© 2000 American Heart Association, Inc.
UltraRapid Communications |
Asynchronous Ca2+ Waves in Intact Venous Smooth Muscle
From the Vancouver Vascular Biology Research Centre, University of British Columbia, St. Paul’s Hospital, Vancouver, BC, Canada.
Correspondence to Cornelis van Breemen, Vancouver Vascular Biology Research Centre, University of British Columbia, St. Paul’s Hospital, Room 292, 1081 Burrard St, Vancouver, BC, V6Z 1Y6, Canada.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsExperimental ProceduresResultsDiscussionReferences |
|---|
Abstract—The rabbit inferior vena cava (IVC) is a large-capacitance vessel that displays typical contractile dose-response curves for caffeine and phenylephrine (PE). Using confocal microscopy on the endothelium-denuded IVC, we undertook experiments to correlate these whole-tissue contractile dose-response curves with changes in subcellular [Ca2+]i signals in the in situ vascular smooth muscle cells (VSMCs). We observed that both caffeine and PE initially elicited Ca2+ waves in individual VSMCs. The [Ca2+]i in cells challenged with caffeine subsequently returned to baseline whereas the [Ca2+]i in cells challenged with PE exhibited repetitive asynchronous Ca2+ waves. These [Ca2+]i oscillations were related to Ca2+ release from the sarcoplasmic reticulum as they were inhibited by ryanodine and caffeine. The lack of synchronicity of the [Ca2+]i oscillations between VSMCs can explain the observed tonic contraction at the whole-tissue level. The nature of these Ca2+ waves was further characterized. For caffeine, the amplitude was all-or-none in nature, with individual cells differing in sensitivity, leading to their recruitment at different concentrations of the agonist. This concentration dependency of recruitment appears to form the basis for the concentration dependency of caffeine-induced contraction. Furthermore, the speed of the Ca2+ waves correlated positively with the concentration of caffeine. In the case of PE, we observed the same characteristics with respect to wave speed, amplitude, and recruitment. Increasing concentrations of PE also enhance the frequency of the [Ca2+]i oscillations. We therefore conclude that PE stimulates whole-tissue contractility through differential recruitment of VSMCs and enhancement of the frequency of asynchronous [Ca2+]i oscillations once the cells are recruited. The full text of this article is available at http://www.circresaha.org.
Key Words: vascular smooth muscle • Ca2+ signaling • smooth muscle contraction • confocal microscopy • Ca2+ oscillation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsExperimental ProceduresResultsDiscussionReferences |
|---|
Smooth muscle contraction is initiated by Ca2+-calmodulin activation of myosin light chain kinase, resulting in crossbridge cycle interaction between actin and myosin filaments. Thus, Ca2+ plays a key role in controlling smooth muscle contractility. In addition, the myofilament sensitivity to Ca2+ is upregulated by contractile agonists and decreased by cyclic nucleotides.
Much of our understanding regarding
[Ca2+]i signaling in
vascular smooth muscle cells (VSMCs) has been based on studies using
enzymatically isolated cells and cultured cells. Isolated and cultured
VSMCs, when stimulated, show cyclic elevations of
[Ca2+]i that progress
over the entire cell length, giving it the appearance of a
wave.1 Although these single-cell experiments have greatly
improved our knowledge of the mechanisms involved in
Ca2+ homeostasis, smooth muscle cells can
present a very different phenotype once
cultured.2 For example, cultured smooth muscle cells
rapidly lose their L-type voltage-operated channels3 and
undergo a reduction in the expression of 
-actin,4 hence
changing from the contractile to the secretory or migrating
phenotype.5 In addition, ubiquitous chemicals
leaching from cell culture plasticware can potently affect the
Ca2+ homeostasis of VSMCs,6 7 and
cell culture media with high glucose concentration can reduce gap
junction expression.8 9 Even when smooth muscle cells are
freshly isolated, but not cultured, it is clear that proteolysis
changes cellular characteristics, which are fundamental to
physiological function. The presence of gap
junctions10 between VSMCs suggests that cells with intact
intercellular communications may behave more like a syncytium and
possibly very different from their behavior as isolated single cells.
It therefore becomes necessary to verify mechanisms deduced from
isolated or cultured cells by data obtained from smooth muscle cells in
situ.
Iino et al11 were the first to examine stimulus-induced VSMC [Ca2+]i signals in situ. They reported that in the intact rat tail artery, which typically responds with tonic contractions, individual smooth muscle cells oscillate asynchronously after electrical and pharmacological stimulation. This has been confirmed by Kasai et al,12 and similar asynchronous [Ca2+]i oscillations have been observed in intact pressurized mesenteric small arteries by Miriel et al.13
Because VSMCs can display considerable heterogeneity in structure and function depending on their location, the question was raised whether the observed asynchronous [Ca2+]i oscillations are also characteristic for veins.
We have previously used the rabbit vena cava to validate new roles for smooth muscle sarcoplasmic reticulum (SR), such as buffering of Ca2+ entry and contributing to Ca2+ extrusion.14 15 In this presentation, we report on yet another function of the SR, which is the generation of cytoplasmic Ca2+ oscillations. We extend the observations of Iino et al11 of asynchronous Ca2+ waves to a correlation of whole vena cava force and [Ca2+]i measurements with single cell [Ca2+]i signaling.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsExperimental ProceduresResultsDiscussionReferences |
|---|
Solutions and Chemicals
All drugs were purchased from Sigma and were of the highest analytical grade. Normal physiological salt solution (PSS) contained (in mmol/L) NaCl 140, KCl 5, CaCl2 1.5, MgCl2 1, glucose 10, and HEPES 5, (pH 7.4 at 37°C). Zero Ca2+ PSS contains no CaCl2 but 1 mmol/L EGTA. Fura-2 AM, Fluo 3-AM, Hoechst 33342, and pluronic were purchased from Molecular Probes and dissolved in dimethly sulfoxide (DMSO), as was ryanodine. Caffeine and phenylephrine (PE) were solubilized in normal PSS. All experiments were performed at 37°C.
Preparation of Tissue
Female New Zealand White rabbits (1.5 to 2.5 kg, obtained
from University of British Columbia animal care) were killed by
CO2 asphyxiation and then exsanguinated in
accordance with local regulations. The inferior vena cava
(IVC) was removed, cleaned of connective and adventitial tissues, the
endothelium removed by gently swiping it with filter
paper,14 15 and the vessel was cut into multiple ring
segments that were 4 to 5 mm wide.
Contraction Studies
Vessel segments attached to isometric force transducers were
equilibrated with 0.4 g of pretension. The solution was
exchanged by rapid draining and refilling within 2 to 3 seconds. Rings
failing to contract after challenge with 80 mmol/L KCl were
excluded from the subsequent exposure to incremental, noncumulative
concentrations of either caffeine or PE to achieve a full
concentration-response curve in each tissue. Data were acquired and
analyzed using Chart v3.4.5 (ADIinstruments).
Measurement of Global [Ca2+]i
The detailed methods to measure global
[Ca2+]i in this
preparation have been described elsewhere.12 13 Briefly,
the tissue was mounted inside a 3-mL cuvette and placed in a
spectrofluorimeter (Spex Fluorolog; Spex Industries, NJ). After
subtraction of autofluorescence, the tissue was loaded with
fura-2 AM (5 µmol/L+5 µmol/L pluronic, 90 minutes at room
temperature) and excited with alternating 340 and 380 nm light. The
emission was collected at 510 nm, and the ratio of these two exciting
wavelengths was taken as an indicator of
[Ca2+]i.
Confocal Studies and Analysis
For confocal experiments, vessel rings were loaded with Fluo
3-AM (5 µmol/L, 90 minutes, 25°C), and left to
equilibrate for 30 minutes. The inverted tissue was isometrically
mounted on a purpose-built microscope stage, and solution change was
achieved by rapid draining and refilling of the bath. Observation of
[Ca2+]i changes was made
using a Noran Oz laser scanning confocal microscope through either an
air x60 (numerical aperture [na] 0.7) or an air x20 (na 0.45) lens
on an inverted Nikon microscope. The tissue was illuminated using the
488-nm line of an argon-krypton laser, and a high-gain photomultiplier
tube collected the emission after it had passed through a 525/52
bandpass filter. All parameters (eg, laser intensity, gain)
were left unchanged during the experiment. Generally, acquisition speed
was set to 66 ms/frame with 2-frame integration, resulting in an
effective frame rate of 133 ms/frame. Comparisons between
recordings made at 66 ms/frame and 133 ms/frame were made when
necessary to exclude sampling artifacts.
All data analysis was performed in ImageProPlus using customized routines written in Visual Basic. To obtain data on recruitment of cells during drug stimulation, a 3-pixel-wide scan line was drawn across multiple cells and propagated through the time stack. The resulting image (Y-t plot) revealed the number of cells responding (expressed as percentage of the cells responding to the highest drug concentration) as well as the degree of heterogeneity between and oscillation frequency within cells. Further analysis of wave parameters was performed using a 3-pixel-wide line along the longitudinal axis of a single cell. The resulting X-t plot revealed the point of origin as well as the progression speed of the apparent "wave." All experimental traces shown represent the averaged fluorescence signals from a 3x3 pixel region (1.36 µm2) in a single cell. Numerical data were analyzed in Excel and Sigma Plot.
|
Experimental Procedures |
|---|
|
TopAbstractIntroductionMaterials and MethodsExperimental ProceduresResultsDiscussionReferences |
|---|
After an initial equilibration time, the tissue was intermittently exposed to increasing concentrations of either caffeine or PE. Between consecutive doses, ample time for recovery was allowed and several experiments were repeated to ensure that the tissue did not desensitize to either treatment.
Statistical Analysis
Numerical data are presented as the mean of at least 3
preparations and the corresponding SEM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsExperimental ProceduresResultsDiscussionReferences |
|---|
Isolation and preparation of the tissue have been an established technique in our laboratory, and previous experiments suggested effective concentration ranges for caffeine from 0.25 to 25 mmol/L and PE from 1.5 nmol/L to 15 µmol/L. In selected experiments, the concentration of PE was increased to as high as 1.5 mmol/L. To confirm endothelial cell removal, 3 denuded and 3 control vessels were incubated with the selective DNA stain Hoechst 33342 (300 µg/mL). Nuclei were visualized using the 360-nm laser illumination. In control vessels, a continuous layer of oblong nuclei, with their long axis oriented parallel to the direction of the flow was observed, indicating an undamaged endothelial cell layer (Figure 1A
). After denudation, the
same staining protocol revealed only elongated nuclei oriented
transversely to the direction of the flow (Figure 1B),
consistent with VSMC nuclei characterized by Daly et
al.16
|
Contraction and Whole-Tissue [Ca2+]i
To characterize the contractile and
[Ca2+]i responses of the
vena cava to caffeine and PE, the tissues were isometrically suspended
and noncumulatively exposed to increasing concentrations of either
caffeine or PE. Representative traces are shown in
Figure 2A (insets). PE (15 µmol/L)
induced a maintained increase in contractile force of 1.79±0.42 g
(mean±SEM, n=8 rings from 4 animals). Caffeine (25 mmol/L) evoked
a transient contraction; 5 seconds after the onset of the response,
tension increased to 0.42±0.09 g (n=11 rings from 4 animals) and
subsequently relaxed completely to the basal level after 120 seconds.
Increasing concentrations of the agonists enhanced the contractile
force until a maximal value was reached. These typical
concentration-response curves are shown in Figure 2A. The
dose-response for PE fell within the concentration range of 0.0015 to
150 µmol/L, whereas caffeine increased force over a much
narrower concentration range of 1 to 10 mmol/L.
|
We further examined how these contraction patterns were related to the
whole-tissue [Ca2+]i
signal. As shown in Figure 2B
, PE (15 µmol/L) induced a
rapid rise in [Ca2+]i.
The peak value, 145±22% of basal F340/380 was reached 62±9 seconds
after the onset of the response (n=4 strips from 4 animals).
[Ca2+]i was maintained;
180 seconds after the maximal level was reached, it remained at
142±19% of the basal F340/380. A similar fast initial rise was
observed with caffeine (25 mmol/L): 136±6% of basal F340/F380
was reached after 53±7 seconds (n=4 strips from 4 animals). In
contrast to PE, the ratio returned to baseline after 180 seconds. Thus,
the patterns of the whole-tissue
[Ca2+]i signals appear to
match those observed in the contraction experiments
Confocal Experiments
Given that both PE and caffeine produce concentration-dependent,
graded levels of contraction in IVC, we designed parallel experiments
to correlate the macroscopic contractile concentration-response curves
with the subcellular
[Ca2+]i signals of
individual VSMCs in situ. Fast laser scanning confocal microscopy of
Fluo 3-AM was used to image subcellular
[Ca2+]i. Initially, both
caffeine (25 mmol/L) and PE (1.5 µmol/L) elicited a rapid
rise in [Ca2+]i that
appeared in the form of a Ca2+ wave propagating
along the longitudinal axis of the VSMCs (Figures 3A and 3B, frame 1). With time,
[Ca2+]i in the in situ
VSMCs challenged with caffeine returned to baseline despite the
continual presence of the drug (Figures 4A through 4C). The transient nature of
the caffeine-induced subcellular Ca2+ signal
agrees with the transient whole-tissue
[Ca2+]i signal and
contraction. In the same vena cava, PE stimulated repetitive smooth
muscle Ca2+ waves that were not synchronized
between adjacent cells (Figure 3A, arrows to cells 1, 2, and 3).
This lack of synchronicity between neighboring VSMCs explains how
summation of the individual-cell
[Ca2+]i
oscillations leads to sustained macroscopic
[Ca2+]i elevation and
tonic contraction of the whole-tissue.
|
|
To understand the cellular basis of the concentration-response curves
illustrated in Figure 2, we analyzed the nature of the
agonist-induced Ca2+ waves. Whenever a
Ca2+ wave was initiated by caffeine, the
amplitude of the [Ca2+]i
signal was constant, irrespective of the concentrations applied (Figure 4D). This indicates an all-or-none phenomenon that excludes
amplitude as a modulator of tissue contractility.
However, we observed that the sensitivity of VSMCs to caffeine varies
between cells, leading to an increased recruitment at higher caffeine
concentrations (Figures 4C). This concentration dependence of
recruitment resembles that of whole-tissue
contractility (Figure 2A) and appears to be the
basis for the concentration dependence of caffeine-induced
contraction.
In the case of PE, we observed similar characteristics with respect to
amplitude and recruitment (Figure 5). The
amplitude displayed an all-or-none character, and the concentration
dependency of recruitment (Figure 5E) appeared to correlate with
the concentration dependency of the PE-induced contraction (Figure 2A) at the lower concentration range of 0.015 to 1.5
µmol/L. However, the continued oscillations of
[Ca2+]i allow for more
elaborate modulation of contractile force. Figure 6 shows that as [PE] was increased, the
frequency of the Ca2+ waves increased. At the
highest concentration used (150 µmol/L PE), the frequency
reached 0.511±0.025 Hz (n=15 cells from 4 animals). As reflected in
the representative traces from Figure 6A, the
increased frequency of
[Ca2+]i
oscillations coincides with shortened interspike intervals.
At high [PE], the interspike
[Ca2+]i level was
elevated above the basal level, but
[Ca2+]i peaks never
fused. The concentration dependence of the frequency of PE-induced
[Ca2+]i
oscillations appears to correlate with the
concentration-response curve of PE-induced contraction at the higher
concentration range of 0.15 to 150 µmol/L.
|
|
Finally, we examined the concentration dependence of the velocity of
agonist-induced Ca2+ waves. The velocity of wave
propagation illustrated in Figure 7 shows
a strong dependence on the concentrations of either PE or caffeine. At
the highest concentrations, wave propagation speeds reached
89.62±7.36 µm/s (n=15 cells from 4 animals) with PE and
126.22±7.31 µm/s with caffeine (n=15 cells from 3 animals). In
addition, as illustrated in Figures 7A and 7B, both PE and
caffeine commonly elicited Ca2+ waves at multiple
foci within a given cell, which then eventually collided.
|
On 1-adrenergic stimulation, the observed
[Ca2+]i
oscillations may derive their Ca2+
from the extracellular space or the SR. To determine the immediate
source of Ca2+ for these PE-induced
[Ca2+]i
oscillations, extracellular Ca2+ was
removed along with the addition of 1 mmol/L EGTA. Under these
conditions, the PE-induced
[Ca2+]i
oscillations persisted for 28.25±2.58 seconds (n=84 cells
from 4 animals) before dissipating. Depletion of caffeine-sensitive and
ryanodine-sensitive Ca2+ stores with either
25 mmol/L caffeine (Figure 8B) or
100 µmol/L ryanodine (Figure 8C) completely abolished
PE-induced oscillations. These findings indicate that the
SR is the immediate source of Ca2+, sustaining
the observed PE-induced
[Ca2+]i
oscillations.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsExperimental ProceduresResultsDiscussionReferences |
|---|
Characteristics of the [Ca2+]i Signals
The vena cava is a large-capacitance vein, whose tonic contractions determine venous return to the heart and therefore modulate the stroke volume and indirectly blood pressure. Our observations on Ca2+ signaling in individual smooth muscle cells of this intact tissue shed new light on how wall tension is regulated in large veins. This response to agonists is typified by repetitive transient elevations in [Ca2+]i, which originate in certain locations and then spread as waves over the length of the cell (Figures 7A
and 7B). The cells respond independently of
each other in that the
[Ca2+]i
oscillations are not synchronized and that cells vary in
their sensitivity to PE. Observing the summation of all smooth muscle
cell activity in the tissue bath reveals typical dose-response curves
for both force development and
[Ca2+]i. Thus, the
asynchronous oscillations of tens of thousands of cells
blend into tonic responses as proposed earlier by Iino et
al11 for the behavior of the rat tail artery. Similar
oscillations were recently reported in the rat mesenteric
artery by Miriel et al.13 In the present study, we
have extended these observations to an actual comparison between the
individual cellular responses and those of the whole vein.
The repetitive [Ca2+]i
waves in the intact vena cava have the following characteristics: (1)
the amplitude has a constant value regardless of the [PE] (Figure 5D), (2) the frequency increases with increasing [PE] (Figure 6C), (3) the velocity increases with increasing [PE] (Figure 7C), and (4) at high [PE], the interspike
[Ca2+]i becomes elevated,
but the peak values remain constant (Figures 5D and 6A).
In addition, the sensitivity of each cell to PE is variable, such
that the recruitment of responding cells increases with increasing
[PE] (Figure 5E).
The mechanism of agonist-induced Ca2+ waves has
been discussed for several cell types. In endothelial
cells and other nonexcitable cells, they are related to
Ca2+ release and reuptake by the
SR.17 This appears to be true also for the IVC, as the
waves were abolished by caffeine and ryanodine but were initially not
affected by removal of Ca2+ from the
extracellular space. The all-or-none amplitude response indicates the
involvement of Ca2+-induced
Ca2+ release (CICR). At this time, we cannot
distinguish between possible involvement of either or both the
ryanodine receptor and the inositol 1,4,5-tris-phosphate
receptor (Ins1,4,5P3R), because both receptors
are sensitive to Ca2+.1 18 A
likely scenario for the agonist-induced Ca2+ wave
begins with elevation of Ins1,4,5P3 and
[Ca2+]i in response to
1-adrenergic activation.
Ins1,4,5P3 sensitizes the
Ins1,4,5P3R to
Ca2+,19 20 and when the
[Ca2+]i reaches a
threshold concentration, the release channels open.21
Ca2+ cascades into the cytoplasm, where it
promotes the opening of adjacent channels, thus starting the
Ca2+ wave, which proceeds down the length of the
smooth muscle cell. As the [PE] is raised, so are the concentrations
of Ins1,4,5P3 and basal cytoplasmic
Ca2+, which clearly shortens the time required
for [Ca2+]i to reach
threshold value for the initiation of the next wave. This mechanism
combined with the fact that Ins1,4,5P3 sensitizes
the Ins1,4,5P3R to Ca2+
ensures that both the frequency and velocity increase with increasing
[PE]. The Ca2+ wave may take origin at a
specific site, because either the [Ins1,4,5P3]
or the [Ca2+]i is
elevated above the remaining cytosolic levels. This could be a
consequence of inhomogeneity of receptor or channel
density.22 However, a more puzzling aspect is that the
oscillations continue even when the
[Ca2+]i rises above the
apparent threshold value. This could be explained by the fact that the
Ca2+ release channels cycle between the closed,
open, and inactivated states23 and that high
[Ca2+]i has an
inhibitory effect on the open
probability.21 24 25 Thus, as the
[Ca2+]i reaches peak
value, the channels close and require some time before opening and
starting another [Ca2+]i
transient on top of the raised background level of
Ca2+.
Correlation of [Ca2+]i Signals With the
Development of Tension
Physiologically, a blood vessel controls
blood flow through its lumen by producing varying degrees of
vasoconstriction. Such regulation is reflected in the classical
dose-response relationship whereby increasing concentration of
endogenous vasoconstrictor agents elicits increasing levels
of contractions at the whole-tissue level. As demonstrated in Figure 2A, rings of rabbit IVC display concentration-dependent
contractions to PE. These graded isometric contractile responses must
be mediated by certain concentration-dependent signaling mechanisms
over the same range of agonist concentration. Although our current
investigation focused on Ca2+-dependent
regulation of VSMC contractility, it is evident from
previous publications that 1-adrenergic
stimulation also enhances myofilament Ca2+
sensitivity in the vena cava and other vessels.26 27 This
is confirmed in the present study in which 0.015 µmol/L PE
initiated low-grade contraction without the associated changes in
[Ca2+]i. On the contrary,
our laboratory has demonstrated previously that caffeine desensitizes
myofilament activation to Ca2+ in the
rabbit IVC.26 This discrepancy could explain the low force
generation by caffeine in comparison to PE (Figure 2A).
With regard to the Ca2+-dependent modulation of
IVC contractility, we have identified distinctive,
concentration-dependent signaling mechanisms both at the tissue level
and at the individual cell level to account for the graded level of
contraction produced. At the tissue level, a concentration-dependent
recruitment of VSMC initiation of Ca2+ signals
was observed in response to both PE and caffeine. The mechanism
responsible for differential recruitment of cells populating the same
tissue is probably based on the intrinsic heterogeneity
of VSMCs.28 More specifically, our observations indicate
that neighboring VSMCs from IVC differ in their sensitivity to PE and
caffeine. These differences may be due to variations in ultrastructure
and/or basal enzyme activities.28 Regulation of
contractile strength by caffeine appears to be mainly related to the
recruitment process. Clearly, the "all-or-none" nature of the
cellular [Ca2+]i signal
is incapable of yielding a graded dose-response curve (Figures 4D and 5D). Thus, the magnitude of the
[Ca2+]i signal at the
cellular level does not encode information to modulate VSMC
contractility. The velocity of the caffeine-induced
Ca2+ wave, however, was found to be concentration
dependent, especially at the higher concentration range for caffeine
between 2.5 mmol/L to 25 mol/L (Figure 7C). This
observation is consistent with the concept that caffeine acts
by increasing the sensitivity of ryanodine-sensitive channels to
Ca2+ in the process of CICR.29 30 31
The Ca2+ wave observed in response to caffeine
reflects regenerative release of Ca2+ through
ryanodine channels downstream from the origin of the
Ca2+ wave. As the concentration of caffeine
applied increases, both Ca2+ sensitivity and open
probability of ryanodine channels are further elevated, resulting in
more rapid wave-like induction of CICR.32 However,
considering that the amplitude of the Ca2+ wave
is constant, it seems unlikely that wave velocity modulates VSMC
contractility. Therefore, given the close resemblance
of the concentration dependency relationships between tension generated
(Figure 2A) and the proportion of cells recruited (Figure 4E), the magnitude of caffeine-induced contraction is mainly
determined by the level of VSMC recruitment.
In contrast to caffeine, a comparison between the [PE]-response
curves for contraction (Figure 2A) and cell recruitment (Figure 5E) reveals a positive correlation between the degree of cell
recruitment and developed force only at the lower concentrations,
ranging from 0.015 to 1.5 µmol/L. This implies that additional
mechanism(s) must be involved in regulating
contractility, especially at the higher concentration
range. We have shown that both frequency of oscillation and
the apparent velocity of the Ca2+ waves are
dependent on [PE] (Figures 6C and 7C), whereas the
amplitude of oscillation is independent of [PE] (Figure 5D). Therefore, information governing the graded level of
contraction at the higher [PE] range is encoded in the frequency and
possibly the velocity domains. As discussed above, it is unclear
whether higher wave velocity can contribute to a greater degree of
contraction. On the other hand, higher frequency of
oscillations can enhance the contractile response by
several possible mechanisms. First, higher frequency of fixed-amplitude
Ca2+ oscillations due to shortening
of the interwave periods results in higher average
[Ca2+]i over time and
thus enhances myofilament activation. Second, increasing frequency of
oscillation may lead to activation of certain
frequency-sensitive enzymes that can potentially affect the level of
contraction. An example of this class of enzyme is
Ca2+-calmodulin kinase II that was
found to be sensitive to the frequency of Ca2+
spikes in vitro.33 Thus, we speculate that
oscillation frequency is an important regulator of
contractility whereas the velocity of the
Ca2+ waves remains of unknown
physiological relevance.
The observations presented and discussed herein emphasize that
the classical concept of an SR-Ca2+ release
contribution to the initial phase of contraction followed by tonic
force dependent on Ca2+ entry from the
extracellular space to the myoplasm does not apply to -adrenergic
constriction of capacitance veins. Instead, the entire tonic
contraction is mediated by the SR beginning with its initial
Ca2+ release followed by repeating cycles of
Ca2+ uptake and release. Because at least part of
the Ca2+ released from the SR is extruded to the
extracellular space,34 the maintained
[Ca2+]i
oscillations have to be supported by stimulated
Ca2+ influx. Given that the
[Ca2+]i in the myoplasm
is regulated by the SR, it follows that the agonist-stimulated
Ca2+ influx raises
[Ca2+]i in a restricted
subplasmalemmal space from where it is taken up by
sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) to be
subsequently directed toward the sites of Ca2+
release near the myofilaments. In this regard, it is of special
interest that our laboratory has presented evidence in support
of redistribution of Ca2+ within the SR
lumen.14 Assuming that the above sequence of
Ca2+ movement is correct, we may speculate on the
advantage that this system provides for smooth muscle cells. In
essence, instead of Ca2+ diffusing from the
plasma membrane through the cytoplasm, it is guided from the plasma
membrane to the myofilaments by the SR. Because the
Ca2+-calmodulin sensitive
myosin light chain kinase is attached to the thin
filaments,35 the function of the SR may be to deliver the
Ca2+ to these activating sites to enhance the
speed and efficiency of the activating mechanism. These novel insights
into venous [Ca2+]i
signaling in relation to contractile modulation will undoubtedly expand
the basis for future therapy of contractile malfunctions such as in
venous pooling during orthostatic hypertension.
|
Acknowledgments |
|---|
Dr Ruehlmann was supported by a grant of the British Columbia/Yukon Heart & Stroke Foundation; Cheng-Han Lee is a recipient of a Medical Research Council MD/PhD studentship. We thank the St. Paul’s Hospital Foundation for their generous support.
|
Footnotes |
|---|
1 Both authors contributed equally to this study.
Received January 7, 2000; accepted February 1, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsExperimental ProceduresResultsDiscussionReferences |
|---|
1. Berridge MJ, Dupont G. Spatial and temporal signalling by calcium. Curr Opin Cell Biol. 1994;6:267–274.[Medline] [Order article via Infotrieve]
2. Thyberg J. Differentiated properties and proliferation of arterial smooth muscle cells in culture. Int Rev Cytol. 1996;169:183–265.[Medline] [Order article via Infotrieve]
3.
Gollasch M, Haase H, Ried C, Lindschau C, Morano I,
Luft FC, Haller H. L-type calcium channel expression depends on the
differentiated state of vascular smooth muscle cells. FASEB
J. 1998;12:593–601.
4.
Campbell JH, Kocher O, Skalli O, Gabbiani G, Campbell
GR. Cytodifferentiation and expression of -smooth muscle actin
mRNA and protein during primary culture of aortic smooth muscle cells.
Correlation with cell density and proliferative state.
Arteriosclerosis. 1989;9:633–643.
5.
Owens GK. Regulation of differentiation of vascular
smooth muscle cells. Physiol Rev. 1995;75:487–515.
6.
Glossmann H, Hering S, Savchenko A, Berger W,
Friedrich K, Garcia ML, Goetz MA, Liesch JM, Zink DL, Kaczorowski GJ. A
light stabilizer (Tinuvin 770) that elutes from polypropylene plastic
tubes is a potent L-type Ca2+-channel blocker.
Proc Natl Acad Sci U S A. 1993;90:9523–9527.
7.
Ruehlmann DO, Steinert JR, Valverde MA, Jacob R, Mann
GE. Environmental estrogenic pollutants induce acute vascular
relaxation by inhibiting L-type Ca2+ channels in
smooth muscle cells. FASEB J. 1998;12:613–619.
8.
Kurjiaka DT, Steele TD, Olsen MV, Burt JM. Gap
junction permeability is diminished in proliferating vascular smooth
muscle cells. Am J Physiol. 1998;275:C1674–C1682.
9. Kuroki T, Inoguchi T, Umeda F, Ueda F, Nawata H. High glucose induces alteration of gap junction permeability and phosphorylation of connexin-43 in cultured aortic smooth muscle cells. Diabetes. 1998;47:931–936.[Abstract]
10.
Little TL, Beyer EC, Duling BR. Connexin 43 and
connexin 40 gap junctional proteins are present in arteriolar
smooth muscle and endothelium in vivo. Am J
Physiol. 1995;268:H729–H739.
11. Iino M, Kasai H, Yamazawa T. Visualization of neural control of intracellular Ca2+ concentration in single vascular smooth muscle cells in situ. EMBO J. 1994;13:5026–5031.[Medline] [Order article via Infotrieve]
12.
Kasai Y, Yamazawa T, Sakurai T, Taketani Y, Iino M.
Endothelium-dependent frequency modulation of
Ca2+ signaling in individual vascular smooth
muscle cells of the rat. J Physiol (Lond). 1997;504:349–357.
13.
Miriel VA, Mauban JRH, Blaustein MP, Wier WG. Local and
cellular Ca2+ transients in smooth muscle of
pressurized rat resistance arteries during myogenic and agonist
stimulation. J Physiol (Lond). 1999;518:815–824.
14. Nazer MA, Van Breemen C. Functional linkage of Na+-Ca2+ exchange and sarcoplasmic reticulum Ca2+ release mediates Ca2+ cycling in vascular smooth muscle. Cell Calcium. 1998;24:275–283.[Medline] [Order article via Infotrieve]
15.
Nazer MA, Van Breemen C. A role for the sarcoplasmic
reticulum in Ca2+ extrusion from rabbit
inferior vena cava smooth muscle. Am J
Physiol. 1998;274:H123–H131.
16. Daly J, Gordon E, McGrath C. The use of fluorescent nuclear dyes for the study of blood vessel structure and function: Novel applications of existing techniques. J Vasc Res. 1992;29:41–48.[Medline] [Order article via Infotrieve]
17.
Isshiki M, Ando J, Korenaga R, Kogo H, Fujimoto T,
Fujita T, Kamiya A. Endothelial
Ca2+ waves preferentially originate at specific
loci in caveolin-rich cell edges. Proc Natl Acad Sci
U S A. 1998;95:5009–5014.
18. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993;361:315–325.[Medline] [Order article via Infotrieve]
19. Streb H, Irvine RF, Berridge MJ, Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5- trisphosphate. Nature. 1983;306:67–69.[Medline] [Order article via Infotrieve]
20. Ferris CD, Cameron AM, Huganir RL, Snyder SH. Quantal calcium release by purified reconstituted inositol 1,4,5-trisphosphate receptors. Nature. 1992;356:350–352.[Medline] [Order article via Infotrieve]
21. Bezprozvanny I, Watras J, Ehrlich BE. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature. 1991;351:751–754.[Medline] [Order article via Infotrieve]
22. Hirose K, Iino M. Heterogeneity of channel density in inositol-1,4,5-trisphosphate-sensitive Ca2+ stores. Nature. 1994;372:791–794.[Medline] [Order article via Infotrieve]
23. Tang Y, Stephenson JL, Othmer HG. Simplification and analysis of models of calcium dynamics based on IP3-sensitive calcium channel kinetics. Biophys J. 1996;70:246–263.[Medline] [Order article via Infotrieve]
24. Iino M, Endo M. Calcium-dependent immediate feedback control of inositol 1,4,5-triphosphate-induced Ca2+ release. Nature. 1992;360:76–78.[Medline] [Order article via Infotrieve]
25. Meissner G, Darling E, Eveleth J. Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+ and adenine nucleotides. Biochemistry. 1986;25:236–244.[Medline] [Order article via Infotrieve]
26. Chen Q, Cannell M, van Breemen C. The superficial buffer barrier in vascular smooth muscle. Can J Physiol Pharmacol. 1992;70:509–514.[Medline] [Order article via Infotrieve]
27.
Bradley AB, Morgan KG. Alterations in cytoplasmic
calcium sensitivity during porcine coronary artery contractions
as detected by aequorin. J Physiol (Lond). 1987;385:437–448.
28.
Frid MG, Aldashev AA, Dempsey EC, Stenmark KR. Smooth
muscle cells isolated from discrete compartments of the mature vascular
media exhibit unique phenotypes and distinct growth
capabilities. Circ Res. 1997;81:940–952.
29.
Rousseau E, Meissner G. Single cardiac sarcoplasmic
reticulum Ca2+-release channel: activation by
caffeine. Am J Physiol. 1989;256:H328–H333.
30.
Sitsapesan R, Williams AJ. Mechanisms of caffeine
activation of single calcium-release channels of sheep cardiac
sarcoplasmic reticulum. J Physiol ( Lond). 1990;423:425–439.
31. Rousseau E, Ladine J, Liu QY, Meissner G. Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds. Arch Biochem Biophys. 1988;267:75–86.[Medline] [Order article via Infotrieve]
32.
Iino M. Calcium-induced calcium release mechanism in
guinea pig taenia caeci. J Gen Physiol. 1989;94:363–383.
33.
De Koninck P, Schulman H. Sensitivity of
Ca2+/calmodulin-dependent protein
kinase II to the frequency of Ca2+
oscillations. Science. 1998;279:227–230.
34. Leijten PA, van Breemen C. The relationship between noradrenaline-induced contraction and 45Ca2+ efflux stimulation in rabbit mesenteric artery. Br J Pharmacol. 1986;89:739–747.[Medline] [Order article via Infotrieve]
35.
Lin P, Luby-Phelps K, Stull JT. Properties of
filament-bound myosin light chain kinase. J Biol Chem. 1999;274:5987–5994.
This article has been cited by other articles:
 |
|
 |
|
  M. Lamboley, A. Schuster, J.-L. Beny, and J.-J. Meister Recruitment of smooth muscle cells and arterial vasomotion Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H562 - H569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Lee, D. Poburko, K.-H. Kuo, C. Y. Seow, and C. van Breemen Ca2+ oscillations, gradients, and homeostasis in vascular smooth muscle Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1571 - H1583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. H. Mauban, C. Lamont, C. W. Balke, and W. G. Wier Adrenergic stimulation of rat resistance arteries affects Ca2+ sparks, Ca2+ waves, and Ca2+ oscillations Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2399 - H2405. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||