How Structure, Ca Signals, and Cellular Communications
Underlie Function in Precapillary Arterioles
L. Borisova, Susan Wray, D. Eisner, T. Burdyga
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Rationale: Precapillary arterioles control blood flow to tissues and their correct function is vital. However, their
small size has limited study and little is known concerning the calcium signals in their endothelial and muscle cells
and how these relate to function.
Objective: We aimed to investigate whether these small vessels are specialized in terms of structure and calcium
signaling.
Methods and Results: Using in situ confocal imaging we have studied the ultrastructure, Ca signaling and
coordination of contraction in precapillary arterioles in ureter and vas deferens. We have compared the data to
that from a small mesenteric artery. In the precapillary arteriole, 1 myocyte covers a ⬇10-␮m length, and
contraction of this single cell can decrease the diameter of this segment. In the mesenteric artery, more than 20
myocytes are required for this. In the precapillary arteriole, Ca signals arise solely from Ca release from the
sarcoplasmic reticulum through phosphoinositol-3,4,5-inositol–induced Ca release and not via ryanodine
receptors. Agonist-induced Ca signals do not require Ca entry into the cell, do not spread or synchronize with
neighboring cells, and are unaffected by endothelial stimulation, thereby allowing local control. This contrasts
with the mesenteric artery, where Ca entry and ryanodine receptors are important and stimulation of the
endothelium removes the Ca signal and contraction.
Conclusions: These data reveal the structural and signaling specializations underlying how blood flow is locally
regulated provide new insight into control of microcirculation, and provide a framework to explain its
vulnerability to disease. (Circ Res. 2009;105:00-00.)
Key Words: calcium 䡲 vascular smooth muscle 䡲 endothelium
P
recapillary arterioles are the final generation of terminal
arterioles. They have diameters ⬍30 ␮m and determine
local blood flow to tissues via capillary beds, as opposed to
the gross distribution of blood and control of pressure,
performed by the larger conducting vessels and resistance
arteries. Little direct investigation of Ca signaling, structure,
and contractility has been possible in live, intact precapillary
arterioles, because their small size virtually precludes dissection. Although vital microscopy and related techniques have
produced useful information,1–3 they are not well suited to
investigations of cellular function, signaling, or morphology.4
A decreased role for the internal Ca store in small vessels has
been suggested,5,6 but generally the paucity of information in
precapillary arterioles has led to the processes found in larger
vessels: integration of neuronal and endothelial signals,3,7–9
being extrapolated to precapillary arterioles. However, consideration of the differences in their function, innervation,
and amount of tone would suggest that the mechanisms and
signaling controlling precapillary arterioles may differ from
those present in other vessels.10,11
In addition to neurohormonal stimulation of myocytes, the
endothelium via Ca signals and NO- and/or EDHF-mediated
effects, can also affect vascular tone.12–15 Little is known
concerning endothelial Ca signals in precapillary arterioles or
their contribution to relaxation; indeed, it is unclear how
relevant flow-, pressure-, or shear-mediated relaxing mechanism are to these smaller vessels.1,11
We now report unique mechanistic signaling data from in
situ precapillary arterioles in 2 host tissues, obtained with
confocal imaging of intracellular Ca, and directly compare
the results with a well-characterized larger vessel, the mesenteric artery (⬇230 ␮m in diameter).16 –19 We show, for the
first time, that Ca signals induced by agonists in precapillary
arteriolar myocytes rely exclusively on Ca release from the
sarcoplasmic reticulum (SR) mediated by phosphoinositol3,4,5-inositol (IP3) receptors (IP3Rs). As the signals do not
pass and coordinate between cells, exquisite local control in
segments of precapillary arterioles is achieved. The endothelium has no effect on agonist-induced tone but abolishes Ca
spikes and contraction produced by L-type Ca channel entry.
Thus, uniquely within the arterial system, in precapillary
arterioles a single myocyte influences a significant part of the
vessel and blood flow to the surrounding region.
Original received November 23, 2006; resubmission received June 17, 2009; revised resubmission received August 5, 2009; accepted August 19, 2009.
From the Physiology Department (L.B., S.W., T.B.), School of Biomedical Sciences, University of Liverpool; and Unit of Cardiac Physiology (D.E.),
University of Manchester, United Kingdom.
Correspondence to D. Eisner, University of Manchester, 46 Grafton St, Manchester M13 9NT, United Kingdom. E-mail [email protected]
© 2009 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.109.202960
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Circulation Research
September 25, 2009
Non-standard Abbreviations and Acronyms
2-APB
8-pCPT-cGMP
CCh
EDHF
ET-1
IP3
IP3R
PhE
SNAP
2-aminoethoxydiphenyl borate
para-chlorophenylthioguanosine-3⬘, 5⬘-cyclic monophosphate
carbachol
endothelium-derived hyperpolarizing factor
endothelin-1
phosphoinositol-3,4,5-inositol
phosphoinositol-3,4,5-inositol receptor
phenylephrine
S-nitroso-N-acetyl-DL-penicillamine
chemicals were from Sigma. Phenylephrine, endothelin-1 (ET-1),
ryanodine, Na nitroprusside, and para-chlorophenylthioguanosine3⬘, 5⬘-cyclic monophosphate (8-pCPT-cGMP) were dissolved in
water; 2-APB, cyclopiazonic acid, S-nitroso-N-acetyl- DL penicillamine (SNAP), U-73122, and cytochalasin D in DMSO; and
nifedipine in ethanol.
Statistics
Values are means⫾SEM, and n is number of vessels or cells.
Experiments were performed on rat: mesenteric small arteries (n⫽5
to 15) and precapillary arterioles in ureter (n⫽5 to 15), with findings
also being verified in vas deferens precapillary arterioles (n⫽4 to 7)
Unless stated otherwise, figures for precapillary arterioles are from
the data obtained in ureteric tissue. Differences were taken as
significant at P⬍0.05 in a Student t test.
Results
Methods
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An expanded Methods section is available in the Online Data
Supplement at http://circres.ahajournals.org.
Calcium and Diameter Measurements
Following euthanasia, strips of rat ureter or vas deferens were
dissected and arterioles within the tissue were studied as described
previously.20 The in situ precapillary arterioles remain patent and
erythrocytes could be seen moving within them, and the myocytes
produced rhythmic activity and vasoconstriction (Online Movies 3
and 4). For comparison small arteries of ⬇230-␮m inner diameter
were dissected form the mesentery (third order branches). Tissues
were loaded with Fluo-4 acetoxymethyl ester (Invitrogen, UK,
15 ␮mol/L; dissolved in DMSO with pluronic acid) for 2 to 3 hours
at 20°C and then transferred to indicator-free solution for ⱖ30
minutes. All tissues were fixed to the bottom of the chamber by
aluminum foil clips, to minimize movement. Preliminary experiments comparing agonist-evoked Ca signals with and without
cytochalasin D demonstrated that movement artifacts were not a
problem in measuring Ca signals. All experiments were performed at
30°C. We used a Nipkow disc, confocal microscope (Ultraview,
Perkin Elmer), connected to an Orca-ER cooled charge-coupled
device camera (Hamamatsu Photonics).20,21 Images were collected at
20 to 30 frames per second using a ⫻60 water objective (NA 1.20)
for best spatial resolution or dry (⫻10, 0.34 NA; and ⫻20, 0.70 NA)
for a larger field of view. To measure elemental events and Ca waves
in myocytes, tangential sections were used, whereas radial sections
through the center of the arteriole were used to measure simultaneously Ca events in both endothelial and smooth muscle cells and
changes in diameter. Mechanical activity of individual smooth
muscle cells was tracked by putting the region of interest close to the
edge of the contracting cell. It was possible to correlate Ca signaling
with contraction of individual myocytes in both radial and tangential
sections. In some experiments, Ca-free solution (2 mmol/L EGTA)
was used. To determine whether this was sufficient to remove
luminal Ca in the face of diffusional limitations, the extracellular Ca
in the lumen of the arterioles (Figure 4C, i) was monitored using
dextran-conjugated Fluo-4 (10 ␮mol/L n⫽3), loaded at room temperature for 60 minutes followed by washing with indicator-free
solution for 20 minutes. In some experiments, vascular myocytes
were stimulated by electric field via focal electrodes: for stimulation
of the nerve endings rectangular pulses of 50V, 1-ms duration and 2
to 20 Hz were used; and for direct stimulation of the muscle,
rectangular pulses of 7 to 12 V, 300 ms in duration and 0.1 Hz were
used.
Solutions
Physiological saline of the following composition was used (mmol/
L): NaCl 120.4, KCl 5.9, MgSO4 1.2, CaCl2 2, glucose 8, and
HEPES 11. 2-Aminoethoxydiphenyl borate (2-APB), ryanodine, and
U-73122, were from Calbiochem (Nottingham, UK), all other
Structure
Figure 1A shows low magnification images of in situ precapillary arterioles in the ureter. Individual myocytes (Figure 1B)
and endothelial cells (Figure 1C) can be resolved more
clearly at higher magnification and their structure determined
(Online Movies 1 and 2). Erythrocytes and fluid moving
within the precapillary arteriole could also be seen (Online
Movie 4). The myocytes of the precapillary arterioles were
100.4⫾4.2 ␮m long and 6.7⫾0.2 ␮m wide (n⫽17) and made
2 or occasionally 3 turns around the endothelium, as could be
seen by tracking a myocyte through the arteriole and highlighted in color in Figure 1B (i and iii). Thus, a single
myocyte covered ⬇10 ␮m (8.5⫾1.4 ␮m) of the length of a
precapillary arteriole segment (Figure 1B).
In the small mesenteric arteries, the same (10-␮m) length
of a vessel segment contained ⬎20 myocytes of length
92⫾3.5 ␮m and width 5.7⫾0.2 ␮m (n⫽11). The myocytes
formed a triple layer of cells in the wall with each cell
contacting around 6 others (Figure 1D, i).
Effect of Single-Cell Activation
The structure described above suggested that activation of a
single myocyte might affect a segment of the precapillary
arteriole. Figure 2A shows examples of Ca rises in single
myocytes as a result of nerve stimulation of the precapillary
arteriole. The transmitted light Online Movie 4 also demonstrates the effects of single-cell rhythmic contraction on the
diameter of the arteriole and blood in motion. An increase of
Ca in a single myocyte causes contraction and a marked local
decrease in the diameter of the precapillary arteriole, as
shown in Figure 2B (i and ii; red circles and Ca traces,
respectively). The luminal diameter of the arterioles in the
area of a contracting cell was reduced to 53.3⫾6.2% of
control, which brought the endothelial cells into close apposition (n⫽10 cells, 5 vessels). Stimulation by phenylephrine
(PhE) or ET-1 produced rhythmic (phasic) changes in the
precapillary arteriole diameter and affected blood flow (Online Movies 3 and 4). In contrast, in mesenteric arteries
contraction of one smooth muscle cell had no effect on the
vessel diameter (Figure 2C, i, and 2B, ii; Online Movie 5).
How Are Myocyte Ca Signals Generated and Are
They Coordinated in Precapillary Arterioles?
Because a single myocyte in precapillary arterioles can
influence the same length of vessel that requires ⬎20 cells in
Borisova et al
Structure and Ca Signal in Precapillary Arterioles
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Figure 1. Architecture of microcirculation in intact rat ureteric wall. A, i and ii, Low- and high-power images of vessels showing: terminal arteriole (TA) (microvessel with inner diameter ⬍50 ␮m) and precapillary arteriole (PA) (the final generation of the terminal arterioles).
B, Images of tangential (i) and radial (ii) sections of precapillary arteriole. Several smooth muscle cells (SMC) have been identified with
colored circles (B, i) and then artificially colored to show the cells making 2 or more turns around the arteriole and the consequent
extensive apposition between parts of the same cell in B, iii. To do this, a series of optical sections from top to bottom of the in situ
vessel was taken (see Online Movie 1). This enabled the tracking of each cell by following its perimeter in every section and allowed
each cell to be followed and colored (see Online Movie 2). EC indicates endothelial cells. C, Images of tangential (i) and radial (ii) sections of a precapillary arteriole showing the abluminal and luminal surfaces of the endothelial cells, respectively. D, Tangential sections
of rat mesenteric artery showing multiple contacts between smooth muscle cells (i) and a luminal tangential view of the monolayer of
endothelial cells (ii). The 40-␮m scale bar applies to B, C, and D.
small diameter arteries, we hypothesized that: (1) the coordination of agonist-induced Ca signals produced by Ca entry,
which is required to control arteries, is redundant in the
precapillary arterioles; and (2) the mechanisms generating
their Ca signals would differ. We therefore compared the role
of the SR to that of Ca entry in generating and coordinating
Ca signals, along with functional effects, in both vessels.
In preliminary experiments, we established that stimulation
of the nerve endings produced asynchronous Ca oscillations,
which were resistant to suramin but inhibited by prazosin or
phentolamine. These data suggested that in precapillary
arterioles noradrenaline is the major neurotransmitter and that
it produces global Ca oscillations. We therefore applied the
long-lasting analog PhE to study the effect of local neurotransmitter release under well-controlled conditions and ET-1
to investigate the effects of a circulating hormonal agonist.
The effects of both agonists were compared for both terminal
arterioles and mesenteric arteries.
Figure 3A and 3B shows Ca measurements from mesenteric arteries stimulated by PhE (10 ␮mol/L) and ET-1 (20
nmol/L). Large, synchronized global oscillations of Ca were
associated with rhythmic activity (vasomotion) and vasoconstriction (Figure 3A, ii; Online Movie 6). Small asynchronous
Ca waves also occurred (* in Figure 3A, i). At low concentrations of PhE (ⱕ1 ␮mol/L), only asynchronous Ca waves
were produced and little or no change in artery diameter
occurred (Online Movie 5). If external Ca was removed or
nifedipine (1 to 10 ␮mol/L) applied, synchronous Ca oscillations rapidly disappeared (3.2⫾0.2 minutes), along with
rhythmic activity and only single Ca waves were produced in
response to agonists (Figure 3B, i; Online Movie 7). These
nifedipine-resistant Ca waves were inhibited by both ryanodine, a blocker of ryanodine receptors (Figure 3B, ii), and the
nonspecific IP3R blocker 2-APB (Figure 3B, iii), confirming
reports that both release channels are involved in their
generation.18 Similar effects of Ca removal and applications
of nifedipine, ryanodine and 2-APB were also seen on the
response to ET-1 (20 nmol/L).
In precapillary arterioles, a very different pattern of Ca
signals was found. At both low and high concentrations,
agonists (PhE, 1 to 100 ␮mol/L; ET-1, 1 to 20 nmol/L) only
produced asynchronous Ca waves (see lack of synchronization between the 2 cells in Figure 3C, i and ii; Online Movie
9), and no role for Ca entry was found (Figure 3D). There was
no transmission of waves to neighboring cells (Figure 3C;
Online Movies 8 and 9). These waves produced rhythmic
activity and local vasoconstriction (Figure 3C, iii). Thus,
unlike mesenteric arteries, irrespective of agonist concentration, Ca waves never synchronized. The Ca waves were
resistant to removal of external Ca (Figure 3D, i) and
nifedipine (not shown) and continued with little decrement
even after 30 to 90 minutes in Ca-free solution. In contrast to
the mesenteric artery, in precapillary arterioles, ryanodine
(which abolished responses to caffeine) did not affect
agonist-induced Ca waves (Figure 3D, ii). However, as was
the case in the mesenteric artery, 2-APB abolished them
(Figure 3D, iii).
Calcium Signals in Endothelium
Endothelial cells could be seen clearly in precapillary arterioles (Figures 1C and 4A; Online Movies 1 and 2). They had
nuclei that protruded into the lumen, an average cell length of
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Circulation Research
September 25, 2009
dine receptor (Figure 4D). Caffeine (3 to 10 mmol/L) did not
stimulate but in fact inhibited spontaneous Ca signaling in the
endothelial cells (Figure 4D, iv). We next investigated the
effect of these endothelial Ca signals on the Ca signaling in
vascular myocytes.
Effect of Endothelial Ca Signals on
Myocyte Function
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Figure 2. Comparison of single-cell activation in precapillary
arterioles and mesenteric arteries. A, Example of activity in a
single myocyte in a precapillary arteriole loaded with Fluo-4 and
stimulated via its nerve. A rise of [Ca2⫹]i can be seen in 1 cell.
This precapillary arteriole was treated with 20 ␮mol/L cytochalasin to reduce tissue movement. B, i, Images showing changes
in Ca signaling and diameter of a segment of the precapillary
arteriole. The lower image shows an increase in [Ca2⫹]i and
contraction of 1 cell (identified by red circle), produced in
response to a low concentration of PhE, so that only asynchronous nonrepetitive Ca waves occur randomly in cells. B, ii, Time
course of changes of [Ca2⫹]i (top) and vessel diameter (bottom)
from the region of the identified cell. C shows data for a mesenteric artery.
61.8⫾8.7 ␮m, and their greatest width was 3.9⫾0.1 ␮m and
height (thickness) 5.2⫾0.9 ␮m (n⫽21, 10 vessels). The endothelial cells of mesenteric artery were longer (80.6⫾5.2 ␮m) and
wider (10.9⫾0.5 ␮m) (n⫽15 cells, 5 vessels; Figure 1D, ii).
In agreement with previous studies,19 the endothelium of
the mesenteric arteries showed no spontaneous activity. The
endothelial cells of precapillary arterioles, however, were
spontaneously active producing Ca puffs: small localized Ca
releases from the ER (Figure 4B; Online Movie 10). Addition
of carbachol (CCh) (50 nmol/L) resulted in Ca oscillations,
the frequency of which was increased at higher concentrations (1 ␮mol/L) (Figure 4B). Figure 4C, i, shows the rapid
time course of extracellular [Ca] decline, as monitored by
dextran-conjugated Fluo-4 fluorescence in the lumen of the
arteriole. The spontaneous intracellular Ca signals continued
with gradual decreases in frequency and amplitude, for up to
30 minutes in Ca-free solutions (Figure 4C, ii; n⫽9). After
the disappearance of spontaneous releases, CCh could still
evoke Ca signals in the endothelial cells (Figure 4C, iii;
Online Movie 11). The Ca signals were abolished by inhibition of SERCA, IP3R, or phospholipase C but not of ryano-
In mesenteric artery, myocytes were stimulated by ET-1 (20
nmol/L) or PhE (10 ␮mol/L) to produce Ca signals and
mechanical responses (rhythmic activity and vasoconstriction) (Figure 5A). Maintaining this stimulation, the endothelium was activated by 1 ␮mol/L CCh: activation of Ca
signaling in the endothelial cells produced inhibition of Ca
signaling in the myocytes and vasodilation (Figure 5A;
Online Movie 12), as reported by others.16,22
In contrast to the arteries, in precapillary arterioles no
effect on Ca signaling or vasoconstriction occurred with
stimulation of the endothelium Ca signaling (Figure 5B;
Online Movie 14). We tested this finding using another
protocol: Ca signaling in the endothelium was activated by
CCh and then PhE (10 ␮mol/L) or ET-1 (10 nmol/L) applied
to stimulate the myocytes. Activation of the endothelium
prevented the normal activation of the myocytes in response
to these agonists in mesenteric arteries (Figure 5C); no
synchronized Ca waves or changes of tone occurred, only
small asynchronous rises of Ca (Figure 5C; Online Movie
13). In contrast, in precapillary arterioles, prestimulation with
CCh did not prevent normal stimulation of myocytes by PhE
or ET-1, ie, Ca waves and tone were unaffected (Figure 5D;
Online Movie 15). In the precapillary myocytes, the frequency of global Ca waves/oscillations evoked by PhE was
0.035⫾0.003 Hz in the presence of CCh compared to
0.036⫾0.002 Hz before CCh (n⫽6). When waves were
evoked by ET-1, their frequency was 0.046⫾0.004 Hz in the
presence and 0.044⫾0.002 Hz in the absence of CCh (n⫽8).
CCh also had no effect on the tone produced by stimulation
with ET-1. With CCh, the mean diameter decreased to
61.4⫾3.4% compared to 64.1⫾3.2% in its absence.
The above data suggested lack of NO- or EDHF-mediated
effects on Ca signaling and changes in vascular tone induced
by agonists in precapillary arterioles. We tested this by
applying the NO donors Na nitroprusside (20 ␮mol/L) or
SNAP (20 ␮mol/L); these also had no significant effect on
the Ca signals or mechanical activity in precapillary arterioles
(Figure 5F; n⫽9). Frequency of oscillations in the presence of
ET-1 was 0.046⫾.0.004 and 0.048⫾.0.004 Hz in the absence
and presence of SNAP, respectively. SNAP or Na nitroprusside (not shown) abolished synchronized Ca oscillations and
rhythmic activity in the mesenteric preparations (n⫽7), mimicking the effects of CCh (Figure 5E). Consistent with the
effects of NO donors, the membrane permeable analog of
cGMP, 8-pCPT-cGMP (100 ␮mol/L), abolished Ca oscillations and rhythmic activity in mesenteric arteries but had no
effect in precapillary arterioles (n⫽4, 2 animals, data not
shown).
The only situation in which the endothelial Ca signals
produced by CCh inhibited precapillary arteriolar myocyte
Ca signaling and tone was when the vessel had been pre-
Borisova et al
Structure and Ca Signal in Precapillary Arterioles
5
Mesenteric artery
A
F/F0
1.0
3.0
B
PhE
3.0
PhE
2.0
*
*
*
i
i
0.8
Average signal
2.6
F/F0
PhE
PhE
Caf
F/F0
ii
1.0
PhE
0-Ca
F/F0
2
Caf
Ry+Nif
ii
1.0
100
%
Vessel diameter
3.6
iii
70
PhE
PhE
Caf
F/F0
Caf
2-APB+Nif
iii
1.0
60 s
60 s
Precapillary arteriole
D
C
ET-1
3.0
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F/F0
2.0
i
Cell 1
1.0
ii
Cell 2
100
%
Vessel diameter
0
iii
20 s
i
Caf
Caf
PhE
F/F0
1.0
2.6
1.0
PhE
0-Ca
F/F0
0.8
2.0
2.0
F/F0
PhE
PhE
Ry+Nif
ii
PhE
PhE Caf
Caf
F/F0
2-APB
iii
1.0
treated with a potassium channel blocker (tetraethylammonium, 5 mmol/L) in the presence of the L-type Ca channel
agonist Bay K-8644 (1 ␮mol/L), so that fast propagating Ca
spikes evoked by direct electric field stimulation were produced (Figure 5G). These electrically evoked Ca spikes,
which depended only on Ca entry in both types of vessels
(Figure 5G, ii)23 and produced transient vasoconstriction, were
reversibly inhibited during activation of endothelial Ca signaling
by CCh (Figure 4G, iii; Online Movie 16) but were resistant to
NO donors, Na nitroprusside or SNAP (Figure 5H).
Discussion
Combining imaging with Ca signaling in intact, in situ
precapillary arterioles has provided unique insight into the
structure and mechanisms that enable them to perform their
specialized function within the circulatory system. The data
show that precapillary myocytes wrap at least twice around
the arterioles, and the activation by Ca signaling of a single
myocyte is sufficient to produce functional effects. The
situation is clearly different in the small arteries, where tens
of myocytes need to be simultaneously activated to bring
about functional change. The control of tone by individual
myocytes enables a very precise response of the microcirculation to local and systemic stimuli.
The use of in situ confocal Ca imagining brings many
advantages to the study of the microcirculation, especially the
maintenance of normal cell architecture, contacts and the
ability to make detailed spatial and temporal measurements,
but it is also the case that the arterioles are not pressurized.
However, (1) the precapillary arterioles we studied main-
Figure 3. Calcium and contraction in precapillary arterioles and mesenteric arteries. A, Measurements in mesenteric vascular smooth muscle cells showing Ca
signaling in a myocyte (i), mean Ca measured over vessel segment (ii), and
changes in artery diameter (iii). Note that
in the individual smooth muscle cell 2
types of Ca oscillations occur: large
amplitude in phase with the average signal; low-amplitude (marked with asterisks)
Ca waves, which did not coincide with
average signal. B, Effects of maneuvers
on Ca signaling in mesenteric myocytes
induced by PhE (10 ␮mol/L). Traces
show: Ca-free solution (i), with a 3 minute
gap between the traces; ryanodine (Ry)
(50 ␮mol/L) and nifedipine (Nif) (1 to
10 ␮mol/L (ii); 30 minutes gap between
the traces; and 2-APB (50 ␮mol/L) (iii). C,
Asynchronous Ca oscillations in 2 sites in
a cell and diameter (bottom trace) in a
precapillary arteriole. D, Effects of Ca-free
solution: nifedipine (i), ryanodine (ii), and
2-APB (iii) on 10 ␮mol/L PhE-induced Ca
signaling in terminal arteriole; 30 minutes
gap between the 2 parts.
60 s
tained their cylindrical shape, were patent and red blood cells
could be seen moving along the lumen (Online Movie 4); (2)
in vivo, these small vessels function at relatively low pressures (⬍20 mm Hg)24; and (3) the precapillary arterioles were
studied with vasoconstriction produced by 2 physiological
agonists. Thus, although not pressurized or perfused, we
consider that, in other respects, near-physiological conditions
were replicated. The fact that we have studied 2 precapillary
vascular beds (ureter and vas deferens) and found no difference between them provides additional confidence in the
generality of our findings.
Our results show that contraction of a single myocyte is
sufficient to nearly occlude a precapillary arteriole. The
question then arises whether this can also occur when the
vessel is pressurized? Using LaPlace’s law and taking force
generation in single vascular myocytes to be 0.5⫻105
N 䡠 m⫺225 and the wall thickness (diameter of the myocyte) to
be 6 ␮m, then in a precapillary arteriole with a radius of
15 ␮m, the pressure generated by a single myocyte is around
15 kPa (112 mm Hg). This will clearly exceed that of the
pressure in these vessels and therefore contraction of 1
myocyte would be expected to be able to stop blood flow. For
the mesenteric artery, using the same values for force generation and cell thickness but a diameter of 120 ␮m, we
calculate a pressure of just 2 kPA (15 mm Hg) is generated by
an individual myocyte, which is clearly insufficient to occlude the artery. Although these calculations are only approximate, they emphasize that activity in a single myocyte in a
precapillary arteriole can have much more functional effect
than is the case in a larger vessel.3
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September 25, 2009
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Figure 4. Ca signaling in endothelial cells of precapillary arterioles. A, Pseudocolor images of endothelial cells showing spontaneous
activity, Ca puffs. B, i and ii, Changes of intracellular Cain 2 regions of cell from A, showing spontaneous Ca activity and the effects of
adding 50 nmol/L and 1 ␮mol/L CCh. iii, Recordings superimposed on an expanded time scale. C, i, Time course of change of extracellular [Ca] monitored by dextran-conjugated Fluo-4 fluorescence in the lumen of the arteriole. ii and iii, Effects of Ca removal on
spontaneous Ca signaling (gap of 20 minutes between traces) and CCh-induced Ca signaling. Note that Ca puffs persist long after
external Ca has been removed (C, ii). D, Effects of various compounds on spontaneous and CCh-induced Ca signaling in endothelial
cells. From top to bottom: the SERCA pump inhibitor cyclopiazonic acid (CPA) (20 ␮mol/L) (i); 2-APB (50 ␮mol/L) (ii); phospholipase C
inhibitor U-73122 (10 ␮mol/L) (ureteric arterioles) (iii); and caffeine (caf) (5 mmol/L) and ryanodine (50 ␮mol/L) (Vas deferens arterioles)
(iv). The gaps in the traces are (in minutes): 20 (i); 30 (ii); 20 (iii); 20 (iv).
We found that a distinct pattern of Ca signals occurs in
precapillary arterioles, with agonists producing only IP3mediated Ca waves. Unlike a previous suggestion,26 these
signals do not require Ca entry, do not synchronize, and
produce only local changes in precapillary arteriolar diameter. Our data reveal that precapillary arterioles do not possess
the coordinated and propagated Ca signals necessary for
recruiting a group of myocytes and synchronizing their
responses to generate vasomotion in larger vessels or the
mechanisms responsible for this. Consistent with this lack of
synchronization, and in agreement with in vivo studies on
some terminal arterioles,27 we did not see the propagated
vasoconstriction responsible for communication with upstream arteries in larger vessels with neurogenic stimulation.
These differences in Ca signaling pathways between the
precapillary arterioles and small arteries, which underlie their
different roles within the circulatory system, presumably
reflect differences in expression of ion channels and pumps.
A gradient in responses and expression of channels and
receptors with vessel diameter has previously been suggested.28
Although poorly defined, earlier studies comparing conduit
and resistance vessels5,6 had led to the suggestion that the SR
plays a less important role in small vessels.29 Our Ca
signaling data have directly tested this in precapillary arterioles and do not support such conclusions. Using Ca-free
solutions, we have shown that in precapillary arterioles, Ca
entry is not required for agonists to produce constriction but
rather IP3-induced SR Ca release is paramount, and this
feature can be regarded as a specialization of these vessels.
Indeed, the continued functioning for over an hour in
Ca-free solution is unique for smooth muscle. This suggests that in precapillary arterioles, there are either differences in plasma membrane Ca extrusion mechanisms or
differences in the SR such as a particularly avid and tightly
controlled Ca reuptake mechanism by the SERCA and its
accessory proteins.
A surprising finding of the present study was that neither
CCh nor NO donors (Na nitroprusside, SNAP) nor a membrane permeable analog of cGMP affected myocyte Ca
signaling induced by agonists in precapillary arterioles. That
NO donors dilate larger vessels more potently than terminal
arterioles has been reported by others,30,31 and has led to the
suggestion that guanylate cyclase or other intermediaries are
absent from some small vessels,32,33 and that EDHF rather
than NO is dominant in small vessels34 (although effects
attributed to NO were found in cremaster microcirculation1).
Our data are consistent with these findings. They also suggest
that the effects of endothelial Ca signaling on arterial myocyte Ca are mediated not via SR Ca release but Ca entry,
because CCh stimulation had no effect on SR Ca release but
inhibited Ca spikes, which were dependent on Ca entry. We
also found the endothelial cells of the arterioles in situ to be
Borisova et al
CCh
B
3.0
Myocyte
1.0
1.7
1.0
3.4
30 s
PhE
PhE
CCh
F/F0
0.8
30 s
PhE
CCh
iii
1.7
Endothelial
cell
1.0
1.0
30 s
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
F
SNAP
2.0
iv
*
*
*
*
*
*
*
*
*
*
*
*
0-Ca
Myocyte
CCh
2.5
*
*
*
*
Myocyte
F/F0
Endothelial
cell
1.0
30 s
10 s
H
SNAP
SNAP
2.5
F/F0
F/F0
Myocyte
1.0
*
2.5
1.0
1.7
Myocyte
*
F/F0
Myocyte
F/F0
2.6
F/F0
PhE
2.4
1.0
2.1
ii
Endothelial
cell
1.0
1.0
E
1.0
1.0
D
2.5
F/F0
1.7
F/F0
TEA+BayK 8644
i
E T -1
3.4
C
G
CCh
E T -1
F/F0
7
Precapillary arteriole
Mesenteric artery
A
Structure and Ca Signal in Precapillary Arterioles
30s
Myocyte
1.0
1.0
10 s
30s
Figure 5. Effects of endothelium activation on agonist induced Ca signaling and vasomotion. A and C, Measurements of Ca in mesenteric myocytes and endothelial cells with ET-1 (20 nmol/L) followed by CCh (1␮mol/L) or PhE (10 ␮mol/L) alone or following CCh. B
and D, The effects in precapillary arterioles of adding ET-1 (10 nmol/L) and then CCh (1 ␮mol/L) or PhE (10 ␮mol/L) alone or following
CCh. E and F, Effect of the NO donor SNAP (20 ␮mol/L) on Ca oscillations induced by PhE in mesenteric (E) and vas deferens precapillary arterioles (F). G, The effect of direct muscle stimulation in the presence of Bay K 8644 (1 ␮mol/L) and tetraethylammonium (TEA)
(5 ␮mol/L) (i). The resulting Ca spikes in myocytes are abolished by Ca-free solution (ii) and by CCh-induced (iii) endothelial Ca signal
(iv). H, The lack of effect of SNAP on Ca spikes.
spontaneously active which agrees with earlier observations,1
which is not the case in larger vessels. Direct investigation of
why NO signaling fails to influence precapillary myocytes is
called for as the ramifications of these data are important for
our understanding of the microcirculation. That the possibility of communication between the endothelial and smooth
muscle cells exists was demonstrated under nonphysiological
conditions of pharmacological enhanced excitability and
electrically driven activity.
In summary, precapillary arterioles have been studied in
more mechanistic detail than previously by using confocal
imaging and Ca measurements. We have shown that both
their structure and Ca signaling contribute to the functional
specialization required in these vessels. The precapillary
arterioles will respond functionally to changes in metabolites,
paracrine factors, or nervous activity with single-cell control,
and this may also explain why the role of the endothelium
appears to be unimportant for controlling tone. It remains to
be elucidated which factors are most important in different
vascular beds and the mechanisms by which they influence
myocyte Ca signaling. Changes in the structure and functions
of the microcirculation underlie the morbidity associated with
conditions such as diabetes, infection, and hypertension.35–38
By revealing differences in the control of function in precapillary arterioles from larger vessels, our work highlights the
need for therapeutic interventions to be targeted to these
newly described mechanisms.
Sources of Funding
The work was supported by grants from the British Heart
Foundation.
Disclosures
None.
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How Structure, Ca Signals, and Cellular Communications Underlie Function in
Precapillary Arterioles
L. Borisova, Susan Wray, D. Eisner and T. Burdyga
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. published online August 27, 2009;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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Supplementary Videos
Click on links to play
Movie 1
Movie 2
Movie 1.
Z-stepping through a Fluo-4 loaded precapillary arteriole. A series of optical
sections using spatial module was taken from a Fluo-4 loaded descending branch of
the ureteric precapillary arteriole showing full perimeter of the blood vessel.
Movie 2.
Z-stepping through a Fluo-4 loaded precapillary arteriole, using colour to reveal
details of myocyte morphology.Movie 2 is a replica of Movie 1 in which four
smooth muscle cells were artificially coloured using Jasc Software Animation Shop
Movie maker to reveal details of their morphology.
Movie 3
Movie 3
Movie 4
Movie 5
Calcium signalling and mechanical response induced by activation of a single
smooth muscle cell, in precapillary arteriole. Precapillary myocytes were stimulated by
1 μM of phenylephrine Vasomotion induced by contraction of individual smooth muscle
cells facilitates blood flow in precapillary arterioles.
Video 4. Vasomotion produced by mechanical oscillations of individual smooth muscle cell in
precapillary arteriole in situ. This movie demonstrates vasomotion produced by
mechanical oscillations of a single smooth muscle cell induced by 1nM ET-1 in ureteric
precapillary arteriole in transmitted light.
Movie 5. Calcium signalling and mechanical response induced by activation of a single
smooth muscle cell, in mesenteric artery. Smooth muscle cells were stimulated by
1 μM of phenylephrine.Vasomotion induced by contraction of individual smooth muscle
cells in mesenteric artery had no effect on the diameter of the blood vessel.
Movie 6
Movie 6.
Movie 7
Asynchronous intracellular and synchronized intercellular Ca oscillations associated
with vasoconstriction/vasomotion evoked by phenylephrine in mesenteric artery myocytes
Mesenteric artery was stimulated by 10 µM phenylephrine.
Movie 7. Inhibition of synchronised Ca oscillations and vasoconstriction/ vasomotion in mesenteric
artery evoked by phenylephrine after exposure to Ca –free solution.Mesentric artery was
stimulated by 10 µM phenylephrine after 10minutes exposure to Ca2+ -free solution with 2mM EGTA.
Movie 8
Movie 9
Movie 8. Asynchronous intracellular Ca waves evoked by phenylephrine in smooth muscle
cells of precapillary arterioles. Tangential section of ureteric precapillary arterioles
stimulated by 1 µM phenylephrine.
Movie 9.
Asynchronous intracellular Ca oscillations evoked by endothelin-1 associated with a
non-propagating vasoconstrictionin a segment of precapillary arteriole. Radial section
of precapillary arteriole stimulated by 10nM endothelin -1.
Movie 10
Movie 11
Movie 10. Spontaneous Ca signalling in endothelial cell of ureteric precapillary arteriole.
Radial section of precapillary arteriole showing spontaneous activity in individual
endothelial cell.
Movie11. Calcium oscillations induced by carbachol in endothelial cells of precapillary
arterioles exposed to Ca-free solution. Precapillary arteriole was pre-treated with
Ca free solution with 2mM EGTA for 60 minutes.
Movie 12
Movie 13
Movie 12. Simultaneous recording of intracellular Ca signalling in endothelial cells and
mesenteric myocytes: effects of carbachol after phenylephrine. This movie
demonstrates that in the rat mesenteric artery activation of endothelial cell Ca signalling
blocks Ca oscillations in smooth muscle cells leading to vasodilation.
Movie 13. Simultaneous recording of intracellular Ca signalling in endothelial cells and
mesenteric myocytes: effects of phenylephrine after carbachol. This movie
demonstrates that in the rat mesenteric artery activation of endothelial cell Ca signalling
prior to agonist stimulation of smooth muscle cells selectively blocks synchronized Ca
oscillations and vasomotion in rat mesenteric artery.
Movie 14
Movie 15
Movie 16
Movie 14. Lack of effect of endothelial cell Ca signalling
on myocyte activity in precapillary
.
.
arterioles: carbachol following endothelin-1. This movie demonstrates that activation of
endothelial cell Ca signalling by carbachol on smooth muscle cells pre-stimulated by agonists
did not terminate agonist – induced Ca oscillations in arteriolar myocytes.
Movie 15. Lack of effect of endothelial cell Ca signalling on myocyte Ca signalling in precapillary
arterioles: endothelin-1 applied after carbachol. This movie demonstrates that activation
of endothelial cell Ca signalling prior to agonist stimulation of arterioles had no effects on
agonists –induced Ca oscillations in arteriolar myocytes.
Movie 16. Inhibitory effects of carbachol induced Ca signalling in endothelial cells on Ca spikes
evoked by electrical field stimulation in the presence of TEA and Bay-K-8644 in the
arteriolar myocytes. This movie demonstrates that activation of Ca signalling in endothelial
cells can inhibit Ca signalling in arteriolar myocytes dependent on Ca entry via voltage gated
Ca channels.