Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688
TRANSITIONS IN CYTOSOLIC CALCIUM
CONCENTRATION ([Ca2+]i) represent an
important physiological signal in cells (1). A rise in
smooth muscle cell [Ca2+]i activates myosin
light chain kinase, which promotes myosin light chain phosphorylation
and actomyosin interaction. However, recent evidence has indicated that
Ca2+-dependent signaling cascades are highly
compartmentalized, suggesting that a nonspecific global rise in
[Ca2+]i per se is insufficient to evoke a
specific physiological outcome. For example, ionomycin-induced rises in
global [Ca2+]i do not regulate
membrane-delimited enzyme function as efficiently as Ca2+
entry across the cell membrane (8, 10, 11, 22). Similarly, large-scale increases in [Ca2+]i do not
always increase myosin light chain phosphorylation (13). These findings illustrate the importance of determining how
compartmentalized Ca2+ signals regulate cell-specific
function. In this issue of the American Journal of
Physiology-Lung Cellular and Molecular Physiology, McDaniel et al.
(24) address the Ca2+ source that is essential
for Gq agonists to contract pulmonary arterioles.
Resting [Ca2+]i is normally maintained near
100 nM, reflecting the constitutive balance of Ca2+ release
from intracellular storage sites (e.g., endoplasmic reticulum and
mitochondria), Ca2+ entry across the plasmalemma,
Ca2+ reuptake into intracellular storage sites, and
Ca2+ extrusion across the cell membrane
(1). This low resting concentration can be rapidly
increased after either Ca2+ release from intracellular
organelles or direct entry across the cell membrane. Multiple
Ca2+ release and entry mechanisms exist, although the
functional significance of each pathway is cell-type specific.
Ca2+ release in smooth muscle cells can be achieved by
three distinct mechanisms, including activation of inositol
1,4,5-trisphosphate [Ins(1,4,5)P3] and
ryanodine/cyclic ADP-ribose receptors (1). Ins(1,4,5)P3-induced
Ca2+ release is relatively well understood. Membrane
receptors coupled to Gq proteins stimulate phospholipase C
and generate Ins(1,4,5)P3, which
promotes Ca2+ release through
Ins(1,4,5)P3 receptors localized
predominantly on the endoplasmic reticulum. Depletion of the
Ins(1,4,5)P3-accessible pool
regulates Ca2+ entry across the plasmalemma through
so-called store-operated Ca2+ (SOC) entry channels. Indeed,
the open probability of SOC entry channels is regulated specifically by
the endoplasmic reticulum Ca2+ concentration. A replete
Ca2+ pool closes SOC entry channels, whereas depletion of
the Ca2+ pool by
Ins(1,4,5)P3 activates plasmalemmal
cation channels that increase [Ca2+]i
(30).
Despite the widespread significance of SOC entry, the molecular
identity of SOC channels remains unclear (16). Transient receptor potential (Trp) gene products are the best
candidates for SOC entry channels. The Drosophila
melanogaster Trp gene was identified in 1989 (reviewed
in Ref. 16) and was later determined to encode a retinal
SOC entry channel. Since 1995, seven related mammalian homologs
have been identified, several of which encode for SOC entry channels
(at least Trp1, Trp2, Trp4, and Trp5) (16, 36). Other Trp homologs, most notably
Trp3, directly interact with the
Ins(1,4,5)P3 receptor (4, 19,
20). Although these findings suggest that individual Trp
proteins exhibit unique regulatory properties, extrapolation of
the findings to endogenously expressed Trp-containing channels is not
evident. Functional Trp channels may be formed by cohesion of four
distinct subunits that assemble as homo- or heteromultimers
(2). The biophysical properties of individual Trp proteins
that are overexpressed to presumably form homotetrameric channels
differ substantially from the biophysical properties observed when
different Trp proteins are overexpressed together and allowed to
coassemble (23). These data suggest that the combination
of Trp proteins expressed may partly determine whether the channels are
store and/or receptor operated. Thus the expression pattern of Trp
proteins may produce cell type-specific channels.
Several agonists have been used to activate SOC entry and to examine
Ca2+ entry through Trp channels. Thapsigargin is a plant
alkaloid that inhibits sarco(endo)plasmic reticulum
Ca2+-ATPase and causes the passive depletion of
endoplasmic reticulum Ca2+ without the confounding
influence of G proteins (33). Studies using thapsigargin
and related compounds [e.g.,
2,5'-di(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid] illustrate that smooth muscle cells possess SOC entry pathways. However, a clear link between SOC entry and smooth
muscle contraction has not been established (3, 14, 34),
perhaps because thapsigargin stimulates endothelial production of
nitric oxide (25). In vivo Gq agonists
represent the prominent stimulus for activation of SOC entry. The
Activation of L-type Ca2+ channels has been clearly linked
to pulmonary and systemic vasoconstriction. Membrane depolarization with high K+-containing physiological salt solutions
rapidly activates these channels, increasing
[Ca2+]i solely through Ca2+ entry
across the plasmalemma. This effect is not limited to
experimental preparations because physiological changes in
PO2 regulate resting membrane potential
(32). Acute hypoxia inhibits voltage-dependent K+ channel activity and reversibly depolarizes the resting
membrane potential sufficiently to activate Ca2+ entry,
resulting in an approximate threefold elevation in
[Ca2+]i (9, 29, 38).
Consequently, hypoxia may cause vasoconstriction by acting directly on
smooth muscle. L-type Ca2+ channels proved to be an
effective pharmacological target for the treatment of systemic and, to
some extent, pulmonary hypertension. However, in considering the
possibility that Indeed, it was this very problem that was addressed by McDaniel et al.
(24). They found that although inhibition of the L-type
Ca2+ channel abolished vasoconstriction induced by
high-K+ buffer, nifedipine and verapamil only reduced the
phenylephrine-induced contraction by 30%. These findings suggested
that either Ca2+-independent mechanisms were evoked by the
Gq agonist or that another Ca2+ source was
required for phenylephrine to sustain a contraction. Recalcification
experiments were performed to address this issue. Phenylephrine was
applied in the absence of extracellular Ca2+ to initiate
Ins(1,4,5)P3-dependent
Ca2+ release. Ca2+ was then readded to the
extracellular buffer to initiate Ca2+ entry in the presence
or absence of Efforts to characterize the SOC entry pathway revealed that store
depletion increased [Ca2+]i in cultured
smooth muscle cells and also activated a cationic current in the whole
cell voltage-clamp configuration. In patch-clamp experiments, the
holding potential was 0 mV to inactivate L-type Ca2+
channels. The cyclopiazonic acid-activated current
(ISOC) was large (approximately McDaniel et al. (24) report expression of Trp1, -2, -4, -5, and -6 in isolated myocytes. These findings implicate Trp proteins in myocyte ISOC but, at the present time, do not
provide direct evidence for which channel or combination of subunits
contributes to the observed nonselective current. Ion selectivity of
Trp proteins is still largely unresolved. Of the channels examined to
date, only Trp4 and Trp5 appear to encode for a
Ca2+-selective channel (26-28, 37), and
even this finding has been inconsistent (31). When
evaluated, nonselective currents have generally been observed after
overexpression of Trp1 (39) and Trp3 (18).
However, my laboratory (Brough G, Wu S, Cioffi D, Moore T, Li M, Dean
N, and Stevens T, unpublished observations) recently examined
the contribution of endogenously expressed Trp1 to a
Ca2+-selective ISOC that resembles
ICRAC (12). Antisense inhibition of
Trp1 reduced this Ca2+-selective current by 50% and did
not left shift the reversal potential from +40 mV. These findings were
interpreted to suggest that although Trp1 may form a structural subunit
of a Ca2+-selective store-operated channel, it is likely
not required to establish the Ca2+ selectivity of the
channel. Thus although it is evident that cation entry occurs
through Trp proteins in response to Ca2+ store depletion,
the endogenous makeup of these channels and their contribution(s) to
nonselective cation and/or Ca2+-selective currents are unclear.
Overall, the findings presented by McDaniel et al. (24)
address an important question and underscore how little is known regarding the fundamental aspects of Ca2+ signaling in the
control of vasoconstriction. As we continue to resolve the detail of
signal transduction pathways, it will be critical to establish more
specifically which Trp proteins coalesce to form functional channels
and to determine how these channels generate nonselective versus
Ca2+-selective currents. It will be equally important to
discern how SOC entry pathways work in combination with L-type
Ca2+ channels to uniformly or discretely regulate vascular
tone. Resolution of these issues may result in the generation of novel
antihypertensive pharmacotherapeutic agents directed against
myocyte-specific, Trp-containing SOC entry channels.
ARTICLE
TOP
ARTICLE
REFERENCES
-adrenoreceptor is such a
Gq-Ins(1,4,5)P3-linked
pathway that regulates the contractile status of blood vessels.
However, studies to date have not clarified whether
-adrenergic
agonists induce vasoconstriction through SOC entry channels
(3) because Gq-linked pathways cause membrane depolarization and can activate voltage-gated Ca2+ channels
that are thought to predominate in smooth muscle (5, 7).
-adrenoreceptor agonists may increase
[Ca2+]i through activation of either
voltage-gated or store-operated Ca2+ channels, the
source of Ca2+ responsible for vasoconstriction
remains unclear.
-adrenoreceptor and L-type Ca2+ channel
blockade. Under these experimental conditions, ~30% of the
vasoconstriction induced by phenylephrine was attributable to SOC entry.
500 pA at
80
mV), reversed near 0 mV, was linear (e.g., was not inwardly
rectifying), and was inhibited by Ni2+ (1 mM) and
La3+ (50 µM). These data resemble the nonselective
currents observed in response to Ca2+ store depletion in
other cell types and do not resemble Ca2+ release-activated
Ca2+ current (ICRAC) found initially
in mast cells and lymphocytes. ICRAC is very
small (less than approximately
50 pA at
80 mV), exhibits a positive
reversal potential (approximately +40 mV), and is strongly inwardly
rectifying. Highly Ca2+-selective channels possess an
anomalous mole fraction effect where monovalent cations are readily
conducted in the absence of Ca2+ but excluded in the
presence of low Ca2+ concentrations (15, 17, 21,
37). In this regard, both ICRAC and
voltage-gated Ca2+ channels exhibit anomalous mole fraction
behavior. The issue of ion selectivity is critical to the development
of an overall understanding of the apparent myocyte Ca2+
entry pathway(s) because it is likely that the molecular makeup of
Ca2+-nonselective and -selective store-operated channels is
distinct. The findings of McDaniel et al. (24) suggest
that at least one smooth muscle cell SOC entry pathway is nonselective,
generally consistent with the recent work of Trepakova et al.
(35).
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ACKNOWLEDGEMENTS |
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I thank Drs. I. F. McMurtry and M. Zhu for helpful comments in preparing this review.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Stevens, Dept. of Pharmacology, MSB 3364, Univ. of South Alabama College of Medicine, Mobile, AL 36688 (E-mail: tstevens{at}jaguar1.usouthal.edu).
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