Differential modulation of caffeine- and
IP3-induced calcium release in
cultured arterial tissue
Karl
Dreja and
Per
Hellstrand
Department of Physiological Sciences, Lund University, S-223 62 Lund, Sweden
 |
ABSTRACT |
To investigate
the Ca2+-dependent plasticity of
sarcoplasmic reticulum (SR) function in vascular smooth muscle,
transient responses to agents releasing intracellular
Ca2+ by either ryanodine
(caffeine) or
D-myo-inositol
1,4,5-trisphosphate [IP3;
produced in response to norepinephrine (NE),
5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptors
in rat tail arterial rings were evaluated after 4 days of organ
culture. Force transients induced by all agents were increased compared
with those induced in fresh rings. Stimulation by 10% FCS
during culture further potentiated the force and
Ca2+ responses to caffeine (20 mM)
but not to NE (10 µM), 5-HT (10 µM), or AVP (0.1 µM). The effect
was persistent, and SR capacity was not altered after reversible
depletion of stores with cyclopiazonic acid. The effects of serum could
be mimicked by culture in depolarizing medium (30 mM
K+) and blocked by the addition
of verapamil (1 µM) or EGTA (1 mM) to the medium, lowering
intracellular Ca2+ concentration
([Ca2+]i)
during culture. These results show that modulation of SR function can
occur in vitro by a mechanism dependent on long-term levels of basal
[Ca2+]i
and involving ryanodine- but not
IP3 receptor-mediated
Ca2+
release.
sarcoplasmic reticulum; organ culture; tail artery; D-myo-inositol
1,4,5-trisphosphate
 |
INTRODUCTION |
THE SARCOPLASMIC RETICULUM (SR) participates in the
regulation of intracellular Ca2+
concentration
([Ca2+]i)
in smooth muscle. Two types of
Ca2+ release receptors have been
identified in the SR. One class (2, 6) is sensitive to
D-myo-inositol
1,4,5-trisphosphate (IP3), produced in response to agonists such as norepinephrine (NE), 5-hydroxytryptamine (5-HT; serotonin), and arginine vasopressin (AVP).
A second class (5, 11) of receptors is sensitive to caffeine and
ryanodine and appears to be involved in the regenerative control of
[Ca2+]i
via Ca2+-induced
Ca2+ release.
The functional role of the SR is probably related to its ability both
to release Ca2+ in response to
receptor activation and to accumulate
Ca2+ via its
Ca2+-activated ATPase activity. In
addition to raising intracellular Ca2+ levels in response to
appropriate stimuli, the SR may contribute to
Ca2+ elimination from the
cytoplasm by virtue of the release of
Ca2+ into a restricted
subplasmalemmal space, thus facilitating extrusion over the cell
membrane via
Na+/Ca2+
exchange (13, 18). Increased
[Ca2+]i
is expected to lead to increased
Ca2+ accumulation in the SR and,
if long-standing, possibly increased capacity of the SR to accumulate
and release Ca2+. Such effects
could contribute to the understanding of its functional role, because
the response to agents acting on the SR would be amplified.
Increased Ca2+ release from the SR
of smooth muscle has been described in animal models of hypertension
and bladder hypertrophy (12, 20). These changes could be hypothesized
to be due to persistent activation, leading to increased
[Ca2+]i
and thus increased load on the SR. To specifically
investigate the role of increased
[Ca2+]i
for this response, an in vitro culture approach would be useful, because this allows the control of experimental conditions to an extent
not attainable in vivo. Dispersed smooth muscle cells in culture
rapidly modulate from a contractile to a synthetic phenotype (3),
confounding the interpretation of results with respect to the
physiological mechanisms operating in intact tissue. Thus an in vitro
culture method preserving the contractile phenotype is needed. The
culture of whole vascular tissue, where smooth muscle cells are kept in
their normal extracellular matrix environment, preserves contractility
and structural integrity for periods of several days (4, 14, 22). In
cultured rat tail artery, we found that the presence of FCS causes
decreased contractility, which may primarily be a consequence of an
increased Ca2+ load and which can
be separated from the growth stimulation caused by FCS (14). In the
present study, the long-term effects of the
Ca2+ load on SR function in rat
tail arterial rings kept in culture for 4 days were investigated.
 |
MATERIALS AND METHODS |
Tissue preparation and culture.
Female Sprague-Dawley rats, weighing ~200 g, were killed by cervical
dislocation. The tail artery was dissected free under sterile
conditions and transferred to culture medium (DMEM/Ham's F-12; 1:1)
containing 1.08 mM CaCl2, 100 U/ml
penicillin, and 100 µg/ml streptomycin. Attached connective tissue
was removed, and the artery was cut into rings under a dissecting
microscope. The width and luminal diameter of the rings did not vary
substantially and were ~0.6 and 0.5 mm, respectively. Rings were
transferred to culture dishes containing the above medium with or
without FCS (10%). To some dishes verapamil (1 µM), EGTA (1 mM), or
KCl and CaCl2 (26 and 1.42 mM,
respectively) were added. The dishes were placed in a
water-jacketed cell incubator at 37°C under 5% CO2 in air.
Experimental procedure.
After 4 days in culture, the rings were bathed in a nominally
Ca2+-free Krebs solution of the
following composition (in mM): 4.7 KCl, 15.5 NaHCO3, 1.2 KH2PO4,
1.2 MgCl2, 11.5 glucose, and 122 NaCl. To remove the endothelium, a thin needle was passed through the
lumen of the ring. The rings were mounted on two stainless steel
wires (diameter 0.2 mm), one connected to a force
transducer (AE 801; SensoNor, Horten, Norway) and the other connected
to an adjustable support. The rings were stretched to a passive tension of 1.2 ± 0.1 mN/mm, which is close to the optimal preload for these
preparations. Rings were immersed in warm physiological salt solution
(PSS) containing (in mM) 135.5 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 11.6 HEPES, and 11.5 glucose. High-K+ (140 mM) solution
was prepared by replacing NaCl with KCl. The solution was contained in
0.4-ml Plexiglas cups fitted into thermostatted (37°C) metal
blocks. For Ca2+ measurements, a
125-µl chamber was perfused by solution at a rate of 1 ml/min. In all
experiments, the rings were allowed to equilibrate for at least 35 min
before being stimulated with
high-K+ solution. After 5 min, the
rings were transferred to
Ca2+-free PSS containing 1 mM
EGTA. After an additional 5 min,
Ca2+-releasing agents were added.
This protocol was repeated with another releasing agent after 15 min of
equilibration in normal PSS or ended with a maximal reference
contraction in a high-K+ solution
supplemented with 10 µM NE. In some experiments
Ca2+ release was also induced
without a preceding contraction in
high-K+ solution. The width of
each ring was measured at the end of the experiments with a dissecting
microscope equipped with an ocular scale. Wall tension was expressed in
units of millinewtons per millimeter by dividing peak force by twice
the ring width.
Ca2+
measurements.
After equilibration, rings were incubated for 90 min at 22°C with
the fluorescent probe fura 2-AM (13.3 µM; Molecular Probes, Eugene,
OR). The Ca2+ release protocol was
the same as that described above. EGTA-containing solution, caffeine,
and NE were applied through a pipette located 1 mm from the ring. This
was done to ensure rapid exposure to the agents necessary to resolve
Ca2+ transients but precluded
heating the applied solution. Therefore, after 30 min of equilibration
at 37°C, the rest of the experiment was performed at room
temperature (22°C). In situ calibration was performed at the end of
each experiment by permeabilization with ionomycin (50 µM) as
described by Himpens et al. (9). [Ca2+]i
was estimated from the ratio of fluorescence at 510 nm for excitation
at 340 and 380 nm, as described by Grynkiewicz et al. (8), with a value
of 224 nM for the dissociation constant of Ca2+ binding to fura 2.
The fluorescence and force measurements were carried out simultaneously
at a sampling frequency of 0.5 Hz, which was increased to 2 Hz for the
resolution of transients, by using an imaging system (IonOptix) mounted
on a Nikon TMD inverted microscope with a Nikon Fluor ×20
objective. To minimize the contribution of adventitial fibroblasts to
the recorded signal, the video image was focused to the medial layer,
45 to 55 µm beneath the adventitial border. This layer of the
arterial wall has a bundled appearance in normal light and does not
contain any fibroblasts (1, 19). Acquisitions were from a zone that
averaged 0.03 mm2. In most
experiments a control zone of the same size was also defined. When
compared, zones from the same experiment showed very small variation in
ratiometric values. The
[Ca2+]i
values obtained just before the induced contractions were subtracted from the ensuing peak value to give the increases in
Ca2+ concentration presented.
Statistics.
Summarized data are expressed as means ± SE. Student's
t-test was used to evaluate
statistical significance. For multiple comparisons, the
Bonferroni correction was used.
 |
RESULTS |
The effects of various
Ca2+-releasing agents on rat tail
arterial rings were tested after 4 days of culture. Identical
experiments were run on fresh preparations from the same animal.
Transient force responses were elicited by the addition of agonists
after incubation in Ca2+-free
solution. Because Ca2+ release
should be highly dependent on the level of
Ca2+ store filling, we
standardized Ca2+ loading by
exposing the rings to high-K+ (140 mM K+) solution for 5 min before
switching to Ca2+-free solution
and inducing Ca2+ release. After
this procedure, responses to releasing agents were clearly greater than
after incubation (>30 min) in normal PSS (caffeine: 145 ± 13%;
NE: 141 ± 5%; n = 8). In all
experiments the releasing agents were applied after 5 min in
Ca2+-free solutions containing 1 mM EGTA to eliminate the inflow of extracellular
Ca2+. This treatment does not
appreciably deplete the SR, because SR
Ca2+ exchanges very slowly in
these preparations. To estimate the rate of loss, we evaluated
NE-evoked force transients after variable time periods in EGTA-buffered
solution. The transient amplitude decreased linearly with time at a
rate of 1.7 ± 0.3%/min (n = 4).
The amplitude of the agonist-evoked force transient was taken as an
indicator of intracellular Ca2+
release. The maximal force output was decreased by culture in the
presence of FCS. Therefore, tension transients were quantitated relative to the maximal force response of the individual ring (Fig.
1). Responses to all agents
were enhanced after culture. This effect of culture varied between
agonists, but there was no apparent difference between caffeine- and
IP3-mediated responses. In
contrast to what was found for all
IP3-inducing agents, responses to
caffeine were significantly greater in rings cultured in the presence
of 10% FCS than in control rings cultured in the absence of
serum. This increase was accompanied by an increased
responsiveness to caffeine at low concentrations, as revealed by
dose-response relationships (Table
1). However, at the standard
concentration of 20 mM used in this study, the responses relative to
the maximum concentration used (40 mM) were equal (0% FCS: 83.7 ± 7.2%; 10% FCS: 79.9 ± 8.0%). Therefore, differing
sensitivities do not account for the increased peak forces in response
to caffeine after culture with FCS. There were no
significant effects of FCS on the sensitivity to NE, 5-HT, or AVP after
culture, and accordingly the concentrations giving maximal tension were
used in Ca2+ release experiments.
Culture itself caused a sensitization to NE, as previously described
(15), probably because of a degeneration of adrenergic nerves (22). In
fresh preparations, caffeine responses were too small to give an
adequate measure of sensitivity.

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Fig. 1.
A: tension responses to maximal
activation [high-K+
solution + 10 µM norepinephrine (NE)] in freshly
prepared and cultured tail arterial rings.
B: force transients in response to
Ca2+-releasing agents [10
µM NE, 10 µM 5-hydroxytryptamine (5-HT), 0.1 µM arginine
vasopressin (AVP), and 20 mM caffeine];
n 6 for all determinations.
* P < 0.05 and ** P < 0.01.
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Ca2+ measurements verified that
the differences in force in response to caffeine and NE observed after
culture reflect differences in the release of intracellular
Ca2+ (Fig.
2 and Table
2). This series of experiments was run
at room temperature (see MATERIALS AND
METHODS), which probably accounts for the low force
development compared with that in the mechanical experiments shown in
Fig. 1. The apparent dissociation between [Ca2+]i
and force on stimulation by NE, in contrast to what was found for
high-K+ solution or caffeine, is
consistent with its
Ca2+-sensitizing effect, first
described by Morgan and Morgan (17). The force elicited by either
high-K+ solution alone or a
mixture of high-K+
solution and NE was lower after FCS culture, whereas the
[Ca2+]i
response was not (Table 2). This confirms that the normalization of
transient responses to maximal force (Fig. 1) is valid in terms of
indicating the magnitudes of agonist-induced
Ca2+ transients. The kinetics of
the Ca2+ transients in response to
the two agents were compared. As shown in Table
3, rings cultured with FCS showed a
significantly faster rising phase of responses to caffeine but there
was no difference in responses to NE. The rate of decline of the
transient was about five times lower than the rate of rise, and
independent of agonist or culture condition.

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Fig. 2.
Simultaneous registrations of tension (mN/mm) and intracellular
Ca2+ concentration
([Ca2+]i;
nM) in rings cultured in presence of 10% FCS
(A) or absence of FCS
(B). Responses to caffeine (caff)
and NE are indicated. K+,
high-K+ solution.
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Table 2.
Basal [Ca2+]i and increase in
[Ca2+]i and force in response to
high-K+ solution and application of caffeine or NE
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Exposure to FCS increases
[Ca2+]i
in the tail artery (14). Because fluorometric measurements of
[Ca2+]i
cannot be done during the actual period of incubation,
the [Ca2+]i-raising
effect of FCS was verified in rings loaded with fura 2 after culture
with FCS and equilibrated in PSS. The addition of FCS raised
[Ca2+]i,
and this effect was antagonized by the addition of EGTA or verapamil
(Fig. 3). Thus no apparent desensitization
to the
[Ca2+]i-raising
action of FCS occurred during culture, and
[Ca2+]i
is likely to have been increased throughout the culture period. Similar
results were obtained with depolarizing
high-Ca2+ medium (30 mM KCl, 2.5 mM CaCl2; data not shown). In
contrast to the direct effect of added FCS, it was found that basal
[Ca2+]i
values in rings loaded with fura 2 were not different after culture
either with or without FCS when the rings were subsequently incubated
in normal FCS-free PSS (Table 2).

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Fig. 3.
Simultaneous measurements of tension and
[Ca2+]i
in one ring after culture for 4 days in 10% FCS. FCS (10%), EGTA (1 mM), and verapamil (1 µM) were added as indicated;
n = 3.
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To test the hypothesis that altered SR
Ca2+ handling is due to increased
[Ca2+]i
during culture, culture media were supplemented with verapamil (1 µM)
or EGTA (1 mM) to lower
[Ca2+]i
(Fig. 3). Both of these treatments significantly inhibited the effects
of FCS on force transients evoked by caffeine but did not affect
NE-evoked transients (Fig.
4A).
Furthermore, the effects of FCS on the
Ca2+ release pattern could be
mimicked by culture in depolarizing high-Ca2+ medium. The effects of
culture with depolarizing medium and with FCS on maximal tension
development were also similar, and both effects were reversible by EGTA
treatment (Fig. 4B).

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Fig. 4.
A: relative tension of caffeine- and
NE-induced transients expressed as percentage of contraction induced by
high-K+ medium + 10 µM
NE. Culture conditions are indicated, as are additions of verapamil (V;
1 µM) and EGTA (E; 1 mM) to medium;
n > 12 for all determinations;
* P < 0.05. B: effects of culture with EGTA (1 mM)
on maximal tension development in rings treated with 0 or 10% FCS or
depolarizing, high-Ca2+ medium.
* P < 0.05 and
** P < 0.01.
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The effect of culture with FCS on agonist-induced force transients was
persistent. It remained after the rings were washed for 30 min with
cold (8°C) Ca2+-free PSS, and
thereafter for at least 2.5 h in PSS without FCS (data not shown).
Pretreatment with ryanodine or the SR
Ca2+ pump inhibitor cyclopiazonic
acid (CPA) abolished the responses to both caffeine and NE. Ryanodine
induced a sustained plateau in force (29 ± 2% of a
contraction induced by high-K+
solution; n = 4),
whereas the contractile response to CPA was more variable. In contrast
to the response to ryanodine, which is irreversible, responses to
caffeine reappeared at their original levels after CPA was washed out
(Fig. 5). This indicates that the increased
caffeine responses after culture reflect an actual increase in the
Ca2+ storage and/or release
capacity of the SR and not merely a persistent shift in ionic
distributions after culture.

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Fig. 5.
Force registration from one ring (of five) cultured in presence of 10%
FCS, showing effect of cyclopiazonic acid (CPA; 10 µM) on caffeine
responses.
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 |
DISCUSSION |
Although the plasticity of intracellular
Ca2+ handling in smooth muscle has
been extensively investigated by using dispersed cells in culture, the
present study is to our knowledge the first investigation of SR release
in intact smooth muscle tissue cultured in vitro. Major differences in
the effect of FCS on SR function in cultured tissue vs. dispersed cells
are found. Most likely, these relate to the phenotypic change occurring
early in cell culture as a result of the disruption of cell-cell and
cell-matrix interactions (3). On the other hand, in vivo models of
hypertension and smooth muscle hypertrophy show effects on SR function
compatible with the findings obtained here (see below). Systemic
compensatory mechanisms active in animal models do not influence
long-term responses in organ culture, and a culture model provides the
ability to apply powerful pharmacological tools not accessible in vivo. Morphologically, rat tail arteries are also well maintained in culture
for at least 2 wk in serum-free medium (22), and thus organ culture
appears to be a valuable model with which to study the long-term
adaptation of SR function as well as other aspects of
excitation-contraction mechanisms (7, 21).
An increased release of Ca2+ from
intracellular stores was found by Rohrmann et al. (20) in the early
phases of urinary bladder hypertrophy caused by outlet obstruction.
Similar changes in SR function were also observed in vascular smooth
muscle cells from spontaneously hypertensive rats (12, 23). The changes
in SR function could be a secondary response to increased sarcolemmal Ca2+ influx or altered
mitochondrial Ca2+ handling but
could possibly also reflect the effects of growth stimulation unrelated
to altered Ca2+ homeostasis.
Notwithstanding the difficulty of relating findings for cultured muscle
to in vivo hypertrophy, results with FCS can give some insight into the
respective effects of growth stimulation and
Ca2+ load on SR function. Culture
with FCS results in a decrease in maximum force, which appears to be a
result of structural effects related to increased
[Ca2+]i
rather than growth stimulation as such (14), and accordingly the
present investigation has focused on the effects of the
[Ca2+]i
increase accompanying culture with FCS. In fact, the growth-stimulating effect of FCS seems severely inhibited by conditions in intact tissue,
in contrast to those in dispersed cells. After culture in the presence
of 10% FCS, responses to caffeine, but not to NE, 5-HT, or AVP, were
increased compared with those in serum-free culture. This was clearly a
Ca2+-dependent effect, because it
could be blocked by the inclusion of verapamil or EGTA in the culture
medium. Furthermore, culture in depolarizing medium (30 mM KCl, 2.5 mM
CaCl2) mimicked the effects of
FCS on caffeine vs. NE responses. The similar effects of depolarizing
medium and FCS on force, and their prevention by EGTA, further show
that the two treatments acted through the same mechanism, namely,
increased
[Ca2+]i.
The unexpected finding that NE-sensitive
Ca2+ release was not affected by
FCS or depolarizing medium suggests that caffeine-sensitive release
mechanisms are preferentially involved in the long-term response to
increased
[Ca2+]i.
The enhanced rate of Ca2+ release
in response to caffeine and the leftward shift of the caffeine
dose-response curve after culture with FCS (Table 2 and Fig. 2) suggest
that the properties or the density of the ryanodine receptors has been
altered. This might reflect a specific role of the ryanodine receptor
pathway in regulating the basal Ca2+ level, e.g., by release into
a restricted subsarcolemmal space for further extrusion via the plasma
membrane (13, 18). However, the relative levels of importance of the
different Ca2+ release pathways
for this mechanism are presently unknown.
Besides the effect of culture with FCS on SR
Ca2+ handling, culture itself,
with or without FCS, caused an increase in
Ca2+ release, affecting both
caffeine- and IP3-mediated
release. This does not seem to be caused by increased
Ca2+ load, because verapamil and
EGTA did not reduce the amplitudes of responses after culture in 0%
FCS. Thus the effect seems to be a trophic response influencing the
Ca2+ storage capacity of the SR,
but the role of FCS for this seems to be minor.
The results presented here are in apparent contrast to some findings
for isolated smooth muscle cells. A decreased ability to release
Ca2+ in response to caffeine or
ANG II was observed in vascular smooth muscle cells after they had been
isolated and placed in culture and had entered a phase of rapid
proliferation under stimulation by FCS (16).
Ca2+ release in response to ANG
II, but not caffeine, is restored when the cultures reach confluence.
However, arrest of the cell cycle by serum-free medium restores the
response to caffeine. Furthermore, basal
[Ca2+]i
and release from the SR have been shown to vary with the stages of the
cell cycle (10). However, in the cultured tail artery there does not
seem to be any correlation between the effect of FCS on basal
[Ca2+]i
and entry into the S phase of the cell cycle, because verapamil does
not affect
[3H]thymidine
incorporation in FCS-stimulated rings (14). Thus comparison with
studies of proliferating cells in culture must be made with caution,
although these studies also suggest that caffeine- and
IP3-sensitive
Ca2+ stores may be separately
regulated by trophic stimuli and
Ca2+.
 |
ACKNOWLEDGEMENTS |
We thank Bengt-Olof Nilsson and Anders Lindqvist for useful discussions.
 |
FOOTNOTES |
This study was supported by the Swedish Medical Research
Council (Project 14X-28), the Medical Faculty, Univ. of
Lund, and AB Astra-Hässle, Mölndal, Sweden.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Hellstrand,
Dept. of Physiological Sciences, Lund Univ., Sölvegatan 19, S-223
62 Lund, Sweden (E-mail: Per.Hellstrand{at}mphy.lu.se).
Received 29 June 1998; accepted in final form 2 February 1999.
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