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* The John B. Pierce Laboratory and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06519, USA; and
Department of Pharmacology, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut 06536, USA
2Correspondence: The John B. Pierce Laboratory, Yale University School of Medicine, 290 Congress Ave., New Haven, CT 06519, USA. E-mail: sssegal{at}jbpierce.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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-nitro-L-arginine (LNA), and
both local and conducted responses were inhibited by 17-octadecynoic
acid (17-ODYA). Microejection of KCl evoked a biphasic response: a
transient conducted vasoconstriction (inhibited by nifedipine),
followed by a conducted vasodilation that was insensitive to LNA,
indomethacin, and 17-ODYA. Phenylephrine evoked focal vasoconstriction
that did not conduct. Perivascular sympathetic nerve stimulation evoked
constriction along arterioles that was inhibited by tetrodotoxin. These
findings indicate that for arterioles in the mouse cremaster muscle,
nitric oxide and endothelial-derived hyperpolarizing factor (as shown
by LNA and 17-ODYA interventions, respectively) mediate vasodilatory
responses to ACh but not to KCl, and that vasomotor responses spread
along arterioles by multiple pathways of cell-to-cell communication.
Hungerford, J. E., Sessa, W. C., Segal, S. S. Vasomotor
control in arterioles of the mouse cremaster muscle.
Key Words: microcirculation • vasodilation • vasoconstriction • conduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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2
3)
. While these models
provide valuable insight into the determinants of cardiovascular
function, the increased emphasis on ‘functional genomics’
necessitates the development of appropriate models to understand
cardiovascular function in normal mice (4)
.
A critical dimension of the cardiovascular system is the control of
blood flow, which underlies the distribution of oxygen and nutrients
throughout the body. The specific cell-to-cell communication pathways
that regulate vasodilation and vasoconstriction in the peripheral
vasculature of the mouse are largely undefined. In conduit vessels as
well as microvessels, vasomotor control appears to be coordinated
within and among vascular segments (5
6
7
8)
. In the
microcirculation, the ‘conduction’ of vasomotor responses reflects
the initiation and spread of electrical signals that travel along
endothelial and smooth muscle cell layers (6
, 9)
. As there
is little information concerning blood flow control in the mouse (with
the exception of cerebral microvessels; ref 10
), it is
unknown whether conduction occurs in the mouse microvasculature, and,
if so, by what signaling pathways.
In the present study, our purpose was to develop a preparation of the
mouse cremaster muscle in order to define constitutive mechanisms of
vasomotor control in arterioles. A spectrum of stimuli that have been
standardized in other systems (6
, 9
10
11
12)
was applied for
this purpose. These included elevated superfusate oxygen to determine
oxygen sensitivity, acetylcholine (ACh) to evoke endothelial-dependent
vasodilation via activation of muscarinic receptors, norepinephrine
(NE) and phenylephrine (PE) to evoke constriction via
-adrenoreceptors on smooth muscle cells, potassium chloride (KCl) to
evoke changes in membrane potential of vascular cells, and electrical
stimulation of perivascular nerves. The goal of these studies was
threefold: to 1) define the properties of vasoconstriction
and vasodilation in arterioles of the mouse cremaster muscle,
2) determine whether vasomotor responses spread along these
vessels, and 3) identify pathways of cell-to-cell
communication involved in the coordination of vasomotor responses
within and among arteriolar branches.
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MATERIALS AND METHODS |
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The left cremaster muscle was prepared in a manner similar to that
previously described for the hamster (13
, 14)
. Briefly, an
incision was made through the skin and the muscle was dissected from
the surrounding connective tissue. The exposed muscle was positioned on
a clear Sylgard (Dow Corning, Midland, Mich.) pedestal and a
longitudinal incision was made on the ventral surface of the muscle
from the apex to the inguinal canal, with care taken to minimize
disruption of the vascular supply. Edges of the muscle were pinned to
the pedestal, then the testis and epididymis were dissected away and
repositioned in the abdominal cavity after severing the deferential
arteriole and venule. The cremaster muscle was spread radially on the
pedestal and pinned at the edges. The preparation was superfused
continuously (5 ml/min) with a bicarbonate-buffered physiological salt
solution (PSS; 34°C, pH 7.4) of the following composition (in mM):
137 NaCl, 4.7 KCl, 1.2 MgSO4, 2
CaCl2, 18 NaHCO3.
Superfusion solutions were equilibrated with 5%
CO2/95% N2 unless noted.
Video microscopy
The preparation was then transferred to the stage of an
intravital microscope (GFL, Zeiss) and viewed using Köhler
illumination (condenser NA = 0.32). The image was acquired using a
Zeiss UD 40 objective (NA = 0.41), then coupled to a video camera
(Toshiba, IKC30A) and monitor (Sony, 1343 MD) with a final
magnification of x1200. Internal vessel diameter, measured from the
internal edges of the vessel lumen (which typically corresponded to the
width of the red blood cell column), was recorded from the video
monitor using a video caliper (Microcirculation Research Institute,
Texas A&M University); spatial resolution was 
1 µm. Data were
acquired at 40 Hz using a PowerLab system (model 800, CB Sciences)
coupled to a Pentium-based personal computer.
Microiontophoresis and pressure microejection of vasoactive
stimuli
Micropipettes were pulled (model P-97, Sutter Instruments) from
borosilicate glass capillary tubes (Warner Instruments, GC120F-10).
Inner tip diameters were ~ 1 µm for microiontophoresis
pipettes and 2 µm for pressure microejection. Micropipettes were
backfilled with filtered (0.2 µm; Acrodisc, Gelman Sciences, Ann
Arbor, Mich.) ACh (1 mol/l), PE (0.5 mol/l), or KCl (1 mol/l) and
positioned with their tips adjacent to the vessel wall using a
hydraulic micromanipulator (Siskiyou Design Instruments, model MX630R)
that was mounted onto the animal platform. The entire preparation was
moved as a unit without disturbing micropipette locations. For
microiontophoresis, micropipettes containing ACh or PE were connected
to a programmer (World Precision Instruments, model 160) via an Ag/AgCl
wire. The programmer was gated by the PowerLab system. An additional
Ag/AgCl wire secured at the edge of the preparation served as a
reference electrode.
Based on preliminary studies, an ejection current of 500 nA and
duration of 500 ms were determined to be just sufficient to elicit a
maximum dilatory response at the local site of stimulation; this was
chosen as the criterion stimulus for ACh microiontophoresis. In a
similar manner, the criterion stimulus for PE microiontophoresis was
determined to be 1000 nA, 500 ms. Retain currents for both agonists
were 200 nA. KCl was delivered using pressure microejection
(15–20 psi, 100–750 ms; model PLI-100, Medical Systems); pulses were
calibrated for each experiment, such that the stimulus did not visually
contract (i.e., depolarize) the surrounding skeletal muscle fibers. The
ejection micropipette was positioned such that superfusion flow
directed KCl across and away from the arteriole to avoid its convection
along the vessel (15)
.
Perivascular nerves
Perivascular nerves were stimulated electrically (1 ms pulse @
32 Hz for 30–50 s, 60–100V; Grass, model S48) with a saline-filled
micropipette (inner diameter 1–2 µm) positioned adjacent to proximal
2A segments (16)
.
Perivascular nerves were visualized by labeling with glyoxylic acid and
counterstaining the muscle with Pontamine sky blue using previously
described methods (17
18
19)
. After staining, the intact
muscle was mounted and viewed on a Nikon E800 microscope using Fluoplan
objectives. Images were acquired with a SPOT digital camera (Diagnostic
Instruments) and processed using Adobe Photoshop.
Reagents
Reagents were purchased from J. T Baker (Phillipsburg, N.J.) or
Sigma (St. Louis, Mo.) unless noted and were prepared as follows: ACh,
NE, PE, KCl, tetrodotoxin (TTX), and -conotoxin GVIA (Auspep) were
dissolved in deionized water;
N-nitro-L-arginine (LNA) was dissolved in PSS,
and indomethacin, nifedipine, and 17-octadecynoic acid (17-ODYA,
BioMol) were dissolved in EtOH; the final concentration of EtOH in the
superfusate was 0.01%.
Experimental procedures
First, second, and third order arterioles (1A, 2A, and 3A) in
the central region of the preparation were chosen for experimentation
in order to minimize the effect of tissue damage (20)
. The
preparation was equilibrated for 20–30 min, during which time the
arteriolar network was scanned and a diagram made to identify sites for
data collection. Resting (baseline) diameters of the chosen vessels
were then measured. To evaluate oxygen sensitivity, the superfusate was
equilibrated with 10% O2 (5%
CO2, balance N2) for 10 min
and the change in diameter was recorded.
Cumulative concentration-response curves were obtained for ACh and NE. Incremental concentrations of ACh or NE were added to the superfusate; each log increase (10-10 to 10-4 mol/l) was equilibrated until diameter reached a steady state (5–10 min). Measurements were obtained for each vessel order (1A, 2A, 3A) in a given preparation. Maximum diameters were taken as the peak response to ACh.
Conduction of vasomotor responses
For studies of conduction, stimuli were delivered to the distal
end of relatively unbranched 2As via micropipettes positioned with
their tips adjacent to the arteriolar wall. One agonist (e.g., ACh or
PE) was studied at a time. To determine the effect of distance on the
amplitude of conducted responses, diameter responses were measured at
the ‘local’ site of stimulation and for at least four successive
upstream locations (350, 700, 1050, 1400 µm, and beyond) along the
arteriole, defined using a calibrated eyepiece reticule. For each
observation along an arteriole, a separate stimulus was delivered at
the local site; the vessel was allowed to recover for 2–3 min between
stimuli to restore resting diameter.
Responses were then re-evaluated in the presence of one or more of the
following inhibitors: LNA (100 µmol/l), indomethacin (10 µmol/l),
nifedipine (10 µmol/l), TTX (1 µmol/l), and -conotoxin (150
nmol/l). Each of these was added to the superfusate to achieve the
desired final concentration and equilibrated for 15–20 min before
testing for effects. The inhibitor was then washed from the preparation
with control PSS. Because 17-ODYA acts as a suicide substrate
(21
, 22)
, the following protocol was used to ‘prime’ the
system by promoting its use by cytochrome P450 enzymes. 17-ODYA (10
µmol/l) was delivered via the superfusate for 45 min. During this
time, superfusate oxygen was cycled between 0 and 21% every 15 min,
and ACh was microiontophoresed (500 nA, 500 ms) onto the vessel every 5
min. The preparation was then re-equilibrated with control PSS for 15
min before the experimental stimuli were repeated to evaluate the
effects of 17-ODYA.
Controls
Controls demonstrated reproducibility with a given stimulus at a
defined distance; tachyphalaxis to ACh, PE, and KCl stimuli were
negligible over the time course of a typical experiment. Moving a
stimulus micropipette more than 100 µm away from the vessel abolished
vasomotor responses, demonstrating that the spread of the response (see
Results) was not due to diffusion of the vasoactive agent along the
arteriole or to its convection in the superfusate (14)
.
Inclusion of 0.01% EtOH in the superfusate and had no effect on either
the baseline diameter or vasomotor responses to stimuli. To control for
the ‘priming’ protocol used with 17-ODYA, oxygen was cycled and ACh
was pulsed intermittently over the preparation in the absence of
17-ODYA, using the same time course as when 17-ODYA was present.
Data analysis
One or two arteriolar networks comprising 1A, 2A, and 3A
branches were studied in each preparation. Each network was treated as
a separate experiment. Data were analyzed with SigmaStat/SigmaPlot
software. All summary data represent 
5 vessels from 5
animals and are presented as mean ± SEM. Paired
t tests (for local responses) and two-way repeated measures
analysis of variance (for conducted responses) were used to evaluate
the effects of treatment and distance along the arteriole. Post hoc
analyses were performed using Tukey’s comparisons when a significant F
ratio was obtained. Results were considered statistically significant
with P 0.05.
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RESULTS |
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). For all three branch orders, raising superfusate oxygen from 0 to
10% significantly reduced resting diameter (Table 1
,
*P<0.05). Concentration-dependent diameter changes
occurred in all branch orders in response to ACh and NE (data not
shown). The concentration of either ACh or NE at 50% maximum diameter
change was taken as the EC50 value (Table 1)
.
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Focal stimulation of arterioles and regulation of conducted
responses
Acetylcholine
ACh microiontophoresis evoked a robust vasodilation at the site of
application (‘local’, Fig. 1A
), which conducted rapidly (< 1 s) along the
arteriole. The amplitude of diameter change decayed with distance along
the arteriole (Fig. 1A
). Summary data demonstrate the
significant decrease in the magnitude of the vasomotor response from
the local site to the first conducted site, 350 µm upstream, which
decayed with distance along the arteriole (Fig. 1B
).
|
LNA depressed the local dilatory effect to ACh significantly
(*P<0.05, Fig. 2A
), but had no significant effect on diameter responses at
conducted sites (P=0.085, Fig. 2A
). Although
exposure to LNA caused a significant decrease in baseline diameter
(Table 2
), vasoconstriction alone (caused by raising superfusate
PO2) did not significantly change responses to
ACh from control values (data not shown). Addition of excess L-arginine
(1 mmol/l) to the superfusate reversed the diameter changes in the
presence of LNA (Table 2)
and restored the local response to ACh
(n=5, data not shown). Indomethacin had no effect on
baseline diameter (Table 2)
or the response to ACh (Fig. 2B
). In contrast, 17-ODYA attenuated both local and
conducted responses to ACh by approximately half (P<0.05,
Fig. 2C
) with no effect on baseline diameter (Table 2)
.
Further, in the presence of 17-ODYA, vasomotor responses to oxygen were
no longer observed, so that cycling between 0% and 21% had no effect
on vessel diameter. Time, protocol, and vehicle controls demonstrated
that the change in response to ACh was specific to the presence of
17-ODYA. Arterioles dilated maximally (to 37±2 µm, n=5)
to SNP (10 µmol/l) in the presence of 17-ODYA, demonstrating that
EC-independent vasodilation was unimpaired.
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Phenylephrine
The local response to PE microiontophoresis was robust; however,
vasoconstriction was constrained to the site of stimulation and did not
spread along arterioles (Fig. 3A, B
). Similar results were obtained with NE (data not
shown).
|
Potassium chloride
KCl evoked a biphasic response, such that a rapid (2–5 s) initial
vasoconstriction was followed by a longer lasting vasodilation (10–20
s; Fig. 4A
). As with ACh, the entire vessel responded synchronously.
Whereas the constrictor component of the response decayed rapidly
(typically by 1 mm) along the arteriole (bottom, Fig. 4B
),
the dilatory component consistently traveled along the vessel (> 1 mm)
with negligible decay (top, Fig. 4B
).
|
Stimulus–response relationships were investigated as a means of
discriminating between the constrictor and dilatory components of
biphasic responses to KCl. Decreasing stimulus duration from 750 to 100
ms significantly decreased the amplitude of both local and conducted
vasoconstriction (P<0.05, respectively) and attenuated the
distance that constriction spread along the arteriole
(P<0.05, bottom, Fig. 4C
). However, neither
local nor conducted dilatory responses varied with KCl stimulus
intensities (top, Fig. 4C
). Furthermore, in some
experiments, a brief pulse of KCl evoked only local and conducted
vasodilation without the initial vasoconstriction (data not shown).
A comparison of the summary data in Fig. 1B
and Fig. 4B
illustrates a striking difference in vasodilatory
responses induced by ACh compared to KCl. In ACh-evoked conduction
(Fig. 1B
), the amplitude of response decayed with distances
encompassing 350 to 2100 µm along arterioles. In contrast, responses
to KCl (Fig. 4B
) showed little decay along arterioles.
Unlike their effects on ACh-induced responses, neither LNA nor 17-ODYA
attenuated either the local or conducted vasodilation evoked by KCl
(data not shown, n=5). Neither vasodilatory nor
vasoconstrictor responses evoked by KCl were altered by TTX or
-conotoxin GVIA (data not shown, n=5).
Constrictions evoked by KCl and PE were compared with respect to the
actions of nifedipine. The addition of this L-type calcium channel
antagonist to the superfusate caused arterioles to dilate
significantly, such that a vasodilatory response to KCl was no longer
observed (Table 2)
. Nevertheless, the amplitude of (local) constriction
to PE was unchanged (data not shown). In contrast, nifedipine
attenuated the local response to KCl by ~ 80% (Fig. 4D
) and abolished conducted vasoconstriction (Fig. 4D
). In control experiments, dilating arterioles with SNP
(10 µmol/l) to the same extent as observed with nifedipine had no
effect on conducted vasoconstriction evoked by KCl (data not shown,
n=5).
Role of perivascular nerves
As demonstrated by glyoxylic acid staining (Fig. 5A, B
), mouse cremaster arterioles are enmeshed in a rich
plexus of sympathetic nerves. A proximal site on 2A branches was
stimulated with a microelectrode. Since the diameter of these vessels
increased progressively between the site of stimulation and the
furthest site of observation, each vasomotor response along the vessel
was normalized to its corresponding baseline diameter. Focal electrical
stimulation resulted in vasoconstriction, which spread along the entire
arteriole (control, Fig. 5C
). Addition of TTX attenuated the
response along the vessel and eliminated responses beyond 800 µm from
the stimulus. Nifedipine nearly abolished responses at distances beyond
the local stimulus, with only slight vasoconstriction remaining at 350
µm upstream. When -conotoxin GVIA was administered in the presence
of nifedipine (n=2, data not shown), only local, attenuated
constriction was observed in response to electrical stimulation.
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DISCUSSION |
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Mechanisms of conduction
In hamster cheek pouch arterioles, the spread of vasodilation and
vasoconstriction reflect the spread of hyperpolarization and
depolarization, respectively, along the vessel wall (9)
.
With the exception of perivascular nerve activation (which results in
regenerative action potentials), the amplitude of diameter change along
mouse cremaster arterioles was therefore taken to reflect the spread of
electrical signals among cells in the vessel wall. Stimuli of short
duration ( 750 ms) were used to minimize convection and/or diffusion
of agonists (23)
.
Vasodilation
ACh activates multiple signaling pathways that can result in
hyperpolarization of both endothelial and smooth muscle cell layers
(24
25
26)
. While hyperpolarization of endothelial cells
reflects ACh binding to muscarinic receptors (26)
, the
subsequent relaxation of smooth muscle cells can be accomplished by one
of several endothelial-dependent mechanisms (22
23
24
25
26)
. The
contribution of these signaling pathways to local or conducted
vasodilation has not been evaluated in the mouse. Therefore, we used
inhibitors to block specific components of purported pathways in order
to determine the mechanism through which ACh evoked vasodilator
responses.
Nitric oxide constitutively suppressed resting tone in all arteriolar
branch orders, as baseline diameter decreased significantly in response
to LNA in the superfusate (Table 2)
. Moreover, local (direct) responses
to ACh decreased significantly when NO synthase was inhibited by LNA.
The reversal of this inhibition with excess L-arginine confirmed the
specificity of NO synthase inhibition. These results are consistent
with observations from other vessels in the mouse (e.g., aorta, carotid
artery, and pial microvessels) that implicate endothelial NO as the
primary mediator of the dilatory response to ACh (10
, 27
28
29)
. Although the conducted response to ACh was slightly
depressed by LNA, it was not significantly different from control (Fig. 2A
). We therefore conclude that the stimulation of NO
release is not integral to the conduction of vasodilation (23
, 30)
.
Prostacyclin can act as a hyperpolarizing agent by opening
ATP-sensitive K+ channels (31)
. In
mice, cyclooxygenase metabolites (including prostacyclin) have been
implicated in the coronary vasodilatory response to ACh
(32)
. However, indomethacin had no effect on either local
or conducted responses in our preparation. Thus, cyclooxygenase
metabolites of arachidonic acid do not contribute to resting tone nor
do they play a role in the conducted response to ACh in mouse cremaster
arterioles (Fig. 2B
).
Given that NO and cyclooxygenase metabolites appeared to have a limited
contribution to the ACh-mediated conducted response, we hypothesized
that smooth muscle hyperpolarization is caused by the action of an
endothelium-derived hyperpolarizing factor (EDHF). Endothelium-derived
cytochrome P450 metabolites, particularly epoxyeicosatrienoic acids
(EETs), act in a paracrine manner to open KCa
channels and thus hyperpolarize and relax smooth muscle cells in a
variety of vascular preparations (33
34
35)
. The suicide
substrate 17-ODYA has been used as an effective specific inhibitor of
this pathway (22
, 36)
. Our studies show that treatment of
mouse cremaster arterioles with 17-ODYA significantly decreased the
local and conducted vasodilatory response to ACh. The remaining
conducted response then more closely resembled that seen with KCl.
These results support the hypothesis that cytochrome P450 metabolites
(most likely EETs (34)
released from arteriolar
endothelial cells) act as an EDHF, resulting in smooth muscle
hyperpolarization in mouse cremaster arterioles.
Whereas the activity of some cytochrome P450 isoforms generates EETs,
the -hydroxylase enzyme has been implicated as an oxygen sensor
within vascular smooth muscle cells of rat and hamster cremaster
arterioles, such that 20-HETE production causes smooth muscle
depolarization and vessel constriction (37
, 38)
. Cremaster
arterioles became insensitive to changes in oxygen during superfusion
of 17-ODYA, although basal tone of the arterioles was uneffected. These
results further reflect a dual role for cytochrome P450-mediated
signaling events in both constriction and dilation along the arteriolar
wall (34
35
36
37
38)
.
Microejection of KCl evoked a biphasic vasomotor response in
which the dilatory component spread further than the constrictor
component along the arteriole. Both endothelium and smooth muscle
layers have been shown to hyperpolarize (as well as depolarize) in
response to KCl. For example, biphasic changes in membrane potential
(an initial depolarization, followed by hyperpolarization) have been
reported in capillary networks in response to K+,
as measured by voltage sensitive dye (39)
. Isolated
cerebral and coronary rat arteries dilate in response to extracellular
K+ in the concentration range of 6–16 mM via
activation of inward rectifier K+ channels and/or
by stimulation of the
Na+/K+ ATPase in smooth
muscle cells (40
, 41)
. The present observations are
consistent with the hypothesis that as the elevation in extracellular
K+ concentration dissipates post-stimulation,
smooth muscle cells hyperpolarize, and this electrical signal then
spreads along the arteriolar wall. If the endothelium is also involved
in mediating conducted vasodilation to KCl, then NO-, prostanoid-, and
cytochrome P450-independent mechanisms are implicated (e.g.,
myoendothelial coupling, see below), as neither addition of LNA,
indomethacin, nor 17-ODYA had an effect on this response. Regardless of
pulse duration, the amplitude of the dilatory response remained the
same and did not decay significantly. In turn, this lack of decay
implies that the cells comprising the conduction pathway are
effectively coupled and may also reflect an active (regenerative)
signaling component intrinsic to the vessel wall (15
, 30)
.
Taken together, these findings suggest that at least two different
communication pathways can mediate conducted vasodilation in mouse
cremaster arterioles. The first is evoked by ACh and involves the
release of EDHFs; the second is evoked by acute changes in the
electrochemical gradient for K+. Moreover, the
ACh-evoked response remaining after treatment with 17-ODYA was similar
in amplitude to the vasodilatory response evoked by KCl (compare Fig. 2C
with Fig. 4B
). As cytochrome P450 metabolites
do not appear to act as an EDHF in all vessel preparations
(42)
, other molecules in addition to EETs have been
implicated (43
, 44)
. Indeed, K+ has
been proposed to act as an EDHF in response to ACh-induced vasodilation
(43)
. Alternatively, there may be direct coupling between
endothelium and smooth muscle, as indicated by the presence of gap
junctions between these cells in arterioles (45
46
47)
.
Additional studies are needed to determine whether these communication
pathways also participate in the conducted response to ACh in mouse
arterioles and how they may be affected by changes in genotype.
Vasoconstriction
Microejection of KCl evoked a transient vasoconstriction that
conducted along arterioles. In contrast, microiontophoretic delivery of
PE (and NE) resulted in constriction that was constrained to the local
site. The latter observations contrast with conducted responses to PE
in arterioles of the hamster cheek pouch (9)
, but agree
with the behavior of arterioles in its retractor muscle
(30)
. In turn, our data are consistent with the hypothesis
that KCl released adjacent to the vessel wall in high concentration
effectively depolarized smooth muscle (and endothelial) cells through a
Nernst effect, which evoked the conduction of vasoconstriction
(9)
. Therefore, it does not appear that the inability of
PE to evoke conducted vasoconstriction was due to a lack of coupling
between the cells that comprise the arteriolar wall.
Once PE acts on smooth muscle
1-adrenoreceptors, contraction occurs through
Ca2+ influx from extracellular sources and/or its
release from intracellular stores (48)
. Although we did
not measure changes in membrane potential in cremaster arterioles, we
evaluated the role of membrane potential by antagonizing
voltage-operated calcium channels that open with depolarization. In the
presence of nifedipine, the local response to KCl was attenuated and
the conducted response to KCl was abolished (Fig. 3D
). In
contrast, nifedipine did not change the vasoconstrictor response to PE.
This lack of effect of nifedipine suggests that the activation of
1-adrenoreceptors evoked the release of
intracellular Ca2+ stores rather than activating
L-type Ca2+ channels in the plasma membrane.
Based on these results, we hypothesize that focal administration of PE
does not cause a change in membrane potential sufficient to evoke
conducted vasomotor responses, despite robust local (i.e., direct)
responses. This interpretation is consistent with observations in
isolated hamster cheek pouch arterioles, where PE often failed to evoke
the conduction of a vasomotor response when electrical signals did not
reach a threshold value (11
, 12)
.
Modulation of vascular tone by perivascular sympathetic nerves
Arterioles in the mouse cremaster are richly invested by
sympathetic nerves, whose activation is blocked by TTX (as seen in the
hamster (16
, 19)
. Whereas sympathetic nerves can affect
vasomotor tone (49)
, TTX had no effect on resting
diameter, which may be attributed to the use of anesthesia and
suppression of basal sympathetic nerve activity. The effects of KCl
were independent of nerve activation, as these responses were not
altered by TTX or by -conotoxin GVIA. In the absence of nerve
activation, vasoconstriction conducted over relatively short distances
(~ 1 mm when elicited by KCl). During electrical stimulation, TTX
significantly reduced, but did not block, the spread of constriction.
This behavior indicates that focal depolarization of smooth muscle
cells resulted in conducted vasoconstriction that was effective for
distances < 1 mm. That TTX effectively blocked vasoconstriction
beyond 1 mm indicates that sympathetic nerve activation was required
for constriction to spread over greater distances. Nifedipine
suppressed the spread of constriction evoked by electrical stimulation,
indicating that the influx of Ca2+ through L-type
channels is integral to the spread of contraction along depolarized
arteriolar smooth muscle. Further, the persistence of local
constriction during electrical stimulation in the presence of
nifedipine argues for the release of neurotransmitter from varicosities
near the microelectrode; this was blocked by inhibiting N-type
Ca2+ channels with -conotoxin GVIA. In
summary, these findings indicate that focal electrical stimulation of
arterioles can depolarize smooth muscle cells directly to evoke
conduction and can activate perivascular sympathetic nerves to
extend the spread of constriction along the arteriolar wall.
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CONCLUSION |
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, 50)
, between vessels of different sizes within a
particular tissue (51)
, and between species. Although
transgenic mouse models now make it possible to study the role of
individual molecules purportedly involved in signal pathways,
meaningful interpretation of data from these animals necessitates the
establishment of a database in the normal mouse. The present studies
are the first to describe the cremaster preparation as a model for
investigating blood flow control in the mouse microcirculation and to
distinguish the conduction of vasomotor responses from those propagated
by sympathetic nerve activity. In so doing, we highlight the importance
of NO and cytochrome P450 metabolites in mediating local vs. conducted
vasodilation in response to ACh, demonstrate a novel biphasic conducted
response evoked by KCl, and establish that perivascular sympathetic
innervation is robust and effectively promotes vasoconstriction along
the arteriolar wall. Our findings illustrate that multiple signaling
pathways coordinate vasomotor responses in arteriolar networks of mouse
skeletal muscle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 3, 1999. Revised for publication October 25, 1999.
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