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Effects of salbutamol on rat diaphragm contractility
H. F. M. VAN DER HEIJDEN, R. H. H. VAN BALKOM, H. T. M. FOLGERING,
C. L. A. VAN HERWAARDEN, AND P. N. R. DEKHUIJZEN
Department of Pulmonary Diseases, University Hospital Nijmegen,
6500 HB Nijmegen, The Netherlands
H. F. M., Van Der Heijden, R. H. H. Van Balkom ,
H. T. M. Folgering, C. L. A* Van H erw aarden, and P. N. R.
Dekhuijzen. Effects of salbutamol on rat diaphragm contrac­
tility. J. AppL Physiol. 81(3): 1103-1110, 1996.—The aim of
this study was to investigate 1 ) the effects and time course of
single doses of salbutamol on isometric contractile properties
of isolated rat diaphragm strips and 2) whether these effects
were caused by a direct effect on the muscle* Two experiments
were performed. In one, salbutamol was administered subcutaneously in doses of 12.5, 25, 50, or 100 pg/kg (25 and
50 pg/kg sc resulted in serum concentrations of —9 and
—15 pg/1, respectively, 0.5 h after injection) and in vitro
contractile properties were determined 0.5, 1, 2, or 4 h after
administration; in the other, salbutamol was added to the
tissue bath in a concentration of ^2, —10, —20, and —80 pg/1.
Twitch force, maximal tetanic force, and twitch force-totetanic force ratio all increased in a dose-dependent way in
both experiments. The increases in force generation were
slightly higher after subcutaneous administration. Forcefrequency curves were shifted upward in both experiments.
No significant effects of time of salbutamol administration
were found, but the increase in force generation was most
pronounced within 2 h after subcutaneous administration. In
conclusion, in vitro force generation can be improved by low
concentrations of salbutamol. The slightly higher increases in
force generation after subcutaneous administration suggest
that in vivo salbutamol may have additional positive inotro­
pic actions on diaphragm contractility besides a direct p 2 adrenergic effect on the muscle itself.
contractile properties; dose response; (3-adrenergic agonists;
respiratory muscles
teers increased Pimax by 7 and 15% after 14 and 21 days
of oral treatment, respectively (23).
Although several of these studies indicate that salbu­
tamol may increase diaphragm force generation under
specific circumstances, the exact effects of salbutamol
on diaphragm contractile properties and its mechanism
of action have not been fully clarified yet. To our
knowledge, the in vitro effects of salbutamol on dia­
phragm contractile properties have not been studied.
The aims of the present study were therefore to 1 )
investigate the dose-response effects and time course of
effects of salbutamol on in vitro isometric contractile
properties of rat diaphragm and 2) investigate whether
these effects can be explained by a direct effect on the
diaphragm. We aimed to study the effects of salbutamol
in low concentrations, which were likely to be of clinical
relevance,
METHODS
Study Design
Protocol A. Saline or salbutamol (Glaxo-Wellcome, The
Netherlands) in a dose of 12.5, 25, 50, or 100 pg/kg was
administered subcutaneously. Salbutamol was dissolved in
0,9% NaCl, and all rats received a volume of 0.5 ml/kg body
wt. At 0.5,1, 2, or 4 h after injection, the animals were killed
and contractile properties of isolated diaphragm strips were
determined,
Protocol B . Saline or salbutamol in a concentration of
0.01 pM ('-'-3 pg/1), 0.03 pM (—10 pg/1), 0.06 pM (-20 p
0.3 pM (—80 pg/1) was dissolved in Krebs-Ringer solution
before each experiment. Subsequently, diaphragm strips were
mounted in the tissue baths containing this Krebs solution to
determine direct effects of salbutamol on contractile proper­
ties.
have indicated that the (^-adrenocep­
tor agonist salbutamol may improve skeletal and respi­
ratory muscle function. In vitro experiments showed Animals and Ti'eatment
that salbutamol hyperpolarized membrane potential in
A total number of 125 adult male outbred Wistar rats, aged
both peripheral skeletal muscles and diaphragm (6, 17-20 wk and weight 556 ± 6 (SE) g, were used in these
36). Salbutamol increased subtetanic force generation experiments. In both protocols, five animals were studied in
in fast-contracting skeletal muscles and decreased force each group. The rats were housed under standard conditions
in slow-contracting muscles (1). This is consistent with and were fed ad libitum. The study was approved by the Animal
Experiments
Committee
of
the
University
of
Nijmegen.
the in vivo effects of catecholamines on skeletal muscles
The salbutamol concentration of 10 pg/1 was based on the
(4). Experiments in dogs showed no significant increase mean human serum concentration reached after a single oral
in transdiaphragmatic pressure (Pdi) or twitch force dose of 4 mg, which was - 1 0 —20 pg/1 (10). The actual serum
(Pt) in fatigued diaphragm after short-term intrave­ salbutamol concentrations reached in protocols A and B were
nous salbutamol administration (11, 29). In contrast, measured in all subcutaneously treated rats and in five
salbutamol did increase diaphragm Pt during compen­ samples obtained from each in vitro concentration. Serum
sated metabolic acidosis (12) and increased shortening samples were obtained from all rats in the study. Blood
samples
were
collected
from
the
thoracic
cavity
immediately
of the canine diaphragm (16). Contradicting effects of
after excision of the diaphragm and dissection of the aorta
salbutamol on respiratory muscle function have also and vena cava. The samples were subsequently centrifuged
been described in humans. Two studies showed no and stored at —80°C until analysis. The analysis was per­
improvement of Pdi (14) or maximal inspiratory mouth formed by using high-performance liquid chromatography
pressure (Pimax) (35) in normal subjects. In contrast, with fluorescence detection (Scotlab Analytical, Coatbridge,
long-term salbutamol administration in healthy volun­ Scotland, UK) (3).
SEVEKAL s t u d i e s
0161-7567/96 $5.00 Copyright © 1996 the American Physiological Society
1103
1104
(*2-ADRENERGIC STIMULATION OF RAT DIAPHRAGM
A pilot study showed no effects of any of the salbutamol
concentrations used on the pH of the oxygenated Krebs
solution. A maximal increase of Pt was reached within 10 min
after addition of salbutamol and lasted for ^15 min. There­
fore, a period of 15 min for thermo equilibration and diffusion
of salbutamol {protocol B ) was applied in both protocols.
The experiments with different doses and time groups were
performed at random. The investigator (H. F. M. Van Der
Heijden) was blinded with regard to dose and time of adminis­
tration.
expressed per cross-sectional area (in kilograms per square
centimeter), and Pt/P0was calculated for each muscle bundle.
Statistics
The data obtained from the two diaphragm strips of each
rat were averaged and used for further analysis. In protocol
A, two-way analysis of variance (ANOVA) was performed
using dose and time of administration as separate factors to
examine salbutamol treatment and time of dosing effects and
possible interaction between these factors. Interaction be­
tween these factors was not significant for any parameter.
Therefore, no linear contrast analysis was performed and
Contractile Properties
main effects of each factor were considered not to be con­
The rats were anesthetized with pentobarbital sodium (70 founded by interaction. The significance of each main effect is
mg/kg ip). A tracheotomy was performed, and a polyethylene reported.
canula was inserted. The animals were mechanically venti­
In protocol B , on e-way ANOVA was performed to determine
lated with a gas mixture of 95% 0 2-5% CO2 (flow of 0.5 m l‘g differences between separate concentrations using Duncan’s
body wt”1■min "1; respiration frequency of 70 breaths/min).
multiple-range test. To evaluate force-frequency characteris­
The diaphragm was quickly cut from the ribs after com­ tics, a two-way ANOVA was performed using dose and
bined laparotomy and thoracotomy and was immediately stimulation frequency as separate factors to assess interac­
submersed in cooled oxygenated Krebs solution at a pH of 7.4. tion between these factors. Pearson’s correlation coefficients
This Krebs solution consisted of (in mM) 137 NaCl, 4 KC1, 2.7 between salbutamol concentration and Pt, P0, and force
CaCl2 , 2 MgCl2 , 1 KH2PO4 , 24 NaHCC>3 , and 7 glucose and 25 generation at 2 5-Hz stimulation frequency were computed,
]uMD-tubocurarine (Sigma Chemical, The Netherlands). From Finally, the salbutamol effects in protocols A and B were
the middle lateral costal region of each hemidiaphragm, a compared using ¿-tests for separate ranges of salbutamol
rectangular bundle was dissected parallel to the long axis of concentrations.
the muscle fibers. Silk sutures were tied firmly to both ends of
The limit of statistical significance was set at P < 0.05. All
the bundle. The bundles were suspended in two tissue baths analyses were performed with the SPSS/PC + package (v5.01)
containing Krebs solution, maintained at 37°C, and perfused (28). All values are means ± SE.
with a 95% 02-5% CO2 mixture. One end was connected to an
isometric force transducer (model 31/1437-10, Sensotec, Co­ RESULTS
lumbus, OH) mounted on a micrometer. Two platinum stimu­
lating electrodes were placed parallel to the bundles. The Salbutamol Concentrations
bundles were placed at their optimal length (L0), defined as
The
mean
serum
salbutamol
concentrations
reached
the length at which peak Pt was obtained, Stimuli were
in
protocol
A
are
presented
in
Fig.
1.
At
0.5
h
after
applied with a pulse duration of 0.2 ms and a train duration of
250 ms and were delivered by a stimulator (ID-electronics, injection, salbutamol concentrations could be mea­
University of Nijmegen, The Netherlands) activated by a sured in all treated rats. Doubling the administered
personal computer. To ensure supramaximal stimulation, the dose approximately doubled the serum concentration.
bundles were stimulated at '—20% above the voltage at which At later time points, the concentrations were either
maximal forces were obtained (~6 V over stimulating elec­ below the lower limit of detection (2 }ig/l) or blank. Only
trodes). Data acquisition and storage of the amplified signal at a dose of 100 ug/kg could salbutamol be detected 2 h
were performed by using a Dash-16 interface on a personal after injection. At 4 h after injection, no salbutamol
computer (Twist-trigger software, ID-electronics, University could be measured,
of Nijmegen).
In protocol A, the diaphragm strip dimensions at L 0 were
— 50- 1
18.02 ± 0.18 X 1.59 ± 0.03 X 0.42 ± 0.01 (SE) mm (length X
_1
width X thickness) and the weight was 24,2 ± 0.6 mg. In
Ui
a.
protocol B , these dimensions were 16.67 ± 0,38 X 1.90 ±
40
0.07 X 0.59 ± 0.01 mm and their mean weight was 28.2 ±
c:
O
1.0 mg. After a thermo equilibration period of 15 min, the
fa
following measurements were performed.
+3 30 c
Twitch characteristics . Two twitches were recorded at L 0 to
a>
determine maximal Pt) contraction time (CT) (time to peak
o
c
Pt), and ha If-relaxation time (RTy2). Average values were
8
20 used for further analysis (7).
"o
M axim al tetanic force (P0). The bundles were stimulated
£
twice at 160 Hz to obtain a maximal plateau in force
5 to
generation. The maximal force was defined as the PQ.
£
Force-frequency characteristics . The bundles were stimu­
<0
CO
o _
lated at 2-min intervals at frequencies of 25, 50, 80,120, and
r
1
----------------1
---------------1
—
-------------1
160 Hz.
0
1
2
3
4
After these measurements* length* thickness, and width of
each muscle bundle were measured at L0. The bundles were
T im e a fte r adm inistration
weighed, and the cross-sectional area was calculated by Fig. 1. Salbutamol concentration curves (protocol A). Values are
dividing diaphragm strip weight (in grams) by strip length (in means ± SE. Time is in hours. O, Dose of 12.5 pg/kg sc; • , 25 P-gA^gsc;
centimeters) times specific density (1.056). Pt and P0 were □, 50 pg/kg sc; ■, 100 pg/kg sc.
-
1105
P2-ADRENERGIC STIMULATION OF RAT DIAPHRAGM
Table 1. Protocol A (subcutaneous administration o f salbutanol): Pt
Time After Subcutaneous Administration
Treatment
0.5 h
lh
2h
4h
Saline
Salbutamol, pg/kg
12.5
25
50
100
0.441 ±0.037
0.420 ±0.042
0.477 ±0.041
0.431 ±0.072
0.553 ±0.050
0.588 ±0.038
0.609 ±0.057
0.735 ±0.025
0.597 ±0.066
0.635 ±0.044
0.681 ±0.082
0.684 ±0.074
0.515 ±0.027
0.541 ±0.040
0.631 ±0.083
0.688 ±0.035
0.520 ±0.025
0.526 ±0.041
0.558 ±0.038
0.594 ±0.067
Values are means ± SE expressed as twitch force (Pt) in kilograms per square centimeter. Significant difference between treatments is found
at P < 0.001 using 2-way analysis of variance (ANOVA). There are no significant differences between times of salbutamol administration.
In protocol B, the in vitro salbutamol concentrations
were below the lower limit of detection in four out of five
samples at 0.01 pM. In the other groups, salbutamol
concentrations of 10.1 ± 0.8, 22.2 ± 1.6, and 78.3 ±
4.0 jig/1 were measured.
Significant dose
effects were found for force generation at stimulation
frequencies of 25 (P < 0,001), 50 (P < 0.01), and 80 Hz
(P < 0.05). No significant effects of time of administra­
tion or interaction were found. Further two-way ANOVÀ
of force-frequency characteristics, with salbutamol dose
Contractile Properties
and stimulation frequency as factors, showed signifi­
Protocol A. TWITCH characteristics a n d P0. Salbuta­ cant effects of salbutamol treatment (P < 0.01 at 0.5, 2,
mol increased Pt in a dose-dependent fashion (P < and 4 h and P < 0.05 at 1 h after administration). No
0.001). No significant effects of time of administration interaction was present between stimulation frequency
were found, and no interaction between these factors and dose, suggesting that force generation was influ­
was found. The effects of salbutamol treatment were enced by salbutamol in a similar way at all stimulation
most pronounced at 0.5 and 1 h after administration frequencies studied. The force-frequency curves ob­
(Table 1). For CT, significant effects were found for dose tained 0.5 h after subcutaneous administration are
(P < 0.01) and time of administration (P < 0.001). The shown in Fig. 2,
Protocol B. twitch characteristics a n d P0. Pt in­
values ranged from 27.6 ± 0.5 to 31,7 ± 0.8 ms. CT was
increased at 2 h after administration in all salbutamol creased in the presence of salbutamol in a concentrationgroups except the 50 pg/kg group. RT1/2 ranged from dependent fashion (Table 3). This effect was significant
22.0 ± 0.9 to 26.7 ± 1.4 ms and was not changed by at a concentration of ~10 jig/1 and higher. CT was
increased at all concentrations; RT1/2 did not change at
salbutamol.
A significant effect of dose on P0was found (P < 0.01). any concentration studied. The values ranged from
P0 increased in a dose-dependent way in all groups 23.8 ± 1.1 to 25.8 ± 0.7 ms. P0 was increased by
(Table 2). No effect of time of administration or interac­ salbutamol in concentrations of ~10 pg/1 or higher;
tion between these factors was observed. For Pt/Pos Pt/Po was not changed.
force - frequency characteristics . Force generation
significant effects of dose and time of administration
(both P < 0.001) were found without interaction. Pt/P0 was increased at all stimulation frequencies (Fig. 3).
values ranged from 0.233 ± 0.009 to 0.310 ± 0.006. This effect was concentration dependent. In a salbuta­
Greatest increases in Pt/P0 were observed 0.5 and 1 h mol concentration of —80 pg/1, significant increases in
after administration of salbutamol. At 4 h after admin­ force production were observed at all stimulation fre­
istration, no effects of salbutamol treatment were found, quencies used. At 25'Hz stimulation, force generation
which is in agreement with the absence of salbutamol was also significantly increased at —10 pg/1 salbutamol.
in serum (Fig. 1).
Two-way ANOVA of force-frequency characteristics with
force - frequency characteristics .
Table 2. Protocol A (subcutaneous administration o f salbutamol): P0
Time After Subcutaneous Administration
Treatment
0 .5 h
lh
2h
4 h
Saline
Salbutamol, pg/kg
12.5
25
50
100
1.885 ±0.130
1.815 ±0.213
1.898 ±0.147
1.810 ±0.187
2.056 ±0.155
2.004±0.197
2.114±0.236
2.375 ±0.112
2.166± 0.231
2.314± 0.093
2.292 ±0.264
2.320 ±0.205
2.051 ±0.092
2.039 ±0.115
2.448 ±0.252
2.448 ±0.153
2.198 ±0.171
2.158 ±0.178
2.393 ±0.202
2.355 ±0.223
Values are means ± SE expressed as maximal tetanic force (P0) in kilograms per square centimeter. Significant difference between
treatments is found at P < 0.01 using 2-way ANOVA. There are no significant differences between times of salbutamol administration.
Pa-ADRENERGIC STIMULATION OF RAT DIAPHRAGM
1106
2.5
-
2.0 -
2.0
CM
I
E
o
ó>
CD
O
w*
-
CM
£
o
OÏ
¿SC
<D
u
1.5 “
1.0
-
1.5 -
1.0
~
o
o
LL.
LL.
0.5 -
0
0.5 -
“ T~
-
1
25
50
0
80
120
160
1
Stimulation frequency (Hz)
50
25
80
120
160
Stimulation frequency (Hz)
Fig. 2. Force-frequency characteristics 0.5 h after subcutaneous
administration of salbutamol. Values are means ± SE. O, Salinetreated animals; • , 12.5 pg/kg; □, 25 pg/kg; ■, 50 pg/kg; A, 100 pg/kg.
Significant difference between treatments is found at P < 0.01 using
dose and stimulation frequency as factors in 2-way analysis of
variance (ANOVA). * Significant difference in treatments for Pt and at
stimulation frequencies of 25 (P < 0.001), 50 (P < 0.01), and 80 (P <
0.05) Hz using time and dose as factors in 2-way ANOVA. No
significant effects of time of administration were found.
salbutamol concentration and stimulation frequency as
factors showed significant effects of salbutamol concen­
tration (P < 0.001). No interaction was found between
stimulation frequency and concentration, suggesting
that force generation was influenced by salbutamol in a
similar way at all stimulation frequencies studied.
Comparison between protocols A and B. Pearson’s
correlation coefficients between salbutamol concentra­
tion and contractile properties (Pt3 P03 and force genera­
tion at 25 Hz) are summarized in Table 4. Significant
correlations for all these parameters were found in
protocol B and at 0.5 h after injection in protocol A. In
the overall analysis in protocol A, only Pt showed a
significant correlation with salbutamol concentration.
At 1 and 2 h after injection, the correlation coefficients
were reduced because the number of samples in which
salbutamol could be detected was reduced.
In Figs. 4 and 5, the effects of specific ranges of
salbutamol are compared between protocols A and 5.
These ranges were set at the lower limit of detection
(2 pg/1) and the upper limit of the therapeutical concen­
tration after oral administration (20 pg/L). In the salinetreated animals, no significant differences in Pt and P0
between both protocols were observed. In the bundles
exposed to salbutamol with concentrations <2 p.g/1, Pt
Fig. 3. Force-frequency characteristics: strictly in vitro effects of
salbutamol (protocol B ). Values are means ± SE. O, Saline control
animals; • , ^2 pg/1; □, ~10 pg/1; ■, ~20 pg/1; A , -80 pg/1.
Significantly different (P < 0.05) using 1-way ANOVA compared with:
* saline; v ^2 pg/1 salbutamol.
and P0 were significantly higher in the in vivo experi­
ment {protocol A). Increases in force generation were
similar at salbutamol concentrations of 2-20 pg/1, but
at concentrations >20 pg/1 group Pt and P0 were again
significantly higher in the in vivo experiment (protocol
A\
D ISCU SSIO N
The present study shows that salbutamol improves
in vitro force generation in rat diaphragm bundles.
Increases in force generation were most prominent
within 2 h after subcutaneous administration and were
dose dependent. Increases in Pt and Pt/P0were found at
clinically relevant salbutamol concentrations. Compa­
rable results were found both after subcutaneous injec­
tion and in a strictly in vitro experiment, although
increases in force generation were highest after subcu­
taneous administration at salbutamol concentrations
<2 pg/1 and >20 pgA.
Based on body weight and systemic availability, a
dose of —25 pg/kg sc used in protocol A is comparable to
a single oral dose of 4 mg for a person weighing 70 kg
(57 pg/kg), since absolute systemic availability in hu­
mans is —50% after oral administration due to first
pass elimination in the intestinal wall (24). This dose
resulted in a plasma concentration of —10-20 pg/1 in
Table 3. Protocol B : strictly in vitro effects of salbutamol on contractile properties
Treatment
Saline
Salbutamol, pg/1
<2
10
20
80
Pt) kg/cm2
CT, ms
Pu, kg/cm“
Pt/P*
0.424 ±0.027
25.6 ± 1.1
1.568 ±0.047
0.270 ±0.011
0.400 ±0.028
0.538 ±0.032*+
0.534±0.059*+
0.611 ±0.026*+
28.8 ±1.0*
28.6 ± 0.8*
29.3 ±0.5*
28.9 ±1.2*
1.491 ±0.110
1.897 ±0.102*1
1.966 ±0.172*t
2.096 ±0.098*t
0.271 ±0.009
0.285 ±0.011
0.271 ±0.012
0.293 ±0.009
Values are means ± SE. CT, contraction time. Significantly different (P<0.05) using 1-way ANOVA compared with: *saline; t ^ 2 pg/l
salbutamol.
1107
p2 -ADRENERGIC STIMULATION OF RAT DIAPHRAGM
Table 4. Pearsons correlation coefficients
between salbutamol concentrations
and contractile properties in protocols A and B
11
r
P
Protocol B (in vitro) 25 0.60 <0.01
Protocol A (in vivo) 100 0.41 <0.001
0.5 h
25 0.68 <0.001
lh
25 0.36
NS
2h
25 0.38
NS
Po
**
P
r
P
0.56 <0.01 0.59 <0.01
0.14 NS 0.22 NS
0.40
0.05 0.54 <0.01
0.22 NS 0.21 NS
0.17 NS 0.42 <0.05
n ~ no. of rats. FF, force frequency; NS, not significant.
*
I---- 1
2.5 -
FF 25 Hz
Po
Pt
3.0 -j
\
E
2.0 -
o
b>
1.5 -
O
CL
1.0 -
Jag
X
0.5 -
0 J
2-20
>20
< 2
humans (10), a range usually regarded as the therapeu­
saline
tic range for bronchodilation (18). In the present experi­
Salbutamol concentration (fJg/L)
ment, a dose of 25 pgAtg sc (serum concentration
9.3 p.g/1) and the concentration of —10 pgA used in Fig. 5. Relationship between salbutamol concentration range and
maximal tetanic force (Pu). Values are means ± SE, Open bars, values
protocol B were within this therapeutic range.
from protocol B; solid bars, values from protocol A . Significantly
Significant effects of time of administration were different using Student’s f-test at: *P < 0.05; !|::i:P < 0.01.
only found for CT and Pt/P0- This increase in CT was
manifest at 2 h after injection. Pt/P0 increase was most
pronounced 0.5 and 1 h after injection. At 4 h after activation in the rat diaphragm coincides with the
injection no increase was noted, which was in agree­ maximal concentration reached in this muscle, which
ment with the absence of salbutamol in serum. For explains the time pattern found in Pt/P0.
The forces obtained in the saline bundles in protocol
other parameters, no significant time effects were
found. The effects on Pt generation found in this study B were slightly lower than those obtained in protocol A,
had a rapid onset of action and were most pronounced although these differences were not statistically signifi­
up to 2 h after subcutaneous administration. There are cant (see Figs. 4 and 5). This may raise concerns about
only limited data available on salbutamol pharmacoki­ the stability of the diaphragm strips during the data
netics in rats. After oral administration, salbutamol is collection. When we examined the data in protocols A
rapidly absorbed and maximal plasma concentration is and B and expressed the force produced in the final
reached within 1-2 h in rats (21). Maximal peripheral 160-Hz stimulation in the force-frequency protocol as a
skeletal muscle concentration was reached at —1—2 h percentage of initial P0, the saline-treated rats reached
after oral administration in rats (21), In the present levels of 100-103% of P0. In the salbutamol-treated
study, the elimination half-life after subcutaneous ad­ animals, these percentages were slightly reduced, e.g.,
ministration appears to be ~0.5 h, The duration of in the 100 jigA^g group these values were 85-102% and
effects in our study is ~2 h, which is in agreement with in protocol B they were 91-103%. This indicates that at
the salbutamol concentrations measured. This could the end of all measurements the diaphragm strip force
indicate that the maximal effect of pa-adrenoceptor production was approximately equal to PQvalues mea­
sured at the start, reensuring the stability of the
diaphragm strips. Furthermore, the diaphragm strip
1.0
dimensions in both protocols were within the critical
radius for 0 2 diffusion into skeletal muscles at 37°C
**
I---- 1
0.8 ~
(—0.6 mm) (31). Therefore, we assume that hypoxia of
the diaphragm strips did not influence force genera­
eIg
tion.
E 0.6
No significant differences in Pt and P0 between the
o
o>
two protocols were found at salbutamol concentrations
of 2—20 pgA. However, at concentrations >20 pgA Pt
0.4 CL
and P0 were significantly higher in the in vivo experi­
ment {protocol A). Also, when we selected the data only
0.2 on salbutamol zero concentrations in the salbutamoltreated animals, Pt and P0 were both significantly
higher in the in vivo experiment. These data, however,
0
saline
<2
2-20
include the serum samples of rats that had salbutamol
>20
concentrations below the limit of detection (0-2 pgA),
Salbutamol concentration (pg/L)
which may have influenced the results. It could also
Fig. 4. Relationship between salbutamol concentration range and indicate that the effect of salbutamol on rat diaphragm
twitch force (Pt). Values are means ± SE. Open bars, values from
contractile
properties
is
not
solely
a
direct
(32-adrenerprotocol B (strictly in vitro experiment); solid bars, values from
protocol A (subcutaneous salbutamol administration).
Signifi­ gic effect on the muscle but is also be mediated by
indirect effects. Subcutaneous administration of salbucantly different at P < 0.01 using Student’s ¿-test.
-
-
1108
(VADRENERGIC STIMULATION OF RAT DIAPHRAGM
tamol may stimulate cardiac p2-adrenoceptors, which
may increase heart rate (26). In addition, activation of
p2-adrenoceptors in skeletal muscle resistance arteri­
oles may dilate diaphragm vessels (20). These effects
may lead to an increased diaphragm blood flow, as was
reported in rats after subcutaneous administration of
the p2-adrenoceptor agonist clenbuterol (30). These
alterations may explain the additional effects found in
vivo but were not specifically explored in the present
study.
Data on the diffusion rate of salbutamol into dia­
phragm muscle strips are not available to our knowl­
edge. In a pilot study, we found that an increase in Pt
was present after a few minutes and that the maximum
effect was reached within 10 min after addition of
salbutamol. The maximal effect of the p2-adrenoceptor
agonist terbutaline was also found within 10-15 min in
a study investigating its in vitro effects on skeletal
muscle contractile properties (5). Therefore, the thermo­
equilibration and diffusion period was set at 15 min in
both protocols. In protocol A, there may have been some
back diffusion from the diaphragm strip into the Krebs
solution, since salbutamol is hydrophilic and rapidly
dissociates from p2-adrenoceptors in rat lung prepara­
tions (27). We did not try to prevent this back diffusion
by adding salbutamol to the Krebs solution because
data on the concentration reached in the rat diaphragm
after subcutaneous administration are not available.
Besides, addition of salbutamol in protocol A would
interfere with our goal to study the mechanism of
action.
Catecholamines exert opposing effects on fast- and
slow-contracting skeletal muscles in vivo (1, 4). In cat
soleus muscle, which contains predominantly type I
(slow-contracting) muscle fibers, intravenously admin­
istered adrenaline depressed Pt? CT, RT1/2, and incom­
plete tetanic force but did not alter P0. However, in
tibialis anterior muscle, a type II (fast-contracting)
muscle, Pt, CT, RTi/2, and incomplete tetanic force were
increased (4). In guinea pig soleus and extensor digitorum longus muscles, similar effects were found with
salbutamol and isoprenaline in vitro (1). A different
response was observed in a study on the effects of
terbutaline on skeletal muscles (5). This in vitro study
reported an increase in Pt both in slow and in fast
skeletal muscle fibers after addition of 10-5 M terbuta­
line. P0 was increased in slow but not in fast muscle
fibers, and BT^ was decreased in both muscle types (5).
Increased force production and CT were also found in
the present study. RTi/2 was not altered in our study,
but force generation at low stimulation frequencies was
increased significantly This indicates that summation
of force was enhanced. These effects could indicate an
increased activation of type II muscle fibers in rat
diaphragm. The diaphragm is a mixed skeletal muscle
and contains -40% type I, 30% type Ha, and 30% type
lib fibers (7). In studies investigating myosin heavy
chain (MHC) distribution, the mature rat diaphragm
consisted of -30% MHC-slow, -30% MHC-2A, -30%
MHC-2X, and —10% MHC-2B fibers (15). In rat skel­
etal muscles, a strong correlation was found between
p-adrenoceptor density and percentage of type I muscle
fibers (22) and for p-adrenoceptor density and succinic
dehydrogenase activity (37). In contrast, 12,i5I-labeled
cyanopindolol-binding affinity was significantly higher ^
in type lib muscle fibers (22). In view of the results
found in the present study, we speculate that p2-agonist
treatment predominantly affects type II fibers in the
rat diaphragm, and thus the diaphragm could be
considered as a fast skeletal muscle in this respect
The design of the present study does not allow a
direct comparison with previous animal studies (2,11,
12, 29). In these in vivo studies in anesthetized dogs,
salbutamol increased twitch Pdi only during compen­
sated metabolic acidosis (12). In fatigued diaphragm,
Pdi was not improved (11, 29). Salbutamol was applied
intravenously in a bolus of 2.5-20 pg/kg (29) or as a
15-min intravenous infusion in a relatively high dose of
—0.6-2.2 pg *kg"1*min“1 (recommended human dose is
0.04-0.3 pg’kg^'m in"1 iv) (11, 12). However, terbuta­
line, in a dose of —25 ]ig/kg iv administered in 5 min,
did increase Pdi in fatigued but not in fresh canine
diaphragm both after direct and phrenic nerve stimula­
tion (2). This effect was blocked by the p-adrenoceptor
antagonist propranolol (2). Likewise, fenoterol and
broxaterol potentiated Pdi in fatigued but not in fresh
canine diaphragm (8, 34). In fresh diaphragm, predomi­
nantly slow motor units are recruited for normal venti­
latory maneuvers (32). In this situation, the lack of
effect of p-agonist treatment is in line with our specula­
tion that the p-agonist treatment predominantly af­
fects type II muscle fibers. Our results might also
suggest improvement of diaphragm contractility in
fatigued diaphragm after p2-adrenoceptor agonist treat­
ment, as indeed was found for terbutaline (2) and
fenoterol (34). However, salbutamol increased Pdi only
during compensated metabolic acidosis (12) and not in
fatigued canine diaphragm (11, 29). Why salbutamol
treatment failed in this respect remains unclear.
The effects of salbutamol on diaphragm function in
healthy humans are contradicting. Javaheri et al. (14)
found no effects of salbutamol on maximal Pdi in fresh
or fatigued diaphragm after 3 days of oral treatment (4
mg 3 times/day). Violante et al. (35) reported that
intravenous administration of —0.05 pg* kg'1*min-1
had no effect on Plmax and ventilatory endurance, but
the duration of treatment was not mentioned (35). In
contrast, Martineau et al. (23) reported a significant
increase in Piraax after 14—21 days of oral treatment
(8 mg 2 times/day). In studies investigating other (32adrenoceptor agonist effects on nonfatigued diaphragm,
similar effects are reported. In patients with chronic
obstructive pulmonary disease (COPD), oral terbuta­
line administration (2.5 mg 3 times/day for 7 days) (33)
as well as terbutaline inhalation (7 days 500-1,500 yig
4 times/day) (13) did not improve Pdi (33) or Piinax (A
33). In healthy adults, a single oral dose of 7.5 or 15 mg
terbutaline (n = 4) or 4 mg tulobuterol (n = 5) also did
not improve Pimax (17). Finally, studies with broxaterol
in COPD patients reported an increase in Pimax after
intravenous administration (200 pg in 10 min) (9) but
no increase in Pinmx after 7 days of oral treatment
p2“ADRENERGIC STIMULATION OF RAT DIAPHRAGM
(0.5 mg 3 times/day) (25), However, this last study did
report an increased endurance time (25). In contrast to
the animal studies, all human studies used maximal
voluntary contractions. Furthermore, the human dia­
phragm has a slightly different fiber type composition,
containing 55 ± 5% type I, 21 ± 6 % type Ila, and 24 ±
3% type lib muscle fibers (19), but the total amount of
oxidative fibers (type I and Ila) is approximately equal
(~70% in rats and ~75% in humans) (7, 19), Further
research is needed to determine the effects of salbuta­
mol on the human diaphragm in patients with COPD
and in the complex situation of respiratory muscle
failure.
In conclusion, in vitro force generation of rat dia­
phragm bundles can be improved by the (Vadrenergic
agonist salbutamol. These effects are present after low
subcutaneous doses and in clinically relevant concentra­
tions in vitro and appear to be concentration depen­
dent. Overall, the increases in force generation were
slightly higher after subcutaneous administration, sug­
gesting that in vivo salbutamol may have additional
positive inotropic actions on diaphragm contractility
besides a direct (32-adrenergic effect on the muscle
itself.
The authors thank Y. Brom for expert biotechnical assistance.
This study was supported by a grant from Glaxo-Wellcome BV, The
Netherlands.
Address for reprint requests: H. F. M. Van Der Heijden, Dept, of
Pulmonary Diseases, Univ. Hospital Nijmegen, PO Box 9101, 6500
HB Nijmegen, The Netherlands.
Received 4 April 1995; accepted in final form 2 April 1996.
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