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Introduction
High K+-induced contraction is often used to examine the smooth muscle contraction function, to standardize the
receptor-mediated contraction, and to determine the optimal resting tension in isolated blood vessel
study[1_10]. There are 2 commonly used techniques to produce high
K+: hyperosmolar addition of KCl (hypertonic
K+) and iso-osmolar substitution of NaCl with KCl (isotonic
K+)[2]. It has been reported that
Ca2+ channel blockers may have differential relaxation effects on
hypertonic and isotonic K+-induced
contractions[7]. Moreover, direct observations suggest different contractions induced
by hypertonic and isotonic K+ in rat
aortas[2] and middle cerebral
arteries[3]. However, it is unknown the differences between
contractions induced by hypertonic and isotonic
K+ at different resting tensions, and whether the differences may lead to
different values of optimal resting tension measured by
hypertonic and isotonic K+. Therefore, the current study
compared the contractions and optimal resting tensions
determined by 2 different high K+ solutions at increasing resting
tensions. Two different kinds of aortic preparations, in which
perivascular adventitial fat was either removed or left
intact[5] from 3 different ages of rats (15, 25, and 62 weeks) were
used for the com-parisons.
Materials and methods
Animals Male Sprague-Dawley rats were purchased from
the Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China)
at the age of 6 weeks and brought up in our animal house
until the age for experiments (15, 25, and 62 weeks,
respec-tively). They were housed in controlled temperature (23_25
°C) and lighting (8:00_20:00), and with free access to tap water
and rat chow. All experimental procedures were performed
in accordance with institutional guidelines for animal care.
Solutions Krebs-Henseleit solution was made using the
following composition (in mmol/L): NaCl 118.4, KCl 4.7,
CaCl2 2.5, MgSO4 1.2,
KH2PO4 1.2,
NaHCO3 25.0, glucose 11.1, and
CaNa2-EDTA 0.026. Isotonic solution of high
K+ (30 mmol/L) was made by iso-osmolar substitution of NaCl with KCl for
Krebs-Henseleit solution and therefore it contained the
following composition (in mmol/L): NaCl 93.1, KCl 30.0,
CaCl2 2.5, MgSO4 1.2,
KH2PO4 1.2,
NaHCO3 25.0, glucose 11.1, and
CaNa2-EDTA 0.026.
Aortic preparations The rat was anesthetized with
sodium pentobarbital (60 mg/kg, ip). After opening the
thoracic cavity, the descending thoracic aorta was immediately
removed into cold Krebs-Henseleit solution aerated with 95%
O2 and 5% CO2, and dissected into 5 mm-wide rings using a
ruler. Four aortic rings were obtained from each rat to make
2 kinds of aortic preparations in which adventitial fat was
either removed or left intact.
Determination of high K+-induced contractions at
increasing resting tensions The aortic rings were suspended
in conventional organ baths filled with 20 mL Krebs-Henseleit
solution maintained at 37 °C and continuously aerated with
95% O2 and 5% CO2. Changes in isometric tension were
recorded with IT1-25 transducers and IOX computerized
system (EMKA Technologies, Paris,
France)[6]. Each aortic ring was allowed to equilibrate for 60 min at a resting tension
of 1.0 g and was then exposed to high
K+ (30 mmol/L). When the high
K+-induced contraction reached a plateau, the rings
were rinsed with Krebs-Henseleit solution and allowed to
re-equilibrate for 30 min at an increased resting tension of
1.5 g. Thereafter, the addition of high
K+ (30 mmol/L), the recording of isometric contraction, and the washing with
Krebs-Henseleit solution were repeated as described
earlier. Increasingly, the resting tension was adjusted to 2.0,
2.5, 3.0, or 3.5 g. At each resting tension, the maximal
contraction induced by high K+ (30 mmol/L) was measured
(Figure 1). The optimal resting tension was obtained from
the resting tension-contraction curve for each aorta and
defined as the resting tension at which the aorta showed a
maximal contraction[4].
In the present study, high K+ (30 mmol/L)-induced
contraction was produced by 2 commonly used techniques in
the separate experiments: (i) hypertonic
K+ (30 mmol/L)-induced contraction was produced by adding KCl to organ
bath containing Krebs-Henseleit solution, the total
concentration of KCl in the bath was 30 mmol/L; and (ii) isotonic
K+ (30 mmol/L)-induced contraction was produced by
replacing Krebs-Henseleit solution in organ bath with isotonic
solution of high K+.
To examine a possible mechanical perturbation caused
by the replacement of solution, we compared the tensions
obtained before and after the change of Krebs-Henseleit
solution using the same time course at the different resting
tensions mentioned earlier. It was found that the change of
solution did not affect the tensions (data not shown).
Statistical analysis Data are reported as means±SEM.
Statistical analysis was performed with ANOVA followed by
two-tailed Student's unpaired t-test. The threshold for
statistical significance was P<0.05.
Results
Rats at 3 different ages (15, 25, and 62 weeks) were used for
the present study; body weight increased with age (Table 1).
The aortic contractions induced by 2 kinds of high
K+ were significantly different in all of the age groups examined
(Figure 2). The different responses to hypertonic and
isotonic K+ were age related. At the age of 15 weeks, isotonic
K+-induced contractions were either greater or tended to be
greater at almost all resting tensions when compared with
The representative original recordings are shown in Figure 3.
Discussion
The main finding of the present study is that there exists
big differences between aortic contractions induced by
hypertonic and isotonic K+ at different resting tensions, and
these differences make the optimal resting tension values
inconsistent when measured by the 2 kinds of high
K+.
The mechanisms for the different responses to
hypertonic and isotonic K+ seem complex. It is well established
that in most smooth muscle, high K+ evokes contraction via
an elevation of cytosolic Ca2+ through the
dihydropyridine-sensitive Ca2+ channel (L-type
Ca2+ channel)[2]. Recent
studies show that high K+ can also cause
Ca2+ sensitization involving the translocation and activation of the RhoA
kinase[8]. In contrast to isotonic
K+, hypertonic K+ causes contraction
not only via the L-type Ca2+ channel, but also by
hyperosmotic action as well as membrane enzyme activation.
For osmotic action, it is well known that alterations in
osmolarity may affect muscle cell volume and membrane
potential[11,12]. Both hyperosmotic and hyposmotic solutions can
change vascular tone. For example, hyperosmotic solution
induced contractions in rat and guinea-pig
aortas[2] and dilation in rat middle cerebral
arteries[3] and skeletal muscle
arteries[13]. Hyposmotic solution induced contractions in rat
tail arteries[14]. For enzymatic action, one study demonstrated
that the dihydropyridine-insensitive component of
hypertonic K+-induced contraction in
Ca2+-free medium was sensitive to protein kinase C inhibitors, H7 and calphostin
C[2]. The results suggested the activation of
Ca2+-independent isozyme of protein kinase C was involved in the hypertonic
K+-induced contraction[2,15]. In addition, it should be noted
whether the factor of low Na+ in the isotonic
K+ solution may influence its effect. Previous studies showed that in rat
aortas without endothelium, increasing
Na+ by the addition of 1_30 mmol/L NaCl to Krebs' solution did not affect the
resting tone, but it changed the phenylephrine-induced
contraction. Increasing Na+ by the addition of 1, 3, and 6
mmol/L NaCl increased the phenylephrine-induced contraction, whereas further increasing
Na+ by the addition of 10, 20, and 30 mmol/L NaCl inhibited the
phenylephrine-induced contraction[16].
Simultaneous Mg2+ withdrawal and
Na+ reduction (to 84 mmol/L) by replacement of NaCl with
isosmolar amounts of sucrose in normal Krebs-Ringer
bicarbonate induced significant increases of basal tone of
denuded rat aortic rings[17]. The reduction of both
Na+ and Cl_ caused hyperpolarization of hamster aortic endothelial cells,
indicating the essential role of endothelial cells in mediating
vascular functions[18]. According to our own results in the
present study, we can not make any conclusions for the
effect of low Na+ on the isotonic
K+-induced contractility. Further experiments need to be performed in the future.
The 2 kinds of high K+ examined in the present study are
commonly used in vascular
studies[1_10,19,20]. Some laboratories usually use hypertonic
K+, which is very easy to produce and manipulate, and others use isotonic
K+, a relatively complicated technique. It should be kept in mind when one
reads the literature reports that hypertonic and isotonic
K+ may induce different results. The previous data and the
present study indicate that isotonic K+ is superior to
hypertonic K+ for most of vascular studies, unless hypertonic
K+ is absolutely necessary for the experiments; for instance,
both high K+ and high osmolarity are needed to mimic the
pathological conditions after traumatic brain
injury[3]. The viewpoint that isotonic
K+ is superior to hypertonic
K+ for most vascular studies is based on the following 3 reasons.
First, isotonic/iso-osmotic solution tallies with the actual
physiological situation. Second, the mechanisms for
isotonic K+-induced contraction are relatively simple compared
with those for hypertonic K+-induced contraction. Third,
the optimal resting tensions measured by isotonic
K+ are almost identical. They are a constant of 2 g in different kinds
of aortic preparations from various age rats. This point is
very important for the comparisons under the same
condition of resting tension in vascular study. In contrast, the
variable values of optimal resting tensions measured by
hypertonic K+ mean that different resting tensions should be
used for various rat aortas, possibly causing inappropriate
results.
We measured high K+-induced contraction at different
resting tensions in aortic rings from 15- to 62-week-old rats.
The resting tension-contraction curve for isotonic
K+ was downshifted with age, while the pattern of the curves was
similar and the value of the measured optimal resting
tensions was almost the same in the different age groups. These
results indicate that the ability of the contraction was easily
affected by the factor of age, while the optimal resting tension,
a property of muscle cell, was relatively constant, at least in
the examined age range from 15 to 62 weeks.
The present study provides detailed data on high
K+-induced contraction and optimal resting tension not only in
a wide range of rat age from 15 to 62 weeks, but also in 2
different kinds of aortic preparations in which adventitial fat
is either removed or left intact. This is because in most
previous isolated artery studies, the adventitial fat is removed
routinely and this manipulation neglects the potential role of
adventitial fat. However, since 2002, several studies have
reported the role of adventitial fat in the regulation of arterial
tone in rat aortas[5,21,22] and mesenteric
arteries[23] as well as in human internal thoracic
arteries[24]. The effects of adventitial fat on high
K+-induced contraction should be evaluated in detail. Our results showed that the adventitial fat
could delay the development of high K+-induced
contractions at different resting tensions, but had little effect on the
maximal contractions in response to high
K+.
Over the last 25 years, the importance of endothelium in
the regulation of vascular tone has become
apparent[25_27]. It is well known that the intimal endothelial cells can produce
and release a variety of vasoactive substances to modulate
medial smooth muscle contraction and relaxation. The
endothelium has inhibitory effect on high
K+-induced contraction. Basal release of endothelial factor(s), such as
NO from rat aortic endothelium, may have a relaxing effect
on smooth muscle, thus reducing the agonist-induced
contraction[6]. It is also reported that vascular smooth muscle
can influence endothelium through myo-endothelial
junctions (gap junctions)[26]. Increases in
Ca2+ in the vascular smooth (which occur with extracellular
K+ concentrations above 20-30 mmol/L) can be transmitted to the
endothelium via the myo-endothelial junctions. Increased
Ca2+ in the endothelium may stimulate the release of NO.
The potential role of adventitial fat in the regulation of
arterial tone was explored recently. Regarding the
interaction between adventitial fat and smooth muscle, some
studies including our unpublished experiments demonstrated that
adventitial fat could inhibit the contraction induced by
several agonists, such as serotonin, phenylephrine, angiotensin II,
and endothelin[5,21_24]. In addition, the bioassay experiments
revealed that adventitial fat could release a relaxing factor,
named as adventitium- or adipocyte-derived relaxing
factor (ADRF)[5,21]. Our results that adventitial fat had little
effect on the maximal contraction in response to high
K+ are consistent with the previous
data[5,23], indicating the inhibitory effect of adventitial fat on vascular contraction is
agonist specific. The slower development of high
K+-induced contraction in aortas with adventitial fat than in aortas
without adventitial fat was possibly due to the concomitant
secretion of ADRF from adventitial fat. The effect of ADRF is
dependent on opening of K+
channels[5,23] which initially suppressed the development of high
K+-induced contraction, but eventually this effect was abolished by high
concentration of extracellular K+[1].
In summary, our experiments provide new evidence about
differential effects of hypertonic and isotonic
K+ on aortic contraction. The optimal resting tensions in different kinds
of aortic preparations from various age rats are almost a
constant determined by isotonic K+, but a variable
determined by hypertonic K+. To our knowledge, this is the first
study to determine the optimal resting tensions in aortas
with adventitial fat, which is meaningful for the future work
on the complex interactions between vascular layers,
especially the study of the paracrine effects of adventitial fat on
vascular tone[28,29].
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