Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calcium entry via voltage-gated L-type channels
is responsible for at least half of the increase in cytosolic calcium
([Ca2+]i)
in afferent arterioles following agonist stimulation. We sought the
presence of capacitative calcium entry in fresh vascular smooth muscle
cells (VSMC) derived from rat preglomerular vessels.
[Ca2+]i
was measured using fura-2 ratiometric fluorescence. Vasopressin V1
receptor agonist (V1R) (107
M) increased
[Ca2+]i
by ~100 nM. A calcium channel blocker (CCB), nifedipine or verapamil
(10
7 M), inhibited the
response by ~50%. V1R in the presence of CCB increased
[Ca2+]i
from 106 to 176 nM, confirming that calcium mobilization and/or entry
may occur independent of voltage-gated channels. In nominally Ca2+-free buffer, V1R increased
[Ca2+]i
from 94 to 129 nM, denoting mobilization; addition of
CaCl2 (1 mM) further elevated
[Ca2+]i
to 176 nM, indicating a secondary phase of
Ca2+ entry. Similar responses were
obtained when CCB was present in calcium-free buffer or when EGTA was
present. In nominally Ca2+-free
medium, the sarcoplasmic reticulum
Ca2+-ATPase inhibitors (SRCAI),
thapsigargin and cyclopiazonic acid (CPA), increased
[Ca2+]i
from 97 to 128 and 143 nM, respectively, and to 214 and 220 nM,
respectively, when 1 mM extracellular
Ca2+ was added. In the presence of
verapamil, the results with CPA acid were nearly identical. In
Ca2+-free buffer, the stimulatory
effect of V1R or SRCAI on the
Ca2+/fura signal was quenched by
the addition of Mn2+ (1 mM),
demonstrating divalent cation entry. These studies provide evidence for
capacitative (store- operated) calcium entry in VSMC freshly isolated
from rat preglomerular arterioles.
calcium signaling; store-operated calcium entry; vascular smooth muscle; renal hemodynamics; afferent arterioles; sarcoplasmic reticulum; calcium adenosinetriphosphatase; voltage-gated calcium channels
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PEPTIDE HORMONE AGONISTS such as angiotensin II (ANG II) and arginine vasopressin (AVP) increase intracellular cytosolic calcium concentration ([Ca2+]i) to regulate cell contraction and vascular resistance in small-diameter renal resistance vessels. Changes in [Ca2+]i are mediated either by mobilization of calcium from intracellular stores or by entry from the extracellular compartment. Afferent and efferent renal arterioles differ from each other in the involvement of Ca2+ entry mechanisms in response to vasoactive agonists. Mobilization from intracellular calcium stores and/or entry via non-L-type channels are the major mechanism in efferent arterioles; in contrast, calcium entry via voltage-gated L-type channels appears to predominate in afferent arterioles (5, 9, 17, 22, 35).
Previous work from our laboratory with rats in vivo (11, 32), in cultured (39) and in freshly harvested (19) vascular smooth muscle cells (VSMC), and isolated vascular segments (33) derived from rat renal resistance vessels has shown evidence for pathways of calcium entry or both calcium mobilization and entry. The in vivo preparations, isolated afferent arterioles, and cultured VSMC exhibit both mechanisms. However, inhibitors of calcium entry through voltage-gated L-type channels (calcium channel blockers, CCB) blocked the ANG II- and AVP-induced increase in intracellular calcium nearly completely in freshly prepared VSMC, suggesting that calcium entry is the major mechanism in this preparation (19).
The concept of capacitative calcium entry (store-operated calcium entry), proposed by Casteels and Droogmans (6) and extensively investigated and refined by Putney (31) in nonexcitable cells and later examined in other cell types (25, 26, 34, 37), depicts a mechanism whereby emptying of intracellular calcium stores stimulates calcium influx. Either receptor activation of inositol 1,4,5-trisphosphate (IP3) or inhibition of sarcoplasmic reticulum (SR)-Ca2+-ATPases (SRCA) induces emptying of SR calcium (1) and subsequent calcium entry into the cytosol via channels that have been named calcium release-activated calcium channels (CRAC) (16). Precisely how emptying of intracellular calcium stores stimulates calcium influx remains uncertain (15).
Evidence for capacitative calcium entry has been demonstrated in cultured VSMC from large conduit vessels, mainly rat aorta and the A-10 or A7r5 cell lines (4, 20, 34, 37). No study has sought this mechanism in either cultured or fresh VSMC derived from rat preglomerular vessels. Utilizing an iron oxide sieving method to isolate fresh VSMC (7, 19), in the present study we employed the sarcoplasmic reticulum Ca2+-ATPase inhibitors (SRCAI), thapsigargin (TG) and cyclopiazonic acid (CPA) (24, 36), and the vasopressin V1 receptor agonist (V1R) (chosen because of its specificity and stability). Responses were assessed in the presence and absence of extracellular calcium and CCBs to show that capacitative calcium entry occurs in VSMC derived from rat preglomerular arterioles.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of preglomerular resistance vessels. We used the iron oxide sieving technique previously described in our laboratory (7, 19) to isolate preglomerular vessels from 120- to 180-g Wistar-Kyoto rats maintained in the Chapel Hill Colony. After intraperitoneal administration of pentobarbital sodium to the rat, a midline incision exposed the abdominal aorta, which was then cannulated below the renal arteries. The aorta was occluded above the renal arteries, the left renal vein was incised, and the kidneys were perfused slowly with 20-40 ml of ice-cold PBS, pH 7.4. In early experiments, PBS with 20 mM phosphate and 5 mM MgCl2 was used (19). Because of concern about the effects of high magnesium concentration on the responsiveness of VSMC to agonists (38), we subsequently employed PBS with the following composition (in mM): 137 NaCl, 2.7 KCl, 0.88 KH2PO4, 6.4 Na2HPO4, and 1.0 MgCl2, adjusted to pH 7.4 at 4°C. One to two milliliters of a 1% suspension of magnetized iron oxide (Fe2O4) in cold PBS was infused until the kidneys turned gray. The modification of the magnetized iron oxide sieving method for isolation of preglomerular vessels has previously been described from our laboratory (7, 19). In later experiments, magnetized polystyrene particles (Spherotech, Libertyville, IL) were used instead of iron oxide. Briefly, thin cortical slices were minced and homogenized, and preglomerular arterioles were separated from the crude homogenate with a magnet. Passage of the suspension through needles of decreasing size disattached vessels from their glomeruli. Application of the suspension to a 100 µM sieve retained vessel segments; the glomeruli and other debris were washed away. The microvessels were washed from the inverted sieve, further purified with another magnet separation, and finally treated with collagenase (Sigma, type 1A) at 37°C. Because of variations in potency from one batch of collagenase to another, time and concentration were adjusted from 0.03-0.1% collagenase and 15-30 min incubation times. The preparation was chilled on ice and then shaken vigorously to disrupt the vessels. Free iron oxide or magnetized microspheres that had floated to the surface were removed with a magnet. Cells were incubated in Hanks' buffered salt solution (HBSS, in mM: 137 NaCl, 5.4 KCl, 0.33 Na2HPO4, 0.44 KH2PO4, 5 glucose, and 1.0 MgCl2) containing 1.6-2 µM of the acetoxymethyl ester of fura 2 (fura 2-AM) for 45-60 min at room temperature in the dark. Room temperature varied from 19-25°C depending on the season of the year. After washing twice with HBSS, cells were kept on ice either in HBSS with 1 mM MgCl2 only (nominally Ca2+-free) or HBSS with 1 mM MgCl2 and EGTA (0.5 mM) (Ca2+-free, <20 nM, measured) or HBSS with 1.0 mM MgCl2 and 1.0 mM CaCl2.
Measurement of cytosolic free calcium
concentration. We measured
[Ca2+]i
using fura 2 as previously described (14, 19, 39). To improve cell
adhesion to the slide, a drop of 1%
poly-L-lysine was
placed on the glass coverslip, allowed to remain for 20 min, and then
vacuum aspirated. A suspension of VSMC (10 µl) was gently aspirated
from the surface of the cell pellet and spread on the coverslip, which
was placed in the optical field of a ×40 oil-immersion fluorescence objective of an inverted microscope (Olympus IX70). VSMC,
identified morphologically by their spindle or crescent shape, have
been shown previously to stain with smooth muscle-specific -actin
and heavy chain myosin SM-1 and SM-2 (19). Care was taken to focus only
on one to three VSMC prior to measurements. HBSS (5 µl) was then
added to ensure that the cells were adhering to the coverslip and would
not be washed away by additions of drugs. As well, if the experiment
were carried out longer than 200 s, more HBSS was added to avoid
potential problems with evaporation. Preliminary studies established
that sequential additions of HBSS had no effect on the measurement of
[Ca2+]i.
Glomeruli and intact tubules were occasionally present in the
suspension and were excluded from the field of focus. The VSMC were
excited alternately with light of 340- and 380-nm wavelength from a
dual-excitation wavelength Delta-Scan equipped with dual monochronometers and a chopper [Photon Technology
International (PTI)]. After passing signals through a barrier
filter (510 nm), fluorescence was detected by a photometer. Signal
intensity was acquired, stored, and processed by an IBM-compatible
Pentium computer and Felix software (PTI). The calibration of
[Ca2+]i
was based on the signal ratio at 340/380 nm and known concentrations of
calcium. The
[Ca2+]i
was calculated according to the formula
![]() |
Manganese (Mn2+) was used as a tool to reveal cation entry by virtue of its ability to quench the fura 2 fluorescent signal. Because Mn2+ has a much higher affinity for fura 2 than Ca2+, the standard 340/380 ratio no longer can quantify [Ca2+]i. The quenching effect of intracellular Mn2+ can be assessed as changes in fluorescent signal at 360 nm, the isobestic point for free and Ca2+-bound fura (8).
All drugs and chemicals were added in a 5-µl volume to the droplet of cells on the coverslip. To study the identical cell(s) with sequential drug additions, it was not possible to wash the cells between additions because of the delicacy of their adherence to the glass coverslip. Calculations of drug concentration were based upon the increasing volumes in the droplet on the coverslip.
Reagents. Collagenase, EGTA, and nifedipine came from Sigma (St. Louis, MO); TG, CPA, and verapamil were from Calbiochem (La Jolla, CA); vasopressin V1 receptor agonist ([Phe2,Ile3,Orn8]vasopressin) was from Peninsula Laboratories (Belmont, CA); iron oxide was from Aldrich Chemical (Milwaukee, WI); and fura 2-AM was from Molecular Probes (Eugene, OR).
Statistics. The data are presented as means ± SE. The data sets were tested with analysis of variance. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We employed the peptide vasoconstrictor V1R at a final concentration of
107 M, except in 5 mM
Mg2+ experiments in which
10
6 M V1R was required to
give a response of similar magnitude. To block L-type voltage-gated
channels, a CCB (nifedipine or verapamil) was added to
achieve a final concentration of
10
7 M
(10
6 M for 5 mM
Mg2+ studies). We wished to assure
ourselves that the class one CCB verapamil acted identically to the
commonly employed dihydropyridines. Because there were no differences
between the effects of nifedipine and verapamil in early experiments,
we chose to use verapamil for most experiments, because we preferred
the water solubility of this agent. Six control experiments showed a
baseline
[Ca2+]i
of 129 ± 14 nM; after addition of verapamil,
[Ca2+]i
was 130 ± 17 nM. Nifedipine addition likewise had no statistically significant effect on baseline
[Ca2+]i.
Stimulation of VSMC with V1R caused a square wave response during the
50-s recording time that is characteristic of the pattern in freshly
isolated VSMC (19) and also in fresh intact afferent arterioles
stimulated with norepinephrine (NE) (33). In less than 5% of
experiments, we viewed a peak plateau pattern, similar to that observed
in cultured VSMC (39). In preliminary experiments, the responses to ANG
II, AVP, and V1R were qualitatively and quantitatively indistinguishable. We chose to focus on V1R because of its specificity and its stability in solution. The response of VSMC to V1R and CCB was
studied under two conditions: with 5.0 or 1.0 mM magnesium in the
buffers used to prepare the cells. Addition of the V1R agonist
(106 M for 5 mM
Mg2+ experiments and
10
7 M for 1 mM
Mg2+ studies) caused an immediate
increase in
[Ca2+]i
at both magnesium concentrations, 114 ± 12 to 215 ± 30 nM and 101 ± 12 to 202 ± 28 nM, respectively. Responses to
CCB, measured 50 s after addition, differed between the two magnesium
concentrations. Verapamil inhibited the response to V1R completely in
the 5 mM magnesium experiments, whereas in the presence of 1 mM
magnesium, CCB diminished
[Ca2+]i
by only 50% (Figs. 1,
A and
B). These studies show that blockade of voltage-gated channels with CCB did not abolish the effect of V1R
stimulation in VSMC prepared with physiological concentrations of
magnesium. In contrast, with higher concentrations of magnesium, CCB
completely inhibited calcium entry as has been seen previously in
studies of fresh VSMC (19). Further confirmation of the ability of
peptide agonist to increase intracellular calcium in the presence of
CCB was achieved by pretreating VSMC with CCB and then adding V1R. All
subsequent experiments were performed in VSMC that had been prepared in
1 mM Mg2+-containing buffer.
|
Figure 2 shows that baseline
[Ca2+]i
of 98 ± 12 nM did not change significantly after the addition of
CCB. Then addition of V1R increased
[Ca2+]i
to 176 ± 21 nM (P < 0.01).
Subsequent experiments to test for calcium entry via non-voltage-gated
L-type channels were performed with CCB present at the outset.
|
To investigate the presence of capacitative calcium entry, VSMC were
maintained in nominally calcium-free buffer throughout isolation and
initial measurements of resting
[Ca2+]i.
Control experiments (n = 7)
demonstrated a resting
[Ca2+]i
of 93 ± 11 nM; addition of
CaCl2 to achieve a final
concentration of 1 mM did not change
[Ca2+]i
(95 ± 12 nM). Figure
3A
shows that when V1R was added to the cells in nominally calcium-free
buffer,
[Ca2+]i
rose from a baseline of 94 ± 8 to 129 ± 9 nM
(P < 0.01). During continued V1R
stimulation, subsequent addition of
CaCl2 (1 mM) caused a further
increase in
[Ca2+]i
to 176 ± 12 nM (P < 0.01),
suggesting that indeed the emptying of intracellular calcium stores
(calcium mobilization) with V1R created the conditions for calcium
entry mechanisms. A set of experiments was performed in which EGTA (0.5 mM) was added to the nominally calcium-free buffer. These results (Fig.
3B) are not different from those
without EGTA. Resting
[Ca2+]i
of 70 ± 4 rose to 97 ± 3 nM after V1R and 161 ± 10 nM
following addition of Ca2+ (1 mM,
measured) (P < 0.01 for both
comparisons). To confirm that voltage-gated L-type channels did not
participate in calcium entry, another set of experiments was done in
the presence of CCB (107 M)
and calcium-free buffer. Figure 3C is
a summation of 10 experiments with nifedipine and verapamil, which were
pooled since the results were similar.
[Ca2+]i
rose from 141 ± 12 to 195 ± 12 nM after the addition of V1R (P = 0.03) in nominally calcium-free
buffer and increased further to 243 ± 16 nM
(P < 0.01) when calcium was added to
a final concentration of 1 mM.
|
To validate that the increase in
[Ca2+]i
after the addition of calcium to the extracellular fluid was indeed a
consequence of calcium entry, we replaced
Ca2+ with
Mn2+ (final concentration, ~1
mM). Influx of Mn2+ should quench
the fura/Ca2+ signal. Figure
4A
shows that in the presence of CCB and calcium-free buffer, V1R
increased
[Ca2+]i
from 116 ± 9 to 163 ± 17 (40% increase), which was then
quenched by the subsequent addition of
Mn2+. The actual intracellular
Ca2+ did not fall, but the
ratiometric calculation of
[Ca2+]i
based on 340- and 380-nm signals suggested an apparent fall (113 ± 15) (P < 0.01 for both). Previous
investigators have shown that Mn2+
can enter via a putative capacitative divalent cation channel(s) (30).
Control experiments showed that addition of
Mn2+ (1 mM) to VSMC (in
calcium-containing or calcium-free buffer) did not alter the ratio of
counts/s at 340 and 380 nm and thus apparent resting
[Ca2+]i
levels (88 ± 11 vs. 85 ± 10 nM). Figure
4B shows the influence of
Mn2+ on fluorescence quantified at
360 nm (8). When Ca2+ was added to
V1R-stimulated cells in EGTA (0.5 mM)
Ca2+-free buffer, subsequent
addition of Mn2+ (~1 mM)
quenched the signal at the calcium-insensitive isosbestic point (360 nm). Resting
[Ca2+]i
of 74 ± 5 nM rose to 98 ± 5 nM after V1R, to 172 ± 11 nM
after Ca2+ (1 mM), and showed an
apparent fall to 109 ± 4 nM with addition of
Mn2+ (~1 mM)
(P < 0.01 for all maneuvers) (Fig.
4C). That
Mn2+ quenched the signal following
influx of Ca2+ suggests that a
capacitative channel was still open at 50 s.
|
TG and CPA, which are SRCAIs, prevent reaccumulation of calcium into
intracellular storage sites. Figure 5 shows
the response of VSMC to TG over time. Baseline
[Ca2+]i
of 90 ± 7 nM rose after addition of TG to 180 ± 20 nM at 50 s
and to 213 ± 27 nM at 150 s. To test the hypothesis that emptying of SR storage sites facilitates calcium entry via a capacitative mechanism, we studied VSMC in nominally calcium-free medium. Under these conditions, when TG was added to VSMC, baseline
[Ca2+]i
rose from 97 ± 10 to 128 ± 14 nM
(P < 0.01); addition of
CaCl2 (1 mM) further elevated
[Ca2+]i
to 214 ± 24 nM (P < 0.01) (Fig.
6A).
In paired experiments, CPA
(106 M) increased
[Ca2+]i
from 97 ± 11 to 143 ± 14 nM (P = 0.01) and then to 220 ± 26 (P = 0.01) following the addition of calcium to the medium (Fig. 6B). The responses to CPA and TG did
not differ statistically. These data suggest that depletion of calcium
stores causes calcium entry via the putative capacitative mechanism
when extracellular calcium is provided. To show that such calcium entry
is independent of L-type voltage-gated channels, we pretreated VSMC
with verapamil prior to replenishing extracellular calcium. Figure
6C shows that baseline
[Ca2+]i
in the presence of verapamil was 101 ± 13 nM. Subsequent addition of CPA caused a rise of
[Ca2+]i
to 147 ± 16 nM, which increased further to 194 ± 20 nM when calcium (1 mM) was added (P < 0.01 for both). These data suggest that emptying of the SR (by inhibiting
refilling) triggers calcium entry that is nearly equal in magnitude to
that produced by V1R stimulation in the presence of extracellular
calcium.
|
|
To substantiate that the increase in
[Ca2+]i
arose from calcium entry, we performed experiments in which
Mn2+ rather than
Ca2+ was added to cells in
calcium-free buffer that had been treated first with CCB and then
SRCAI. Figure
7A
shows the quenching effect of Mn2+
(1 mM) on the Ca2+/fura signal.
Baseline
[Ca2+]i
in the presence of CCB was 99 ± 3 nM; it rose to 145 ± 10 nM (P < 0.01) after the addition of
SRCAI. The ratiometric fura signal was reduced, causing an apparent
fall of
[Ca2+]i
to 96 ± 8 (P < 0.01) when
Mn2+ was added. Furthermore, in
experiments in which VSMC in nominally calcium-free buffer were treated
with V1R and then calcium, Mn2+
still quenched the fura signal, suggesting that the capacitative channel remained open for longer than 50 s. Figure
7B demonstrates that baseline
[Ca2+]i
of 111 ± 11 rose to 157 ± 16 nM after CPA
(106 M) stimulation and
further to 206 ± 19 after the addition of calcium. The apparent
[Ca2+]i
value then fell to 111 ± 18 nM when
Mn2+ was added as well
(P < 0.01 for all 4 maneuvers).
These experiments were repeated in the presence of EGTA (0.5 mM)
Ca2+-free buffer (Fig.
7C). Baseline
[Ca2+]i
of 71 ± 5 increased to 90 ± 5 nM with TG and to 171 ± 22 nM after addition of Ca2+ (1 mM,
measured). Fluorescence declined following introduction of
Mn2+ (~1 mM)
(P < 0.01 for all comparisons).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have explored the possibility that capacitative (store operated) calcium entry mechanisms (2, 15, 21, 31), previously demonstrated only in nonexcitable cells, adrenal glomerulosa cells, and VSMC derived from large conduit vessels (4, 20, 34, 37), might be operative in preglomerular arterioles. The discrepancy in relative contribution of calcium entry via L-type, voltage-gated channels between in vivo experiments (11, 32), isolated renal preglomerular vessels (33), or cultured VSMC (39) and studies with the isolated hydronephrotic kidney (35), juxtamedullary blood-perfused kidney (5, 17), or freshly isolated VSMC (19) led us to investigate reasons for these differences and to consider whether the operation of capacitative influx mechanisms might provide insight into these differences. CCBs, given after peptide agonist stimulation of freshly isolated VSMC in 1 mM magnesium buffer in the present study, reduced [Ca2+]i by ~50%; neither did pretreatment of VSMC with CCB block the ability of peptide agonists to increase [Ca2+]i. Studies of these cells in nominally calcium-free buffer or EGTA-containing calcium-free buffer further demonstrated the presence of non-L-channel entry pathway(s). In calcium-free buffer, VSMC responded to V1R or SRCAI with an increase in [Ca2+]i, indicating mobilization of calcium from intracellular stores. Addition of 1 mM calcium to the extracellular fluid even in the presence of a CCB further increased [Ca2+]i, suggesting that emptying of intracellular calcium storage sites had facilitated capacitative calcium entry via non-voltage-gated channels. Finally, in calcium-free buffer, Mn2+ quenched the fura signal after V1R or SRCAI, supporting the concept of divalent cation influx after emptying of intracellular Ca2+ stores. These observations provide the first evidence that capacitative calcium entry mechanisms may play a role in the regulation of resistance vessels and specifically preglomerular arterioles.
Redundancy in biological systems is a not uncommon phenomenon. For a resistance vessel that requires sustained contraction (sustained elevation of intracellular calcium), the presence of more than one calcium entry pathway might be anticipated. Thus one could postulate that peptide receptor activation could cause discharge of calcium from the SR (mobilization), opening of voltage-gated L-type channels, and entry as well via CRAC. Such a system could maintain tonic contraction as the SR filled and emptied in a dynamic fashion.
Studies of renal blood flow (RBF) in anesthetized rats in which vasoactive agents were injected directly into the renal artery examined the relative contributions of calcium entry and calcium mobilization (11, 32). Importantly, the animals were pretreated with indomethacin to eliminate the confounding effects of endogenous prostaglandins. The CCB nifedipine blocked only 50% of the maximal ANG II response (32) and 35% of the AVP response (10); 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a blocker of the IP3 receptor and calcium mobilization, inhibited reactivity to ANG II and AVP 50-65% in a dose-dependent fashion. Although the effects of the two inhibitors were additive, together they inhibited ANG II- or AVP-induced vasoconstriction by only 85%, raising the possibility of the presence in renal resistance vessels of a non-L-channel calcium influx pathway such as the putative capacitative entry mechanism.
In the isolated hydronephrotic kidney model (22, 23), nifedipine
dose-dependently reversed the changes in lumen diameter of afferent
arterioles constricted with 30 mM KCl. A nifedipine concentration of
106 M was required to
achieve full inhibition; each concentration of nifedipine was given
over a period of 10 min. More recent studies of the hydronephrotic
kidney demonstrate that 10
6
M nifedipine reversed afferent arteriolar constriction induced by ANG
II (0.3 × 10
9 M)
(35). In calcium-free medium, ANG II elicited transient afferent
vasoconstriction lasting ~2 min, providing evidence for calcium
mobilization. Pretreatment with TG blocked afferent arteriolar responses to ANG II. These studies of afferent arterioles (which contain intact endothelium) were conducted in the absence of
cyclooxygenase inhibitors.
Studies of the effect of NE on RBF in vivo and on changes in
[Ca2+]i
in microdissected afferent arterioles (33) showed that nifedipine attenuated the NE response by up to 50% in a dose-dependent manner. Response of
[Ca2+]i
to NE gave a square wave pattern. A nominally calcium-free solution,
however, blocked 80-90% of the
[Ca2+]i
response to NE. These results indicate that both calcium entry and
calcium mobilization mechanisms operate in renal afferent arterioles
and that non-L-channel pathways for calcium entry exist. In contrast,
application of verapamil (5 × 105 M) or diltiazem
(10
5 M) to afferent
arterioles in the in vitro blood-perfused juxtamedullary preparation
(5) abolished the vasoconstrictive effect of ANG II. To examine the
contribution of calcium mobilization in afferent and efferent
arterioles in the blood-perfused juxtamedullary preparation (17), the
responses to ANG II and NE with and without intracellular calcium
storage depletion with 10
6
M TG were measured. TG treatment shifted the afferent arteriolar ANG II
and NE response curves significantly to the right, demonstrating that
mobilization is an essential component of the response to these agents.
As well, TG treatment did not impair L-channel activity at this concentration.
Afferent arteriolar VSMC maintained in tissue culture responded to ANG II with a peak plateau pattern; both nifedipine and verapamil inhibited the response to ANG II by ~50% (Ref. 39; and K. E. Purdy, unpublished data). These observations, like those in the in vivo RBF studies and isolated afferent arteriole experiments, imply the presence of calcium mobilization and possible entry via non-L-type channels.
Inscho et al. (18) reported that single fresh VSMC prepared by an iron
oxide microdissection and sieving method responded to KCl and ANG II in
a peak plateau pattern. Responses to KCl were abolished in a
calcium-free medium. Diltiazem
(105 M), added prior to
agonist, diminished the stimulatory effects of KCl and
ANG II on
[Ca2+]i
by 80-90%. These findings are consistent with some contribution of a non-L-channel pathway for calcium entry. In contrast, in studies
in fresh VSMC (prepared in 5 mM magnesium buffer) in which CCB was
added during the sustained square wave phase of stimulation, nifedipine
(10
8 M) totally inhibited
ANG II-induced increases in
[Ca2+]i
both immediately and at 50 s (19). However, when these investigators employed AVP as the agonist, nifedipine inhibited the
[Ca2+]i
response by ~80% at 50 s. When they added EGTA during the square wave response to either ANG II or AVP, the effect was abolished (19).
Addition of EGTA to resting VSMC lowered baseline
[Ca2+]i
from 200 nM to 100 nM, and subsequent addition of agonist was without
effect under these conditions.
The findings in the current study with fresh VSMC prepared in 5 mM
magnesium buffer are consistent with those of Iversen and Arendshorst
(19). However, VSMC prepared in 1 mM magnesium buffer differ in
response to CCB with only a 50% inhibition of peptide agonist-induced
increases in
[Ca2+]i;
furthermore, pretreatment of these VSMC with CCB did not abolish significant responses to V1R. These results are consonant with studies
of cultured cells, microdissected afferent arterioles, and whole animal
experiments (11, 32, 33, 39). The reasons for the differences between
the two studies of fresh VSMC may be attributable to the difference in
magnesium concentration of the buffers used in preparing the VSMC. Our
experiments in which CCB was given prior to peptide agonist were all
performed in buffer containing 1 mM magnesium, whereas 5 mM magnesium
was employed previously (19). High concentrations of magnesium (5 mM)
have been shown to inhibit capacitative calcium entry in cultured rat aortic smooth muscle cells (39). In the present study, we routinely measured responses in only one to three cells at a time, whereas previously (19), a larger population density was typically used. Mechanism of effects of ANG II and AVP may be dose dependent. In
cultured rat aortic VSMC, ANG II > 108 M and AVP > 10
7 M differed from lower
concentrations in being able to stimulate an increase in
[Ca2+]i
in calcium-free medium. Whether differences in concentration of peptide
agonist in studies of fresh VSMC can account for variance among
investigations is not known.
Sodium/calcium exchange pathways have been demonstrated in arterioles of the rabbit kidney (13, 29). These investigators believe that "the Na-Ca exchanger normally plays a significant role in restoring [Ca2+]i to basal levels during recovery from vascular smooth muscle activation" (13). Reverse operation of the pathway, that is, calcium entry and sodium exit, can be stimulated by bathing vessels in a medium in which sodium has been replaced by N-methyl-D-glucamine. There is no reason to consider significant calcium entry via reverse operation of the Na/Ca exchanger in the present study, in which there was no change in extracellular sodium nor were the VSMC treated with ouabain. Furthermore, control experiments in the present study show that addition of external Ca2+ to VSMC maintained in nominally calcium-free buffer did not change baseline [Ca2+]i significantly.
Broad investigations have substantiated the hypothesis that emptying of intracellular calcium storage sites leads to calcium entry via a capacitative or store-operated influx mechanism (30). Present in many different cell types, capacitative entry can be stimulated either by inhibition of SRCA or by receptor activation followed by IP3-induced Ca2+ release from the SR (1). The channel through which Ca2+ entry occurs has been termed the CRAC (15). Although extensively described in nonexcitable cells initially, the capacitative model has subsequently been shown to operate in such VSMC as rat A7r5 or A-10 VSMC lines (4, 20, 34) and cultured rat aortic VSMC (37). A recent review (15) discusses the hypothetical models for capacitative Ca2+ entry signaling between intracellular storage sites and the plasma membrane. Considered mechanisms include presence of a diffusible factor, conformational coupling or channel coupling, or a combination of these. No previous study has provided evidence for the capacitative entry model in fresh VSMC derived from renal preglomerular vessels.
A potential criticism of the present study is that TG at concentrations
of 106 M may have an effect
on voltage-gated L-type channels. Because we routinely used
concentrations of <10
6 M,
we attempted to avoid this problem. However, CPA, which does not
interact with L-channels, had effects that were indistinguishable from
those of TG in our study.
In summary, we have shown, for the first time, evidence for capacitative (store-operated) calcium entry in VSMC freshly isolated from rat resistance vessels, specifically, preglomerular arterioles. Our observations of less than total inhibition of V1R-induced increases in [Ca2+]i by CCB are concordant with those in whole animal studies (11, 32), cultured renal VSMC (39), and isolated rat renal arterioles (33). In calcium-free buffer, both V1R and inhibition of SRCA with TG or CPA stimulated intracellular calcium mobilization; subsequent addition of calcium to the extracellular medium caused a further rise in [Ca2+]i via non-voltage-gated channels. As well, addition of Mn2+ following either V1R or SRCAI quenched fura fluorescence, further substantiating that emptying of the SR promoted divalent cation influx.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported in part by National Heart, Lung, and Blood Institute Grant R01-HL-02334.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. K. Fellner, Dept. of Cell and Molecular Physiology, CB 7545, Rm. 171, Medical Sciences Research Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: sfellner{at}med.unc.edu).
Received 16 November 1998; accepted in final form 21 May 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berridge, M. J.
Capacitative calcium entry.
Biochem. J.
312:
1-11,
1995[Medline].
2.
Blatter, L. A.
Depletion and filling of intracellular calcium stores in vascular smooth muscle.
Am. J. Physiol.
268 (Cell Physiol. 37):
C503-C512,
1995
3.
Buryi, V.,
N. Morel,
S. Salomone,
S. Kerger,
and
T. Godfraind.
Evidence for a direct interaction of thapsigargin with voltage-dependent Ca2+ channel.
Naunyn Schmiedebergs Arch. Pharmacol.
351:
40-45,
1995[Medline].
4.
Byron, K. L.,
and
C. W. Taylor.
Vasopressin stimulation of Ca2+ mobilization, two bivalent cation entry pathways and Ca2+ efflux in A7r5 rat smooth muscle cells.
J. Physiol. (Lond.)
485:
455-468,
1995[Abstract].
5.
Carmines, P. K.,
and
L. G. Navar.
Disparate effects of Ca channel blockade on afferent and efferent arteriolar responses to ANG II.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F1015-F1021,
1989
6.
Casteels, R.,
and
G. Droogmans.
Exchange characteristics of the noradrenaline-sensitive calcium store in vascular smooth muscle cells of the rabbit ear artery.
J. Physiol. (Lond.)
317:
263-279,
1981[Abstract].
7.
Chatziantoniou, C.,
and
W. J. Arendshorst.
Angiotensin receptor sites in renal vasculature of rats developing genetic hypertension.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F853-F862,
1993
8.
Chiavaroli, C.,
G. Bird,
and
J. W. Putney.
Delayed "all-or-none" activation of inositol 1,4,5-trisphosphate-dependent calcium signaling in single rat hepatocytes.
J. Biol. Chem.
269:
25570-25575,
1994
9.
Conger, J. D.,
and
S. A. Falk.
KCl and angiotensin responses in isolated rat renal arterioles: effects of diltiazem and low calcium medium.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F134-F140,
1993
10.
Conger, J. D.,
S. A. Falk,
and
J. B. Robinnette.
Angiotensin II-induced changes in smooth muscle calcium in rat renal arterioles.
J. Am. Soc. Nephrol.
3:
1792-1803,
1993[Abstract].
11.
Feng, J. J.,
and
W. J. Arendshorst.
Calcium signaling mechanisms in renal vascular responses to vasopressin in genetic hypertension.
Hypertension
30:
1223-1231,
1997
12.
Fleming, J. T.,
N. Parekh,
and
M. Steinhausen.
Calcium antagonists preferentially dilate preglomerular vessels of hydronephrotic kidney.
Am. J. Physiol.
253 (Renal Fluid Electrolyte Physiol. 22):
F1157-F1163,
1987
13.
Fowler, B. C.,
P. K. Carmines,
L. D. Nelson,
and
P. D. Bell.
Characterization of sodium-calcium exchange in rabbit renal arterioles.
Kidney Int.
50:
1856-1862,
1996[Medline].
14.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
15.
Holda, J. R.,
A. Klishin,
M. Sedova,
J. Huser,
and
L. A. Blatter.
Capacitative calcium entry.
News Physiol. Sci.
13:
157-163,
1998.
16.
Hoth, M.,
and
R. Penner.
Depletion of intracellular calcium stores activates a calcium current in mast cells.
Nature
355:
353-356,
1992[Medline].
17.
Inscho, E. W.,
J. D. Imig,
and
A. K. Cook.
Afferent and efferent arteriolar vasoconstriction to angiotensin II and norepinephrine involves release of Ca2+ from intracellular stores.
Hypertension
29:
222-227,
1997
18.
Inscho, E. W.,
M. J. Mason,
A. C. Schhroeder,
P. C. Deichmann,
K. D. Stiegler,
and
J. D. Imig.
Agonist-induced calcium regulation in freshly isolated renal microvascular smooth muscle cell.
J. Am. Soc. Nephrol.
8:
569-579,
1997[Abstract].
19.
Iversen, B. I.,
and
W. J. Arendshorst.
Angiotensin II and vasopressin stimulate calcium entry in freshly isolated afferent arteriolar smooth muscle cells.
Am. J. Physiol.
274 (Renal Physiol. 43):
F498-F508,
1998
20.
Iwasawa, K.,
T. Nakajima,
H. Hazama,
A Goto,
W. S. Shin,
T. Toyo-oka,
and
M. Omata.
Effects of extracellular pH on receptor-mediated Ca2+ influx in A7r5 rat smooth muscle cells: involvement of two different types of channel.
J. Physiol. (Lond.)
503:
237-251,
1997[Abstract].
21.
Kwan, C. Y.,
H. Takemura,
J. F. Obie,
O. Thastrup,
and
J. W. Putney.
Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells.
Am. J. Physiol.
258 (Cell Physiol. 27):
C1006-C1015,
1990
22.
Loutzenhiser, R.,
and
M. Epstein.
Renal microvascular actions of calcium antagonists.
J. Cardiovasc. Pharmacol.
12, Suppl. 16:
S48-S52,
1989.
23.
Loutzenhiser, R.,
K. Hayashi,
and
M. Epstein.
Divergent effects of KCl-induced depolarization on afferent and efferent arterioles.
Am. J. Physiol.
257 (Renal Fluid Electrolyte Physiol. 26):
F561-F564,
1989
24.
Lyttone, J.,
M. Westlin,
and
M. R. Hanley.
Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.
J. Biol. Chem.
266:
17067-17071,
1991
25.
Madge, L.,
I. C. B. Marshall,
and
C. W. Taylor.
Delayed autoregulation of the Ca2+ signals resulting from capacitative Ca2+ entry in bovine pulmonary artery endothelial cells.
J. Physiol. (Lond.)
498:
351-369,
1997[Abstract].
26.
Mene, P.,
F. Pugliese,
and
G. A. Cinotti.
Regulation of capacitative calcium influx in cultured human mesangial cells: roles of protein kinase C and calmodulin.
J. Am. Soc. Nephrol.
7:
983-990,
1996[Abstract].
27.
Murphy, C. T.,
C. T. Poll,
and
J. Westwick.
The whoosh and trickle of calcium signalling.
Cell Calcium
18:
245-251,
1995[Medline].
28.
Nabika, T.,
P. A. Velletri,
W. Lovenberg,
and
M. A. Beaven.
Increases in cytosolic calcium and phosphoinositide metabolism induced by angiotensin II and [Arg]vasopressin in vascular smooth muscle cells.
J. Biol. Chem.
260:
4661-4670,
1985[Abstract].
29.
Nelson, L. D.,
N. A. Mashburn,
and
P. D. Bell.
Altered sodium-calcium exchange in afferent arterioles of the spontaneously hypertensive rat.
Kidney Int.
50:
1889-1896,
1996[Medline].
30.
Parekh, A. B.,
and
R. Penner.
Store depletion and calcium influx.
Physiol. Rev.
77:
901-930,
1997
31.
Putney, J. W., Jr.
Capacitative calcium entry revisited.
Cell Calcium
11:
611-624,
1990[Medline].
32.
Ruan, X.,
and
W. J. Arendshorst.
Calcium entry and mobilization signalling pathways in ANG II-induced renal vasoconstriction in vivo.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F398-F405,
1996
33.
Salomonsson, M.,
and
W. J. Arendshorst.
Calcium recruitment in the renal vasculature: NE effects on blood flow and cytosolic calcium concentration.
Am. J. Physiol.
276 (Renal Physiol. 45):
F700-F710,
1999
34.
Skutella, M.,
and
U. T. Ruegg.
Studies on capacitative calcium entry in vascular smooth muscle cells by measuring 45Ca2+ influx.
J. Recept. Signal Transduct. Res.
171:
163-175,
1997.
35.
Takenaka, T.,
H. Suzuki,
K. Fujiwara,
Y. Kanno,
Y. Ohno,
K. Hayashi,
T. Nagahama,
and
T. Saruta.
Cellular mechanisms mediating rat renal microvascular constriction by angiotensin II.
J. Clin. Invest.
100:
2107-2114,
1997
36.
Thastrup, O.,
P. J. Cullen,
B. K. Drobak,
M. R. Hanley,
and
A. P. Dawson.
thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase.
Proc. Natl. Acad. Sci. USA
87:
2466-2470,
1996[Abstract].
37.
Xuan, Y.,
O. Wang,
and
A. R. Whorton.
Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1258-C1265,
1992
38.
Yoshimura, M.,
T. Oshima,
H. Matsuura,
T. Ishida,
M. Kambe,
and
G. Kajiyama.
Extracellular Mg2+ inhibits capacitative Ca2+ entry in vascular smooth muscle cells.
Circulation
3:
2567-2572,
1997.
39.
Zhu, Z.,
and
W. J. Arendshorst.
Angiotensin II-receptor stimulation of cytosolic calcium concentration in cultured renal resistance arterioles.
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F1239-F1247,
1996