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Am J Physiol Renal Physiol 289: F127-F136, 2005. First published February 8, 2005; doi:10.1152/ajprenal.00397.2004
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Calmodulin mediates norepinephrine-induced receptor-operated calcium entry in preglomerular resistance arteries

Carie S. Facemire and William J. Arendshorst

Department of Cell and Molecular Physiology and Program in Integrative Vascular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Submitted 1 November 2004 ; accepted in final form 2 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although L-type voltage-dependent calcium channels play a major role in mediating vascular smooth muscle cell contraction in the renal vasculature, non-L-type calcium entry mechanisms represent a significant component of vasoactive agonist-induced calcium entry in these cells as well. To investigate the role of these non-voltage-dependent calcium entry pathways in the regulation of renal microvascular reactivity, we have characterized the function of store- and receptor-operated channels (SOCs and ROCs) in renal cortical interlobular arteries (ILAs) of rats. Using fura 2-loaded, microdissected ILAs, we find that the L-type channel antagonist nifedipine blocks less than half the rise in intracellular calcium concentration ([Ca2+]i) elicited by norepinephrine. SOCs were activated in these vessels using the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors cyclopiazonic acid and thapsigargin and were dose dependently blocked by the SOC antagonists Gd3+ and 2-aminoethoxydiphenyl borate (2-APB) and the combined SOC/ROC antagonist SKF-96365. Gd3+ had no effect on the non-L-type Ca2+ entry activated by 1 µM NE. A low concentration of SKF-96365 that did not affect thapsigargin-induced store-operated Ca2+ entry blocked 60–70% of the NE-induced Ca2+ entry. Two different calmodulin inhibitors (W-7 and trifluoperazine) also blocked the NE-induced Ca2+ entry. These data suggest that in addition to L-type channels, NE primarily activates ROCs rather than SOCs in ILAs and that this receptor-operated Ca2+ entry mechanism is regulated by calmodulin. Interestingly, 2-APB completely blocked the NE-induced non-L-type Ca2+ entry, implying that SOCs and ROCs in preglomerular resistance vessels share a common molecular structure.

receptor-operated channel; store-operated channel; vascular smooth muscle

VASCULAR SMOOTH MUSCLE CELLS (VSMCs) comprising the preglomerular resistance vessels (interlobular artery and afferent arteriole) mediate changes in luminal diameter, thereby regulating renal vascular resistance, renal blood flow, and glomerular filtration rate. The vasoactive effects of circulating and locally produced vasoconstrictor agents such as angiotensin II, vasopressin, endothelin-1, and norepinephrine (NE) are mediated by G protein-coupled receptors (GPCRs), whose activation cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in preglomerular VSMCs. Elevated [Ca2+]i (via calmodulin) activates myosin light chain kinase, which phosphorylates the regulatory myosin light chain, leading to VSMC contraction.

Activation of preglomerular arterial {alpha}1-adrenoceptors by NE leads to increased VSMC [Ca2+]i by stimulating calcium mobilization from intracellular stores and calcium entry from the extracellular fluid (39). Although inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine receptor calcium release channels are both present in VSMCs, NE-induced calcium mobilization appears to be mediated primarily by IP3 receptors in isolated afferent arterioles (39) and interlobular arteries (ILAs) (Facemire CS and Arendshorst WJ, unpublished observations). A sustained rise in [Ca2+]i is generated via calcium entry across the plasma membrane. In many resistance vessels, including those of the renal microcirculation, it is known that vasoactive agonist-induced calcium entry occurs partially through dihydropyridine-sensitive L-type calcium channels (42). However, it has been shown recently in rats that nifedipine, an antagonist of L-type channels, attenuates the NE-induced reduction in renal blood flow by only 50%. Nifedipine exerts a similar degree of inhibition on the NE-induced rise in [Ca2+]i in isolated afferent arterioles (39), indicating that non-L-type calcium entry mechanisms contribute to the [Ca2+]i response to NE in renal resistance vessels. These could include voltage-dependent channels such as T- or P/Q-type channels (21, 22) and/or voltage-independent store- and receptor-operated channels.

Studies in VSMCs using 45Ca indicated the presence of a Ca2+ influx mechanism that was activated when sarcoplasmic reticular Ca2+ content had been depleted (7), and several groups including our own (12–14) have confirmed the presence of these "store-operated channels" (SOCs) in various types of smooth muscle cells (for a review, see Ref. 2). Intracellular signaling mechanisms linking store depletion to SOC opening in SMCs are poorly understood. In aortic VSMCs, a "calcium influx factor" activates SOCs (54, 55), and recently it has been shown that Ca2+-independent phospholipase A2 is required for SOC activation in these cells (44). In portal vein myocytes, {alpha}-adrenoceptor activation of SOCs by store depletion requires PKC-mediated phosphorylation of proteins, although specific proteins have not been identified (1, 2). The few studies addressing this question suggest that SOC activity is governed by multiple inputs, whose physiological importance is associated with cell type.

The term "receptor-operated channel" (ROC), which was introduced by Bolton (4) and van Breemen et al. (56), encompasses both the P2X1 ligand-gated channel and channels that are opened as a result of GPCR activation, independent of store depletion, in smooth muscle cells (26). Neurotransmitters such as NE and acetylcholine, as well as several hormones including histamine, endothelin-1, and vasopressin, have been reported to activate nonselective cation channels (NSCCs) in VSMCs (for reviews, see Refs. 26 and 31). ROCs have been shown to be activated by vasoactive agonists in coronary (60), mesenteric (8, 25), and cerebral (18) resistance arteries. An essential role for Gi/Go proteins has been demonstrated in gastrointestinal and airway smooth muscle, and ROC currents in other smooth muscle preparations are modulated by calcium, myosin light chain kinase, tyrosine kinase, and PKC (31). However, further details of the signaling pathways responsible for GPCR-mediated activation of ROCs in SMCs have not been well defined.

Recently, canonical transient receptor potential (TRPC) proteins have been shown to mediate store- and receptor-operated calcium entry in various cell types, including VSMCs (24, 59). TRPC channels belong to the superfamily of hexahelical cation channels, which includes voltage-operated calcium and potassium channels as well as other TRP-related channels (36). We have recently demonstrated that five of the seven known TRPC proteins (TRPC1, 3, 4, 5, and 6) are expressed in freshly isolated preglomerular resistance vessels and that TRPC3 and TRPC6 are much more abundant than any other TRPC proteins in these vessels (11). These two proteins belong to the TRPC3/6/7 subfamily, which is thought to form store-independent ROCs on homo- or heterotetramerization (23, 53). TRPC3 and TRPC6 each have a calmodulin/IP3R binding (CIRB) domain in their COOH terminus, and calmodulin (CaM) binding to a CIRB homologous region has been demonstrated for all members of the TRPC family (47). Furthermore, CaM has been implicated in regulating TRPC3 and TRPC6 activity in stably transfected cells (5, 53).

Little is known about the function of non-L-type calcium channels and TRPC proteins in native VSMCs of resistance arteries/arterioles and less is known regarding the role of these calcium entry pathways in the renal vasculature. In the present study, we utilize pharmacological tools known to act on TRPC channels to study the physiological regulation of non-L-type calcium entry in the renal microcirculation. We demonstrate for the first time the relative contributions of SOCs and ROCs to the NE-induced [Ca2+]i response and investigate intracellular signaling mechanisms that regulate these calcium entry pathways in isolated preglomerular resistance vessels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Microdissection of ILAs. ILAs (40- to 60-µm outer diameter) were isolated from kidneys of 6- to 7-wk-old male Sprague-Dawley rats (175–250 g) from our Chapel Hill breeding colony. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and in compliance with the guidelines and practices of the UNC-CH Institutional Animal Care and Use Committee. For each experiment, one rat was anesthetized with pentobarbital sodium (50 mg/kg ip) and the left kidney was quickly removed via a lateral abdominal incision and placed into ice-cold physiological saline solution (PSS; composition in mM: 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 5 D-glucose) supplemented with BSA (5 mg/ml, Sigma). Several thin (0.5–1 mm) slices were cut from the middle region of the kidney and transferred to a dissection dish containing PSS+BSA. Sharpened forceps and a knife blade were used for the microdissection procedure under Wild (Heerbrugg, Switzerland) microscopic visualization. An arcuate artery was localized and a wedge-shaped segment, consisting of glomeruli, blood vessels, and tubular structures, was cut from the slice. The tubules were carefully stripped away using forceps, and segments of ILAs were cut using a sharpened knife blade. To obtain a homogeneous population of vessels, the most superficial cortical segments of ILAs were isolated.

Measurement of [Ca2+]i. Fura 2-AM (Molecular Probes) and Pluronic F-127 (Molecular Probes) were mixed with PSS+BSA to a final concentration of 1 µM and 0.005%, respectively. Isolated ILAs were incubated in this loading solution for 20–45 min at room temperature (23–25°C) in the dark. The vessels were then transferred to a dish containing PSS+BSA at room temperature and incubated for an additional 15–30 min in the dark to allow for deesterification of the dye. A single ILA was transferred to a chamber containing PSS on the stage of an inverted microscope (Olympus IX 70) using a micropipette. The two ends of the vessel were gently aspirated into fire-polished glass holding pipettes using syringes connected to the back of the pipettes to generate negative pressure.

For [Ca2+]i measurements, the ILA was centered in the optical field of a x40 quartz oil-immersion objective and excited alternatively with ultraviolet light of 340- and 380-nm wavelengths from a DeltaRAM high-speed multiwavelength illuminator (Photon Technology International, South Brunswick, NJ). Images were captured using an intensified CCD video camera (PTI IC-300), and fluorescent signals were recorded using ImageMaster acquisition software (PTI) after passing through a 510-nm band-pass filter. A "region of interest" (area containing the vessel preparation) was selected from the visual field using the acquisition software, enabling selective recording of a fluorescent signal from only the region of interst. [Ca2+]i was calculated based on the fluorescence ratio at 340/380 nm, according to the equation described by Grynkiewicz et al. (17): [Ca2+]i = Kd·[(R – Rmin)/(Rmax – R)]·(Sf/Sb), where Kd is the dissociation constant of fura 2 for calcium, Sf and Sb are the 380-nm fluorescence intensities at saturating and zero calcium concentrations, respectively, and Rmin and Rmax are values of R (fluorescence ratio at 340/380 nm) at low and saturating calcium concentrations, respectively. Values for Kd, Rmin, Rmax, Sf, and Sb were determined by in vitro calibration, which has been described previously (41).

All experiments were performed at room temperature (23–25°C). The volume of fluid in the experimental chamber (~400 µl) was maintained at a constant level using a vacuum suction system. Experimental solutions were added in large volume (1 ml), which allowed total exchange of the bathing solution in the chamber within 5 s. Calcium-free solution was prepared by adding 0.5 mM EGTA to PSS and replacing CaCl2 with NaCl. The viability of each vessel preparation was tested by exposing it to a short (50–60 s) pulse of NE (1 µM). If there was no immediate [Ca2+]i response, the preparation was discarded. In experiments involving channel antagonist pretreatment or removal of extracellular calcium, ILAs were exposed to antagonist or calcium-free PSS for 1 min before addition of NE (in the presence of the pretreatment solution). Mean [Ca2+]i values for the control period were obtained between 5 and 10 s before the addition of NE. Initial peak and sustained plateau [Ca2+]i values were measured between 1 and 15 s and between 30 and 35 s, respectively, after NE stimulation. To establish reversibility and to exclude any potential prolonged action of pretreatments, experimental periods were performed in random order.

For experiments involving SERCA inhibitors, ILAs were pretreated with either cyclopiazonic acid (CPA; Calbiochem) or thapsigargin (Sigma) in calcium-free PSS for 5 min before addition of extracellular calcium. In studies resolving the mobilization and entry components of the NE calcium response (see Figs. 2–7), the vessel preparation was exposed to calcium-free PSS for 1 min and then stimulated with NE in calcium-free PSS to mobilize SR calcium (mobilization response shown in Fig. 2A). Extracellular calcium (1 mM) was then added (in the continued presence of NE and nifedipine) 1 min after the initial NE stimulation, once [Ca2+]i had returned to baseline, to produce the calcium entry response. Vehicle controls consisted of dimethylsulfoxide (CPA and thapsigargin experiments) and deionized water (NE experiments).



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  		Fig. 2. Dose dependency of NE-induced calcium mobilization and non-L-type calcium entry in isolated ILAs. A: composite tracings showing NE-induced transient mobilization responses followed by sustained calcium entry responses upon addition of extracellular calcium over a 100-fold range of NE concentrations (n = 6). B: dose-response curves for NE-induced calcium mobilization and non-L-type calcium entry. Mobilization and entry values were measured as the peak change in [Ca2+]i within 15–20 s after addition of NE or 1 mM [Ca2+]e, respectively. *P < 0.05 vs. baseline (0.1 µM NE) level of mobilization or entry; n = 6.

 


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  		Fig. 7. Effect of calmodulin (CaM) inhibition on CPA- and NE-induced calcium entry. A: blocking effect of 50 µM W-7 on CPA-induced store-operated calcium entry. B: W-7-mediated inhibition of NE-induced non-L-type calcium entry. C: inhibition of NE-induced calcium entry by trifluoperazine (TFP; 5 µM). *P < 0.05 vs. NE alone.

 
Quantification of calcium entry responses and statistical analysis. Calcium entry responses were quantified by calculating the area under the curve for the first 100 (NE experiments) or 230 s (CPA, thapsigargin experiments) of the response after addition of extracellular calcium. For all studies, no more than two vessels from any one animal were used in a given data set. Data are presented as means ± SE. SigmaStat (SPSS, Chicago, IL) software was used for statistical analysis. Statistical significance was evaluated by one-way ANOVA plus a Holm-Sidak post hoc test for multiple comparisons, and a value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In isolated rat afferent arterioles, NE activates both calcium mobilization and entry to produce a rise in [Ca2+]i consisting of an immediate peak followed by a sustained plateau (39). To confirm that isolated rat ILAs do not respond differently than afferent arterioles, we performed studies using NE and calcium-free PSS to eliminate calcium entry. Figure 1, A and B, shows that in the presence of extracellular calcium, NE caused a peak rise in [Ca2+]i of 107 ± 12 nM and a sustained plateau of 69 ± 4 nM, whereas a calcium-free solution inhibited ~50% of the peak (49 ± 12 nM) and 90% of the plateau (8 ± 2 nM). To determine the contribution of voltage-operated L-type calcium channels to the NE-induced rise in [Ca2+]i, we used nifedipine at a concentration (1 µM) that completely blocks the depolarization-induced rise in [Ca2+]i stimulated by 100 mM KCl (Fig. 1C, P < 0.01). Nifedipine blocked ~30% of the peak (108 ± 11 vs. 71 ± 10 nM, P < 0.01) and 40–50% of the sustained plateau (78 ± 9 vs. 43 ± 8 nM, P < 0.001) phases of the NE-induced calcium response (Fig. 1C). We also tested the T-type and P/Q-type voltage-operated channel blockers mibefradil (0.1 µM) and {omega}-agatoxin (10 nM) and found them to cause no detectable change in the NE- or KCl-induced rise in [Ca2+]i (in nM: 109 ± 22 NE peak vs. 112 ± 22 mibefradil peak, 71 ± 5 NE plateau vs. 75 ± 7 mibefradil plateau, P > 0.2; 194 ± 44 KCl peak vs. 203 ± 45 {omega}-agatoxin peak, 111 ± 25 KCl plateau vs. 99 ± 20 {omega}-agatoxin plateau, P > 0.1).



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  		Fig. 1. A: representative tracing showing the intracellular Ca2+ concentration ([Ca2+]i) response to norepinephrine (NE) stimulation in the presence of 1 mM extracellular calcium. Inhibitory effects of removal of extracellular calcium (B; n = 13) and 1 µM nifedipine (Nif; C; n = 14) on the 100 mM KCl (K100)- and NE-induced rise in [Ca2+]i in isolated intralobular arteries (ILAs). Peak values represent the maximum change in [Ca2+]i achieved during the first 15 s after stimulation with NE or K100, and plateau values represent the sustained change in [Ca2+]i 30–35 s after stimulation. *P < 0.05 vs. NE or K100 alone.

 
To examine the non-L-type calcium entry activated by NE, we separated the mobilization and entry components of the NE-induced rise in [Ca2+]i using the maneuver shown in Fig. 2A. In the presence of nifedipine and calcium-free PSS, NE stimulated a transient mobilization response, and upon addition of 1 mM extracellular calcium stimulated an immediate and sustained calcium entry response representing non-L-type calcium entry (Fig. 2A). The NE-induced calcium mobilization transient typically reached peak magnitude within 2–3 s and returned to baseline within 30–35 s of stimulation. The calcium entry response elicited by NE generally reached its peak within 3–5 s and was sustained above baseline for at least 2 min after addition of extracellular calcium. A gradual decline in [Ca2+]i was observed in some preparations after an initial peak response to extracellular calcium; however, all maintained some level of elevated [Ca2+]i for 2 min after initiation of the calcium entry response. The magnitudes of both the mobilization and entry responses increased dose dependently between 0.1 and 10 µM NE (Fig. 2B).

To generate a pharmacological profile of the non-L-type, presumably store- or receptor-operated, calcium entry channels activated by NE in ILAs, we first activated SOCs independently of receptor stimulation by passive store depletion to characterize the effects of putative SOC antagonists in these vessels. As shown in Fig. 3A, pretreatment with the SERCA inhibitor CPA (25 µM) caused an immediate and sustained calcium entry response upon addition of 1 mM extracellular calcium, demonstrating that SOCs are present in ILAs. This store-operated calcium entry was dose dependently blocked by Gd3+ (Fig. 3A). Another SERCA inhibitor, thapsigargin, also activated SOCs in ILAs, and the inhibitory effect of the combined SOC/ROC antagonist SKF-96365 was also dose dependent (Fig. 3B). 2-Aminoethoxydiphenyl borate (2-APB), a drug traditionally used as an IP3 receptor or SOC antagonist, dose dependently blocked thapsigargin-induced store-operated calcium entry as well, with 30 µM 2-APB producing an essentially complete blockade (Fig. 3C).



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  		Fig. 3. Pharmacology of store-operated channels (SOCs) in ILAs. A: effects of increasing concentrations of Gd3+ on cyclopiazonic acid (CPA)-induced store-operated Ca2+ entry. B: dose-dependent inhibition of thapsigargin (Thaps)-induced store-operated Ca2+ entry by SKF-96365. C: blocking effects of increasing concentrations of 2-aminoethoxydiphenyl borate (2-APB) on thapsigargin-induced store-operated Ca2+ entry. ILAs were pretreated with 25 µM CPA or 10 µM thapsigargin in the absence of extracellular Ca2+ for 5 min before the addition of 1 mM extracellular Ca2+.

 
To determine whether SOCs are activated by NE in ILAs, we tested the effects of a high concentration of Gd3+ (250 µM) on the non-L-type calcium entry activated by NE. This concentration of Gd3+ blocked nearly all CPA-induced store-operated calcium entry (Fig. 3A) but had no significant effect on the non-L-type calcium entry activated by 1 µM NE when analyzed as area under the curve (Fig. 4A, P > 0.5). In contrast, 5 µM NE activated non-L-type calcium entry that was significantly inhibited by Gd3+ (Fig. 4B, P < 0.05).



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  		Fig. 4. Effects of high-dose Gd3+ on NE-induced Ca2+ entry. A: composite tracing (left) and group means (right) showing the effect of 250 µM Gd3+ on non-L-type calcium entry activated by 1 µM NE. B: inhibitory effect of 250 µM Gd3+ on 5 µM NE-induced non-L-type calcium entry. [Ca2+]e, extracellular Ca2+ concentration; *P < 0.05 vs. NE alone.

 
We next investigated the contribution of ROCs to NE-induced non-L-type calcium entry using 2 µM SKF-96365 as a ROC-specific antagonist. This concentration of SKF-96365 had no significant effect on thapsigargin-induced calcium entry (Fig. 3B; P > 0.08 vs. thapsigargin alone) but blocked 60–70% of the non-L-type calcium entry activated by 1 µM (Fig. 5A, P < 0.02) and 5 µM NE (Fig. 5B, P ≤ 0.001). The magnitude of the SKF-96365-insensitive portion of the response was not significantly different from that in the absence of NE (Fig. 5, A and B; P > 0.2). 2-APB (30 µM) completely blocked the non-L-type calcium entry activated by 1 µM NE (Fig. 6, P < 0.01). 2-APB also completely blocked 5 µM NE-induced non-L-type calcium entry (data not shown).



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  		Fig. 5. Blocking effect of receptor-operated channel (ROC)-selective concentration of SKF-96365 on NE-induced non-L-type Ca2+ entry. Composite tracings (left) and group means (right) showing inhibition of 1 (A) and 5 µM (B) NE-induced non-L-type Ca2+ entry by 2 µM SKF-96365. *P < 0.05 vs. NE alone.

 


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  		Fig. 6. Effect of 2-APB on NE-induced non-L-type Ca2+ entry. Composite tracing (top) and group means (bottom) showing the blocking effect of 30 µM 2-APB on 1 µM NE-induced Ca2+ entry. *P < 0.05 vs. NE alone.

 
CaM is known to regulate the activity of a variety of ion channels (for a review, see Ref. 38) and has recently been reported to be a key regulatory molecule involved in TRPC channel gating and activity (5, 47, 61). To examine a potential role for CaM in activation of non-L-type calcium entry downstream of {alpha}1-adrenoceptors, we tested the effects of two different CaM inhibitors, W-7 (50 µM) and trifluoperazine (TFP; 5 µM), on NE-induced calcium entry in our ILA preparation. W-7 had only a small effect (<25% block) on store-operated calcium entry activated by CPA (Fig. 7A). In contrast, W-7 inhibited ~60% of the non-L-type calcium entry activated by 1 µM NE (Fig. 7B, P < 0.03), and TFP blocked the response almost entirely (Fig. 7C, P < 0.001).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Voltage-independent calcium entry pathways have been studied extensively in nonexcitable cell types, as they represent the primary route(s) of calcium influx in these cells. Only recently have these calcium entry mechanisms been comprehensively investigated in native smooth muscle cells, where they appear to have major roles in regulating contraction and proliferation (2, 26). Interest in these non-voltage-dependent channels has increased dramatically in recent years, motivated primarily by the identification of mammalian TRP homologs, which have proven to be likely candidates for forming voltage-independent cation channels in a vast number of cell types (for reviews, see Refs. 3 and 31). A persistent problem in the study of store- and receptor-operated calcium entry has been a lack of highly specific pharmacological antagonists. Blockers such as lanthanides (La3+, Gd3+), SKF-96365, and 2-APB, whose effects were originally characterized based on functional classifications of voltage-independent cation channels (SOC, ROC, NSCC) (16, 31), have now been demonstrated to have effects on TRPC channels (20, 24, 30), further supporting the proposal of TRPC subunits forming both SOCs and ROCs. This potentially common subunit structure between SOCs and ROCs, combined with differential TRPC expression patterns in various tissues and the probability of heteromultimerization of TRPC channel subunits (23), may explain the inconsistent effects of these SOC and ROC blockers among different cell types and different laboratories.

Our laboratory has previously demonstrated that SOCs are functional in single VSMCs freshly dispersed from preglomerular resistance vessels (12–14). In the present study, we demonstrate for the first time that SOCs are functional in isolated preglomerular resistance vessels and have characterized them pharmacologically. One limitation of using microdissected vessel segments rather than single dispersed cells is that the endothelium is presumably present in these ILAs, and SOCs are abundant in vascular endothelial cells (36). We are confident, however, that the calcium responses we observe using fura 2-loaded ILAs, including store-operated calcium entry signals, originate from VSMCs rather than endothelial cells. The justification for this assumption has been outlined previously by others using similar vessel preparations and includes the fact that VSMCs comprise a larger mass within the vessel wall such that even if endothelial cells load with fura 2 their fluorescence emissions contribute very little to the overall calcium signal (6, 9). Furthermore, in this isolated ILA preparation the vessel ends are sealed off by glass holding pipettes, preventing the bathing solution from reaching the vessel lumen. Additionally, we have observed nearly identical results with SERCA inhibitors in cultured VSMCs from preglomerular resistance vessels (Ref. 37 and Facemire CS and Arendshorst WJ, unpublished observations) as well as freshly dispersed preglomerular VSMCs (12, 14).

We show here that SOCs are activated in isolated ILAs by passive store depletion using two different SERCA inhibitors, CPA and thapsigargin (Figs. 3 and 7A). Calcium entry through these SOCs was blocked by Gd3+, although a relatively high concentration was required for complete blockade (Fig. 3A). While low micromolar concentrations of Gd3+ are reported to block SOCs in many cell types, higher (0.1–1 mM) concentrations of Gd3+ have been required for SOC inhibition in some vascular preparations, including renal arterial VSMCs (58) and cerebral arterioles (15). SOCs in pulmonary VSMCs (58) and small intrapulmonary arteries (19) are reported to be unaffected by 100 and 10 µM Gd3+, respectively. Anococcygeus smooth muscle SOCs are not affected by concentrations of Gd3+ up to 400 µM (57), and studies in the isolated, perfused rat kidney suggest that SOCs in juxtaglomerular granular cells are insensitive to Gd3+ (43). This variation in Gd3+ sensitivity of SOCs may be explained, in part, by the recent observation that lanthanides block TRPC channels more effectively from the cytosolic side of the plasma membrane, and therefore the rate of cellular uptake of Gd3+ ions may largely affect the apparent IC50 value upon extracellular application (20).

We also demonstrate that SOCs in isolated ILAs are dose dependently blocked by two other agents, SKF-96365 and 2-APB (Fig. 3, B and C). The imidazole derivative SKF-96365 is also reported to block ROCs (31) and is one of very few commercially available compounds exhibiting this property. We therefore used a low concentration of SKF-96365, one that did not affect thapsigargin-induced calcium entry via SOCs, as a selective ROC antagonist. The large blocking effect of 2 µM SKF-96365 observed in isolated ILAs (Fig. 5A), combined with the lack of effect of 250 µM Gd3+ (Fig. 4A), on 1 µM NE-induced non-L-type calcium entry strongly suggests that this entry is mediated primarily by ROCs rather than SOCs. Notably, there is evidence in cultured VSMCs that noncapacitative calcium entry (i.e., ROCs) and capacitative calcium entry (i.e., SOCs) are activated in a strict temporal sequence in response to receptor activation by vasopressin. Only the noncapacitative entry pathway is active in the presence of AVP, whereas the capacitative pathway is only active after removal of AVP (33, 34).

Previous studies have demonstrated the effects of SKF-96365 on renal efferent arteriolar calcium responses and constriction (29, 46). These authors demonstrated that slightly higher concentrations (≥10 µM) of SKF-96365 are required to significantly inhibit store-operated calcium entry or constriction stimulated by angiotensin II; however, the contribution of ROCs was not specifically examined in these studies. These studies report IC50 values for SKF-96365 that are similar to those originally described for this drug (8–12 µM) (32) and are also in the range of concentrations we show here to block half the thapsigargin-induced store-operated calcium entry in ILAs (5–30 µM, Fig. 3B). Our data demonstrating a blocking effect of 2 µM SKF-96365 on NE-induced non-L-type calcium entry in the absence of an effect on thapsigargin-induced store-operated calcium entry supports evidence from the literature that this drug affects both SOCs and ROCs and provides the first evidence, to our knowledge, that these two classes of channels are inhibited in a concentration-dependent manner.

At 5 µM NE, the consistent level of blockade elicited by SKF-96365 (60–70%, Fig. 5B) combined with the moderate inhibitory effect of Gd3+ (~40%, Fig. 4B) indicates that SOCs may be recruited at higher agonist concentrations where greater degrees of store depletion occur. This suggests that SOCs may play a more significant role in modulating renal vascular reactivity under pathophysiological conditions when plasma catecholamines and/or sympathetic activity is chronically elevated. In support of this hypothesis is the observation that calcium entry through SOCs is exaggerated in VSMCs from preglomerular resistance vessels of young spontaneously hypertensive rats (14).

Based on the observed effects of Gd3+ and SKF-96365 in our ILA preparation, we were surprised to find that 2-APB, an agent commonly used as a SOC antagonist, completely blocked the NE-induced receptor-operated calcium entry. There is evidence in the literature for 2-APB blockade of store-independent calcium entry, including that activated by acetylcholine in PC12 adrenal chromaffin cells (49), by carbachol in murine gastric smooth muscle (27), and by {alpha}1A adrenoceptor agonists in prostate cancer epithelial cells (45, 50). 2-APB also inhibits receptor-activated, store-independent calcium entry mediated by TRPC3 overexpressed in HEK293 cells (28, 52). These studies suggest that 2-APB may not be as selective a SOC antagonist as originally thought, and rather that it is selective for a specific channel subunit that may comprise both SOCs and ROCs. In this study, we have characterized the effects of reported SOC and ROC antagonists on both SOC-mediated calcium entry (activated by SERCA inhibitors) and NE-induced non-L-type calcium entry in an effort to determine the selectivity of these drugs in the isolated rat ILA preparation. This novel approach examines concentration-dependent effects and validates the actions of these drugs in this particular system.

An intracellular signaling complex, or "signalplex," regulating TRP channel activity in the Drosophila photoreceptor has been identified and includes CaM and the scaffold protein INAD (for inactivation but no afterpotential D), both of which bind TRP. INAD also anchors the Drosophila homologs of PKC, PLC, and Gq{alpha} within the signalplex, allowing for highly efficient light-induced signal transduction (35). Recently, all members of the mammalian TRPC family have also been shown to bind CaM (47). CaM has subsequently been found to inhibit TRPC3 (61) and to activate TRPC6 as well as other TRPCs (5, 47). Taken together, these TRPC overexpression data combined with the pharmacological profile we have generated for SOCs and ROCs in ILAs and the relative abundance of TRPC3 and TRPC6 in preglomerular resistance vessels (11) suggest that TRPC3 and TRPC6 may be components of ROCs and/or SOCs in the renal microcirculation. We therefore used two CaM inhibitors, W-7 and TFP, to gain insight into the possible TRPC subunit composition of these channels in ILAs. Our observations that these calmodulin inhibitors block NE-induced non-L-type calcium entry and have little effect on CPA-induced store-operated calcium entry suggest that CaM is a mediator of ROC activation and that TRPC6 may be a component of these channels in ILAs (Fig. 7). In support of this conclusion, TRPC6 has been identified as the essential component of the store-independent, {alpha}1-adrenoceptor-activated NSCC in portal vein myocytes (24). Our results indicate a novel role for CaM as a direct regulator of calcium entry in preglomerular VSMCs that is distinct from its well-documented sensitization effects on the contractile apparatus via activation of myosin light chain kinase.

Although NE activates all classes of adrenergic GPCRs ({alpha}1, {alpha}2, {beta}1, and {beta}2), renal adrenergic vasoconstriction in the rat is virtually exclusively mediated by {alpha}1-adrenoceptors (40). Furthermore, expression of {alpha}2- and {beta}-adrenoceptors is very low in the rat renal microcirculation (10). NE-induced increases in [Ca2+]i and vasoconstriction in rat renal resistance vessels can therefore be presumed to occur through activation of {alpha}1-adrenoceptors. Based on our present study and evidence from the literature investigating Drosophila TRP and mammalian TRPC regulation, we propose a model whereby CaM activates a TRPC6-containing ROC downstream of activated {alpha}1-adrenoceptors, independent of IP3-mediated store depletion. It is likely that the receptor, channel, and associated regulatory proteins are colocalized in specific regions of the plasma membrane, possibly caveolae (28, 51), by a scaffolding protein similar to that found in Drosophila. Indeed, the mammalian scaffold protein Na+/H+ exchanger regulatory factor binds TRPCs as well as PLCs and has been proposed to organize an analogous supramolecular complex (48).

In conclusion, we present evidence that although SOCs are present in ILAs, they do not contribute significantly to the calcium entry response elicited by physiological concentrations of NE. Rather, NE activates a store-independent ROC that appears to be regulated my CaM. This regulation by CaM, along with the observation that 2-APB blocks both SOCs and ROCs in isolated ILAs, supports the hypothesis that TRPC proteins comprise these voltage-independent channels in preglomerular resistance vessels. Our studies suggest that non-L-type calcium entry mechanisms play a larger role in regulation of preglomerular VSMC [Ca2+]i than previously thought. Elucidation of the molecular structure and physiological regulation of the specific channels involved will lead to further insight into the regulation of renal vascular reactivity and the pathophysiological progression of hypertension and renal disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
These studies were supported by National Heart, Lung, and Blood Institute Grants HL-02334 and HL-69768.


   ACKNOWLEDGMENTS
 
W. J. Arendshorst is a member of the Carolina Cardiovascular Biology Center at UNC-CH.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. J. Arendshorst, Dept. of Cell and Molecular Physiology, Univ. of North Carolina at Chapel Hill, CB #7545, School of Medicine, 6341B Medical Biomolecular Research Bldg., Chapel Hill, NC 27599-7545 (e-mail: arends{at}med.unc.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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