple monophasic effect of
[Ca2+]i.
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Fig. 6.
Effect of increases in cytosolic
Ca2+ levels on cAMP accumulation induced by NE.
A, brown adipocytes were incubated with 1 µM
NE in the presence of the indicated concentrations of cirazoline.
Points are means from two experiments: 100% corresponded to 37 ± 14 pmol of cAMP/well. The EC50 for cirazoline was 33 nM and the maximal inhibition was 22%. B, as in
A, except that ionomycin was added. Mean ± S.E. from
three experiments: 100% corresponded to 28 ± 6 pmol of
cAMP/well. The EC50 for ionomycin was 0.4 µM
and the maximal inhibition was 80%. C, inhibition of
agonist-induced cAMP accumulation as a function of cytosolic
Ca2+ levels. The curves are constructed by combining the
[Ca2+]i levels compiled in Fig. 4B
with the corresponding cAMP levels compiled in Figs. 5B and
6B. However, to the [Ca2+]i data
from Fig. 4D, the estimated [Ca2+]i
of 300 nM for NE has been added (see text). In parallel,
the cAMP values from B are recalculated based on the maximal
values revealed by prazosin (Fig. 1B) (see text). refer
to the CGP-12177 data and 
to the NE data. The curve is drawn for
best simple Michaelis-Menten fit of the data and has an
EC50 of 86 nM
[Ca2+]i.
80% according to Fig.
5B).
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Fig. 7.
Effect of increase in
[Ca2+]i on the kinetics of cAMP accumulation in
brown adipocytes. Brown adipocytes were stimulated 1 µM forskolin in the presence (A) of 100 µM IBMX or in its absence (B), in the further
presence ( 
) of 1 µM ionomycin or in its absence ().
Results are mean ± S.E. of three experiments. The data points
were fitted to a first-order kinetic equation.
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Fig. 8.
Effect of phosphodiesterase inhibitors on
cAMP accumulation. A, effect of different PDE
inhibitors on NE-induced cAMP accumulation. The effect of 0.1 µM NE alone (n = 3) in each experiment
was set to 100%, corresponding to 25 ± 7 pmol of cAMP/well, and
in different experiments, the following inhibitors were added 5-10 min
before the indicated concentrations of NE: 100 µM
8-MM-IBMX ( ); 0.5 µM OPC-3911 (
); 15 µM Ro 20-1724 (
). B, effect of the
calmodulin antagonist calmidazolium on the dose-response curve for
NE-induced cAMP accumulation. The effect of 0.1 µM NE
alone in each experiment was set to 100% corresponding to 18 ± 2 pmol of cAMP/well. Results are mean ± S.E. from two
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-adrenoreceptor (11) gene expression in cultured brown adipocytes from mice. These kinetics of gene expression are reflections of the norepinephrine-induced cAMP levels, which thus also display these characteristic kinetics (6). Thus, to understand the regulation
of UCP1 and 1-adrenoreceptor gene expression, the mechanism behind the distinctive non-Michaelis-Menten kinetics of cAMP
accumulation should be understood.
-component was inherently much larger than normally
observed. The inhibitory component was 1-adrenergic in
nature, with a relatively high EC50, explaining the
bell-shaped kinetics of the resultant. The inhibition was
intracellularily mediated via an increase in
[Ca2+]i; the protein kinase C pathway was not
involved. The Ca2+ mediation was very sensitive, in that a
[Ca2+]i increase of less than 100 nM was sufficient to mediate the effect. The increased
[Ca2+]i stimulated a calmodulin-sensitive
phosphodiesterase, possibly of the PDE-1 type, the activity of which
was thus of sufficient magnitude to more than halve the outcome of full
adenylyl cyclase activation. The acquirement of this process is
apparently a part of the differentiation program in the brown adipocytes.
1-adrenoreceptor gene expression. Furthermore, as cAMP is a second messenger with a crucial
role in the control of both acute thermogenesis (33), cell
proliferation (8), apoptosis (34), and cell differentiation (3, 6) in
the brown adipocytes, knowledge of the exact mechanism of cAMP control
may be of significance for understanding the control of other cellular
processes and may help in devising pharmacological agents to control
brown adipocyte recruitment and activity.
-adrenergic response, leading to stimulation of adenylyl cyclase and
a monophasic increase in cAMP level. As a consequence of the analysis,
it was realized that the inherent capacity of this system was much
higher than that revealed by norepinephrine stimulation. In traditional
investigations of adenylyl cyclase activity in plasma membrane
preparations from brown adipose tissue, only monophasic responses to
norepinephrine have been reported (35). Although this is thus valid for
the isolated adenylyl cyclase system, it may, as understood from the
present investigation, not be representative for the actual effect of norepinephrine on the integrated cAMP system, as the final and determining cAMP value is substantially influenced by the inhibitory component.
-adrenergic stimulation may be reduced due to the effect of
other hormones, which may act via activation of phosphodiesterase, as
is the case for insulin (38). Conversely, when other hormones stimulate
cAMP accumulation, norepinephrine may interact negatively, via
1-receptors and activation of a
Ca2+/calmodulin-sensitive phosphodiesterase (39).
1 effect was small and the
resultant curve was still a monophasic Michaelis-Menten relationship.
1-pathway has a high capacity but also a relatively high
EC50, compared with the stimulatory pathway, and through this, it shapes the characteristic dose-response curve and
substantially influences the maximal cAMP levels reached.
[Ca2+]i--
The
inhibitory component was mediated via an increase in
[Ca2+]i. The very low EC50 for
[Ca2+]i for this effect, less than 100 nM, is remarkable. It is much lower than maximum
norepinephrine-induced increases in [Ca2+]i which
may easily reach peak levels at least 10-fold higher, and there thus
seems to be a high redundancy in the norepinephrine signal. However,
considering the kinetics of the norepinephrine-induced [Ca2+]i response, with its high negative time
dependence, the high sensitivity may be understood as allowing for a
sustained response: the phosphodiesterase system will remain activated
until Ca2+ levels approaching basal have been reached. This
high sensitivity of the response also means that agents with effects on
[Ca2+]i that seem trivially small may still be of
significance for the outcome. The effective increase, a
[Ca2+]i of <100 nM, seems low
also when related to the basal level of [Ca2+]i
which has been estimated in this and most earlier investigations to be
in the order of 200 nM. A technical complication may
perhaps explain this. In the present investigation, as in many others,
the Rmin used for the calibration of the
fluorescence signal necessary for determination of
[Ca2+]i has been established with
Mn2+, which functions to eradicate the fluorescence. This
may lead to some overestimation of the basal level, and the relative
increase in [Ca2+]i may therefore be higher than
anticipated from the values given here.
[Ca2+]i
may also explain the somewhat unexpected results concerning
isoprenaline. Isoprenaline is routinely used in receptor and receptor
response studies as a selective -agonist, and it is generally
anticipated that the response does not include effects of
-receptors. The data presented here (Figs. 2 and 4) clearly
demonstrate, however, that at commonly used isoprenaline
concentrations, -pathways are stimulated in brown adipocytes. In
this respect, isoprenaline-induced responses are therefore principally
different from those of selective 3-agonists, for which
we see no indication of an 1 effect. It is possible that
this 1-component may explain several recent observations
on brown fat cells where the response to isoprenaline deviated from
that to selective 3-stimulation or direct adenylyl cyclase activation (41-43). That isoprenaline seems to influence the
1-pathway to a larger extent in brown fat cells than in
many other tissues may be due to the very high level of expression of
1-receptors in brown adipose tissue, compared with the
levels in other tissues (44). If this is parallelled by a similar high level of 1-receptors, the functional affinity of brown
fat cells for 1-stimulation would be high, and agents
with only a small 1 activity, such as isoprenaline,
would manifest their 1 action particularly in brown adipocytes.
1-receptors that mediate the inhibitory component (both gene expression levels (44, 45) and receptor number
(46, 48)), whereas expression of the stimulatory
3-receptors is down-regulated (47, 48), as is the
activity of some of the mediating steps (49).
3-adrenergic receptor has as one of its features, in
comparison to 1/2-receptors, the absence
in its primary structure of the serine residues that are involved in
classical desensitization (50). Although this inability to classically
desensitize was originally considered the feature of choice of
3-receptors, it would seem that concerning this
receptor, nature utilizes two alternative pathways to achieve a
functional desensitization: both an intensive down-regulation of
3-receptor gene expression (47, 48), and the activation
of an 1-mediated inhibitory pathway, as demonstrated here.
1/3-Interaction in Brown Adipose
Tissue Function--
The function of brown adipose tissue is highly
regulated via -adrenergic processes, mediated through an increase in
cytosolic cAMP levels. This is so concerning both the acute thermogenic response and stimulation of cell proliferation, apoptosis, and cell
differentiation. Thus, based solely on the data reported here,
1-stimulation should be associated with a suppressing
effect on the function of brown adipose tissue.
1- and -stimulation. Thus,
1-stimulation enhances the ability of a given level of
cAMP to induce thermogenesis in the cells (33), and
1-stimulation may, at least in brown fat cells from
certain species, have some thermogenic effects in itself (51).
Furthermore, and perhaps most importantly, 1-stimulation has additive or synergistic effects on the gene expression of a series
of very significant genes for brown adipose tissue function, such as
c-fos (10), thyroxine 5'-deodinase (52), lipoprotein lipase
(53), and the uncoupling protein (UCP1) itself (3, 4).2
- and
1-adrenergic signals, some positive, some apparently
negative. We have clearly still not reached a sufficient depth of
knowledge to allow us to understand the intricacies of the complex
network found in this cell system, unique in its dependence upon
adrenergic stimulation for cellular growth and function.
ACKNOWLEDGEMENT
FOOTNOTES
On leave from the Institute of Cell Biophysics, Russian Academy of
Sciences, Pushchino, Russia. Present address: Centre for Molecular
Biology and Medicine, Epworth Hospital, 89 Bridge Rd., Richmond
(Melbourne), 3121 Australia.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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