1 Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm; and 2 Department of Anaesthesiology and Intensive Care Medicine, Karolinska Hospital and Institute, SE-171 76, Stockholm, Sweden
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ABSTRACT |
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The pathway
for adrenergic relaxation of smooth muscle is not fully understood. As
mitochondrial uncoupling protein-1 (UCP1) expression has been reported
in cells within the longitudinal smooth muscle layer of organs
exhibiting peristalsis, we examined whether the absence of UCP1 affects
adrenergic responsiveness. Intestinal (ileal) segments were obtained
from UCP1-ablated mice and from wild-type mice (C57Bl/6, 129/SvPas, and
outbred NMRI). In UCP1-containing mice, isoprenaline totally inhibited
contractions induced by electrical field stimulation, but in intestine
from UCP1-ablated mice, a significant residual contraction remained even at a high isoprenaline concentration; the segments were threefold less sensitive to isoprenaline. Also, when contraction was induced by
carbachol, there was a residual isoprenaline-insensitive contraction. Similar results were obtained with the 3-selective
agonist CL-316,243 and with the adenylyl cyclase stimulator forskolin.
Thus the UCP1 reported to be expressed in the longitudinal muscle layer
of the mouse intestine is apparently functional, and UCP1, presumably through uncoupling, may be involved in a novel pathway leading from
increased cAMP levels to relaxation in organs exhibiting peristalsis.
carbachol; isoprenaline; forskolin
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INTRODUCTION |
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SMOOTH MUSCLE TONE IS
ACHIEVED by a balance between the actions of contraction- and
relaxation-inducing agents. Intracellular signaling pathways for the
corresponding processes have been proposed (for reviews see Refs.
38, 43, 53, 65,
73). The pathway from the muscarinic acetylcholine
receptors to enhanced contraction clearly involves an increase in
cytosolic Ca2+ levels. The corresponding pathway leading
from stimulation of -adrenergic receptors and increases in cAMP to
relaxation of phasic smooth muscle (36, 24, 73) is
not fully understood. It involves a reduction in Ca2+
levels but also a reduction in the effectiveness of Ca2+ in
invoking contraction; the mechanism behind this reduced effectiveness has not been clarified.
A novel possibility for explaining the reduced Ca2+ effectiveness has recently arisen unexpectedly based on observations originating from studies in an apparently unrelated field, that of nonshivering thermogenesis. The phenomenon of classic adaptive nonshivering thermogenesis is presently understood molecularly as being the outcome of the activity of the archetypal mitochondrial uncoupling protein-1 (UCP1; thermogenin) (reviewed in Refs. 16, 35, 51, 60) originally identified in brown adipose tissue (20, 40) and demonstrated to be the only protein able to mediate nonshivering thermogenesis (17). UCP1 functions as a regulated transporter for proton equivalents over the mitochondrial membrane, leading to energy being released as heat instead of being captured in ATP. Until recently, UCP1 has been considered to be expressed only in brown adipose tissue (3, 25), to the extent that its occurrence in a given adipose cell has become the established criterion distinguishing brown and white adipose tissue (4). Conversely, no indication of UCP1 expression in other tissues has existed that could not be understood as being caused by contamination of extracts with brown adipocytes or being explainable based on the existence of the recently identified closely related proteins UCP2 or UCP3 (2). However, in a recent investigation, Nibbelink et al. (52) have reported that UCP1 is expressed very specifically in cells that seem to be included among the longitudinal smooth muscle layer of a series of organs, all characterized by exhibiting peristaltic motion: stomach, eosophagus, small intestine, ureter origin, ductus deferens, and uterus. In the cells that apparently contain UCP1, the level of UCP1 protein was of a magnitude similar to that in brown adipocytes (52). UCP1 was not found in arterial walls; and although arterial walls contain smooth muscle cells, they do not demonstrate peristaltic movements.
Functionally, the UCP1 could be present in these organs for the same
reason that it is in brown adipose tissue, i.e., for thermogenesis.
However, in classic experiments, Depocas (8, 9) showed
that norepinephrine-induced thermogenesis in cold-acclimated rats was
unaffected by functional evisceration, demonstrating that at least at
the systemic level, no contribution was observable from any thermogenic
process in the abdomen. It is therefore an interesting possibility
that, in these organs, UCP1 has a functional role that is not
thermogenic. In brown adipose tissue, the activation of UCP1
(thermogenesis) is induced by -adrenergic receptors and increased
cAMP (75, 76). In different organs exhibiting peristaltic motion,
-adrenergic receptors also have a role: to induce
relaxation/inhibition of contraction via an increase in cAMP levels
(22, 24, 26, 36, 68, 73). Other relaxing agents [VIP,
pituitary adenylate cyclase-activating polypeptide (PACAP)] have also
been suggested as inducing relaxation through this cAMP-dependent
pathway (for reviews see Refs. 43 and 53).
Because activated UCP1 in the mitochondria of smooth muscle cells may
functionally explain the lowered effectiveness of Ca2+ in
inducing contraction observed under these conditions (see DISCUSSION), we have here utilized UCP1-ablated mice to
examine whether a UCP1-dependent component exists in
-adrenergically induced relaxation.
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MATERIALS AND METHODS |
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Animals
The UCP1-ablated [UCP1(Tissue Collection
Routinely on each experimental day, one wild-type and one UCP1(Organ Bath Studies
Routinely, eight ileal segments (4 from each type of mice) were mounted vertically in 25-ml water-jacketed organ baths made of glass (Section of Engineering, Mayo Clinic) filled with the salt solution detailed above, maintained at 37°C, and bubbled continuously with 5% CO2 in O2. During the experiments, the solution in the baths was changed at 20- to 30-min intervals by superfusion of aerated solution preheated to 37°C. The segment preparations were given an initial isometric load of 2.5 mN (250 mg weight) and were allowed to equilibrate for 2-3 h before the experiments were started. We observed that the time required to obtain stable responses to electrical field stimulation (EFS) (see EFS) appeared to be longer in segments from UCP1(EFS
Isolated ileal segment preparations were submitted to EFS by use of a direct current amplifier (Section of Engineering, Mayo Clinic) triggered by a stimulator (model S44, Grass Medical Instruments) via two parallel platinum electrodes (10 × 50 mm) 8 mm apart. This type of stimulation leads to contraction; this contraction may be due to a direct electrical effect on the muscles or due to stimulation of the nerves (that may be both excitatory and inhibitory for contraction). In methodological preexperiments, pulse duration response, and voltage response curves were, therefore, produced to determine optimal stimulation variables and to identify the nerve-mediated stimulation (that is sensitive to Na+ channel block). For the pulse duration response study, EFS was applied at 18 V, 3 Hz in 5-s trains every 2 min, and the pulse duration was increased gradually from 0.01 to 5.0 ms. This was repeated in the presence of 0.3 µM of the fast neuronal sodium channel blocker tetrodotoxin. At 0.2-ms pulse duration, the responses were maximum, with negligible responses remaining in the presence of tetrodotoxin. Because no response was observed when voltage-sensitive Na+ channels were blocked, these responses were regarded as nerve mediated, and this stimulation duration was used throughout the study. For the voltage-response study, the EFS was applied at various voltages (5, 10, 18, 30 V) at 3 Hz in 5-s trains every 2 min. Pulse duration was increased gradually from 0.1 to 3.0 ms at each voltage. Responses to EFS with 0.2 ms and 18 V were regarded to be (supra)maximal and were used in this study. Thus, for the experiments reported here, EFS of 0.2-ms pulse duration, 3-Hz frequency, and 15-V amplitude (as measured in the tissue baths, 18 V at the output) were applied in 5-s trains every 2 min.Effects of Relaxing Agents on EFS-Evoked Contraction
Ileal segments were exposed to EFS as described above for 30-60 min before agonists were added. The mean of the amplitudes of the stable responses to EFS (the last 4 before additions) for each segment was considered as the control value and set to 100%. Investigated agents (as indicated in the relevant figure legends) were added cumulatively to the tissue baths during the pause between contractions. The mean of the amplitudes of the two contractions after the addition of each drug concentration was measured and expressed in percentage of the control response. On the basis of these normalized segment responses, the mean values from all segments (3-4) from each mouse were calculated.Effects of Relaxing Agents on Carbachol-Induced Contraction
Ileal segments were contracted submaximally with 1 µM of the acetylcholinesterase-resistent muscarinic receptor agonist carbachol. The contraction reached a stable level within 8 min and maintained a stable plateau for >30 min when not further affected (not shown). To investigate the relaxant action, specified agonists (as indicated in the figure legends) were added cumulatively during this plateau contraction to the organ baths with 4 min allowed for stabilization of the relaxation response to each concentration. At the end of each cumulative dose-response curve, the ileal segments were maximally relaxed with 15 µM of the nonspecific smooth muscle relaxant papaverine. Total carbachol-induced contraction was defined as the difference between the stable carbachol value and the value after papaverine and was set to 100% for each segment, and all responses were expressed as a percentage of this carbachol response. On the basis of these normalized segment responses, the mean values from all segments (3-4) from each mouse were calculated. After full relaxant response had been obtained, tissue was rinsed at 10-min intervals forData Analysis
Mean dose-response data obtained from each mouse as described above (mean of 3-4 segments) were analyzed for simple Michaelis-Menten kinetics with the curve-fit option of the KaleidaGraph application using the equation V(x) = 100%Chemicals
Isoproterenol hydrochloride (isoprenaline), papaverine hydrochloride, tetrodotoxin, carbamylcholine chloride (carbachol), and forskolin were purchased from Sigma (St. Louis, MO). CL-316,243 was a gift from E. Danforth. Isoprenaline was dissolved in 0.1 mM ascorbic acid. All other drugs were dissolved in distilled water, except forskolin, which was dissolved in DMSO. Drugs were added to the baths in volumes <1% of the total bath volume. The final concentration of DMSO itself (0.5%) showed no significant biological effects. ![]() |
RESULTS |
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A UCP1-Dependent Component in Isoprenaline-Induced Intestinal Relaxation
EFS.
In wild-type mice, EFS induced stable and reproducible contractions in
segments of small intestine (Fig.
1A); according to our
methodological preexperiments (see MATERIALS AND
METHODS), these contractions represented the outcome of
nervous stimulation of the segments. The general -adrenergic agonist
isoprenaline dose dependently and reversibly inhibited these
contractile responses to EFS; at high isoprenaline concentrations, the
contractile responses were virtually abolished (Fig. 1A).
Data for seven mice are compiled in Fig.
2A. As seen, isoprenaline was
consistently able to fully inhibit the electrically induced contraction
in wild-type mice: at 1 µM isoprenaline, only 2 ± 1% remained
(mean value from 7 mice), and based on Michaelis-Menten analysis of
data points, the remaining contraction at infinite isoprenaline
concentration was
2 ± 2%, with an IC50 for
isoprenaline of 23 ± 3 nM (mean ± SE from 7 animals, each
individually analyzed as illustrated for the mean values in Fig.
2A).
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Carbachol-induced contraction.
Stimulation of intestine from UCP1(+/+) mice with the stable
acetylcholine analog carbachol led, as expected (19), to
persistent intestinal contraction, as exemplified in Fig.
3A. Addition of increasing concentrations of isoprenaline
led, again as expected (19), to full relaxation of the
carbachol-induced contraction, to the extent that addition of the
general relaxant papaverine did not elicit any further relaxation. As
compiled in Fig. 3C, full relaxation (4 ± 3%) was
always reached at infinite isoprenaline concentrations, and
isoprenaline had an IC50 of 27 ± 9 nM
(n = 5), i.e., a value identical to its
IC50 value for inhibition of EFS-stimulated contractions.
3-Adrenergic Stimulation
The 3-specific agonist CL-316,243 was not fully as
efficient as the general
-agonist isoprenaline in inhibiting
EFS-induced intestinal contraction; a residual response of 22 ± 1% remained in wild-type mice (Fig.
4A) even at infinite
isoprenaline concentrations (mean from 5 mice). The affinity
(IC50) for CL-316,243 was 19 ± 4 nM (Fig.
4A). When CL-316,243 was used to inhibit EFS-induced contraction in intestine from UCP1(
/
) mice, the residual
contraction was significantly greater (41 ± 6%;
P < 0.04) than in wild-type mice; the IC50
was 21 ± 6 nM (Fig. 4A). [We examined whether there was any effect of UCP1 ablation on the expression of the
3-receptors in the intestine, principally performed as
in (1), but found no difference in expression (not
shown)].
Similar observations were made when relaxation of
carbachol-precontracted ileal segments was induced with CL-316,243
(Fig. 4B). Here the residual contraction was 16 ± 3%
in intestine from wild-type mice [principally in agreement with
earlier data (11, 23)], and as high as 35 ± 6% in
intestine from UCP1(/
) mice (P < 0.03; means ± SE from 5 mice); the IC50 was 33 ± 9 in the wild-type and 34 ± 8 in the UCP1(
/
) mice.
Thus the absence of UCP1 resulted in a similar degree of lack of
relaxation potency, whether the relaxation was induced by a general
-agonist or by a
3-selective agonist.
cAMP-Induced Relaxation Involves a UCP1-Dependent Component
To investigate whether the UCP1-dependent relaxation necessitated directThus, as expected, in wild-type mice, forskolin could inhibit
EFS-induced contractions, and at infinite forskolin
concentrations, these contractions were fully eliminated (1 ± 3% remaining) (Fig. 5). In contrast, as
seen in Fig. 5, in ileal segments from UCP1(
/
) mice, forskolin was
only able to partly inhibit the EFS-induced contractions. Even when
tested in independent experiments at 100 µM forskolin concentration,
full inhibition was not obtained (not shown), and the Michaelis-Menten
analysis indicates that even at infinite forskolin concentrations
11 ± 3% of the contractions would be remaining
(P < 0.05; n = 3). Forskolin also
had a nearly threefold lower apparent affinity in these segments
[IC50 was 159 ± 32 nM in intestine from UCP1(+/+)
mice and 393 ± 92 nM in intestine from UCP1(
/
) mice
(P < 0.05, n = 3) (Fig.
5)].
It was therefore concluded that the difference between intestine from
UCP1(+/+) and UCP1(/
) mice was not due to an alteration in
-receptor function but was located in the pathway between cAMP and
muscle relaxation. Correspondingly, the presence of the UCP1-dependent
relaxation was not dependent on the
-receptor itself being
stimulated; rather, any receptor, the function of which is mediated via
cAMP, would be expected to demonstrate an UCP1-dependent component.
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DISCUSSION |
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In the present investigation, we have demonstrated the existence
of a UCP1-dependent component in the pathway for -adrenergically induced relaxation in intestine. Whereas the presence of this component
may be an indirect consequence of the absence of UCP1 in brown adipose
tissue, it is more likely that it is explainable by the absence of UCP1
in intestinal cells. Thus a UCP1-dependent mechanism involved in the
transduction pathway from
-adrenergic receptor stimulation and cAMP
increases to intestinal relaxation may exist. A hypothesis for a
functional role of UCP1 in contraction/relaxation control is
presented below. In extension of these studies, it may be envisaged
that the existence of a UCP1-dependent component in relaxation may not
be restricted to adrenergic control of intestinal contraction but may
be of significance for the understanding of the function of other
organs apparently expressing UCP1 and exhibiting peristalsis.
-Adrenergically Induced Relaxation Versus Other Relaxing Agents
Functional Consequences of Absence of UCP1 in the Intestine
For evident reasons, all dedicated investigations of phenotypical alterations in UCP1(It may be important to consider whether the existence of the
UCP1-dependent adrenergically induced intestinal relaxation
demonstrated here challenges the conclusions from earlier studies on
the phenotype of the UCP1(/
) mice. However, the conclusions of
studies published to date (7, 10, 17, 21, 46-49)
would not seem to be influenced by this new aspect: the reported
effects are adequately understood based on a functional defect in brown
adipose tissue alone. However, the situation may be different regarding
the analysis of so-called brown-adipose-tissue-deficient animals
(13, 18, 41, 44). In these transgenic animals, diphtheria
toxin is expressed under the UCP1 promotor. Thus any cell that normally
expresses UCP1 will also express diphtheria toxin and this toxin will
kill the cell. This means that the intestinal cells expressing UCP1
will be eliminated, which may have broader consequences than the
partial deterioration of their function reported here. Thus certain
phenotypic features of the so-called brown adipose tissue-deficient
mice may be ascribable to e.g., intestinal effects.
Mechanism of -Adrenergically Induced Intestitinal Relaxation
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In the contraction process induced by acetylcholine binding
to the muscarinic receptors and intracellularly mediated via an increase in cytosolic Ca2+ levels, ATP is used
(31, 32, 65). This ATP is of mitochondrial origin. Adrenergic stimulation through -receptors can be
expected to lead to an increase in cAMP levels, and this could
subsequently lead to activation of UCP1. This would lead to
mitochondrial uncoupling, i.e., some of the protons pumped out from the
mitochondria by the respiratory chain would return to the mitochondrial
matrix through UCP1, i.e., without passing through the ATP synthase, and less ATP would thus be formed. If this process is of sufficient magnitude, the ATP/ADP ratio in the cells will decrease, and less ATP
will, therefore, be available for the contraction process. Thus a
graded counteraction of induced contraction would be caused by
adrenergic stimulation, although the Ca2+ level may be
unaffected, i.e., a given level of Ca2+ may be less
effective in inducing contraction, as has been found earlier
(73). The fact that relaxation takes place, although not
to the same extent, in the absence of UCP1, implies that other mechanisms are responsible for the major part of the relaxation process, but the UCP1-dependent relaxation would seem to have a higher
sensitivity to
-adrenergic stimulation/cAMP levels.
In brown adipocytes, the link between increased cAMP levels and the associated increase in protein kinase A activity (14) is associated with phosphorylation (activation) of hormone-sensitive lipase (63) and perilipin (6), leading to stimulated hydrolysis of triglycerides, and the released fatty acids, either in themselves or in some metabolite form, activate UCP1 (16, 35, 51, 60). Stores of triglyceride are apparently not observable in the longitudinal muscle layer of the intestine, and there are no reports that hormone-sensitive lipase or perilipin are expressed in this layer (although this may be due to the very small fraction of total tissue that constitutes the cells of interest). In the apparent absence of the established pathway for UCP1 activation, an interesting question as to the nature of the intracellular physiological activator of UCP1 in these cells arises. However, the mitochondria containing UCP1 are only a very small fraction of all mitochondria in intestinal tissue, and to examine the function of UCP1 in intestinal mitochondrial preparations is probably unfeasible, due to high dilution by mitochondria from other parts of the tissue.
Perspectives
The present studies were restricted to analyzing the effect of UCP1 ablation on relaxation of ileal segments. Evidently, the question may be raised as to whether similar mechanisms are involved in the relaxation of other types of smooth muscle. Because UCP1 is not present in the smooth muscle layers of blood vessels (52), the mechanism discussed here is not relevant in the control of vessel contractility and thus not for blood pressure in general. This distinction may be paralleled by the presence of distinct forms of myosin in vascular and peristaltic smooth muscle (30, 64).However, the results presented here may be of interest concerning the function of other organs in which UCP1 has been indicated: stomach, eosophagus, ureter origin, epididymus, ductus deferens, and uterus. It is noteworthy that in most of these organs, a cAMP-mediated relaxation is seen [uterus (39, 56); vas deferens (27), urinary bladder (58), detrusor (57), distal colon (33), ileum (61), esophagus, and ureter (54)]. It is reasonable to assume that this relaxation could partly be UCP1-dependent, as demonstrated here for the ileal segments. Furthermore, it is also noteworthy that these organs all exhibit peristaltic motion. Regulated phasic contractile activity is coordinated, at least in the intestine, by the pacemaker cells, the intestinal cells of Cajal (reviewed in Refs. 62, 67), which are smooth muscle cells characterized e.g., by a high mitochondrial content (71). Similar pacemaker cells have at least been identified in the ureter (34) and discussed concerning the uterus (74). It is therefore not unlikely that UCP1 expression, cAMP-mediated relaxation, pacemaker cells, and peristalsis may be functionally linked in several organs, in addition to the intestine directly studied here.
Peristalsis, in addition to being required for the handling of food
through the gastrointestinal system, is important in such functions as
semen transfer, uterine function, and urine production. In the absence
of dedicated observations and experiments, it may not be unexpected
that no other phenotypic alterations have as yet been reported in
UCP1(/
) mice than those related to thermogenesis and now intestinal
relaxation. However, the present observations may stimulate new types
of dedicated studies of phenotypic manifestations of UCP1 ablation.
Establishment of the significance of UCP1 in these contexts adds new
challenges to the study of this unique mammalian protein.
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ACKNOWLEDGEMENTS |
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We thank Anette Ebberyd and Annelie Brolinson for technical help, Martin Nedergaard for animal observations, and Dana Hutchinson and L. P. Kozak for advice and contributions.
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FOOTNOTES |
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This study was supported by a grant from The Swedish Science Council.
Address for reprint requests and other correspondence: J. Nedergaard, Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden (E-mail: jan{at}metabol.su.se).
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.
August 7, 2002;10.1152/ajpgi.00193.2002
Received 21 May 2002; accepted in final form 30 July 2002.
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