Capacitative calcium entry in smooth muscle cells from preglomerular vessels

Susan K. Fellner and William J. Arendshorst

Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) (10-7 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = [(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)] × (S<SUB>f</SUB>/S<SUB>b</SUB>) × <IT>K</IT><SUB>d</SUB>
where R is the ratio of the 340/380 nm fluorescence signal, Rmax is the 340/380 ratio in the presence of saturation calcium, Rmin is the 340/380 ratio in calcium-free buffer containing 10 nM EGTA, and Sf/Sb is the ratio of the 380 nm fluorescence measured in a calcium-free buffer to that measured in a calcium replete solution (14). Baseline values of [Ca2+]i varied depending on cell density and time and temperature of loading of fura 2-AM.

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We employed the peptide vasoconstrictor V1R at a final concentration of 10-7 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 (10-6 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.



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Fig. 1.   A: baseline cytosolic calcium concentration ([Ca2+]i) () and effect of treatment with vasopressin V1 receptor agonist (V1R) (10-6 M) and subsequent addition of the calcium channel blocker (CCB) verapamil (10-6 M) on vascular smooth muscle cells (VSMC) freshly derived from preglomerular arterioles in buffer containing 5.0 mM magnesium (n = 8). In 5 mM magnesium buffer, CCB completely inhibited calcium entry. B: VSMC prepared and maintained in 1 mM magnesium buffer (n = 8) (); V1R (10-7 M) followed by verapamil (10-7 M) inhibited the response by ~50%. Data are means ± SE. P < 0.01 for baseline vs. V1R and V1R vs. verapamil, both for 5 and 1 mM magnesium.

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.


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Fig. 2.   Baseline [Ca2+]i in fresh VSMC isolated from preglomerular vessels followed by addition of verapamil (10-7 M) (n = 11). In presence of verapamil (10-7 M), V1R (10-7 M) increased [Ca2+]i by 70 ± 20 nM. Data are means ± SE. P = 0.01 for baseline vs. verapamil. P < 0.01 for V1R vs. verapamil.

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 (10-7 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.




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Fig. 3.   A: temporal responses of [Ca2+]i to V1R (10-7 M) in freshly isolated preglomerular VSMC in nominally calcium-free buffer. Results are means ± SE for 10 experiments. Cells responded to V1R (10-7 M) in absence of extracellular calcium ([Ca2+]e); addition of calcium to medium caused a further increase in [Ca2+]i in a stepwise fashion (P < 0.01 for all comparisons). B: in presence of calcium-free buffer containing EGTA (0.5 mM), VSMC (n = 11) responded to V1R (10-7 M) and addition of extracellular calcium in a manner similar to VSMC in nominally calcium-free buffer without EGTA (P < 0.01 for all comparisons). C: response of VSMC to V1R (10-7 M) in nominally calcium-free buffer pretreated with CCB (5 experiments each with nifedipine and with verapamil, 10-7 M). P < 0.01 for all comparisons.

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.




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Fig. 4.   A: freshly isolated VSMC from preglomerular vessels (n = 7), pretreated with 10-7 M CCB (4 with nifedipine, 3 with verapamil) in nominally calcium-free buffer, responded immediately to V1R (10-7 M). Addition of Mn2+ (1 mM) gradually reduced the 340/380 nm ratio and thus the apparent estimate of [Ca2+]i. P < 0.01 for both comparisons. P = 0.70 for verapamil vs. nifedipine experiments. B: VSMC in EGTA calcium-free buffer containing nifedipine (10-7 M) and V1R (10-7 M) at the isosbestic point (360 nm). Addition of external calcium (1 mM) permitted calcium entry but did not change counts/s at this wavelength; however, addition of Mn2+ (~1 mM) did quench the fura signal. C: VSMC (n = 8) in presence of EGTA responded nearly identically to the same concentrations of V1R and Ca2+. Subsequent addition of Mn2+ produced a fall in the Ca2+/fura signal. P < 0.01 for all comparisons. Data are means ± SE.

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 (10-6 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.


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Fig. 5.   Time course of response of fresh preglomerular arteriolar VSMC (n = 6) to thapsigargin (TG, 10-7 M) in buffer containing calcium (1 mM). Data are means ± SE. P < 0.001, TG vs. baseline.





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Fig. 6.   A: sequential addition of TG (10-7 M) and calcium (1 mM) to fresh preglomerular VSMC (n = 8) in nominally calcium-free buffer (P = 0.01 for both comparisons). B: experiment identical to A, except cyclopiazonic acid (CPA, 10-6 M) was used instead of TG (n = 7) (P < 0.05 for baseline vs. CPA; P < 0.01 for CPA vs. Ca2+). There are no differences between responses to TG and CPA. C: response to CPA (10-6 M) in VSMC (n = 6) pretreated with verapamil (10-7 M) in nominally calcium-free buffer followed by addition of extracellular Ca2+ (P < 0.01 for both comparisons); these results with verapamil are not different from those in B performed in absence of verapamil. Data are means ± SE.

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 (10-6 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).




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Fig. 7.   A: fresh VSMC from preglomerular vessels (n = 6) in nominally calcium-free buffer responded immediately to sarcoplasmic reticulum Ca2+-ATPase inhibitors (SRCAI: CPA, 10-6 M; or TG, 10-7 M). Mn2+ (1 mM) addition diminished the Ca2+/fura signal (P < 0.01 for both comparisons). Although the ratio of 340/380 fluorescence declines, it is no longer a measurement of actual [Ca2+]i, because of the quenching effect of Mn2+ on fura fluorescence. B: VSMC (n = 5) in nominally calcium-free buffer to which have been added consecutively CPA (10-6 M), Ca2+ (1 mM), and Mn2+ (1 mM) (P < 0.01 for all comparisons). C: VSMC (n = 6) in presence of EGTA-containing (0.5 mM) calcium-free buffer are sequentially treated with TG (10-6 M), Ca2+ (1 mM), and Mn2+ (~1 mM) (P < 0.01 for baseline vs. TG and Ca2+ vs. Mn2+; P = 0.02 for TG vs. Ca2+). Data are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-6 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 × 10-5 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 (10-5 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 > 10-8 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 10-6 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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