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Introduction
The autonomic nervous system controls the activity of smooth muscles in the cardiovascular-renal system,
gastrointestinal system, and urogenital system. Afferent and efferent (sensory) nerves release a `cocktail' of neurotransmitters that
control the contractile and trophic state of the smooth muscle
cells[1]. These neurotransmitters activate several cell signaling
systems, including the ubiquitous intracellular
Ca2+ signaling systems[2]. Yet, neurogenic
Ca2+ signaling in smooth muscle (that elicited by neurally released transmitters, as opposed to exogenously applied receptor agonists) has been investigated
so far only in a few types of arteries, the vas deferens, the bladder and the uterus. Until now, this remarkable state of affairs
reflected the difficulty of observing intracellular
(Ca2+) in the muscle cells of intact organs in which nerves can be stimulated.
Our intent in this article is to review what is known about the changes in intracellular
(Ca2+) that are elicited by sympathetic
nerves, and to identify the important remaining questions.
Many factors are involved in regulating arterial diameter including the central and peripheral nervous systems,
endothelial cells, circulating hormones, locally released substances, blood pressure, blood flow, and intrinsic mechanisms of smooth
muscle. In arteries that contribute significantly to total peripheral resistance, such as mesenteric small
arteries[3,4] (which control blood flow to the intestine), control of smooth muscle contraction by the sympathetic nervous system is of major
importance. This control is complex, involving multiple neurotransmitters and receptors, as well as complex patterns of nerve
fiber activity. It is known that at least 3 different sympathetic neurotransmitters [ATP, norepinephrine (NE), and
neuropeptide Y (NPY)] are released from sympathetic varicosities, and that their effects vary with the pattern of nerve fiber activity.
Actions of the transmitters to elicit
Ca2+ signaling in smooth muscle are believed to be synergistic, although detailed
information is lacking. Finally, the probability of transmitter release at a single
sympathetic nerve terminal is low (~0.05) and the pattern of sympathetic nerve activity
in vivo is complex, consisting of single action potentials (AP) or bursts of AP superimposed on a background of tonic
activity[5,6].
All 3 sympathetic cotransmitters, NE, ATP, and NPY contribute to sympathetically mediated vasoconstriction.
Stimulation of the nerves supplying the rat mesenteric arterial bed (the tissue with which we are mainly concerned in this review)
elicits an increase in perfusion pressure that can be blocked completely only by the combined administration of prazosin,
suramin and BIBP 3226
((R)-N2-(diphenacetyl)-N-(4-hydroxyphenyl)
methyl)-D-arginineamide) (a selective
a1-adrenoceptor antagonist, a non-selective antagonist of all P2X receptor subtypes, and a selective antagonist of the NPY receptor subtype,
Y1), respectively[7]. Neurogenic contractions of isolated mesenteric small arteries are typically bi-phasic (Figure 1), with the
small, initial transient component being attributed to purinergic (P2X) receptors, and the later, slow, large component being
attributed to neurally released NE. However, the relative importance of each type of receptor depends on the size and type
of the artery, as well as on the pattern of sympathetic nerve activity. In mesenteric arteries, the purinergic component of the
contraction is relatively large in very small arteries (compared to the
a1-receptor-mediated component), and the purinergic
component predominates during brief bursts of sympathetic nerve fiber
activity[8]. Indeed, it has recently been concluded
that, "all three cotransmitters contribute significantly to vascular responses and their contribution varies markedly with
impulse numbers"[9]. The varying contribution of the transmitters under different conditions is the result of both pre- and
post-synaptic factors.
Our goal in this review is to summarize recent new information on sympathetic neuromuscular transmission and the
resultant Ca2+ signaling in the smooth muscle cells of small arteries during neurogenic contractions.
Ca2+ signaling during neurogenic contractions activated by trains of sympathetic nerve fiber action potentials is in fact significantly different from
that elicited by the simple application of exogenous neurotransmitters (both ATP and NE) to isolated arteries (or single
isolated smooth muscle cells). We end by identifying important questions remaining in our understanding of sympathetic
neuromuscular transmission and the physiological regulation of arterial smooth muscle contraction by the sympathetic
nervous system.
Sympathetic nerves in isolated small arteries
The perivascular nerve fibers present in rat mesenteric small arteries are of 2 major types: sympathetic and `sensory'. The
latter, which will not be discussed further here, are the so-called non-adrenergic, non-cholinergic (NANC) or `sensory'
nerves, whose cell bodies are located in the dorsal root ganglia. The perivascular sympathetic nerves that remain with
isolated arteries consist of the neuroeffector system, that is, the post-ganglionic fibers (cell bodies located in paravertebral
ganglia) and their nodal areas of axoplasmic specialization, that is, varicosities and synaptic junctions with the smooth
muscle. Experimentally, the perivascular nerves of an isolated artery can easily be electrically stimulated to release transmitters.
The function of NANC nerves may be blocked through the use of
capsaicin[10], thus permitting selective activation of the
sympathetic nerves.
We next review very briefly the salient features of current concepts on the pre-synaptic and post-synaptic mechanisms
that are involved in sympathetic neuromuscular transmission at such junctions, particularly those involved in the differential
release of sympathetic cotransmitters. A major physiological phenomenon to be explained is that the effects of ATP are
predominant at low frequencies of sympathetic nerve fiber activity, while those of NE are predominant at high frequencies.
Synaptic vesicles in sympathetic varicosities
Varicosities on the sympathetic perivascular nerves in rat mesenteric small arteries contain several different types of
synaptic vesicles, probably containing different proportions of NE, ATP and NPY. These vesicles may have different origins
(cell body vs formation at synapse) and mechanisms of exocytosis (including regulation by varicosity
Ca2+). Rat mesenteric arteries adrenergic nerve terminals contain 3 types of vesicles: large dense-cored vesicles (LDCV; ~100 nm diameter), small
dense core vesicles (SDCV; ~50 nm diameter) and small clear vesicles (SCV; ~50 nm
diameter)[11_13]. LDCV comprises 5% of the total
vesicles in a varicosity, with the remainder being small vesicles
(SV)[10]. The majority of the SV have (88%) dense cores
(SDCV); the remainder without, SCV[10]. NE and ATP are thought to be stored in all vesicle types, but in different proportions.
It is generally agreed that the ATP and NE are released from different nerve terminal
stores[12,14] although the type of vesicles
involved is not generally agreed upon. The exact vesicular origin of the released transmitters remains to be elucidated and
may be species and tissue specific.
Post junctional receptors
Post-synaptic receptors for ATP
Receptors for purine (ATP, ADP) and pyrimidine (UTP, UDP) nucleotides are presently
divided into 2 families: ionotropic P2X receptors
(7 cloned subtypes) and metabotropic P2Y receptors (6 cloned
subtypes)[15]. In the cardiovascular system, P2X receptors are
expressed predominantly on smooth muscle (but are present on endothelial cells), and P2Y receptors are predominantly
expressed on endothelial cells (also present on smooth muscle cells). P1 receptors will not be considered here except as they
may be activated by adenosine produced by endothelial nucleotidase
activity[16,17]. Neurally-released ATP activated P2X
receptors (ligand-gated ion channels) on smooth muscle to produce the excitatory junction potentials, and activated P2Y
receptors on endothelium (G protein-coupled receptors) to produce endothelium dependent hyperpolarizing factor. In rat
mesenteric arteries, the predominant P2X receptor is
P2X1[18].
Neurally-released ATP binds to both P2X and P2Y receptors. While `overflow' of ATP may be quantified chemically, only
the effect of ATP on P2X receptors is detectable with electrical and optical methods. There is no doubt that neurally-released
ATP produces the excitatory junction currents (EJCs), junctional
Ca2+ transients (jCaT) and in vas deferens, the neuroeffector
Ca2+ transients (NCT). jCaT and NCT are post-junctional changes in
Ca2+ of smooth muscle cells in response to
neurally-released ATP. In mouse vas deferens, EJC are of 2 types: (1) large and fast; and (2) small and slow. The 2 types are
differentially affected by external
(Ca2+) and osmotic
pressure[12] and sensitivity to
heptanol[19]. Considering the work of these authors and the `dual vesicle' hypothesis of
Stjarne[12], it may be speculated that the `large and fast' EJC arise from the
release of the `big quanta' type of SV, and that the `small and slow' EJC arise from the `small quanta' type of SV. jCaT and
NCT would arise from the `big quanta' SV; the extent to which work in the smooth muscle of vas deferens may apply to that
of arteries is not known.
Post-synaptic receptors for NE
The second sympathetic cotransmitter, NE, binds to adrenoceptors, the classification
and function of which have been reviewed
recently[20]. At least 9 subtypes of vascular adrenoceptors have been
identified[21]: α1A,
α1B, α1D, α2A/D,
α2B, α2C, β1,
β2, and β3. There is also believed to be a low affinity (to prazosin) state of
α1A-adrenoceptors, known as the
α1L-adrenoceptors, and they are found in various vascular
beds[22]. A useful heuristic generalization is that
a1-adrenoceptors are located post-junctionally on vascular smooth muscle and have a primary role in controlling arterial tone,
particularly in small resistance arteries. This accounts for the predominant use of the synthetic
α1-adrenoceptor agonist, phenylephrine (PE), in the majority of experimental studies. Exogenous activation of adrenoceptors using PE and the use of
an α1B-knockout mouse has permitted the conclusion that
α1D is most important in conduit arteries, while
α1A is most important in small arteries and
α1B was reported to have a minor
contribution[23]. Recent studies using nerve-evoked contraction,
also in small arteries isolated from
α1D-knockout mouse, have shown the importance of
α1A-adrenoceptors as the predominant
subtype and a1D having a small, but significant
role[24]. Pre-junctionally, transmitter release at sympathetic varicosities
(neuromuscular junctions) is importantly regulated by
a2A/D and a2C. Endothelial cells have at least 5 subtypes of adrenoceptors:
a2A/D, a2C, b1,
b2, and b3. Adrenergic signaling mechanisms in arterial smooth muscle have been reviewed
recently[25], with the emphasis on
Ca2+ activation of contraction and
Ca2+-sensitizing mechanisms activated by bath-applied phenylephrine.
Neurally-released NE has been detected classically by chemical methods in the effluent of neurally stimulated arteries
(`overflow experiments') or by amperometric methods, in which the oxidation of NE on the surface of a carbon fiber electrode
is detected as an electrical current. Most significantly, the release of single quanta of NE has been detected from the surface
of rat mesenteric small arteries through the use of carbon fiber microelectrodes
(CFmE). These microelectrodes are 7 mm in diameter and are believed to detect the release of NE quanta from a distance of 8
mm[14]. Furthermore, spontaneous oxidation
currents (SOC) were also recorded. These authors speculated that the SOC arose from NE released from large dense-cored
vesicles (LDV). An important observation was that
a-latrotoxin increased the frequency of SOC about 4-fold, but increased
the frequency of spontaneous excitatory junction potentials (which monitor packeted or quantal ATP release) by 30-fold.
This observation supports the suggestion that SEJP (spontaneous excitatory junction potentials activated by released ATP
) and SOC (NE release) occur through different synaptic vesicles, under these conditions.
In most vascular beds,
α1-adrenoceptors play an important role in vasoconstriction. The relative importance of the
different a1-adrenoceptor subtypes in regulation of peripheral resistance and systemic arterial blood pressure is not clear, as
the contribution of different subtypes to
vasoconstrictiondiffers with the mode of activation and
species[26]. For example, studies in rat mesenteric small arteries using exogenous agonists have revealed the role of
α1A-[27,28] or
α1B-[29] or
α1L-adrenoceptors[30]. In contrast, nerve-evoked contractions in rat mesenteric arteries were predominantly mediated by
α1A-adrenoceptors[27,28]. Further, studies in mesenteric small arteries using
α1B-adrenoceptor knockout mouse have revealed the
predominance of α1A-adrenoceptors
in vasoconstriction to
phenylephrine[23] and nerve-evoked contractions showed the predominance of
α1B-adreno-ceptors[31]. Therefore, it is important to study
in vivo preparations to understand the clinical relevance of the
α1-adrenoceptor subtypes involved in vasoconstriction.
Post-synaptic receptors for NPY
The third sympathetic cotransmitter, NPY, is believed to enhance the effects of both
ATP and NE, by acting on post-junctional
NPY-Y1 receptors[32]. Five distinct NPY receptors
(Y1, Y2, Y4,
Y5, and Y6) have been
cloned[33]. Y1 receptors are believed to be the major type present post-junctionally in the cardiovascular system and to
mediate the response to NPY, although the possible involvement of pre- and post-junctional
Y2 receptors has been suggested. NPY receptors act via pertussis toxin-sensitive G proteins. A major effect of their activation is the inhibition of adenylyl
cyclase. NPY shifts agonist dose-response curves to the
right[34]. It has been suggested that NPY activates mesenteric small
arteries through 2 different mechanisms: activation of non-selective cation channels and consequent
Ca2+ entry, and the inhibition of the hyperpolarization produced by
cAMP[35] (cyclic adenosine monophosphate).
Differential release of ATP and NE
There is good evidence that ATP is the predominant sympathetic effector in response
to `single' action potentials, while at high frequencies of activation, NE is the predominant sympathetic effector. This may
result from the pre- and post-synaptic mechanisms discussed earlier. Low frequency electrical field stimulation (EFS; eg 1 Hz)
elicits relatively small, brief, phasic contraction of rat mesenteric arteries, whereas higher frequency EFS (eg 16 Hz) elicits
larger, sustained contractions. The irreversible adrenergic antagonist, phenoxy-benzamine, abolished the response to
bath-applied norepinephrine, but reduced the response to a single nerve stimulus by only
20%[36]. Conversely, the stable ATP analogue,
a,b-methylene ATP (which desensitizes P2X receptors), reduced the response to a single pulse by 70%, while
reducing the contraction to high frequency stimulation only 10%. Similarly, in the rabbit ear artery, it has been concluded that
`short pulse bursts at low frequency favor the prazosin-resistant (purinergic) component of the
response'[37].
Post-synaptic
Ca2+ signaling
Recent work has revealed in detail the
Ca2+ signals in smooth muscle cells during sympathetically-mediated neurogenic
contractions of small arteries[38] and vas
deferens[39]. There are differences between these 2 tissues (which have very
different functions), and we will focus here on what is known about sympathetic neuromuscular transmission and neurogenic
contractions of vascular smooth muscle. The goals of the current research in this area are to attribute different types of
post-synaptic Ca2+ signals to the activation of specific receptors, channels, and organelles, and to determine to what extent the
different post-junctional Ca2+ signals actually activate contraction. Here, we summarize the recent studies in which
Ca2+ signals, attributable to both neurally-released ATP and neurally released NE, have been observed.
jCaT: post-synaptic
Ca2+ signals activated by neurally released ATP
Neurally-released ATP activates a specific, localized,
Ca2+ transient in arterial smooth muscle
cells[40] that has been termed a `jCaT', for junctional
Ca2+ transient. It was shown that these Ca transients arise in the vicinity of perivascular
nerves[38] or even directly beneath single visualized nerve
fibers[40]. The confocal images of jCaT in Figure 2 were obtained in pressurized (70 mmHg) rat mesenteric small arteries subjected to
EFS. Low frequency, low voltage stimulation (0.67 Hz, 0.2 ms) excited nerve fibers without causing an appreciable contraction.
This was referred to this as `sub-threshold' EFS, as it is sub-threshold for muscle contraction. Thus, in these experiments,
motion did not occur and the characteristics of the jCaT could be studied in detail. Figure 2 illustrates the basic appearance
of jCaT in line-scan images and demonstrates that: (1) nerve fibers are being excited by each EFS pulse; (2) a jCaT occurs
nearly simultaneously with an EFS pulse; (3) jCaT occur near nerve fibers; and (4) jCaT are events of very low probability.
Spatio-temporal characteristics of jCaT
JCaT are larger in spatial spread and last longer than spontaneous
Ca2+ sparks. JCaT always occur with brief latency to the EFS pulse. The spatio-temporal differences between the jCaT and the sparks are
obvious in published records[40]: the jCaT is larger in space, lasts longer, and occurs at the time of the stimulus pulse. These
characteristics and their pharmacology are how jCaT are distinguished from sparks. The vast majority of jCaT occurred
within 12 ms (4 scan lines) after the stimulus pulse. The evidence linking the occurrence of jCaT to the stimulus seems
unequivocal. The spatial full-width-at-half-maximum (FWHM) for jCaT is 4.8 µm, and the time taken to fall to half-amplitude,
t1/2, (from the peak) is 145 ms. The means of these distributions are quite different from those of sparks in smooth muscle
(sparks: t1/2, 48_56 ms; FWHM, 2.4
µm)[41]. Some jCaT occurred before the stimulus, and some much after; we hypothesize
that these are associated with spontaneous neurotransmitter release.
Pharmacology of jCaT Pharmacological studies of
jCaT[40] have provided strong evidence that they arise from the activation
of purinergic receptors. JCaT persist, apparently unchanged, in the presence of capsaicin, and are thus not dependent on
sensory nerves. They are completely absent in the presence of the purinergic receptor blocker, suramin (300 µmol/L). They
persist in the presence of a1-adrenergic blocker prazosin (10 µmol/L), sufficient to block neurogenic adrenergic responses
completely. They are also largely unaffected by ryanodine (30 µmol/L), while
Ca2+ sparks are abolished. Although we favor
the hypothesis that jCaT are due to ATP and
P2X1 receptors, further studies are required, particularly to be sure that it is the
P2X1 receptor subtype. NPY is also reported to activate non-specific cation
channels[36] and thus it could contribute to jCaT.
This possibility could be tested using specific
Y1 receptor antagonists (BIBP 3226).
In our recent
study[42] using P2X1 knockout
animals[43], we clearly showed that jCaT represent
Ca2+ that enter vascular smooth muscle cells through
P2X1 receptors activated by neurally-released ATP. The
P2X1 knockout models also showed the importance of the involvement of ATP-mediated
P2X1 activation in the initial rapid component of the nerve-evoked
contraction[42].
Ca2+ waves: post-synaptic
Ca2+ signals activated by a1-adrenergic agonists
The Ca2+ signaling elicited by adrenergic
agonists has been studied mainly through the use of exogenous, bath-applied synthetic catecholamines such as the
a1-adrenoceptor specific agonist, PE. An early study was that of Zang and
colleagues[44], in which it was shown that PE elicited
asynchronous propagating Ca2+ waves in rat mesenteric arteries. The basis of the dose-response to PE was increasing
recruitment of individual smooth muscle cells to produce
Ca2+ waves, and to produce them at higher and higher frequencies.
As expected, asynchronous propagating
Ca2+ waves seem also to be the response elicited by neurally released NE. This is
discussed in more detail later in relation to the activation of contraction by neurally released NE, during neurogenic
contractions of small arteries.
Neurogenic contractions of small arteries
The detailed studies on jCaTs described above were performed in pressurized small arteries that did not contract because
the electrical stimulation was `sub-threshold' for contraction. In this section, we review the studies in which jCaT and
Ca2+ waves have been observed during isometric neurogenic contractions. For these studies, the arteries were mounted in a
myograph that permitted simultaneous (i) high-speed confocal imaging of fluorescence from individual smooth muscle cells;
(ii) electrical stimulation of perivascular nerves; and (iii) recording of isometric tension. Sympathetic neuro-muscular
transmission was achieved by EFS (frequency, 10 Hz; pulse voltage, 40 V; pulse duration, 0.2 ms) in the presence of capsaicin and
scopolamine (to inhibit `sensory' and cholinergic nerves, respectively). As shown in Figure 3, during the first 20 s of EFS,
force rose to a small peak, then declined, similar to that recorded
previously[45]. During this time, jCaT were present at a
relatively high frequency. Propagating asynchronous
Ca2+ waves, previously associated with bath-applied
a1-adrenoceptor agonists, were not initially present. During the next 2.5 min of EFS, force rose slowly, and asynchronous propagating
Ca2+ waves appeared. The selective
a1-adrenoceptor antagonist, prazosin, abolished both the slowly developing contraction and
the Ca2+ waves, but reduced the initial transient contraction by only ~25%.
Purinergic component of neurogenic contraction
In order to study selectively the arterial contractions generated by
neurally-released ATP, arteries were exposed to prazosin (1_10 µmol/L) to block
a1-adrenergic receptors (Figure 4).
Others[8] have shown that purinergic receptor antagonists, such as suramin, abolish the small contractions that remain in prazosin. We
found that after prazosin treatment 73.7%±14.0%
(n=7) of the initial transient contraction remained and 5.00%±0.98%
(n=5) of the maintained contraction. We then sought to characterize the changes in frequency and amplitude of jCaT that might occur
during the EFS, and which activate contraction. Because the jCaT is a local
Ca2+ signal, it was not necessarily clear that jCaT
would activate contraction effectively. Confocal imaging of Fluo-4 fluorescence at 30
images·s-1 was performed for 3 periods
of 20 s in the beginning (0_20 s), middle (80_100 s), and end (160_180 s) of 3 min EFS (periods
indicated by bars in Figure 4A). The frequency of jCaT declined markedly during the 3 min of EFS (Figure 4B, iv_vi). In contrast to the frequency, the peak
amplitude of the jCaT changed little during 3 min EFS. JCaT occurred in sufficient numbers during the first 20
s of EFS to produce a detectable elevation of average
(Ca2+; fluorescence ratio), which paralleled the transient contraction that occurred
during this time. On the other hand, jCaT occurred at a very low frequency later in the EFS, when contractile force fell to very
low levels. Thus, it seems reasonable to attribute the contractile activation to jCaT, despite their limited spatial extent and
frequency.
Adrenergic component of neurogenic contraction
Later during neurogenic contractions, asynchronous
Ca2+ waves propagated within individual smooth muscle cells of the arterial wall. A representative
Ca2+ signal, obtained as the average
fluorescence with an area of interest (AOI; 1.35 mm square) within a single smooth muscle cell is shown in Figure 5A (lower
trace, right hand scale). For this data, images were obtained at 2
s-1; a rate which is too slow to resolve the
jCaT[40] generated during the initial purinergic component. For the analysis of the adrenergic component of the experiment illustrated in Figure
5, 74 individual smooth muscle cells were identified and an AOI placed on each. In these 74 cells, 809
Ca2+ waves were detected and the time of onset and peak amplitude of each was determined, and the data presented as a histogram.
Different roles of ryanodine (RyR) and inositol(1,4,5)-trisphosphate receptors
(InsP3R) in neurogenic contractions
The role of the sarcoplasmic reticulum (SR) and its
Ca2+-release channels (RyR,
(Ins(1,4,5)P3R) of individual smooth muscle
cells of the arterial wall and in these
Ca2+ signals is not completely known. Zang
et al[44] used confocal laser scanning
microscopy and Fluo-4 to visualize Ca2+ transients within individual smooth muscle cells of rat resistance arteries during
a1-adrenoceptor activation. They noticed that in the presence of PE, caffeine also elicited a massive release of
Ca2+, at a time when Ca2+waves had died away completely, or when further responses to PE would have been much diminished. This result
shows that caffeine-sensitive Ca2+ stores are not depleted of
Ca2+ in the presence of PE and further indicates that the
a1-agonist-releasable Ca2+ store and the caffeine releasable
Ca2+ store are different[44]. More recent experiments by our group
(Lamont and Wier, 2004)[46] showed that
Ins(1,4,5)P3R are essential for adrenergically-induced asynchronous
Ca2+ waves and the associated steady vasoconstriction, but RyR are not appreciably opened during adrenergic activation (because PE did
not facilitate the development of the effects of ryanodine). Also
Ins(1,4,5)P3R are not essential for
Ca2+ sparks. This provides an explanation of the fact that adrenergic stimulation decreases the frequency of
Ca2+ sparks (previously reported) while
simultaneously increasing the frequency of asynchronous propagating
Ca2+ waves; different SR
Ca2+-release channels are
involved[46].
Both the contraction and the underlying
Ca2+ signals during the adrenergic component of the neurogenic isometric
contraction are also distinctly different from those occurring during externally applied
a1-adrenoceptor agonist (typically PE). After the initial purinergic component, the adrenergic component of the neurogenic contractions, even at maximally
effective EFS, rises much more slowly than does the contraction in response to bath-applied
a1-adrenoceptor agonist. Maximally effective concentrations of exogenous PE elicit an initial synchronous release of
Ca2+, followed by asynchronous propagating
Ca2+ waves, both in
veins[45] and in the
arteries[34,44]. Thus, the initial rapid rise in force in response to externally
applied PE appears to be generated by a synchronous release of
Ca2+ from intracellular stores. This does not occur during
neurogenic contractions, possibly because neuronally released NA does not initially reach the uniformly high levels that are
achieved rapidly after external application.
Influence of myogenic tone
Another significant complication that arises from arteries
pressurized at 70 mmHg above room temperature is the
development of tone. Earlier studies (eg Mauban
et al[47]), were at room temperature, which is not conductive for the development of
myogenic tone. The only previous study in which spatially resolved imaging was performed in mesenteric small arteries at 37
ºC[48] was complicated by the development of adrenoceptor-mediated vasomotion, preventing clear observation of
Ca2+ signaling in individual smooth muscle. However, mouse mesenteric small arteries at 32
ºC showed the development of myogenic tone and also rarely resulted in vasomotion on stimulation by PE. In such arteries, PE elicited only a spatially
uniform increase in Ca2+ with little or no
Ca2+ waves, suggesting that the rise in
Ca2+i leads to the development of myogenic
tone and also inactivation of IP3
receptors[49].
Conclusion
The confocal microscope, along with other new technologies has provided much new information on the physiology and
pharmacology of sympathetically evoked
Ca2+ signaling in arterial smooth muscle. We have previously advanced a scheme
to explain the Ca2+ signals and isometric contraction elicited by electrical field stimulation of perivascular sympathetic nerves
of a rat mesenteric small artery (Figure 6). Early during a train of nerve fiber action potentials, smooth muscle contraction is
activated mainly by jCaT induced by neurally released ATP. JCaT are localized to the post-junctional region, and arise from
Ca2+ that has entered via
P2X1 receptors. At this time, sympathetic varicosities may release mainly synaptic vesicles that
contain a relatively high concentration of ATP (the relatively few `big' quanta proposed by
Stjarne[12]). Later during a train of nerve fiber action potentials, jCaT are rare, and contraction is activated by
Ca2+ waves that arise from SR.
Ca2+ release from SR is activated by
InsP3, produced after the binding of NE to
a1-adrenoceptors. At this time, sympathetic varicosities may release
small synaptic vesicles (the more numerous `small' quanta, green; Figure 6) that contain a relatively high concentration of NE.
We stress that the mechanisms accounting for differential release of ATP and NE are quite speculative at this time.
JCaT are distinct from
Ca2+ transients activated in isolated venous myocytes by exogenously applied
ATP[50]. Previous studies of the effects of ATP on small arteries utilized spatially averaged measurements of
Ca2+ [51] and we can not determine therefore whether jCaT might be produced by bath-applied ATP or not. In rat mesenteric small arteries similar to those used
here, low (0.01_1 mmol/L) concentrations of exogenous ATP caused `global'
Ca2+ transients that seemed to involve
Ca2+ influx through channels sensitive to nifedipine and the putative blocker of receptor operated channels, SKF
96365[52], whereas higher concentrations (1_3 mmol/L) caused a release of
Ca2+ from intracellular stores (Ca2+
transients were elicited by high ATP in the absence of external
Ca2+). When applied to isolated venous myocytes, low concentrations of ATP (0.1 µmol/L) induced
rather uniform increases in Ca2+ (which started from the edges of the cell), and at
higher concentrations (1 µmol/L), propagating
Ca2+ waves[50].
In the arteries studied here in the presence of prazosin, no propagating
Ca2+ waves were ever observed during EFS. We
interpret this to mean that neurally-released ATP does
not evoke significant release of Ca2+ from intracellular stores, a result in
agreement with previous pharmacological studies on rat mesenteric small
arteries[7]. We speculate that the differences between
the effects of bath-applied ATP and neurally-released ATP are due to a markedly different spatio-temporal pattern of ATP on the
smooth muscle cell in the 2 cases.
Both the contraction and the underlying
Ca2+ signals during the adrenergic component of the neurogenic isometric
contraction are also distinctly different from those occurring during externally
applied a1-adrenoceptor agonist (typically
phenylephrine, PE). After the initial purinergic component, the adrenergic component of the neurogenic contractions, even at
maximally effective EFS, rises much more slowly than the contraction in response to bath-applied
a1-adrenoceptor agonist. Maximally effective concentrations of exogenous PE elicit an initial synchronous release of
Ca2+, followed by asynchronous propagating
Ca2+ waves, both in
veins[53] and
arteries[44,47]. Thus, the initial rapid rise in force in response to externally applied
PE appears to be generated by a synchronous release of
Ca2+ from intracellular stores. This does not occur during neurogenic
contractions, possibly because neurally-released NE does not initially reach the uniformly high levels that are achieved
rapidly after external application.
Acknowledgement
We gratefully acknowledge the assistance of Becky SAUNDERS in the preparation of this manuscript.
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