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Am J Physiol Renal Physiol 294: F212-F219, 2008. First published October 31, 2007; doi:10.1152/ajprenal.00244.2007
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Angiotensin II-stimulated Ca2+ entry mechanisms in afferent arterioles: role of transient receptor potential canonical channels and reverse Na+/Ca2+ exchange

Susan K. Fellner and William J. Arendshorst

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

Submitted 25 May 2007 ; accepted in final form 26 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In afferent arterioles, the signaling events that lead to an increase in cytosolic Ca2+ concentration ([Ca2+]i) and initiation of vascular contraction are increasingly being delineated. We have recently studied angiotensin II (ANG II)-mediated effects on sarcoplasmic reticulum (SR) mobilization of Ca2+ and the role of superoxide and cyclic adenosine diphosphoribose in these processes. In the current study we investigated the participation of transient receptor potential canonical channels (TRPC) and a Na+/Ca2+ exchanger (NCX) in Ca2+ entry mechanisms. Afferent arterioles, isolated with the magnetized polystyrene bead method, were loaded with fura-2 to measure [Ca2+]i ratiometrically. We observed that the Ca2+-dependent chloride channel blocker niflumic acid (10 and 50 µ M) affects neither the peak nor plateau [Ca2+]i response to ANG II. Arterioles were pretreated with ryanodine (100 µM) and TMB-8 to block SR mobilization via the ryanodine receptor and inositol trisphosphate receptor, respectively. The peak [Ca2+]i response to ANG II was reduced by 40%. Addition of 2-aminoethoxydiphenyl borane to block TRPC-mediated Ca2+ entry inhibited the peak [Ca2+]i ANG II response by 80% and the plateau by 74%. Flufenamic acid (FFA; 50 µM), which stimulates TRPC6, caused a sustained increase of [Ca2+]i of 146 nM. This response was unaffected by diltiazem or nifedipine. KB-R7943 (at the low concentration of 10 µM) inhibits reverse (but not forward) mode NCX. KB-R7943 decreased the peak [Ca2+]i response to ANG II by 48% and to FFA by 38%. We conclude that TRPC6 and reverse-mode NCX may be important Ca2+ entry pathways in afferent arterioles.

renal microcirculation; voltage-gated calcium entry; vascular smooth muscle cell

A RISE IN INTRACELLULAR Ca2+ concentration ([Ca2+]i) typically initiates vascular contraction. Over the past several years, we have studied Ca2+ signaling in freshly isolated afferent arterioles to gain a better understanding of mechanisms that control renal microvascular function and that may be important in the development of hypertension. In particular, we have examined events that lead to mobilization of Ca2+ from the sarcoplasmic reticulum (SR) via the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) and have demonstrated the importance of superoxide generation and adenine diphosphoribose cyclase (ADPR cyclase) activation (12–14). As well, we have shown that Ca2+ entry via L-type voltage-gated Ca2+ channels (VGCC) stimulates Ca2+-induced Ca2+ release (15). Less well understood are the mechanisms involved in the activation of Ca2+ entry pathways following stimulation of afferent arterioles with angiotensin II (ANG II), endothelin (ET-1), or norepinephrine (NE).

It is well accepted that following ANG II stimulation of preglomerular microvessels, Ca2+ entry via L-type VGCC is a major pathway. In contrast, cortical efferent arterioles have little or no L-type expression (23) or activity (33). The mechanism by which ANG II causes depolarization sufficient to activate VGCC has remained a puzzle, largely because of the lack of specific pharmacological probes. Some investigators believe that a rapid increase in [Ca2+]i (likely more from mobilization of Ca2+ from the SR than from entry) stimulates a Ca2+-dependent chloride channel (ClCa) with subsequent Cl– efflux and membrane depolarization (19, 30). Functional support for this premise was provided by experiments in which removal of Cl– from the bath abolished the contractile response to ANG II in microperfused rabbit arterioles (28). In studies of renal blood flow, intrarenal infusion of the nonselective Cl– channel blocker 4,4-diiosthioyanostilbene-2,2' disulfonic acid (DIDS), but not IAA-94 or niflumic acid (NFA), inhibited the vasoconstrictor response to ANG II (50). These same investigators measured [Ca2+]i in afferent arterioles in the presence of NFA or IAA-94 and found no inhibition of the [Ca2+]i response to ANG II (50). Both DIDS and diphenylamine-2-carboxylic acid (DPC), another nonspecific blocker of Cl– channels, suppressed the peak and plateau [Ca2+]i responses to ANG II in fresh preglomerular vascular smooth muscle cells (VSMC) (19). The lack of specificity of each of these ClCa blockers limits interpretation of their effects.

Although opening of VGCC may be a major pathway for Ca2+ entry in response to ANG II, other Ca2+ entry mechanisms occur as well. In particular, nonselective cation channels (NSCC) of the transient receptor potential (TRP) families of ion channels, the TRP canonical (TRPC) channels (see reviews in Refs. 1, 45, 52) have been suggested to play a role in Ca2+ entry in VSMC. TRPC3, TRPC6, and TRPC7 proteins share ~75% identity and are activated by diacylglycerol (DAG) but not by protein kinase C (PKC). The homo- or heterotetramers are six membrane-spanning units. TRPC6 has been associated with receptor-operated Ca2+ entry (ROC) in several VSMC types and in response to such vasoconstrictor agonists as vasopressin, ANG II, and phenylephrine. In A7r5 cells stimulated with vasopressin, Ca2+ entry is markedly suppressed by small interference RNA directed against TRPC6 (49). In another study of A7r5 cells in which expression of TRPC3 was not found, heteromultimeric TRPC6-TRPC7 channels contributed to a vasopressin-induced cation current (37). Both vasopressin and ET-1, as well as flufenamic acid (FFA), likewise stimulate cation currents in A7r5 cells (29). ANG II at low concentrations activates a cation conductance in mesenteric artery VSMC with TRPC6 channel properties (46). TRPC6 was found to be an essential component of a Ca2+-permeable nonselective cation channel in cultured rabbit portal vein VSMC stimulated with phenylephrine (26). Pharmacological studies have demonstrated TRPC6 stimulation by FFA and inhibition by Gd3+ and SKF-96365 (26). Similar results were reported in mesenteric artery VSMC stimulated with phenylephrine and FFA (24).

Our laboratory has characterized the expression and abundance of transient receptor TRPC in cells derived from preglomerular vessels (11). Quantitative RT-PCR showed the presence of TRPC1, TRPC3, and TRPC6 mRNA (11). Protein levels of TRPC6 were approximately sevenfold greater in preglomerular VSMC than in endothelium-denuded aortic VSMC (11). Stimulation of fura-2-loaded renal interlobular arteries with NE in the presence of nifedipine (to block VGCC) caused an increase in [Ca2+]i that could be distinguished from store-operated Ca2+ entry (SOC). The pharmacological profile of the [Ca2+]i response was consistent with a ROC mechanism (10).

Given that TRPCs exist in renal resistance vessels (10) and that NSCC occurs through some of these channels, we explored the possibility of a relationship between cation entry via ROC and activation of VGCC. Because of the substantial Na+ as well as Ca2+ entry following activation of TRPC6 (1.5–5 Ca2+:1 Na+), we considered that depolarization might occur sufficient to activate VGCC (8, 26, 49). We also asked whether entry of Na+ might activate Na+/Ca2+ exchange (NCX) in the reverse direction, that is, with Ca2+ entering rather than exiting the cell, as has been suggested in cardiomyocytes and aortic VSMC (9, 32). We used pharmacological agents to stimulate or inhibit Ca2+ entry channels and to block SR receptors.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All studies were approved by and performed in compliance with the guidelines and practices of the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.

Preparation of fresh afferent arterioles. We used the magnetized polystyrene microsphere-sieving technique as previously described in our laboratory to isolate afferent arterioles (<20 µm in diameter) from 5-wk-old (90–125 g) male Sprague-Dawley rats maintained in the Chapel Hill Colony (16). Phosphate-buffered saline (PBS), with the following composition (in mM): 137 NaCl, 4.1 KCl, 0.66 KH2PO4, 3.4 Na2HPO4, 2.5 NaHCO3, 1.0 MgCl2, and 5 glucose, was adjusted daily to pH 7.4 at 4 and 23°C. The vessel segments in PBS containing 0.1% bovine serum albumin (BSA) were treated with collagenase type IV (Worthington; 374 U/mg, 4–5 µg/ml) for 18 min at 34°C. Arterioles were loaded with fura-2 AM (3 µM) and 0.1% BSA for 55 min at 23°C in the dark. After arterioles had been washed with PBS, the suspension was kept in Ca2+ (1.1 mM)-containing buffer on ice.

Measurement of [Ca2+]i. We measured [Ca2+]i as previously described (13). Afferent arterioles were identified by their morphology and measured external diameter of 15–20 µm. Also, we required visualization of microspheres in the lumen of the afferent arteriole or in the proximal branch of an interlobular artery from which it arose, to exclude the possibility that the vessel was an efferent arteriole. A random segment of an afferent arteriole was centered in a small window of the optical field that was free of glomeruli or tubular fragments.

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), Birmingham, NJ]. After signals had been passed through a barrier filter (510 nm), fluorescence was detected using a photomultiplier tube. Signal intensity was acquired, stored, and processed using an IBM-compatible Pentium computer and Felix software (PTI). Background subtraction was performed in all studies. There was no interruption in the recording during the addition of reagents to the chamber. A video camera projected images of afferent arterioles onto a video monitor, permitting visualization of vascular contraction. We have previously demonstrated that application of fura-2 and pharmacological agents on the abluminal side of the afferent arteriole results in no detectable contribution to the [Ca2+]i signal from endothelial cells (12).

Reagents. We purchased ANG II, nifedipine, diltiazem, FFA, NFA, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), and ryanodine from Sigma Aldrich (St. Louis, MO), KB-R7943 and 2-aminoethoxydiphenyl borane (2-APB) from CalBiochem (San Diego, CA), fura-2 AM from Molecular Probes (Eugene, OR), and magnetized microspheres from Spherotech (Libertyville, IL).

Concentrations of inhibitors were based on previous data from the literature. Lower concentrations of NFA block the ClCa channel, whereas higher concentrations (≥50 µM) also stimulate the Ca2+-activated K+ (KCa) channel (21, 31, 41, 42). Ryanodine (100 µM) and TMB-8 (10 µM) block the RyR and IP3R, respectively (13). 2-APB (<100 µM) not only blocks the TRPC but also is an inhibitor of the IP3R (34, 35). FFA stimulates TRPC6 and inhibits TRPC3 (2, 24, 26, 29) and, at higher concentrations (>100 µM), stimulates the KCa channel (41) and inhibits L-type channels (48). Thus we chose a concentration of 50 µM to avoid the latter actions. KB-R7943 selectively inhibits reverse-mode NCX at a concentration of 10 µM; concentrations of 30 µM or greater inhibit forward mode (27).

Statistics. The data are means ± SE. Each data set was derived from afferent arterioles originating from at least three separate experiments, with two rats (4 kidneys) per experiment. Individual arterioles were studied only once and then discarded. Paired data for arterioles before and after agonist stimulation were tested with Student's paired t-test. Unpaired t-tests were employed for comparisons of responses between two groups.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
[Ca2+]i response to ANG II. Afferent arterioles respond to ANG II (1 µM) with a sharp peak response followed by a sustained plateau (Fig. 1). Based on the methods employed in this study, the measured mean increase in peak [Ca2+]i was 135 ± 11 nM, and that of the plateau was 49 ± 6 nM (n = 30, P < 0.01, peak and plateau vs. baseline; Fig. 2B).


Figure 1
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  		Fig. 1. Representative tracing of the cytosolic Ca2+ concentration ([Ca2+]i) response of an isolated afferent arteriole to angiotensin-II (ANG II).

 

Figure 2
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  		Fig. 2. [Ca2+]i responses of afferent arterioles to ANG II in controls and in the presence of niflumic acid (NFA). A: representative tracing showing the lack of inhibition by NFA (10 µM). B: summary data showing that there was a nonsignificant increase in baseline [Ca2+]i (shaded bars) and that neither 10 or 50 µM NFA affected the peak (solid bars) or plateau [Ca2+]i responses (open bars).

 
Does NFA alter the [Ca2+]i responses to ANG II in afferent arterioles? NFA has been considered to be among the most potent blockers of ClCa channels and therefore has been widely used to assess their role in the control of vascular tone (22, 31). As noted above, NFA inhibits the channels at low concentrations (≤10 µM) and activates the channels at higher concentrations (≥50 µM) and after prolonged (minutes) exposure (25, 31). We studied two concentrations of NFA, 10 and 50 µM.

NFA (10 µM) pretreatment caused a small, nonsignificant increase in [Ca2+]i compared with baseline (14 ± 2 nM, n = 11, P = 0.3). In the presence of NFA, ANG II increased peak [Ca2+]i by 103 ± 18 nM and plateau by 59 ± 5 nM (P = 0.2 and 0.4 respectively, vs. control). NFA (50 µM) likewise caused a small, nonsignificant increase in baseline [Ca2+]i (14 ± 5 nM, n = 7). The increase in peak [Ca2+]i after addition of ANG II was 122 ± 24 nM, and the increase in plateau was 58 ± 7 nM (P = 0.6 and 0.4, respectively, vs. control, Fig. 2). These results confirm those of Steendahl et al. (50), who showed a lack of effect of NFA (50 µM) on [Ca2+]i responses to ANG II in isolated rat afferent arterioles.

[Ca2+]i entry in the absence of mobilization. The combination of a high concentration of ryanodine to block the RyR and of TMB-8 to block the IP3R prevents release of [Ca2+]i from the SR (13). In the presence of these two agents, the [Ca2+]i response to ANG II should represent Ca2+ that has entered the cell from the extracellular space. When we pretreated afferent arterioles with ryanodine (100 µM) and TMB-8 (10 µM), baseline [Ca2+]i was unchanged. The peak [Ca2+]i response to ANG II was reduced to 83 ± 12 nM (39% inhibition) (n = 11, P <0.02 vs. control), whereas the plateau [Ca2+]i was not different from control (59 ± 5 nM, P = 0.6, Fig. 3).


Figure 3
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  		Fig. 3. [Ca2+]i responses of afferent arterioles to ANG II in the presence of ryanodine and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) to prevent [Ca2+]i mobilization via the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R) and in the presence of ryanodine and 2-aminoethoxydiphenyl borane (2-APB) to block the RyR, IP3R, and nonselective cation entry of TRPC. A: representative tracing of the inhibitory effect of ryanodine and TMB-8 on the ANG II-mediated [Ca2+]i response. B: typical tracing of the additional inhibitory effect of 2-APB on the ANG II response. C: summary data showing the inhibitory effect on [Ca2+]i peak (solid bars) and plateau [Ca2+]i responses (open bars). *P < 0.01 vs. control. #P < 0.01 vs. ryanodine + TMB-8.

 
Pretreatment of afferent arterioles with both ryanodine (100 µM) and 2-APB (50 µM) will additionally diminish [Ca2+]i entry through TRPC channels, thereby preventing Ca2+ entry via a ROC mechanism. Under these conditions, we found that the peak [Ca2+]i response to ANG II was further reduced to 27 ± 4 nM (80% inhibition) (n= 10, P < 0.01 vs. ryanodine + TMB-8), and the plateau was reduced to 13 ± 1 nM (P = 0.01, Fig. 3). To further document that ryanodine and 2-APB effectively prevent mobilization of Ca2+ from the SR, we prepared afferent arterioles in nominally Ca2+-free buffer. In the absence of external Ca2+, the peak [Ca2+]i response to ANG II was 9 ± 2 nM and the plateau was 4 ± 3 nM (n = 5, P > 0.7 and 0.8, respectively, vs. baseline). These results suggest that opening of TRPC channel contributes to a substantial fraction of [Ca2+]i entry in afferent arterioles stimulated with ANG II.

FFA is a putative stimulator of TRPC6. Based on the results with ryanodine and 2-APB suggesting that there is a Ca2+ entry mechanism that is independent of mobilization but inhibited by 2-APB, we studied the drug FFA, which has been shown to stimulate TRPC6 and to inhibit TRPC3 in VSMC (2, 24, 26, 29). Stimulation of afferent arterioles with FFA (50 µM) caused a prompt and sustained rise in [Ca2+]i of 145 ± 18 nM (n = 12, P < 0.01). Addition of ANG II in the continued presence of FFA promoted a further increase in [Ca2+]i (peak, 54 ± 14 nM, P < 0.03 vs. FFA; plateau, 43 ± 13 nM; Fig. 4), demonstrating that ANG II subsequently increases Ca2+ by TRPC6-independent mechanisms such as activation of the NADPH oxidase, superoxide, ADPR cyclase pathway (13, 14). To further support the premise that FFA activates TRPC6 in afferent arterioles, we pretreated the vascular segments with 2-APB (50 µM) to block TRPC channel activity. The [Ca2+]i response to FFA was reduced by 74% and contrasts with the 33% inhibition caused by KB-R 7943 (vide infra) (n = 4, P < 0.01 vs. control).


Figure 4
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  		Fig. 4. [Ca2+]i responses of afferent arteriolar to flufenamic acid (FFA) followed by ANG II in the continued presence of FFA. A: representative tracing showing that ANG II caused a second [Ca2+]i peak and plateau response following that of FFA. B: effects of the voltage-gated L-type Ca2+ channel (VGCC) blockers nifedipine or diltiazem. The VGCC blockers had no effect on the FFA (shaded bars) or subsequent ANG II peak [Ca2+]i responses (black bars) but did inhibit the ANG II plateau (open bars). *P < 0.01 vs. control.

 
Activation of TRPC6 channels in A7r5 VSMC with oleoyl-2-acetyl-sn-glycerol (OAG) not only causes Ca2+ entry via the NSCC itself but also causes sufficient depolarization to open VGCC that are inhibited by voltage-gated Ca2+ channel blockers (CCB) in A7r5 cells (49). In contrast to studies that show a response to OAG in fresh or cultured VSMC, we have been unable to get reproducible responses to this agent in fresh, intact afferent arterioles. Furthermore, because FFA inhibits TRPC3, we could narrow our focus to TRPC6. We do not know the extent to which OAG may inhibit TRPC6 by causing an increase in PKC. Thus we studied the [Ca2+]i response of afferent arterioles to FFA in the presence and absence of the CCBs nifedipine (n = 6) or diltiazem (n = 5) (10 µM both). The findings for the two inhibitors were not different from each other, and therefore the data have been pooled. In the presence of CCB, the [Ca2+]i response to FFA was 102 ± 24 nM [P = nonsignificant (NS) vs. control]. Hence, we could not demonstrate a linkage between presumed TRPC6 activation with FFA and opening of L-type channels contributing to the [Ca2+]i response under these experimental conditions. However, the fact that FFA may block ClCa channels may confound our results.

The subsequent peak [Ca2+]i response to ANG II in the continued presence of FFA and CCB was 52 ± 6 nM, the same as in the absence of CCB. As one would anticipate, the plateau [Ca2+]i level (largely representing Ca2+ entry) was reduced ({Delta}21 ± 5 nM, P < 0.01 vs. control, Fig. 4).

The Na+ entry that accompanies Ca2+ entry following activation of TRPC3 or TRPC6 (nonselective cation entry) has been shown to activate reverse direction NCX in cardiomyocytes and aortic VSMC (9, 32). To investigate whether activation of TRPC6 with FFA in afferent arterioles is associated with operation of reverse-mode NCX, we pretreated the vessels with KB-R7943 (10 µM). In the presence of the inhibitor, the [Ca2+]i response to FFA was 91 ± 12 nM (37% inhibition) (n = 9, P < 0.05 vs. control; Fig. 5). In a similar fashion, we studied the [Ca2+]i response to ANG II in the presence of KB-R7943. The peak [Ca2+]i response was reduced to 71 ± 12 nM, and the plateau was reduced to 23 ± 7 nM (n = 9, peak P = 0.01 and plateau P < 0.05 vs. control; Fig. 5). These results suggest that entry of Na+ via TRPC6 activates reverse-mode NCX following either FFA or ANG II.


Figure 5
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  		Fig. 5. KB-R7943 (10 µM), a blocker of reverse-mode Na+/Ca2+ exchange (NCX) at this concentration, inhibited the ANG II peak (solid bars) and plateau [Ca2+]i responses (open bars) to ANG II and the [Ca2+]i responses to FFA (shaded bars). *P < 0.05 vs. FFA. **P < 0.01; #P <0.04 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Nonspecificity of blockers of ClCa. Interpretation of results obtained using pharmacological agents to probe mechanisms of Ca2+ signaling in biological tissues is often limited by lack of specificity of the agent. A major case in point is the attempt to block the ClCa channel. It has traditionally been proposed that an increase in [Ca2+]i activates this channel, causing membrane depolarization of sufficient magnitude to open VGCC. Because a major fraction of Ca2+ entry in afferent arterioles is believed to be the result of activation of L-type VGCC, documentation of the role of the role of ClCa channels is an important issue. Experiments in Cl–-free buffers tend to substantiate the relevance of this concept, but changes in intracellular pH (from lack of Cl–/HCO3– exchange) may cloud the interpretation of the results (28).

Not one of the pharmacological agents utilized to block ClCa channels in VSMC has absolute specificity. The fenamates (NFA, FFA) variably stimulate KCa channels, stimulate TRPC6, and inhibit TRPC3 in a dose-dependent fashion (21, 43). DPC has been reported to block L-type VGCC, fast Na+ channels, and NSCC in a variety of cells (6, 39). DIDS, which is membrane impermeant, can inhibit Ca2+-Mg2+-ATPase, inhibit Cl–/HCO3– exchange, and stimulate Cl– conductance by elevating [Ca2+]i (5). Because NFA, reportedly the most potent among the ClCa blockers (21, 25), appears to have minimal effects on KCa channels at a concentration of 10 µM, we studied the effect of both 10 and 50 µM on the [Ca2+]i response to ANG II in afferent arterioles. At neither concentration was there a change in the peak or plateau [Ca2+]i responses. These results agree with those of others obtained in afferent arterioles (50). In contrast, in single VSMC derived from preglomerular vessels, high concentrations of DPC (100 and 500 µM) are reported to inhibit both the peak and plateau responses to ANG II (19).

Further complicating the interpretation of experiments designed to test the magnitude of Ca2+ entry via VGCC is the fact that examples of all three classes of CCB (nifedipine, diltiazem, and verapamil) inhibit the ability of nicotinic acid dinucleotide phosphate (NAADP) to affect lysosomal [Ca2+]i release (20, 36, 56). Preliminary work in our laboratory suggests that ANG II as well as ET-1 stimulates the formation of NAADP to influence Ca2+ signaling in afferent arterioles.

TRPC channels in VSM. The recent explosion of interest in TRPC channels as mediators of Ca2+ entry in VSMC led our laboratory to study the distribution and expression of TRPC in VSMC of preglomerular vessels (11). As well, the role of TRPC in NE-induced ROC was studied in interlobular arteries (10). As noted above, TRPC3, -6, and -7 can form mono- or heterotetramers, are stimulated by DAG, and are considered to be NSCC (8). Thus agonist stimulation of G protein-coupled receptors in VSMC that result in formation of phospholipase C (PLC) can initiate formation not only of IP3-mediated Ca2+ signaling events but also DAG activation of this group of TRPC. TRPC6 and often TRPC3 have been shown to be present in some large arteries (coronary, aorta, main renal artery) (11, 24, 53, 55) and in some A7r5 cell lines (38). TRPC3 may be the predominant subtype in endothelial cells (44, 55), whereas small arteries and arterioles appear to contain largely TPRC6 (54). Preglomerular VSMC have six to eight times more TRPC 6 protein than aortic VSMC (11). One cannot exclude the possibility that fresh cellular preparations of small vessels contain endothelial cells that contribute to the presence of TRPC3 (11).

Study of TRPC channel function in fresh, intact arterioles presents unique challenges that are not present in work done in cultured VSMC or in fresh single cells. Previous pharmacological maneuvers have proven to provide reliable data regarding Ca2+ signaling events in our fresh preparation of the rat. The extensive literature supporting the effect of FFA to stimulate TRPC6 and to inhibit TRPC3, as well as the effect of low concentrations of KB-R7943 to block reverse-mode NCX-1, substantiate use of these agents in studies of afferent arteriolar VSMC. Nonetheless, we appreciate the limitations that use of pharmacological agents may impose. One approach would be the use of gene targeting in mice to examine more specifically one TRPC subtype compared with another. Such experiments were done in TRPC6–/– mice (7). Surprisingly, the TRPC6–/– mice are hypertensive, and contractility in response to phenylephrine is increased in aortic and mesenteric artery rings. Analysis of mRNA with PCR shows more than doubling of TRPC3 in aortic and cerebral arteries. They conclude that in these larger arteries, TRPC3 and TRPC6 are functionally nonredundant. They postulate that TRPC6 may suppress a high basal activity of TRPC3, important for the tight regulation of the NSCC complex in the regulation of vascular tone (7). These important issues need to be addressed in future studies of resistance arterioles.

Another approach is the use of blocking or inhibitory antibodies. In studies of rabbit mesenteric VSMC, anti-TRPC6 antibodies raised against putative intracellular epitopes reversed the Icat1 (store depletion-independent cation current) activity of ANG II (1 nM) when applied to the cytoplasmic surface of inside-out patches (46). In this same study, FFA (100 µM), which activates TRPC6, stimulated an Icat1 that potentiated the current (46). To our knowledge, use of inhibitory antibodies has not been employed in fresh preparations of renal resistance vessels.

Ca2+ entry in afferent arterioles. To address the issue of Ca2+ entry pathways in afferent arterioles independently of mobilization events, we blocked the RyR and IP3R with high concentrations of ryanodine and TMB-8. The peak [Ca2+]i response to ANG II was diminished by ~40%, but the plateau was unchanged. We then sought to assess the contribution of TRPC in Ca2+ entry via NSCC. Although OAG, the cell membrane-permeant analog of DAG, has been used to stimulate TRPC3 and TRPC6 in a variety of isolated cell types, we were unable to obtain responses to OAG of sufficient magnitude or reproducibility in our preparation of afferent arterioles. As noted above, FFA has been reported to stimulate TRPC6 and to inhibit TRPC3 in aortic and mesenteric VSMC and in human embryonic kidney cells (2, 24, 26, 29, 46). Thus FFA (at concentrations of ~100 µM) is a useful tool to evaluate TRPC6 function in afferent arterioles.

We found that FFA caused a substantial (145 nM) increase in [Ca2+]i and that addition of ANG II in the continued presence of FFA caused a second peak, presumably via a Ca2+ mechanism different from TRPC3 or TRPC6. Other entry pathways could include SOC, activation of VGCC, or NCX operating in the reverse mode. Because some investigators have suggested that activation of TRPC3 or TRPC6, and thus entry of both Na+ and Ca2+, might result in membrane depolarization (9, 32), we examined the [Ca2+]i response to FFA in the presence of the CCB nifedipine or diltiazem at concentrations known to block Ca2+ entry via L-type VGCC. The results with FFA plus CCB were not different from those with the FFA control, but, as one would expect, the plateau phase of the subsequent ANG II response was diminished by CCB. Therefore, in our preparation of afferent arterioles utilizing pharmacological probes, we did not find functional evidence that FFA causes depolarization sufficient to activate VGCC. These results are in agreement with findings in pig interlobular VSMC (51). A caveat is that if FFA were to simultaneously stimulate KCa channels, the hyperpolarizing effect would act in the opposite direction (41). NFA and FFA have nearly identical stimulatory effects on KCa in coronary VSMC (mean fractional increase in open probability of ~0.3 at a concentration of 100 µM). Neither tetraethylammonium nor charybdotoxin, classic inhibitors of the channel, interfere with the change in open probability produced by NFA (41).

NCX in the renal microcirculation. NCX transports Ca2+ across the plasma membrane based on the transmembrane electrochemical gradient of Na+ and Ca2+ (4). The exchangers may operate in the so-called forward mode (3 Na+ entry and 1 Ca2+ exit) or the reverse mode (3 Na+ exit and 1 Ca2+ entry). Because of this 3 Na+:1 Ca2+ relationship, [Ca2+]i is largely related to changes in cytosolic Na+ concentration ([Na+]i) (4, 57, 58). The equation to describe this correlation is [Ca2+]i = [Ca2+]o x ([Na+]i/[Na+]o)3 x eEmFRT, where Em is the membrane potential, F is the Faraday constant, T is absolute temperature and R is the gas constant (57). Thus, when extracellular Na+ and Ca2+ are constant, [Ca2+]i entry via reverse-mode NCX is proportional to the third power of ([Na+]i) (4, 57, 58).

Evidence for the presence of a NCX has been demonstrated in afferent and efferent arterioles of rabbit and rat (18, 40). Traditionally, the NCX has been thought to operate in the forward direction to facilitate Ca2+ exit from the VSMC following increases in [Ca2+]i. Studies performed in low-extracellular Na+ medium, while showing the presence of the exchanger, are examining the reverse mode of transport. When extracellular Na+ is reduced, or when intracellular Na+ is increased, Ca2+ enters the cell (reviewed in Ref. 3). In rabbit afferent and efferent arterioles attached to a glomerulus, exposure to a nominally Na+-free bath caused an increase in [Ca2+]i that was nearly twice as large in the afferent compared with the efferent arteriole. The change in [Ca2+]i was not blocked by the CCB diltiazem but was inhibited by the nonspecific inhibitor of NCX, Ni2+ (18). The functional role of NCX has been assessed in the isolated perfused rat kidney. Reduction of perfusate [Na+] in a graded manner caused increases in renal vascular resistance (RVR), presumably via reverse-mode NCX (47). KB-R7943 inhibits NCX reverse mode at concentrations of 10 µM or less; higher concentrations (>30 µM) inhibit the forward mode as well (27). KB-R7943 (50 µM) caused an increase in RVR in the isolated rat kidney (47). It is likely that at this concentration, KB-R7943 is blocking Ca2+ exit when NCX is operating in forward mode.

In the current study, we employed KB-R7943 at a concentration (10 µM), reported to block only reverse-mode NCX. The peak [Ca2+]i response to ANG II was reduced by 50%, and that to FFA was reduced by 37%. These data suggest that reverse-mode NCX plays a part in the [Ca2+]i response to ANG II. Furthermore, the data suggest that ANG II-induced activation of TRPC6 (or FFA stimulation of TRPC6) causes sufficient Na+ entry to accomplish reverse-mode NCX characteristics. Similar conclusions have been made in a study of aortic VSMC in which KB-R7943 inhibited the [Ca2+]i response to ATP (32) and in cardiomyocytes stimulated with ANG II (9). If KB-R7943 had been blocking NCX in the forward rather than the reverse mode in our preparation of afferent arterioles, one would expect an increase rather than a decrease in [Ca2+]i following stimulation with ANG II.

In summary, we present new data showing the complex relationships among Ca2+ entry pathways in afferent arterioles. Investigation of any of these Ca2+ pathways must be viewed as a snapshot of a much larger picture in which intricate interactions, often occurring within milliseconds, work in concert to achieve a change in [Ca2+]i that precedes vascular contraction. The pharmacological agents used to explore the mechanisms by which ANG II stimulation of afferent arterioles causes depolarization and opening of VGGC may also affect TRPC and KCa channels. Thus it has been difficult to unravel the way in which L- and T-type channel opening occurs, utilizing pharmacological maneuvers. We present evidence that stimulation of TRPC6 with ANG II or FFA causes Ca2+ entry that is in part the consequence of NCX operating in the reverse mode. Because TRPC channels have been implicated in the pathophysiology of hypertension (17), our findings in afferent arteriolar VSMC should open new avenues of exploration in the causes of genetic hypertension and potential involvement of the renal microcirculation.


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This work was supported in part by an award from the Thomas H. Maren Foundation and from National Heart, Lung and Blood Institute Grant HL-02334.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. K. Fellner, Dept. of Cell and Molecular Physiology, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (e-mail: sfellner{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.


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