1 The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden; and 2 Klinik für Neurologie, Universität Magdeburg, D-39120 Magdeburg, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The
nature of the sustained norepinephrine-induced depolarization in brown
fat cells was examined by patch-clamp techniques. Norepinephrine (NE)
stimulation led to a whole cell current response consisting of two
phases: a first inward current, lasting for only 1 min, and a sustained
inward current, lasting as long as the adrenergic stimulation was
maintained. The nature of the sustained current was here investigated.
It could be induced by the 1-agonist cirazoline but not
by the
3-agonist CGP-12177A. Reduction of extracellular
Cl
concentration had no effect, but omission of
extracellular Ca2+ or Na+ totally eliminated
it. When unstimulated cells were studied in the cell-attached mode,
some activity of
30 pS nonselective cation channels was observed. NE
perfusion led to a 10-fold increase in their open probability (from
0.002 to
0.017), which persisted as long as the perfusion was
maintained. The activation was much stronger with the
1-agonist phenylephrine than with the
3-agonist CGP-12177A, and with the Ca2+
ionophore A-23187 than with the adenylyl cyclase activator forskolin. We conclude that the sustained inward current was due to activation of
30 pS nonselective cation channels via
1-adrenergic
receptors and that the effect may be mediated via an increase in
intracellular free Ca2+ concentration.
cirazoline; calcium; patch-clamp technique
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NOREPINEPHRINE HAS WIDE-RANGING EFFECTS on brown fat cells, spanning from acute stimulation of thermogenesis to regulation of cellular proliferation, growth, and differentiation (for review, see Ref. 19). Among the least understood effects of norepinephrine on brown fat cells are those on the cell membrane potential. That norepinephrine has such effects is well documented (5-7, 9, 10, 15, 30). However, no definite physiological role (in, for example, thermogenesis) has as yet been ascribed to these membrane potential alterations.
There is general agreement that three phases can be discerned in the
brown fat cell membrane response to adrenergic stimulation (as earlier
summarized by Refs. 2 and 8). 1) The first phase, a rapid
depolarization, is mediated via 1-adrenergic
receptors and results from a transient Cl
efflux
mediated by an increase in intracellular free Ca2+
concentration ([Ca2+]i) (4, 6, 15,
21). 2) The second phase, a short-lived hyperpolarization, is also evoked (directly or indirectly) via an
1-adrenergic pathway and results from the activation of
apamin-sensitive Ca2+-activated K+ channels
(13, 15, 17, 18) and probably also of voltage-sensitive K+ channels (14, 23). 3) The third
phase, the sustained depolarization, has until now generally been
suggested to be mediated via a
-adrenergic pathway (9,
15); the channels involved have until now not been identified.
The sustained depolarization was observed already in the first
recording of cell membrane potentials in brown fat cells, performed
with sharp penetrating microelectrodes (7), and it has
been observed in several subsequent studies with such techniques
(5, 6, 9, 10, 30) but not with patch-clamp techniques.
Because the nature of this sustained, and therefore potentially most
important, phase of the membrane depolarization events has remained
unresolved, we have attempted here to characterize it with patch-clamp
techniques, both at the cellular and at the single-channel level.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Isolation and Maintenance
The brown fat cells used for this study were prepared principally as earlier described (11, 12, 23, 26, 28). Young male (80-200 g) Sprague-Dawley rats (reared at 21°C with free access to food and water) were killed by CO2 followed by decapitation. The interscapular fat depots were excised, and the pooled and minced brown fat depots were dispersed into single cells by collagenase digestion (Sigma, type II, 5 mg/ml) for 35 min in extracellular solution (see below) in a shaking water bath at 37°C. The floating cells (i.e., the mature brown fat cells) were separated from the collagenase suspension by centrifugation (150 g, 10 min) and kept in 6-well dishes in an incubator at 37°C, in an 8% CO2-92% air atmosphere in DMEM, supplemented with newborn-calf serum (10% vol/vol), insulin (4 nM), Na-ascorbate (25 µg/ml), glutamine (4 mM), penicillin (50 IU/ml), and streptomycin (50 µg/ml). After addition of the cells to a well, a Heraeus-Biofoil-25 membrane was placed on the surface of the medium. The floating brown fat cells spontaneously adhered to the hydrophilic side of this membrane. The medium was exchanged after 24 h, and the cells on the membrane were then examined, as we will describe. Routinely, cells were examined during the first 1-3 days, but occasionally they were kept in the well for as many as 7 days; we did not observe any difference in response during this time, nor did the cells change their multilocular appearance.Patch-Clamp Techniques
For both the perforated-patch studies and the cell-attached-mode studies, the biofoil with the attached brown fat cells was placed in a chamber with a volume ofPipettes were pulled from borosilicate glass and had resistances
between 6 and 3 M. The pipette solution consisted of (in mM): 60 KCl, 80 K gluconate, 10 NaCl, 1 CaCl2, 1 EGTA, and 10 MOPS
(pH 7.2 adjusted with KOH, corresponding to
5 mM K+).
The choice of the rather high level of Cl
was based on
implications from 36Cl
efflux experiments
indicating that the cytosolic Cl
level is higher than the
equilibrium level for the resting membrane potential (4)
(which would be ~50 mM) and from experiments (21)
showing that the Cl
currents induced by norepinephrine
stimulation had a reversal potential of
20 mV, corresponding to an
estimated cytosolic concentration of
70 mM. The free
Ca2+ concentration in the pipette was calculated to be 10 µM, according to the computer program BAD3; the presence of 80 mM
gluconate, which may have Ca2+-chelating properties, may
have lowered this level further. To the pipette solution,
0.24-0.32 µg of amphotericin B (dissolved in DMSO) was added per
milliliter. In a few cases, pluronic F-127 (0.08%) was also added to
the pipette solution (to facilitate perforation), but no clear effects
were seen. The reference electrode was an AgCl-pellet electrode
connected to the chamber through a 150 mM NaCl agar bridge (50 k
).
A few minutes after a giga seal had formed, the membrane potential was
read off in the current-clamped mode. The voltage read off was about
20 mV, i.e., identical to the zero-current voltage determined in the
voltage ramps (see below). The pipette was then voltage-clamped to
50
mV, and the capacitance (5-7 pF) of the pipette was electronically
compensated. Access resistance was monitored from frequent 40-ms 5-mV
hyperpolarizing steps. The recording was started after 15-35 min,
when the access resistance had stabilized and had usually dropped below
50 M
. Access resistance was not compensated in the results shown.
The junction potential of the pipette solution vs. the standard
extracellular solution was experimentally determined to be +6 mV; i.e.,
the potential is 6 mV more negative than that indicated on the axes.
The junction potentials in the buffers with altered Cl
concentration were not markedly different. Routinely, the cell capacitance in the resting state (
30 pF) was compensated by the circuitry in the amplifier, and no further capacitance compensation was
performed during the experiment. Each cell responded qualitatively in
the same way to repeated agonist stimulation for up to 2 h, but in
general there was a tendency to a decrease in the magnitude of the
responses on successive stimulations.
For the cell-attached-mode studies, the pipettes had resistances
between 8 and 12 M, and the pipette medium was here identical to the
extracellular solution. Each experiment was finalized by excising the
patch and studying it as an inside-out patch; these excised patches
were thus formed and studied in the extracellular solution.
Recordings and Data Analysis
The currents were recorded with an L/M EPC 7 patch-clamp amplifier. During the whole cell experiments, voltage ramps were frequently run under manual initiation. In control experiments (not shown), we found that the current at each voltage tested was stable with time, except for those at very high depolarizations (more positive than 0 mV), which became inactivated, in accordance with our earlier observations (23). Therefore, we surmise that the voltage-dependent conductance changes observed during voltage ramps represented time-independent values. In each voltage ramp, the holding potential ofIn the recordings in the cell-attached mode, the number of
simultaneously open channels was low (1-3), making it
possible routinely with the pCLAMP program to calculate the open and
closed times and the observed open probability
(Po,obs) directly from lists of events,
according to a 50% amplitude threshold crossing criterion. To obtain
the Po,obs values, data were collected over 60
s in the control state (to yield Po,obs,basal)
and 240 s during stimulation (to yield
Po,obs,stim). Each observation was finalized with the excision of the patch, which led to the spontaneous activation of several channels. The maximal number of channels observed in the
excised inside-out patch was denoted Ntot. The
Po,tot values were obtained by dividing the
Po,obs values by the
Ntot/Nobs value. The
current amplitudes of the single channels were calculated from
amplitude histograms fitted to Gaussian distributions by the pCLAMP program.
In the studies in the cell-attached mode, potentials applied to the pipette are stated as holding potentials, relative to the rest of the outside of the cells (because the true cell membrane potential was not simultaneously measured). In the displayed recordings, downward deflections denote currents corresponding to cellular inward currents.
Chemicals
EGTA, K-gluconate, L-norepinephrine bitartrate, Na-aspartate, and L-phenylephrine HCl were directly dissolved in the extracellular solution, and amphotericin B, forskolin, and A-23187 were stock-dissolved in DMSO (0.1% in final solution); all were from Sigma. CGP-12177A was a gift from Ciba-Geigy, cirazoline was from Research Biochemicals International, and pluronic F-127 (dissolved in DMSO) was from Molecular Probes. The adrenergic agonists were protected from light in the perfusion system. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present experiments, the response of brown fat cells to sustained adrenergic stimulation was analyzed first as whole cell currents, with the perforated-patch technique. To directly identify the channels responsible for the currents observed, the cell-attached patch-clamp technique was subsequently used.
Whole Cell Currents
In the perforated-patch whole cell studies, the cell membrane potential was clamped atThe pipette solution was composed so as to be close to expected
cytosolic ion levels. Because amphotericin makes the membrane permeable
to Na+, K+, and Cl ions
(22), the monovalent ions from the pipette probably
diffused sufficiently to in reality determine the intracellular levels. [Because amphotericin does not make the patch permeable to
Ca2+ (22), the pipette Ca2+
concentration was not expected to influence cytosolic Ca2+
levels.] The expected equilibrium potentials were therefore
82 mV
for K+, +68 mV for Na+, and
18 mV for
Cl
.
Whole cell currents were determined when a stable access membrane
resistance had been attained. The resting cell capacitance was 35 ± 1 pF (n = 26). During the experiments, we frequently followed the current response to voltage ramps (as is seen, for example, in Fig. 1). These ramps allowed
for determination of membrane potential (i.e., the zero current
potential) and were also used to measure total cellular conductance at
50 mV (referred to as Gm,
50) (i.e., the
conductance at the clamped potential) and at +15 mV
(Gm,+15).
|
The unstimulated state.
In unstimulated cells, a stable resting current (Fig. 1A)
was observed. On the basis of voltage ramps performed during this period (as exemplified in Fig. 1B, current 1),
the mean resting membrane potential was estimated to be 21 mV (Table
1); this was also the voltage observed in
the current clamp mode immediately before the setup was switched to the
voltage clamp mode. The membrane conductance at the holding potential
of
50 mV (Gm,
50) was 0.7 nS (Table 1). As is
also seen from the current ramp in Fig. 1B, the membrane
conductance at positive membrane potentials was much higher; at +15 mV
(i.e., Gm,+15), it was 14 nS.
|
Effects of norepinephrine. After initiation of perfusion with 1 µM norepinephrine, the cells responded with characteristic successive alterations in transmembrane current, despite the constant presence of norepinephrine. Two major response types could be distinguished. In ~75% of the cells, a relatively simple type of response, such as that illustrated in Fig. 1, was observed. In the other cells, an oscillatory response was observed (not shown). The present analysis concerns only the response pattern of the majority of cells.
CURRENTS. In the current response elicited by norepinephrine, there were two phases (Fig. 1). Both phases consisted of inward currents, i.e., both currents would probably be depolarizing in the unclamped cell. The "first inward current" (referred to throughout in this way) was transient, with a rapid initiation and a rather slow inactivation. It was large, with a mean maximal current value of 367 pA (Table 1). The mean total length of this current phase, i.e., the time from the current deviation from resting level to its attainment of new plateau corresponding to the next phase, was 58 ± 5 s (n = 13). This current was followed by a "sustained inward current" (referred to throughout in this way), which persisted unabated and nonoscillatory for as long as the norepinephrine stimulation lasted. This sustained inward current amounted to 44 pA. When norepinephrine was washed out, the cell membrane current returned to the initial resting levels. Because the sustained phase (which is the one under investigation here) of necessity always was preceded by the first inward current, our results concerning the first inward current are also briefly reported here, but a full analysis is not attempted. MEMBRANE POTENTIALS. On the basis of the voltage ramps, the alterations in membrane potential (Em) under the present conditions and in conductances were followed during the entire response. Membrane potential changes are depicted in Fig. 2A and summarized in Table 1. As seen, after norepinephrine had reached the cell, a depolarization occurred during the first inward current, from
|
Effects of the adrenergic subtype-selective agonists cirazoline and
CGP-12177A.
To determine which subtype of adrenergic receptor was responsible for
the different phases in the norepinephrine-induced current response, we
studied the effects of the 1-selective agonist
cirazoline and the
3-specific agonist CGP-12177A, known
selective activators of the indicated adrenergic subtypes in brown fat
cells (31). We compared the responses to each of these
selective agonists with the responses to the endogenous agonist
norepinephrine described above; norepinephrine is expected in itself to
be able to activate all types of adrenergic receptors.
|
Effects of extracellular Ca2+ removal during
1-stimulation.
1-Adrenergic stimulation is associated with an increase
in [Ca2+]i in brown fat cells (1, 13,
27, 29), and this increase is believed to be one of the
intracellular mediators of
1-adrenergic stimulation. To
investigate to what extent the observed
1-effects were
dependent on Ca2+, we examined the responses to adrenergic
stimulation in a buffer that did not contain Ca2+ but
instead contained 1 mM of the Ca2+ chelator EGTA. Such
conditions lead to a diminished norepinephrine-induced increase in
[Ca2+]i (see Ref. 29).
|
|
Identification of ions involved.
To establish which ion(s) carry the inward current, especially the
sustained inward current, experiments were performed with omission of
extracellular Cl or Na+.
|
|
A Large-Conductance NSC Channel
During norepinephrine perfusion, we often (in 14 of the 21 cells tested) observed currents apparently resulting from single channel activity. Such currents are quite prominent in Fig. 3B and are discernible in Fig. 1A. In Fig. 5A, we show an enlargement of such current events. The events clearly represented inward currents from a single but large ion channel. In most cases, only one active channel per cell was detected, maximally two. A preliminary characterization of these channels could be made from their response to the conditions tested above.
|
The large-conductance channel currents were never observed in
unstimulated cells but were frequently observed during norepinephrine and cirazoline stimulation. CGP-12177A (tested in 4 cells) was not able
to activate the channel, even in a cell in which both norepinephrine and cirazoline could activate it. The activity disappeared upon washout of the adrenergic agonists. It was
occasionally possible to observe activity during a voltage ramp (Fig.
5B); when it opened during the sustained inward current
phase, it temporarily doubled the current. Extrapolation to zero
current voltage indicates that the cell depolarized ~10 mV during
each such opening. It is also clear from this and similar recordings
(not shown) that the activity was not observable at potentials more
positive than 10 mV.
In some experiments, we recorded the conductance and the activity of the large-conductance channel at different membrane potentials. An example of this is given in Fig. 5A, and data from one such study are collected in Fig. 5, C and D. In Fig. 5C, the current-voltage relationship is shown. The activity corresponded to a single-channel conductance of 270 pS, which was fully ohmic within the voltages tested and which had a reversal potential very close to 0 mV. This would indicate that this large-conductance channel was an NSC channel. The open probability (Po) showed an unusual dependence on the membrane potential, being, if anything, activated by hyperpolarization (Fig. 5D).
Omission of extracellular Ca2+ did not affect the
conductance of the channel (the current was 13 pA at 50 mV both
before and during Ca2+ removal) but impressively increased
the open probability sevenfold, from 0.05 to 0.33 (not shown but cf.
Fig. 3B). Reduction of extracellular Cl
to 30 mM did not
affect the single-channel amplitude or the activity of the channel (not
shown). However, removal of extracellular Na+ reversibly
abolished the large-conductance channel activity (not shown),
demonstrating that it was a Na+-conducting channel.
Thus the large-conductance channel was identified as an NSC channel with unusual regulatory properties. The physiological role of this type of channel in brown fat cells remains unknown, but a similar type of channel has been observed in rat basophilic leukemia cells; that channel was suggested to regulate exocytosis (20).
The properties of the large-conductance channel, especially the unusual Ca2+ dependence characteristics, clearly indicated that it was not this channel that carried the current during the sustained inward current phase. We therefore proceeded to the cell-attached mode to identify the ion channels carrying the depolarizing current.
Adrenergic Activation of the 27 pS NSC Channel
To identify the ion channels responsible for the sustained depolarization of brown fat cells during adrenergic stimulation, patch-clamp experiments in the cell-attached mode were performed. In the whole cell studies above, the current to be sought was established to be elicited byEffects of norepinephrine on single channel activity.
NOREPINEPHRINE ACTIVATES SINGLE CHANNEL ACTIVITY.
Figure 6A shows a trace of the
current through the cell-attached pipette after a gigaohm seal had been
formed. The recording was started while the cell was perfused with only
extracellular solution; as seen, some spontaneous ion channel activity
was observed during this time. Such channel activity in unstimulated
brown fat cells has earlier been observed and demonstrated to be
mediated via 27 pS NSC channels (28) (and see below).
During the time indicated by the horizontal line, the cell was perfused
with the same solution, now containing 1 µM norepinephrine; as seen,
this resulted in a dramatic increase in channel activity in the patch.
It was considered likely that this prominent activity could be
responsible for the sustained inward current; further analysis detailed
below supported this view. Because the perfused norepinephrine did not
come in direct contact with the channels investigated, it was clear
that the activation must have been mediated through an intracellular mediator.
|
|
|
|
Effects of selective 1- and
3-adrenergic agonists on ion channel activity.
On the basis of the data shown (above) from whole cell current studies,
the channel responsible for the sustained inward current should be
1-adrenergically activated. To examine whether this was
the case for the single channel activity observed here, we examined the
ability of two subtype-selective adrenergic agonists, the
3-agonist CGP-12177A and the
1-agonist
phenylephrine, to elicit a channel activation similar to that elicited
by norepinephrine.
Identity of the second messenger regulating the ion channel
activity.
On the basis of the fact that the sustained inward current observed in
the whole cell studies was 1-adrenergic and
Ca2+ dependent, it would be expected that the single
channel activity responsible for the mediation of this current would be
activated, directly or indirectly, through an increase in
[Ca2+]i. To investigate whether this was the
case, we examined whether an increase in cAMP, or, as would be
expected, an increase in [Ca2+]i, would be
able to increase the single channel activity. We used the adenylyl
cyclase activator forskolin to increase intracellular cAMP levels and
the ionophore A-23187 to increase [Ca2+]i.
Identity of the channel mediating the sustained inward current.
It is clear from the patch-clamp studies in the cell-attached mode that
the single channel activity observed fulfilled the criteria for being
the channel activity behind the sustained inward current observed in
the whole cell studies: time course of activation, nonselective nature
of current, adrenergic receptor involvement, and Ca2+
dependence of activation. With respect to Ca2+ dependence,
and especially on consideration of the observed channel size of 30
pS, it is very notable that, in excised patches from these cells, an
NSC channel with exactly these properties has been well characterized
(11, 12, 23, 26, 28). It is therefore very likely that it
is this NSC channel, the 27 pS channel, that mediates the sustained
inward current.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We show here that norepinephrine induced two inward current phases
in brown fat cells: a transient current, which lasted for ~1 min, and
a sustained current that persisted as long as norepinephrine stimulation persisted. The experiments conducted had as their main goal
to further the understanding of the nature of the sustained current,
which we could characterize fairly well, and which we conclude is an
1-adrenergically induced Ca2+-dependent
activation of 27 pS NSC channels.
Concerning the first inward current phase, it was, of course,
unavoidable to obtain experimental results during the experiments conducted here; they were also briefly reported above. The elucidation of these events was not the prime goal of the present investigations, and a full understanding of the events has not been reached here. However, on the basis of these observations and literature data, a
tentative interpretation of the plasma membrane events after norepinephrine stimulation of brown fat cells is given below, in which
we discuss in succession the resting state, the first inward current,
and the sustained inward current (Fig.
9).
|
The Resting State
The mean capacitance of the brown fat cells investigated here was 35 pF (Fig. 8A). Based on the general value of 1 µF/cm2 cell membrane, this capacitance corresponds to the surface of a sphere with a diameter of 33 µm.The Gm,50 value of 0.7 nS was much smaller
than whole cell conductances observed in classical microelectrode
experiments in brown adipose tissue [~700 nS (7)] and
even somewhat smaller than those earlier observed with patch-clamp
techniques, in classical whole cell studies (14), or in
perforated-patch studies (2-8 nS) (15).
On the basis of the zero current during voltage ramps, the resting
membrane potential was estimated to be about 21 mV. Because the cells
studied probably had a clamped intracellular ion level determined by
the pipette concentration, it may be questioned whether this potential
represents that present under innate conditions. However, the estimates
in the cell-attached mode, in which intracellular ion concentrations
were not affected, confirmed this rather low value under these conditions.
The identity of the ion permeabilities that determine the resting
membrane potential is not immediately evident. The measured potential
is far from the expected K+ equilibrium potential of 83
mV. This deviation could be due to fairly high permeabilities for
Cl
or Na+. Alterations in extracellular
Cl
concentration did not markedly influence the resting
membrane potential, but the membrane potential became more negative
when extracellular Na+ was exchanged with
NMDG+. However, in the presence of mefenamic acid, which
fully inhibits the 27 pS NSC channel (11), the observed
resting Em did not come closer to
85 mV. This
also implies that the low basal activity of the NSC channels (cf. Table
5) is not sufficient to significantly influence the resting membrane
potential. Thus the identities of the ion permeabilities responsible
for the low resting membrane potential are not known, but a
Na+ permeability not mediated by the 27 pS NSC channels is
likely involved.
The First Inward Current
Adrenergic receptor type involved.
The selective 1-adrenergic agonist cirazoline was able
to induce the first inward current in a manner qualitatively similar to
norepinephrine, but the response was only two-thirds of that observed
with norepinephrine (Fig. 8, B and C). The
difference may be due to effects mediated via
3-receptors, because CGP-12177A generally elicited some
membrane currents during this phase. The
1-induced
inward current became much smaller in Ca2+-free buffer,
implying that the
1-signal is intracellularly mediated via an increase in Ca2+ levels.
Ionic characteristics of the first inward current.
The first inward current phase probably involves currents of more than
one ion species, mediated through more than one channel. The fact that
the currents during voltage ramps were changed as an effect of
reduction of extracellular Cl levels is in agreement with
results from earlier ion flux (4) and patch-clamp
(21) studies concluding that Cl
currents
probably were induced in this phase.
Ion channels involved.
The identities of the ion channels involved in the first inward current
have not been established. A 50 pS Cl channel has been
observed in brown fat cells (24), but there is no
functional evidence to associate it with the norepinephrine-induced Cl
current. At the single channel level, a
Ca2+-activated K+ channel has not been
identified in the tissue.
The Sustained Inward Current
Norepinephrine could consistently activate a sustained inward current (membrane depolarization) in the rat brown fat cells (Fig. 9D). The existence of a norepinephrine-induced sustained depolarization is well established from classical microelectrode studies in brown adipose tissue (5-7, 9, 10, 30). The events during this phase have now become clear.Adrenergic receptor involved.
The sustained inward current and the depolarization could be induced by
the 1-agonist cirazoline to the same extent as by norepinephrine;
3-stimulation was without effect. As
mentioned in the introductory comments, the third-phase sustained
depolarization in brown fat cells has generally been thought to be
-adrenergic in nature (reviewed in Refs. 2 and 8), and the present
data are therefore in contrast to that tenet. However, a reexamination of the literature prompted by the present results revealed that the
-adrenergic nature of the sustained-phase depolarization is not well
established experimentally. In several earlier investigations, a
poststimulation recovery response, after a short pulse of adrenergic or
nervous stimulation, is in reality what has been analyzed (6, 9), i.e., a situation that is principally different from the persistent stimulation studied here. In contrast, several
investigations with persistent adrenergic stimulation implied both an
- and a
-induced depolarization of about equal magnitude
(5, 10). The published
-results are thus in good
agreement with the outcome of our studies. The question is therefore
why a marked
-induced depolarization has often been observed
previously, whereas in our studies, the
-adrenergic pathway seems to
be of no importance.
Ionic character of the sustained current.
Partial substitution of aspartate for Cl was without
effect on the sustained inward current, but the current was completely abolished when extracellular Na+ was replaced with
NMDG+. Our observations therefore also agree with the
observation that the corresponding depolarization is Na+
dependent (8). The current is thus clearly a
Na+ current, but because it is
1-adrenergic,
it cannot be related to the
-adrenergically induced Na+
influx earlier reported in brown fat cells (3). Because
the
1-induced sustained current had a zero current
potential close to 0 mV, it is unlikely that it represented a current
through selective Na+ channels. This zero current potential
is rather what is expected for a current mediated through an NSC channel.
The sustained depolarization may be ascribed to the activation of
the 27 pS NSC channels.
Based on the studies in the cell-attached mode, we concluded that the
NSC channels with a conductance of 27 pS, earlier observed in brown
fat cells (11, 12, 23, 26, 28), had all the properties
required to ascribe to them the role of being the channels responsible
for the adrenergically induced sustained inward current in
voltage-clamped brown fat cells and the sustained membrane depolarization in nonclamped cells. Their activity was increased as an
effect of norepinephrine stimulation; there was a 1-min delay before
full activation was reached, but the adrenergically induced NSC channel
activity then persisted as long as the adrenergic stimulation lasted.
The activated channels had an
30 pS slope, and the reversal
potential in the cell-attached configuration was identical to the known
membrane potential of the brown fat cells under these conditions.
Furthermore, the activation was mainly
1-adrenergic in
nature and occurred through a second messenger, probably
[Ca2+]i. In all these respects, the NSC
channel exhibited exactly the properties required if these channels
should be the mediators of the sustained current.
Is the NSC channel activity sufficient?
In the whole cell studies, the total cell conductance (at the clamped
voltage of 50 mV) was increased by 0.7 nS during the sustained inward
current phase (Table 1). If the NSC channels observed here are to be
the sole channels responsible for this increase in conductance, the sum
of their conductances must be of this magnitude. From the values
obtained here, it is possible to estimate this combined conductance.
With a single channel conductance of 30 pS (Table 6) and the increase
in Po of 0.03 in the norepinephrine-stimulated state (Table 5), each norepinephrine-activated channel corresponds to a
virtual conductance of 0.9 pS. To estimate the corresponding whole cell
conductance, a number of 1.2 observed channels per patch, an estimated
patch area of 1 µm2 (25), and the estimate
of the cell being a sphere with a diameter of 33 µm were used. Thus,
on the basis of the single channel activities, the estimated increase
in total cell conductance during the sustained phase would be 3.7 nS
(or
2 nS if it is supposed that channels on the foil-adhering part
of the cell are not functional). This estimated value thus even exceeds
the conductance increase actually observed in the whole cell system
(0.7 nS), and this calculation therefore indicates that the NSC
channels together have the capacity to be the channels mediating the
sustained inward current.
The Physiological Role of the Sustained Depolarization
The immediate effect of the increased membrane conductance underlying the sustained current described here would be a decrease in membrane potential, as was indeed observed. It may be that this depolarization in itself is the major physiological significance of the sustained increase in membrane conductance. Many cellular uptake or release processes are dependent on the magnitude of the membrane potential, and it has, for example, been demonstrated that depolarization of the brown fat cell membrane (with high extracellular K+) diminishes the [Ca2+]i level observed in the cytosol during adrenergic stimulation (13). Thus the sustained increase in membrane conductance may protect the cells from excessively high increases in [Ca2+]i.Because the described increase in conductance is associated with inward
Na+ currents, a direct connection between Na+
influx and thermogenesis could be postulated, as was indeed originally suggested by Girardier et al. (7). However, this is
unlikely, because substitution of NMDG+ for Na+
has only marginal effects on norepinephrine-induced thermogenesis (3). However, the sustained inward current would represent a Na+ influx that, in the new steady state, would have to
be counteracted by the Na+-K+-ATPase. In
agreement with this, a fraction of 1-adrenergically stimulated respiration can be inhibited by the
Na+-K+-ATPase inhibitor ouabain
(16). It should, however, be realized that this fraction
represents only a very small fraction (
5%) of the total oxygen
consumption (thermogenesis) that is induced by norepinephrine; the main
part is through
-adrenergic pathways. Furthermore, as an effect of
the increased Na+ permeability, a new steady-state
cytosolic Na+ level would be established, somewhat higher
than the resting level; there could be direct effects of such
alterations in cytosolic Na+ levels.
The above effects are all of an acute nature. However, there is now
extensive evidence that adrenergic stimulation of the brown fat cell
promotes cell proliferation and cell differentiation (19).
In these processes, 1-adrenergic stimulation may be
important. Indeed, the expression of c-fos, for example,
which is supposedly involved in regulation of cell differentiation, is
under strong synergistic control by
1- and
-adrenergic pathways (27). It is an interesting
possibility that it is in processes like these that the
1-induced depolarization, the ensuing influx of
Na+ ions, and the possible increase in cytosolic
Na+ levels are of importance.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Stig Sundelin for general assistance with the electronic equipment and to Barbara Cannon for valuable discussions.
![]() |
FOOTNOTES |
---|
Financial support from the Swedish Natural Science Research Council, the Hasselblad Foundation, and the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
Address for reprint requests and other correspondence: A. Koivisto, Orion Corporation, Orion Pharma Preclinical Research, PO Box 425, FIN-20101 Turku, Finland (E-mail: Ari-Pekka.Koivisto{at}Orionpharma.com).
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.
Received 27 January 2000; accepted in final form 30 May 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bronnikov, GE,
Zhang SJ,
Cannon B,
and
Nedergaard J.
A dual component analysis explains the distinctive kinetics of cAMP accumulation in brown adipocytes.
J Biol Chem
274:
37770-37780,
1999
2.
Connolly, E,
Dasso L,
and
Nedergaard J.
Adrenergic regulation of ion fluxes in brown adipocytes.
In: Thermoregulation: Research and Clinical Applications, edited by Lomax P,
and Schönbaum E. Basel: Karger, 1989, p. 31-34.
3.
Connolly, E,
Nånberg E,
and
Nedergaard J.
Norepinephrine-induced Na+ influx in brown adipocytes is cyclic AMP-mediated.
J Biol Chem
261:
14377-14385,
1986
4.
Dasso, L,
Connolly E,
and
Nedergaard J.
1-Adrenergic stimulation of Cl
efflux in isolated brown adipocytes.
FEBS Lett
262:
25-28,
1990[ISI][Medline].
5.
Fink, SA,
and
Williams JA.
Adrenergic receptors mediating depolarization in brown adipose tissue.
Am J Physiol
231:
700-706,
1976[ISI][Medline].
6.
Girardier, L,
and
Schneider-Picard G.
- and
-Adrenergic mediation of membrane potential changes and metabolism in rat brown adipose tissue.
J Physiol (Lond)
335:
629-641,
1983[Abstract].
7.
Girardier, L,
Seydoux J,
and
Clausen T.
Membrane potential of brown adipose tissue. A suggested mechanism for the regulation of thermogenesis.
J Gen Physiol
52:
925-940,
1968
8.
Horwitz, B,
Hamilton JS,
Lucero MT,
and
Pappone PA.
Catecholamine-induced changes in activated brown adipocytes.
In: Living in the Cold, edited by Malan A,
and Canguilhem B. London: Libbey, 1989, p. 377-386.
9.
Horwitz, BA,
and
Hamilton J.
Alpha-adrenergic-induced changes in hamster (Mesocricetus) brown adipocyte respiration and membrane potential.
Comp Biochem Physiol
78C:
99-104,
1984[ISI].
10.
Horwitz, BA,
Horowitz JM,
and
Smith RE.
Norepinephrine-induced depolarization of brown fat cells.
Proc Natl Acad Sci USA
64:
113-120,
1969[Abstract].
11.
Koivisto, A,
Klinge A,
Nedergaard J,
and
Siemen D.
Regulation of the activity of 27 pS nonselective cation channels in excised membrane patches from rat brown-fat cells.
Cell Physiol Biochem
8:
231-245,
1998[ISI][Medline].
12.
Koivisto, A,
and
Nedergaard J.
Modulation of calcium-activated non-selective cation channel activity by nitric oxide in rat brown adipose tissue.
J Physiol (Lond)
486:
59-65,
1995[Abstract].
13.
Lee, SC,
Nuccitelli R,
and
Pappone PA.
Adrenergically activated Ca2+ increases in brown fat cells: effects of Ca2+, K+, and K channel block.
Am J Physiol Cell Physiol
264:
C217-C228,
1993
14.
Lucero, MT,
and
Pappone PA.
Voltage-gated potassium channels in brown fat cells.
J Gen Physiol
93:
451-472,
1989[Abstract].
15.
Lucero, MT,
and
Pappone PA.
Membrane responses to norepinephrine in cultured brown fat cells.
J Gen Physiol
95:
523-544,
1990[Abstract].
16.
Mohell, N,
Connolly E,
and
Nedergaard J.
Distinction between mechanisms underlying 1- and
-adrenergic respiratory stimulation in brown fat cells.
Am J Physiol Cell Physiol
253:
C301-C308,
1987
17.
Nånberg, E,
Connolly E,
and
Nedergaard J.
Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance of 1-adrenergic stimulation.
Biochim Biophys Acta
844:
42-49,
1985[ISI][Medline].
18.
Nånberg, E,
Nedergaard J,
and
Cannon B.
-Adrenergic effects on 86Rb+ (K+) potentials and fluxes in brown fat cells.
Biochim Biophys Acta
804:
291-300,
1984[ISI][Medline].
19.
Nedergaard, J,
Herron D,
Jacobsson A,
Rehnmark S,
and
Cannon B.
Norepinephrine as a morphogen?its unique interaction with brown adipose tissue.
Int J Develop Biol
39:
827-837,
1995[ISI].
20.
Obukhov, AG,
Jones SVP,
Degtiar VG,
Lückhoff A,
Schultz G,
and
Hescheler J.
Ca2+-permeable large-conductance nonselective cation channels in rat basophilic leukemia cells.
Am J Physiol Cell Physiol
269:
C1119-C1125,
1995
21.
Pappone, PA,
and
Lee SC.
-Adrenergic stimulation activates a calcium-sensitive chloride current in brown fat cells.
J Gen Physiol
106:
231-258,
1995[Abstract].
22.
Ramos, H,
Valdivieso E,
Gamargo M,
Dagger F,
and
Cohen BE.
Amphotericin B kills unicellular leishmanias by forming aqueous pores permeable to small cations and anions.
J Membr Biol
152:
65-75,
1996[ISI][Medline].
23.
Russ, U,
Ringer T,
and
Siemen D.
A voltage-dependent and a voltage-independent potassium channel in brown adipocytes of the rat.
Biochim Biophys Acta
1153:
249-256,
1993[ISI][Medline].
24.
Sabanov, V,
and
Nedergaard J.
Chloride channels in brown adipocyte plasma membranes: candidates for mediation of 1-adrenergic depolarization?
Biochem Biophys Res Commun
211:
639-647,
1995[ISI][Medline].
25.
Sakmann, B,
and
Neher E.
Geometric parameters of pipettes and membrane patches.
In: Single-Channel Recording, edited by Sakmann B,
and Neher E. New York: Plenum, 1995, p. 637-650.
26.
Siemen, D,
and
Reuhl T.
Non-selective cationic channel in primary cultured cells of brown adipose tissue.
Pflügers Arch
408:
534-536,
1987[ISI][Medline].
27.
Thonberg, H,
Zhang SJ,
Tvrdik P,
Jacobsson A,
and
Nedergaard J.
Norepinephrine utilizes 1- and
-adrenoreceptors synergistically to maximally induce c-fos expression in brown adipocytes.
J Biol Chem
269:
33179-33186,
1994
28.
Weber, A,
and
Siemen D.
Permeability of the non-selective channel in brown adipocytes to small cations.
Pflügers Arch
414:
564-570,
1989[ISI][Medline].
29.
Wilcke, M,
and
Nedergaard J.
1- and
-Adrenergic regulation of intracellular Ca2+ levels in brown adipocytes.
Biochem Biophys Res Commun
163:
292-300,
1989[ISI][Medline].
30.
Williams, J,
and
Matthews E.
Membrane depolarization, cAMP and glycerol release by brown adipose tissue.
Am J Physiol
227:
987-992,
1974[ISI][Medline].
31.
Zhao, J,
Cannon B,
and
Nedergaard J.
1-Adrenergic stimulation potentiates the thermogenic action of
3-adrenoceptor-generated cAMP in brown fat cells.
J Biol Chem
272:
32847-32856,
1997