1 Department of Physiological Sciences, Lund University, SE-221 Lund, Sweden; 2 Department of Vegetative Physiology, University of Cologne, D-50923 Cologne, Germany; and 3 Department of Physiology, University of Aarhus, DK-8000 Aarhus, Denmark
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ABSTRACT |
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Agonist-induced activation of smooth
muscle involves a rise in intracellular Ca2+ concentration
and sensitization of myosin light chain phosphorylation to
Ca2+. Sr2+ can enter through Ca2+
channels, be sequestered and released from sarcoplasmic reticulum, and
replace Ca2+ in activation of myosin light chain
phosphorylation. Sr2+ cannot replace Ca2+ in
facilitation of agonist-activated Ca2+-dependent
nonselective cation channels. It is not known whether Sr2+
can replace Ca2+ in small G protein-mediated sensitization
of phosphorylation. To explore mechanisms involved in
-receptor-activated contractions in smooth muscle, effects of
replacing Ca2+ with Sr2+ were examined in rat
portal vein. Norepinephrine (NE) at >3.0 × 10
7 M
in the presence of Ca2+ resulted in a strong sustained
contraction, whereas this sustained component was absent in the
presence of Sr2+; only the amplitude of phasic contractions
increased. Pretreatment with low (~0.05 mM) free Ca2+
followed by 2.5 mM Sr2+ resulted in a sustained component
of the NE response. In
-escin-permeabilized preparations,
phenylephrine in the presence of GTP or guanosine 5'-O-(3-thiotriphosphate) alone induced sensitization to
Sr2+. In conclusion, a Ca2+-regulated
membrane/channel process is required for the sustained component of NE
responses in rat portal vein. Sensitization alone is not responsible
for the sustained phase of the NE contraction.
vascular smooth muscle; calcium sensitization
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INTRODUCTION |
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AGONIST STIMULATION OF SMOOTH muscle initiates a complex series of events leading to activation of the contractile proteins. For most agonists, this involves both membrane depolarization, with an associated influx of Ca2+, and activation of membrane potential-independent processes [i.e., the electromechanical and pharmacomechanical coupling pathways (38)]. The main cellular event in activation is a rise in free Ca2+ concentration ([Ca2+]), initiating myosin light chain phosphorylation (14). However, agonist stimulation of smooth muscle also involves a sensitization of the contractile process to Ca2+ (2). The sensitization of the contractile proteins to Ca2+ was originally described in experiments on intact smooth muscle (12, 23, 25, 34), where force was correlated with measured levels of free Ca2+. The mechanisms of Ca2+ sensitization have been further examined in skinned preparations, where the free [Ca2+] can be controlled. Sensitization to Ca2+ has substantial effects on force at low free [Ca2+] (18) and has been suggested to strongly contribute to the tonic (or sustained) phase of agonist-induced contractions via small G protein-associated pathways (37). Consistent with this view, inhibition of G protein (rhoA)-mediated Ca2+ sensitization results in loss of sustained force development in the rabbit portal vein activated by norepinephrine (9) and in the guinea pig ileum activated by carbachol (23, 28). Recently, a Ca2+-independent kinase, associated with myofilaments, was found and postulated to contribute to sensitization and Ca2+-independent contraction (41). This raises the following question: Are mechanisms associated with sustained [Ca2+] increase required, or is the sensitization alone sufficient for activation of the sustained contraction phase in the intact smooth muscle?
Sr2+ can replace Ca2+ in many biological systems. In smooth muscle, Sr2+ can activate myosin light chain phosphorylation and contraction of chemically skinned muscle fibers (11, 13, 35, 36), although the concentration-force relationship is shifted toward higher concentrations in the presence of Sr2+ than in the presence of Ca2+. In intact muscle, Sr2+ can support spontaneous contractile activity and contractions induced by membrane depolarization (1, 11, 40). These results are consistent with electrophysiological studies showing that Sr2+ can enter through voltage-gated L-type channels (10) and thus replace Ca2+ in the electromechanical coupling.
It has been shown that one event in pharmacomechanical coupling is nonfunctional in the presence of Sr2+; the agonist-activated nonselective cation channel is facilitated by Ca2+, but not by Sr2+ (15); and Ca2+, as well as the agonist, is required for the channel to open. When open, the channel conducts several cations, including Sr2+ and Ca2+. This channel has been shown to have a very high conductance (200 pS) in the smooth muscle of the rat portal vein (22). In this tissue, norepinephrine-induced contractions, in contrast to high-K+ contractions, are greatly attenuated if Sr2+ is substituted for Ca2+ (1). This attenuation can reflect the effects of Sr2+ on the channel (see above). It can also indicate that sensitization, the main process in the pharmacomechanical coupling, is not functional in the presence of Sr2+.
The aim of this study was to determine whether sensitization of the
phosphorylation process is the only mechanism of physiological importance in control of the sustained phase of the agonist-induced contraction in intact tissue. We used the rat portal vein and examined
norepinephrine-induced contractions in intact tissue in the presence of
Sr2+ and Ca2+. To study the sensitization, we
determined responses to guanosine 5'-O-(3-thiotriphosphate)
(GTPS) and norepinephrine/GTP in permeabilized preparations at fixed
concentrations of Ca2+ and Sr2+.
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METHODS |
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Muscle fiber preparations. Female Sprague-Dawley rats (150-200 g) were obtained from Möllegaard (M & B, Ry, Denmark). The animals were killed by cervical dislocation, and the portal veins were removed and dissected free of connective tissue.
Experiments on intact preparations. The portal vein was opened longitudinally and used whole or split longitudinally into two equal parts. The strips, 7-9 mm long, were mounted in open organ baths at 37°C, using thin silk thread, between a stainless steel pin on an adjustable stand and a transducer (model FT03, Grass Instrument, Freeport, IL) for recording of isometric force. The preparations were equilibrated in physiological saline solution (PSS; see composition below) with 2.5 mM Ca2+ for 1 h at a passive tension corresponding to optimal length for active force (24). In each muscle preparation, a reference contraction was recorded after addition of 80 mM KCl from a 3 M stock solution to the PSS with 2.5 mM CaCl2. This activation method, which depolarizes in a nonisotonic solution, enables a fast change in KCl concentration without solution exchange and gives reproducible contractions. These contractions were stronger than those obtained using isosmolar substitution of Na+ for K+, probably due to cell swelling shown to occur after Na+/K+ isosmotic replacement (17). We used the plateau force of the 80 mM KCl in 2.5 mM CaCl2 contractions as a reference for normalization of all later force responses.
We performed Sr2+ and Ca2+ experiments in varying order on the same fiber. Previous reports have shown that the sarcoplasmic reticulum is depleted after 5-6 min in Ca2+-free solution at 20°C (6). To ensure that all intracellular stores were emptied, preparations were kept in Ca2+/Sr2+-free PSS for 10 min, and thereafter the muscle was challenged three times with 10Experiments on -escin-permeabilized preparations.
The portal vein was prepared as described above, and then four to six
5- to 7-mm-long longitudinal strips were cut from each vein. The strips
were mounted horizontally on small metal clips between the extended arm
of a force transducer (Scientific Instruments, Heidelberg, Germany) and
a steel pin, enabling length adjustment. The experiments were carried
out at ambient temperature (~23°C) in 1-ml cups. The strips were
equilibrated for ~45 min in HEPES-buffered PSS (see below) before a
norepinephrine (10
5 M) test contraction was made. The
preparations were chemically permeabilized using
-escin essentially
as described previously (19, 28). The strips were
thoroughly relaxed in Ca2+-free HEPES-buffered PSS for 10 min. After the preparations were transferred to relaxing solution for 5 min, they were treated with relaxing solution plus 50 µM
-escin
for 35 min. Finally, the preparations were kept for 10 min in
-escin-free relaxing solution.
Solutions for intact preparations. The PSS contained (in mM) 122 NaCl, 4.7 KCl, 1.2 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11.5 glucose. Ca2+- and Sr2+-containing solutions were made by addition of CaCl2 or SrCl2, respectively. High-K+ solutions, for K+ dependence experiments, were made by isosmotic substitution of K+ for Na+. Two different isosmolar high-K+ solutions, with 60 and 128 mM K+, were used. The reference contraction in 80 mM K+ was made by addition of KCl to the PSS with 2.5 mM CaCl2. All solutions were continuously gassed with 95% O2-5% CO2 during the experiments, giving a pH of 7.4 at 37°C.
Solutions for chemically permeabilized preparations.
The HEPES-buffered PSS contained (in mM) 118 NaCl, 5 KCl, 1.2 Na2HPO4, 1.2 MgCl2, 1.6 CaCl2, 24 HEPES, and 10 glucose, with pH adjusted to 7.4. The Ca2+-free PSS had the same composition, except
CaCl2 was replaced by 2 mM EGTA. Double-concentrated
solutions were used for skinned preparations and were stored at
20°C until use. The relaxing solution contained (in mM) 20 imidazole, 7.5 Na2ATP, 10 EGTA, 10 magnesium acetate, 10 phosphocreatine, 2 dithioerythritol, 5 NaN3, 0.001 leupeptin, and 0.0015 calmodulin. Ionic strength was adjusted to 150 mM
with potassium methanesulfonate, and pH was adjusted to 7.0 at room
temperature. The Sr2+ and Ca2+ contraction
solutions were made by addition of 10 mM CaCl2 or, alternatively, 10 mM SrCl2. Solutions with intermediate
free [Sr2+] and [Ca2+] were made by mixing
the relaxing and contracting solutions to achieve different ratios of
total SrCl2/CaCl2 to EGTA. Free
[Ca2+] and [Sr2+] were calculated as
described previously (8) using the apparent binding
constants for Ca2+ and Sr2+ to EGTA provided
elsewhere (11).
Statistics. Values are means ± SE, with the number of observations in parentheses.
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RESULTS |
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Spontaneous contractile activity and
high-K+-induced force in
Ca2+ and
Sr2+ solutions in intact muscle.
All effects of Sr2+ and Ca2+ in intact muscle
were fully reversible and independent of whether Sr2+
experiments were performed before or after the Ca2+
controls. The rat portal vein exhibited spontaneous contractile activity in PSS with 2.5 mM CaCl2. In PSS containing 2.5 mM
SrCl2, the spontaneous contractile activity was also
present, although with a slightly more regular pattern (Fig.
1, left). In
SrCl2, the frequency of contractions was 1.0 ± 0.1 min1 and the force amplitude was 69 ± 6%
(n = 6) of the "reference force" (see
METHODS) in response to activation with 80 mM
K+ in 2.5 mM CaCl2. The corresponding values
for spontaneous activity in CaCl2 were 2.2 ± 0.3 min
1 and 54 ± 10% (n = 6).
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Norepinephrine-induced force responses in the presence of
Ca2+ and
Sr2+ in intact muscle.
In the presence of Ca2+,
108-10
7 M norepinephrine increased the
amplitude and frequency of the spontaneous activity. After activation at higher norepinephrine concentrations (above ~3.0 × 10
7 M), a sustained component appeared. If
Sr2+ was substituted for Ca2+, responses at
lower norepinephrine concentrations were essentially unaltered.
However, no sustained component appeared at high norepinephrine concentrations; instead, the amplitude and frequency of the phasic contractions increased (Fig. 1, right). In experiments not
shown here, we increased the SrCl2 concentration to 5 and
10 mM and observed no sustained contraction. In Fig.
3, summarizing the results at 2.5 mM
CaCl2 and SrCl2, the amplitude of the phasic (i.e., baseline to peak force) and sustained (i.e., baseline to sustained force) components are plotted against the norepinephrine concentration. The phasic component reached half-maximal amplitude at
~3 × 10
8 M norepinephrine in the presence of
Ca2+ and at 1 × 10
7 M norepinephrine in
the presence of Sr2+. The sustained component reached
half-maximal amplitude at ~1 × 10
6 M
norepinephrine in the presence of Ca2+. It was absent in
the presence of Sr2+.
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Ca2+ dependence of the sustained
phase during norepinephrine-induced contraction in intact
muscle.
The experiments described above to study activation with norepinephrine
were performed in the continuous presence of Sr2+ or
Ca2+. In an attempt to provoke a sustained contraction
phase in the presence of Sr2+, we added 2.5 mM
Ca2+ 3 min after activation with 105 M
norepinephrine in 2.5 mM Sr2+. This induced irregular
contractions but without a sustained phase. This suggests a competition
between Sr2+ and Ca2+ in a step involved in the
activation of the sustained norepinephrine force. To overcome this
interaction but still examine the Ca2+ dependence of the
sustained contraction, we performed experiments where the muscle
preparation initially was depleted of Ca2+ and
Sr2+ using 1 mM EGTA for 10 min. Thereafter, the muscle was
exposed to a low concentration of EGTA (0.1 mM) and challenged with
10
5 M norepinephrine. This did not cause a contraction.
If 2.5 mM Sr2+ was introduced, phasic contractions without
any sustained force gradually developed in a manner similar to that
described above (Fig. 4,
left). If, instead, 2.5 mM Ca2+ was used, a
sustained contraction developed rapidly (Fig. 4, right). In
the presence of low [Ca2+] (0.1 mM EGTA and 0.15 mM
CaCl2), norepinephrine did not cause a contraction, showing
that this low free [Ca2+] (~50 µM) did not support
contraction by itself (Fig. 4, middle). If 2.5 mM
Sr2+ was added in this situation (Fig. 4,
middle), a strong contraction developed and was sustained
for
1 min. Thereafter, the sustained force returned to baseline and a
phasic contraction pattern, similar to that observed in the presence of
Sr2+ alone, appeared. These results show that a very low
free [Ca2+], which by itself cannot support contraction,
can act as a switch permitting a sustained phase in combination with
Sr2+. In addition, Sr2+ appears to compete with
Ca2+ and inhibit this process.
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Ca2+ and
Sr2+ dependence of G protein-mediated
sensitization of contraction in -escin-permeabilized preparations.
To examine whether the lack of sustained force in the
norepinephrine-induced contraction in the presence of Sr2+
was due to a failure of G protein-mediated sensitization of the contractile machinery, we performed experiments on
-escin-permeabilized portal veins. Figure
5, top, shows contractile
responses to Ca2+ and Sr2+ in a thin
permeabilized preparation of a portal vein. At intermediate [Sr2+], addition of 100 µM GTP
S induced a pronounced
increase in force. Transfer to saturating [Sr2+] resulted
in a force equaling that induced at maximal [Ca2+].
Similar sensitization effects of GTP
S were observed at intermediate [Ca2+]. Figure 5, bottom, summarizes the data
from permeabilized muscles. The force responses were dependent on free
[Ca2+] and [Sr2+], and GTP
S caused a
leftward shift (~0.2 pCa and 0.3 pSr unit) of the Ca2+-
and Sr2+-force relationship, i.e., sensitization. To
examine whether the receptor-mediated responses were present in
Sr2+-containing solution, we examined the
phenylephrine-induced increase in force in the presence of GTP in
-escin-skinned preparations. The muscles were activated with
Ca2+ or Sr2+ in the presence of GTP to give
~30% of maximal Ca2+-induced force: 29 ± 7%
(n = 8) and 32 ± 7% (n = 6) in
the presence of Ca2+ and Sr2+, respectively. In
the presence of Ca2+ and Sr2+, addition of
10
4 M phenylephrine resulted in an increase in force
[14 ± 4% (n = 8) and 21 ± 3%
(n = 6) in Ca2+ and Sr2+,
respectively], showing that receptor-coupled pathways are functional in the presence of Ca2+ and Sr2+.
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DISCUSSION |
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A rise in intracellular free [Ca2+] and sensitization of the contractile machinery to Ca2+ are key features in receptor-mediated agonist activation of smooth muscle (27). Clear evidence has been presented that the sustained phase of responses to norepinephrine and carbachol is influenced by sensitization (18, 23, 28). We show here that the sustained phase of norepinephrine-induced contraction in a vascular smooth muscle is dependent also on Ca2+-specific regulation of a membrane-associated process and that the agonist-induced sensitization of the contractile proteins is not the only mechanism determining the sustained phase.
The sustained phase of norepinephrine-induced contraction was
selectively removed when Sr2+ was substituted for
Ca2+. In the presence of Ca2+, the peak and
sustained components of the norepinephrine contraction in the portal
vein had different norepinephrine dependencies (cf. Fig. 2). On
replacement of Ca2+ with Sr2+, the phasic
responses at low norepinephrine concentration were similar. Although
the concentration-response relationship of the phasic component was
shifted slightly toward higher norepinephrine concentrations in the
presence of Sr2+, the response at saturating norepinephrine
concentration reached the same amplitude as in the presence of
Ca2+. In contrast, no sustained response appeared, even at
very high norepinephrine concentrations (3 × 10
5
M). Thus the lack of sustained contraction in the presence of Sr2+ is not due to a shift in the norepinephrine
dose-response relationship but, rather, reflects a failure in the
adrenergic activation pathway in the presence of Sr2+.
Norepinephrine-induced activation involves Ca2+ release from the sarcoplasmic reticulum (30). The role of the released Ca2+ is dual: to activate the contractile machinery directly and to regulate Ca2+-sensitive channel activity, which alters the membrane potential and Ca2+ channel influx (16, 27, 31). The sarcoplasmic reticulum in phasic muscle, such as the portal vein, is poorly developed (7). Ca2+ release from the sarcoplasmic reticulum primarily contributes to the fast initial phase of contraction (30). Sr2+ can be sequestered by the sarcoplasmic reticulum (33, 39) in smooth muscle and released (4). Sr2+ can induce Ca2+ release from the sarcoplasmic reticulum (10). These results suggest that impaired release from the sarcoplasmic reticulum is not responsible for the loss of sustained contraction in the presence of Sr2+.
Previous studies on chemically permeabilized smooth muscle have shown
that Sr2+ can activate the contractile proteins and induce
myosin light chain phosphorylation (11, 13, 36), although
the concentration dependence is shifted toward higher concentrations in
the presence of Sr2+ than in the presence of
Ca2+. Our data from the -escin-treated portal veins are
consistent with these results. Because [Ca2+] and
[Sr2+] dependencies of force were similar in
high-K+-activated muscle and because spontaneous activity
was present in Sr2+-containing PSS, depolarization of the
membrane can lead to sufficient activation of L-type channels, ion
influx, and activation of the contractile machinery in the presence of
Sr2+ and Ca2+. This is consistent with previous
results from K+-depolarized swine carotid media
(11) showing that Sr2+ can substitute for
Ca2+ in the activation of intact smooth muscle. The
phosphorylation-stress relationships of depolarized intact smooth
muscle have been shown to be similar in the presence of
Sr2+ and Ca2+ (11). These results,
together with our finding that the spontaneous activity (Fig. 1) and
high-K+ induced force (Fig. 2) were of high amplitude in
the presence of Sr2+, show that the level of
phosphorylation can reach the same levels in the presence of
Sr2+ and Ca2+. The presence of a very small
[Ca2+] can activate the sustained phase in the presence
of Sr2+ (Fig. 4). We find that the effect of
Ca2+ in the presence of Sr2+ depended on the
order in which the ions were introduced; adding Ca2+, even
at 2.5 mM, after Sr2+ did not result in a sustained phase.
In addition, the increase in [Ca2+] that resulted in a
sustained phase in the presence of Sr2+ (Fig. 4) was very
low, on the order of 50 µM, which, by itself, was too low to induce
force, even in fully depolarized muscle (Fig. 2). We can therefore
exclude the possibility that a difference in concentration dependence
for activation of the contractile proteins between Sr2+ and
Ca2+ is responsible for the lack of sustained force after
norepinephrine activation in the presence of Sr2+. Instead,
we propose that Ca2+ has a switchlike action at the
membrane level, allowing [Sr2+] to be high for a
sufficient amount of time to support a sustained phase.
We show, using GTPS and GTP/phenylephrine in permeabilized
preparations, that the receptor-coupled G protein-mediated
sensitization pathway is present in Sr2+-containing
solutions. The increase in force of the phasic component (cf. Figs. 2
and 3) and the slowed relaxation after activation with norepinephrine
in the presence of Sr2+ are consistent with an inhibition
of the phosphatase. Sensitization via G protein-mediated inhibition of
the phosphatase is an important mechanism after receptor activation
(20, 28). The sensitization is considered a major
mechanism for the induction of the sustained phase (21,
37). Theoretically, it is possible that norepinephrine-induced sustained force could be the result of the inhibition of the myosin light chain phosphatase alone, independently of an increase in free
Ca2+. If a constitutively active
Ca2+-independent light chain kinase is present, inhibition
of the phosphatase alone would be sufficient. In a recent study
(41), a Ca2+-independent light chain kinase,
distinct from the myosin light chain kinase, was demonstrated in rat
caudal arterial smooth muscle strips and shown to be associated with
myofilaments in isolated chicken gizzard. It was suggested that this
kinase could have a role in Ca2+ sensitization and
Ca2+-independent contraction of smooth muscle in response
to stimuli that act via Ca2+-independent pathways and
inhibition of the phosphatase. If this kinase is operating in the rat
portal vein, we can exclude the possibility that it has any
physiological significance in preserving the sustained phase in
norepinephrine-induced contraction, since we can induce G
protein-mediated sensitization without eliciting a sustained component
in the presence of Sr2+.
Because the responses of the permeabilized preparations were similar in
the presence of Ca2+ and Sr2+, the lack of a
sustained phase in the presence of Sr2+ must be attributed
to a membrane-associated mechanism. Two Ca2+-regulated
channels have been suggested to contribute to membrane depolarization
after norepinephrine activation in the portal vein (5):
the agonist-activated, Ca2+-facilitated nonspecific cation
channel and the Ca2+-activated Cl channel. As
a consequence of the depolarization, L-type channels would open and
permit a large inflow of Ca2+. A failure to open the
agonist-activated nonselective Ca2+-facilitated cation
channel and/or the Ca2+-activated Cl
channel
could be responsible for the lack of a sustained phase after
norepinephrine activation in the presence of Sr2+.
The nonselective Ca2+-facilitated cation channel is
sensitive to intracellular Ca2+ and possibly also to
external Ca2+ (5). That the agonist-activated
nonselective Ca2+-facilitated cation channel is
Ca2+ specific [i.e., could not be facilitated by
Sr2+ (15, 29)], is active in the right time
scale for the sustained phase (29), and has a substantial
conductance (200 pS) in the portal vein (22) suggests that
this channel may be important for the sustained phase of the
norepinephrine-induced contraction in the intact portal vein. The
sustained phase after activation with norepinephrine is mainly
dependent on prolonged depolarization activating the L-type
Ca2+ channels (26). This depolarization is
postulated to be maintained primarily by the agonist-activated cation
channel, possibly in cooperation with the Ca2+-activated
Cl channel (32).
In conclusion, norepinephrine causes sensitization of the myosin phosphorylation in the presence of Ca2+ and Sr2+. The sensitization, via small G proteins and inhibition of phosphatase, is activated without the appearance of a sustained contraction in the presence of Sr2+. Our interpretation of the lack of sustained phase is that the activities of myosin light chain kinase and/or other myosin phosphorylating kinases are insufficient to reach the contractile threshold of light chain phosphorylation, even in the sensitized myofilaments. The results show that sensitization is not sufficient for the sustained phase of agonist-induced contraction.
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ACKNOWLEDGEMENTS |
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This study was supported by Swedish Medical Research Council Grants 04x-8248 and 04x-12584 (A. Arner and U. Malmqvist) and Deutsche Forschungsgemeinschaft Grant Pf 226/4-2 (G. Pfitzer). Collaboration between Lund University and the University of Köln was supported by grants from the Deutsche Akademische Austauchdienst. H. Nilsson is a Nordisk Forskerutdanningsakademi guest professor at Lund University.
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FOOTNOTES |
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Part of these results has been presented in preliminary form (3).
Address for reprint requests and other correspondence: A. Arner, Dept. of Physiological Sciences, Lund University, BMC F11, Tornavägen 10, SE-221 84 Lund, Sweden (E-mail: Anders.Arner{at}mphy.lu.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.
10.1152/ajpcell.00191.2001
Received 29 August 2001; accepted in final form 19 September 2001.
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