This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Loutzenhiser, K. Articles by Loutzenhiser, R. Search for Related Content PubMed PubMed Citation Articles by Loutzenhiser, K. Articles by Loutzenhiser, R.

(Circulation Research. 2000;87:551.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Angiotensin II–Induced Ca²⁺ Influx in Renal Afferent and Efferent Arterioles

Differing Roles of Voltage-Gated and Store-Operated Ca²⁺ Entry

Kathy Loutzenhiser, Rodger Loutzenhiser

From the Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, University of Calgary, Alberta, Canada.

Correspondence to Rodger D. Loutzenhiser, PhD, Department of Pharmacology and Therapeutics, The University of Calgary, Health Sciences Centre, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1, Canada. E-mail rloutzen{at}ucalgary.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Angiotensin II (Ang II)–induced Ca²⁺ signaling was studied in isolated rat renal arterioles using fura-2. Ang II (10 nmol/L) caused a sustained elevation in [Ca²⁺]_i, which was dependent on [Ca²⁺]_o in both vessel types. This response was blocked by nifedipine in only the afferent arteriole. Using the Mn²⁺ quench technique, we found that Ang II stimulates Ca²⁺ influx in both vessels. Nifedipine blocked the Ang II–induced Ca²⁺ influx in afferent arterioles but not in efferent arterioles. In contrast to Ang II, KCl-induced depolarization stimulated Ca²⁺ influx in only the afferent arteriole. Cyclopiazonic acid (CPA, 30 µmol/L) was used to examine the presence of store-operated Ca²⁺ entry in myocytes isolated from each arteriole. In efferent myocytes, CPA induced a sustained Ca²⁺ increase that was dependent on [Ca²⁺]_o and insensitive to nifedipine. This mechanism was absent in afferent myocytes. SKF 96365 inhibited Ang II–induced Ca²⁺ entry in efferent arterioles and CPA-induced Ca²⁺ entry in efferent myocytes over identical concentrations. Our findings thus indicate that Ang II activates differing Ca²⁺ influx mechanisms in pre- and postglomerular arterioles. In the afferent arteriole, Ang II activates dihydropyridine-sensitive L-type Ca²⁺ channels, presumably by membrane depolarization. In the efferent arteriole, Ang II appears to stimulate Ca²⁺ entry via store-operated Ca²⁺ influx.

Key Words: renal microcirculation • renal hemodynamics • nifedipine • cyclopiazonic acid • SKF 96365

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The renal afferent and efferent arterioles regulate glomerular inflow and outflow resistances, thereby controlling the pressure within the intervening glomerular capillaries (PGC). Through their influence on PGC, these 2 vessels exert independent and opposite effects on glomerular filtration rate (GFR). Knowledge of the smooth muscle mechanisms of the renal arterioles is critical in understanding how PGC and GFR are regulated and how regulation is disrupted in disease states. Vasoactive agents do exert differing effects on these 2 vessels. For example, atrial natriuretic peptide dilates the afferent arteriole and constricts the efferent arteriole to increase GFR,¹ whereas thromboxane preferentially constricts the afferent arteriole, decreasing GFR.² The relative reactivity of the afferent versus efferent arterioles to vasoconstrictors is also modified by physiological or pathophysiological conditions. For example, angiotensin II (Ang II), which is capable of constricting both vessels,³ preferentially constricts the efferent arteriole in the setting of renal arterial stenosis, thereby maintaining PGC and GFR in the face of reduced perfusion pressure.⁴ Diabetes is another condition in which reactivity is selectively altered, as a reduced afferent-versus-efferent reactivity is thought to contribute to the elevation in PGC, hyperfiltration, and proteinuria associated with this disorder.^{5 6} Although information on the signaling mechanisms regulating afferent versus efferent tone is of critical importance, our current knowledge in this area is quite limited.

Alterations in membrane potential and voltage-mediated changes in L-type Ca²⁺ channel activity play a prominent role in smooth-muscle excitation-contraction coupling and are especially important for the regulation of tone in resistance vessels.⁷ The efferent arteriole is a notable exception. L-type Ca²⁺ channel blockers have no effect on this vessel (reviewed in Reference ⁸ ), and efferent tone is refractory to depolarizing and hyperpolarizing stimuli.^{9 10} Moreover, we have shown that Ang II–induced afferent vasoconstriction is closely coupled to membrane depolarization, whereas the contractile response of the efferent arteriole to Ang II is fully dissociated from changes in membrane potential.¹¹ Thus, although evidence implicates membrane depolarization and voltage-gated Ca²⁺ entry in afferent vasoconstrictor responses, the mechanisms mediating efferent vasoconstriction and the role of Ca²⁺ influx in this vessel are unknown.

The present study was undertaken to determine whether Ang II activates distinct Ca²⁺ entry mechanisms in the afferent and efferent arteriole. To address this issue, we used fura-2 to assess Ca²⁺ signaling in freshly isolated single renal arterioles, and we used the manganese quench technique to assess Ca²⁺ influx in this preparation. The possible role of store-operated Ca²⁺ influx was investigated using myocytes freshly dispersed from individually isolated afferent and efferent arterioles.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adult male Sprague-Dawley rats were anesthetized (halothane), and the left kidney was perfused in vivo with warmed DMEM. The kidney was then excised, perfused with agarose solution (2% in DMEM, 37°C), and chilled (4°C). Cortical slices were incubated in Ca²⁺-free, modified DMEM containing 5 mmol/L HEPES and 3.9 mmol/L HCO₃ and treated with collagenase IV, dispase II, and DNAse I to dissociate the microvessels. Isolated arterioles (Figure 1A) were attached to coverslips and equilibrated in DMEM containing (in mmol/L) Ca²⁺ 1.8, H₂CO₃ 25, and HEPES 5 and 5% CO₂ (95% air).

View larger version (156K): [in this window] [in a new window]   Figure 1. Photomicrograph of isolated renal arteriole and arteriolar myocytes. A, Afferent arteriole. Note presence of intraluminal gel. B, Myocyte from afferent arteriole. C, Myocyte from efferent arteriole. Note bifurcations at each pole.

Arterioles were loaded in 5 µmol/L fura 2--acetoxymethyl ester at room temperature, warmed to 37°C, washed, and placed on a heated microscope stage. Fluorescence was measured using a Nikon inverted microscope equipped with a dual-image module and 535-nm dichroic splitter. The vessels were transilluminated with red light (650 nmol/L) for imaging and epi-illuminated using alternating 340/380 nmol/L (dye ratio) or 360 nm light (Mn²⁺ quench studies). Fluorescence (510 nmol/L) was measured at 10 to 15 Hz, and data were averaged over 1-second intervals. Medium was pumped through the perfusion chamber (2 mL/min), which was maintained at 37°C and equilibrated with 5% CO₂ (95% air).

Initial studies assessed the contribution of the endothelium to the responses of the intact arterioles. As shown in Figure 2A, acetylcholine attenuated the response to Ang II, indicating a functional endothelium and a predominant reporting of the smooth muscle [Ca²⁺]. When added alone, acetylcholine did elicit a Ca²⁺ increase (Figure 2B). However, Ang II (10 nmol/L) did not alter [Ca²⁺] in endothelial cells isolated from efferent arterioles (Figure 2C). We conclude that although the endothelium is present, it does not contribute directly to the Ang II response. Nevertheless, the presence of these 2 differing cell types precluded the calculation of [Ca²⁺]_i from the fura 2 ratio. Thus, the ratio itself was used as a qualitative index of [Ca²⁺]_i. Ang II elicited concentration-dependent responses over 0.1 to 10 nmol/L (see online Figure 1; data supplement available at http://www.circresaha.org).

View larger version (27K): [in this window] [in a new window]   Figure 2. Representative tracings illustrating endothelium-dependent responses. A, In intact afferent arteriole, acetylcholine (ACh, 1.0 µmol/L) decreases Ca2+ response to Ang II (10 nmol/L), reflecting the actions of endothelium-derived relaxing factors. B, When administered alone, acetylcholine increases Ca2+. C, In endothelial cells isolated from an efferent arteriole (EA), Ang II had no effect on [Ca2+]i, whereas acetylcholine elicited a robust response.

The Mn²⁺ quench technique was used to investigate Ca²⁺ entry in fura-2–loaded arterioles. Fluorescence was monitored in nominally Ca²⁺-free DMEM. MnCl₂ (100 µmol/L) and the experimental agent (Ang II, KCl) were then added sequentially. The rate of decline in fluorescence (percentage per second) was calculated by linear regression. An increase in the quench rate was interpreted as an increase in Ca²⁺/Mn²⁺ entry.

Single myocytes were used to study store-operated Ca²⁺ influx (SOCI). Myocytes were isolated from individual arterioles, attached to coverslips, and loaded with fura-2, as described above (see expanded Materials and Methods online; data supplement available at http://www.circresaha.org). The morphology of the 2 myocyte types was quite different. Afferent myocytes exhibited a typical spindle-shape morphology (Figure 1B), whereas efferent myocytes were characterized by a bifurcation at each pole (Figure 1C). To assay for SOCI, myocytes were treated with 0.1 µmol/L nifedipine, followed by 30 µmol/L cyclopiazonic acid (CPA) to deplete [Ca²⁺]_i stores. The dependence of the resultant response on [Ca²⁺]_o was then assessed.

All data are expressed as the mean±SE. Data were analyzed by ANOVA and paired or unpaired Student t test. P<0.05 was considered to be significant.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II elicited sustained increases in [Ca²⁺]_i in both afferent and efferent arterioles. In the afferent arteriole (Figure 3), Ang II evoked peak and sustained responses corresponding to 225±16% and 200±12% of the basal fura-2 ratio. Removal of [Ca²⁺]_o abolished the sustained component. When [Ca²⁺]_o was replenished, the fura-2 ratio returned to 182±10% of the basal ratio (P<0.001, n=10). Treatment with 0.1 µmol/L nifedipine reduced the peak response by 60% (to 152±7% of basal, P<0.001) and the [Ca²⁺]_o-dependent component by 90% (to 109±3% of basal, P>0.025, n=9). When Ang II was administered in the absence of [Ca²⁺]_o, an initial transient Ca²⁺ response was still observed (online Figure 2; data supplement available at http://www.circresaha.org), supporting our interpretation that this nifedipine-insensitive component represents the release of Ca²⁺ from the sarcoplasmic reticulum (SR).

View larger version (34K): [in this window] [in a new window]   Figure 3. Afferent arteriolar response to Ang II. Top, Tracings obtained in absence (left) and presence (right) of 0.1 µmol/L nifedipine. Ang II elicited a sustained Ca2+ signal that was dependent on [Ca2+]o. Bottom, Nifedipine (hatched bars) reduced the initial peak response by 60% and the sustained component by 90%. Basal fura-2 ratios for controls (n=10) and nifedipine-treated vessels (n=9) are shown on lower left panel.

The Ca²⁺ response of the efferent arteriole to Ang II was more biphasic in character (Figure 4). In this vessel, the peak and sustained components corresponded to 144±5% (P<0.001) and 122±5% (P<0.01) of the basal value. As seen with the afferent arteriole, the sustained component was dependent on [Ca²⁺]_o. However, in contrast to the afferent arteriole, nifedipine did not alter either the peak or sustained component of the efferent response to Ang II (n=9). As seen with the afferent arteriole, after removal of [Ca²⁺]_o, the application of Ang II elicited only a phasic Ca²⁺ response (see online Figure 2; data supplement available at http://www.circresaha.org), reflecting the release of SR Ca²⁺ stores.

View larger version (31K): [in this window] [in a new window]   Figure 4. Efferent arteriolar response to Ang II. This vessel exhibited a biphasic response to Ang II, characterized by an initial peak followed by a sustained component. The sustained component was dependent on the presence of [Ca2+]o but was not affected by nifedipine. Top, Tracing obtained in the presence of 0.1 µmol/L nifedipine. Bottom, Mean data for controls (n=8) and nifedipine-treated vessels (hatched bars, n=9).

Our finding that the sustained Ca²⁺ response of both arterioles depends on [Ca²⁺]_o implicates a role of Ca²⁺ influx in each vessel type. To further evaluate this postulate, we used the Mn²⁺ quench technique. This approach takes advantage of the fact that Mn²⁺ binding eliminates fura-2 fluorescence. Thus, the addition of extracellular Mn²⁺ triggers a decay in fluorescence that reflects the rate of Mn²⁺ entry. Accordingly, a stimulation of divalent cation influx increases the Mn²⁺ quench rate. As shown in Figure 5, Ang II stimulated divalent influx in both arterioles, increasing the quench rate from 0.18±0.03% to 0.43±0.7% per second (n=7, P<0.025) in afferent arterioles and from 0.18±0.03% to 0.41±0.07% per second (n=8, P<0.005) in efferent arterioles. This corresponds to increases to 276±60% and 243±36% of the respective controls. As further depicted in Figure 5, pretreatment with 0.1 nmol/L nifedipine blocked the afferent arteriolar response to Ang II (126±11% of basal, from 0.06±0.01% to 0.08±0.01% per second, n=6, P>0.05). However, nifedipine did not alter the efferent response to Ang II. Thus, Ang II increased influx to 284±60% of control in the presence of nifedipine (from 0.18±0.01% to 0.53±0.13% per second, n=7, P<0.05), a value similar to that seen in untreated vessels (246±36%, P>0.50).

View larger version (40K): [in this window] [in a new window]   Figure 5. Effects of Ang II on Mn2+ quench rate in afferent and efferent arterioles. Top, Quench rate increases on addition of 100 µmol/L Mn2+ (open arrow) and increases further on addition of 10 nmol/L Ang II (solid arrow). Bottom, Mean data illustrating significant responses in both afferent (n=7) and efferent (n=8) arterioles. Quench rate is expressed as percentage of control in bottom right. Pretreatment with 0.1 µmol/L nifedipine (hatched bars) abolished Ang II responses of afferent arterioles (n=6) but did not alter responses of efferent arterioles (n=7). *P<0.05 vs basal quench rate.

We used KCl-induced depolarization to examine the role of voltage-gated Ca²⁺ entry in each vessel (Figure 6). In the afferent arteriole, elevating KCl from 5 to 30 or 80 mmol/L increased the quench rate from 0.16±0.03% to 0.31±0.03% per second (n=5, P<0.01) and from 0.16±0.03% to 0.56±0.10% per second (n=5, P<0.025), respectively. This corresponds to influx rates of 214±31% and 367±56% of control. In contrast, KCl reduced influx by 25±6% and 37±13% (n=5, P<0.05 versus basal) in the efferent arteriole. Thus, voltage-activated Ca²⁺ influx, seen in the afferent arteriole, is absent in the efferent arteriole.

View larger version (24K): [in this window] [in a new window]   Figure 6. Effects of KCl-induced depolarization on Mn2+ quench. Tracings (top) illustrate increase and decrease in quench rate in afferent (left) and efferent (right) arterioles by 30 mmol/L KCl. Mean data (bottom, expressed as percentage of basal rate) illustrate stimulatory effects of 30 and 80 mmol/L KCl on the afferent arteriole (n=5 for both groups, P<0.05 vs basal) and depressant effects on the efferent arteriole (n=5, P<0.05). Thus, voltage-gated Ca2+ entry is present in the afferent arteriole but is absent in the efferent arteriole.

We next examined the role of store-operated Ca²⁺ influx (SOCI). SOCI is insensitive to dihydropyridines and is activated by depletion of SR/endoplasmic reticulum Ca²⁺ stores.¹² To test for the presence of SOCI, we sought to determine whether the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) inhibitor CPA (30 µmol/L) elicited a sustained Ca²⁺ response that was dependent on [Ca²⁺]_o but insensitive to nifedipine. Because SOCI is known to be present in the endothelium (see References ¹² and ¹³ ), these studies were conducted using single myocytes isolated from each arteriole type. Figure 7 depicts signal-averaged fura-2 tracings and mean data. In afferent myocytes, CPA elicited only a phasic response, suggesting a lack of SOCI in this vessel. In contrast, CPA elicited a sustained Ca²⁺ signal in efferent myocytes that was clearly dependent on the presence of [Ca²⁺]_o.

View larger version (27K): [in this window] [in a new window]   Figure 7. Effects of store depletion (30 µmol/L CPA) on myocytes obtained from single afferent and efferent arterioles. Top, Signal-averaged tracings from 6 myocytes. In efferent myocytes, CPA elicited a biphasic response, characterized by an initial peak followed by a sustained component that was dependent on [Ca2+]o. In afferent myocytes, CPA elicited only a phasic response, suggesting the absence of SOCI in this vessel.

To further characterize Ca²⁺ influx in the efferent arteriole, we examined the effects of SKF 96365 both on the response of the intact vessel to Ang II and on the response of the efferent myocyte to CPA. SKF 96365 inhibits a wide range of Ca²⁺ influx mechanisms, including SOCI. The protocol is illustrated by the tracing presented in Figure 8A. Arterioles or myocytes were treated with either Ang II or CPA, and the effects of SKF 96365 on the [Ca²⁺]_o-dependent responses were assessed. As shown in Figure 8B, SKF 96265 caused an identical blockade of the efferent responses to CPA and Ang II over concentrations of 1 to 100 µmol/L. This finding is consistent with the postulate that Ang II, by depleting SR Ca²⁺ stores, activates SOCI in the efferent arteriole.

View larger version (20K): [in this window] [in a new window]   Figure 8. Effects of SKF 96365 on efferent responses to Ang II and CPA. A, Tracing illustrating effects of SKF 96365 on Ang II response of single efferent arteriole. B, Comparison of the effects of SKF 96365 on Ang II responses in intact arterioles (, n=5 for each concentration) and CPA responses of isolated efferent myocytes (•, n=3 to 4 for each concentration).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study is the first to demonstrate that Ang II activates differing Ca²⁺ entry mechanisms in afferent and efferent arterioles. Our findings are thus consistent with evolving concepts concerning the segmental heterogeneity of activation mechanisms within the renal microvasculature. In the afferent arteriole, Ang II stimulates Ca²⁺ influx via dihydropyridine-sensitive and voltage-activated L-type Ca²⁺ channels, an activating mechanism that is absent in the efferent arteriole. In the efferent arteriole, Ang II stimulates Ca²⁺ influx through a signaling pathway that is nifedipine-insensitive and is not voltage-activated. Store depletion with CPA activates a nifedipine-insensitive Ca²⁺ entry in efferent myocytes that has a sensitivity to SKF 96365 identical to that of the Ca²⁺ influx activated by Ang II in the intact arteriole. This store-operated Ca²⁺ entry mechanism is absent in the afferent arteriole.

Our findings agree with those of previous studies assessing renal microvascular contractile responses, which also suggest divergent signaling mechanisms in these 2 vessels. We previously reported that the afferent vasoconstriction elicited by Ang II is closely coupled to membrane depolarization, whereas the efferent vasoconstriction is not.¹¹ Contractile studies using a variety of experimental approaches consistently show that L-type Ca²⁺ channel blockers inhibit afferent vasoconstriction without affecting the efferent arteriole (eg, Reference ¹⁴ ). Functional studies, assessing the actions of Ca²⁺ antagonists on GFR and renal hemodynamics in the intact kidney, also indicate a preferential preglomerular action (see Reference ⁸ ). In addition, afferent and efferent arterioles exhibit differing responses to hyperpolarizing and depolarizing stimuli. We found pinacidil, a K-channel opener, to act preferentially on the afferent arteriole¹⁰ and KCl-induced depolarization, an experimental means of directly activating L-type Ca²⁺ channels, to elicit preferential afferent vasoconstriction.⁹ These findings are consistent with the current observation that KCl stimulates Ca²⁺ influx in afferent, but not efferent, arterioles. However, in contrast to the above observations, Conger et al¹⁵ and Inscho et al¹⁶ reported efferent constrictor responses to KCl. The reasons for the latter findings are not clear, because Ca²⁺ antagonists do not effect efferent responses to agonists in the preparations used by these investigators (see Reference ¹⁴ ).

Our findings are also largely consistent with previous investigations of Ang II–induced Ca²⁺ signaling in renal arterioles, although some discrepancies in this regard must also be noted. Conger et al¹⁷ found that Ang II increased Ca²⁺ in both afferent and efferent arterioles and that diltiazem selectively blocked the afferent response. Conger et al¹⁵ also found that removal of [Ca²⁺]_o resulted in a phasic response to Ang II in both vessels and that dantroline abolished this component, suggesting Ang II–induced release of SR Ca²⁺ in each vessel type. Iversen and Arendshorst¹⁸ also reported that nifedipine and [Ca²⁺]_o removal inhibited the responses of preglomerular arteriolar segments to Ang II. However, in contrast to the above study, these authors did not observe phasic Ca²⁺ transients after treatment with either nifedipine or [Ca²⁺]_o removal. The present findings agree with those of Conger et al,¹⁵ in that we observed transient Ang II responses in [Ca²⁺]_o-free media in both afferent and efferent arterioles, indicating that Ang II releases Ca²⁺ from SR stores in both vessels. Interestingly, in the afferent arteriole, 0.1 µmol/L nifedipine reduced this initial phasic Ca²⁺ signal by 50%, suggesting a possible contribution of both Ca²⁺-induced Ca²⁺ release and inositol 1,4,5-triphosphate–dependent Ca²⁺ release.

In contrast to the above studies, Helou and Marchetti¹⁹ reported that 1.0 µmol/L nifedipine only partially blocked Ang II signaling in the afferent arteriole (ie, by only 40%). These authors found 2 populations of efferent arterioles, thin-walled vessels from the outer cortex that were insensitive to nifedipine and thick-walled vessels from the inner cortex that were partially sensitive to nifedipine. The reasons for these discrepancies are unclear, but differences in experimental conditions may be involved. Our studies are conducted at 37°C and use physiological levels of PCO₂ and HCO₃. These conditions are essential for normal pH_i regulation. Ang II can cause alkalinization in the absence of physiological HCO₃, thereby altering smooth muscle Ca²⁺ signaling (reviewed in Reference ²⁰ ). The studies by Helou and Marchetti,¹⁹ Iversen and Arendshorst,¹⁸ and Fellner and Arendshorst²¹ used atmospheric PCO₂ and low [HCO₃] and were conducted at room temperature.

The effects of KCl on renal arteriolar Ca²⁺ signaling have also been studied by other laboratories. Carmines et al²² reported that KCl-induced depolarization increased [Ca²⁺]_i in the afferent arteriole and reduced [Ca²⁺]_i in the efferent arteriole, observations similar to our findings. The attenuated rate of Mn²⁺ quenching in the efferent arteriole seen on the addition of KCl likely reflects the reduction in the electrical gradient induced by depolarization. In contrast to both our findings and those of Carmines et al,²² Helou and Marchetti¹⁹ found that KCl increased [Ca²⁺]_i in both vessel types.

A major finding of the present study is that Ang II activates a distinct Ca²⁺ influx mechanism in the efferent arteriole. Previous studies have shown that Ang II elicits a sustained elevation of Ca²⁺ in the efferent arteriole.^{17 19 23} Previous reports that SERCA inhibitors block Ang II–induced efferent vasoconstriction prompted suggestions that Ca²⁺ released from the SR accounts for the observed increase in efferent [Ca²⁺]_i.^{15 16} However, although Ca²⁺ release accounts for the initial Ca²⁺ transient, this mechanism alone cannot support the sustained, [Ca²⁺]_o-dependent response. Moreover, SERCA inhibition can block vasoconstriction indirectly, as this manipulation activates the endothelium to release endothelium-derived relaxing factors^{12 13} and elicits NO-dependent renal vasodilation.²⁴ Our findings indicate that the sustained Ca²⁺ response of the efferent arteriole is not due to Ca²⁺ release per se but rather is mediated by Ca²⁺ influx. A role of Ca²⁺ influx was previously suggested by Takenaka et al,²⁵ who demonstrated that removal of [Ca²⁺]_o or application of MnCl₂ attenuated the efferent arteriolar contractile responses to Ang II.

Our findings further suggest that the Ca²⁺ influx mechanism in the efferent arteriole is store operated. Calcium influx linked to membrane depolarization and activation of voltage-gated Ca²⁺ channels, such as is seen in the afferent arteriole, is a common signaling event in excitable tissues, including most vascular smooth muscle. In nonexcitable cells, such as leukocytes and endothelial cells, membrane depolarization is not involved in cell signaling, and voltage-gated Ca²⁺ channels are not expressed. In these tissues, nifedipine-insensitive SOCI contributes to Ca²⁺ signaling (reviewed in Reference ¹² ). Efferent arteriolar smooth muscle can be considered as a nonexcitable tissue, in that membrane depolarization is not linked to contraction^{9 11} and voltage-gated Ca²⁺ entry appears to be absent. Our finding that CPA elicits a sustained Ca²⁺ signal and that this response depends on [Ca²⁺]_o but is insensitive to nifedipine suggests that SOCI is present in efferent myocytes. Ang II would activate this pathway in the intact vessel through 1,4,5-triphosphate–mediated Ca²⁺ release and depletion of SR Ca²⁺ stores. Our finding that the efferent myocyte response to CPA and the response of the intact efferent arteriole to Ang II were inhibited in an identical manner by SKF 96365 supports this interpretation. Although there are currently no pharmacological agents that exhibit absolute selectivity for SOCI, SKF 96365 has been shown to inhibit this pathway over the concentrations we used.²⁶ We recognize that our studies do not rule out the possibility that Ang II may activate a Ca²⁺ entry mechanism in the efferent arteriole that differs from SOCI but is similarly inhibited by SKF 96365. For example, Ang II might activate a nonselective cation channel or a novel Ca²⁺ channel in this vessel. Given that the reversal potential for nonselective cation channel is more positive than the resting membrane potential, the activation of these channels would evoke depolarization. We have previously shown that Ang II–induced efferent vasoconstriction is not associated with depolarization,¹¹ arguing against this possibility.

Ang II also releases SR stores in the afferent arteriole and should activate SOCI in this vessel, if this mechanism is present. However, we found that nifedipine completely abolished Ang II influx in this vessel. Furthermore, unlike efferent myocytes, afferent myocytes exhibited only a phasic Ca²⁺ response to CPA. We interpret the 2 findings as indicating that SOCI is not present in the afferent arteriole. In contrast, Fellner and Arendshorst²¹ reported that SOCI is present in preglomerular vessels obtained by iron oxide sieving. Curiously, these Ca²⁺ signals were reported to be sustained even in the absence of [Ca²⁺]_o, suggesting unusual characteristics of their assay. Furthermore, this same laboratory had previously reported that nifedipine fully blocked the response of this preparation to Ang II¹⁸ whereas, as discussed above, SOCI would not be affected by dihydropyridines. It is important to note that endothelial cells express SOCI, and thus the presence of contaminating endothelial cells would contribute to erroneous findings. For this reason, our studies were conducted with single myocytes.

Activation of Ca²⁺ entry by SERCA inhibition is the defining feature of SOCI. However, the intervening mechanisms and the identity of the store-operated Ca²⁺ channels are not presently defined (see Reference ¹² ). Products of the trp gene family remain the most promising candidates for the latter (see Reference ²⁷ ). Clearly, a resolution of these issues is required to further define the role of SOCI in the efferent arteriole. Future studies characterizing the nature of Ca²⁺ currents and the expression of SOCI signaling genes in efferent myocytes will be needed. This question is of significant therapeutic interest, as the identification of the efferent Ca²⁺ influx mechanism could contribute to the development of an efferent-specific vasodilator. In settings such as diabetes, efferent vasodilation is a potential therapeutic approach for reducing glomerular hypertension, a primary factor in the progression of diabetic nephropathy.

In conclusion, we have demonstrated that Ang II activates differing Ca²⁺ influx mechanisms in renal afferent and efferent arterioles. In afferent arterioles, Ang II activates L-type Ca²⁺ channels, as does KCl-induced depolarization. This Ca²⁺ influx mechanism is absent in the efferent arteriole. In the efferent arteriole, Ang II activates a voltage-independent and nifedipine-insensitive Ca²⁺ influx, as does the SERCA inhibitor CPA. This CPA-activated SOCI is absent in afferent myocytes. We suggest that Ang II–induced depletion of SR Ca²⁺ stores activates SOCI in the efferent arteriole. Our finding that SKF 96365 produces identical concentration-dependent blockade of Ang II– and CPA-induced responses supports this interpretation. Because the efferent arteriole lacks electromechanical coupling, we suggest that this vessel is a unique example of a nonexcitable vascular smooth muscle and that SOCI plays a prominent role in its activation.

	Acknowledgments

 
This study was supported by an operating grant from the Kidney Foundation of Canada. R.L. is a Senior Medical Scholar of the Alberta Heritage Foundation for Medical Research.

Received April 12, 2000; revision received July 31, 2000; accepted August 23, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Loutzenhiser R, Hayashi K, Epstein M. Atrial natriuretic peptide reverses afferent arteriolar vasoconstriction and promotes efferent arteriolar vasoconstriction in the isolated perfused rat kidney. J Pharmacol Exp Ther. 1988;246:522–528.[Abstract/Free Full Text]

2. Hayashi K, Loutzenhiser R, Epstein M. Direct evidence that thromboxane mimetic U44069 preferentially constricts the afferent arteriole. J Am Soc Nephrol. 1997;8:25–31.[Abstract]

3. Navar LG, Inscho EW, Majid DSA, Imig JD, Harrison-Bernard LM, Mitchell KD. Paracrine regulation of the renal microcirculation. Physiol Rev. 1996;76:425–536.[Abstract/Free Full Text]

4. Hricik DE, Browing PJ, Kopelman R, Goorno WE, Madias NE, Dzau VJ. Captopril-induced functional renal insufficiency in patients with bilateral renal-artery stenosis or renal-artery stenosis in a solitary kidney. N Engl J Med. 1983;308:373–376.[Medline] [Order article via Infotrieve]

5. Hayashi K, Epstein M, Loutzenhiser R, Forster H. Impaired myogenic responsiveness of the renal afferent arteriole in streptozotocin-induced diabetic rats: role of eicosanoid derangements. J Am Soc Nephrol. 1992;2:1578–1586.[Abstract]

6. Inman SR, Porter JP, Feming JT. Reduced renal microvascular reactivity to angiotensin II in diabetic rats. Microcirculation. 1994;1:137–145.[Medline] [Order article via Infotrieve]

7. Nelson MT, Patlak JB, Worley JF, Standen NB. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol. 1990;259:C3–C18.[Abstract/Free Full Text]

8. Loutzenhiser R, Epstein M, eds. The renal hemodynamic effects of calcium antagonists. In: Calcium Antagonists and the Kidney. Philadelphia, Pa: Hanley and Belfus; 1990:33–75.

9. Loutzenhiser R, Hayashi K, Epstein M. Divergent effects of KCl-induced depolarization on afferent and efferent arterioles. Am J Physiol. 1989;257:F561–F564.[Abstract/Free Full Text]

10. Reslerova M, Loutzenhiser R. Divergent mechanisms of KATP-induced vasodilation in renal afferent and efferent arterioles: evidence for both L-type calcium channel-dependent and -independent actions of pinacidil. Circ Res. 1995;77:1114–1120.[Abstract/Free Full Text]

11. Loutzenhiser R, Chilton L, Trottier G. Membrane potential recordings of afferent and efferent arterioles in the in vitro perfused hydronephrotic rat kidney: actions of angiotensin II. Am J Physiol. 1997;273:F307–F314.[Abstract/Free Full Text]

12. Parekh AB, Penner R. Store depletion and calcium influx. Physiol Rev. 1997;77:901–930.[Abstract/Free Full Text]

13. Fasolato C, Nilius B. Store depletion triggers the calcium release-activated calcium current in macrovascular endothelial cells: a comparison with Jurkat and embryonic kidney cell lines. Pflugers Arch. 1998;436:69–74.[Medline] [Order article via Infotrieve]

14. Carmines PK, Navar LG. Disparate effects of Ca channel blockade on afferent and efferent arteriolar responses to ANG II. Am J Physiol. 1989;256:F1015–F1020.[Abstract/Free Full Text]

15. Conger JD, Falk SA. KCl and angiotensin responses in isolated rat renal arterioles: effects of diltiazem and low-calcium medium. Am J Physiol. 1993;264:F134–F140.[Abstract/Free Full Text]

16. Inscho EW, Imig JD, Cook AK. Afferent and efferent arteriolar vasoconstriction to angiotensin II and norepinephrine involves release of Ca from intracellular stores. Hypertension. 1997;29:222–227.[Abstract/Free Full Text]

17. Conger JD, Falk SA, Robinette JB. Angiotensin II-induced changes in smooth muscle calcium in rat renal arterioles. J Am Soc Nephrol. 1993;3:1792–1803.[Abstract]

18. Iversen BM, Arendshorst WJ. ANG II and vasopressin stimulate calcium entry in dispersed smooth muscle cells of preglomerular arterioles. Am J Physiol. 1998;274:F498–F508.[Abstract/Free Full Text]

19. Helou CM, Marchetti J. Morphological heterogeneity of renal glomerular arterioles and distinct [Ca]_i responses to ANG II. Am J Physiol. 1997;273:F84–F96.[Abstract/Free Full Text]

20. Smith GL, Austin C, Crichton C, Wray S. A review of the actions and control of intracellular pH in vascular smooth muscle. Cardiovasc Res. 1998;38:316–331.[Abstract/Free Full Text]

21. Fellner SK, Arendshorst WJ. Capacitative calcium entry in smooth muscle cells from preglomerular vessels. Am J Physiol. 1999;277:F533–F542.[Abstract/Free Full Text]

22. Carmines PK, Fowler BC, Bell PD. Segmentally distinct effects of depolarization on intracellular [Ca²⁺] in renal arterioles. Am J Physiol. 1993;265:F677–F685.[Abstract/Free Full Text]

23. Kornfeld M, Gutierrez AM, Gonzales E, Salomonsson M, Persson AE. Cell calcium concentration in glomerular afferent and efferent arterioles under the action of noradrenaline and angiotensin II. Acta Physiol Scand. 1994;151:99–105.[Medline] [Order article via Infotrieve]

24. Trottier G, Loutzenhiser R. Cyclopiazonic acid elicits endothelium-dependent vasodilation in the renal afferent arteriole. J Am Soc Nephrol. 1999;10:389A.

25. Takenaka T, Suzuki H, Fujiwara K, Kanno Y, Ohno Y, Hayashi K, Nagahama T, Saruta T. Cellular mechanisms mediating rat renal microvascular constriction by angiotensin II. J Clin Invest. 1997;100:2107–2114.[Medline] [Order article via Infotrieve]

26. Merritt JE, Armstrong WP, Benham CD, Hallam TJ, Jacob R, Jaxa-Chamiec A, Leigh BK, McCarthy SA, Moores KE, Rink TJ. SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J. 1990;271:515–522.[Medline] [Order article via Infotrieve]

27. Montell C. New light on TRP and TRPL. Mol Pharmacol. 1997;51:755–763.[Abstract/Free Full Text]

This article has been cited by other articles:

				F. Helle, C. Jouzel, C. Chadjichristos, S. Placier, M. Flamant, D. Guerrot, H. Francois, J.-C. Dussaule, and C. Chatziantoniou Improvement of renal hemodynamics during hypertension-induced chronic renal disease: role of EGF receptor antagonism Am J Physiol Renal Physiol, July 1, 2009; 297(1): F191 - F199. [Abstract] [Full Text] [PDF]

				B. Ponnuchamy and R. A. Khalil Cellular mediators of renal vascular dysfunction in hypertension Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2009; 296(4): R1001 - R1018. [Abstract] [Full Text] [PDF]

				F. Helle, M. Hultstrom, T. Skogstrand, F. Palm, and B. M. Iversen Angiotensin II-induced contraction is attenuated by nitric oxide in afferent arterioles from the nonclipped kidney in 2K1C Am J Physiol Renal Physiol, January 1, 2009; 296(1): F78 - F86. [Abstract] [Full Text] [PDF]

				X. Wang, K. Takeya, P. I. Aaronson, K. Loutzenhiser, and R. Loutzenhiser Effects of amiloride, benzamil, and alterations in extracellular Na+ on the rat afferent arteriole and its myogenic response Am J Physiol Renal Physiol, July 1, 2008; 295(1): F272 - F282. [Abstract] [Full Text] [PDF]

				K. Takeya, K. Loutzenhiser, M. Shiraishi, R. Loutzenhiser, and M. P. Walsh A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles Am J Physiol Renal Physiol, June 1, 2008; 294(6): F1487 - F1492. [Abstract] [Full Text] [PDF]

				Q. Zhang, C. Cao, Z. Zhang, W. G. Wier, A. Edwards, and T. L. Pallone Membrane current oscillations in descending vasa recta pericytes Am J Physiol Renal Physiol, March 1, 2008; 294(3): F656 - F666. [Abstract] [Full Text] [PDF]

				L. Chilton, K. Loutzenhiser, E. Morales, J. Breaks, G. J. Kargacin, and R. Loutzenhiser Inward Rectifier K+ Currents and Kir2.1 Expression in Renal Afferent and Efferent Arterioles J. Am. Soc. Nephrol., January 1, 2008; 19(1): 69 - 76. [Full Text] [PDF]

				S. K. Fellner and W. J. Arendshorst Angiotensin II-stimulated Ca2+ entry mechanisms in afferent arterioles: role of transient receptor potential canonical channels and reverse Na+/Ca2+ exchange Am J Physiol Renal Physiol, January 1, 2008; 294(1): F212 - F219. [Abstract] [Full Text] [PDF]

				T. L. Thai, S. K. Fellner, and W. J. Arendshorst ADP-ribosyl cyclase and ryanodine receptor activity contribute to basal renal vasomotor tone and agonist-induced renal vasoconstriction in vivo Am J Physiol Renal Physiol, October 1, 2007; 293(4): F1107 - F1114. [Abstract] [Full Text] [PDF]

				X. Wang, J. Breaks, K. Loutzenhiser, and R. Loutzenhiser Effects of inhibition of the Na+/K+/2Cl- cotransporter on myogenic and angiotensin II responses of the rat afferent arteriole Am J Physiol Renal Physiol, March 1, 2007; 292(3): F999 - F1006. [Abstract] [Full Text] [PDF]

				A. M. Langager, B. E. Hammerberg, D. L. Rotella, and H. M. Stauss Very low-frequency blood pressure variability depends on voltage-gated L-type Ca2+ channels in conscious rats Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1321 - H1327. [Abstract] [Full Text] [PDF]

				F. Helle, O. B. Vagnes, and B. M. Iversen Angiotensin II-induced calcium signaling in the afferent arteriole from rats with two-kidney, one-clip hypertension Am J Physiol Renal Physiol, July 1, 2006; 291(1): F140 - F147. [Abstract] [Full Text] [PDF]

				D. Andreasen, U. G. Friis, T. R. Uhrenholt, B. L. Jensen, O. Skott, and P. B. Hansen Coexpression of Voltage-Dependent Calcium Channels Cav1.2, 2.1a, and 2.1b in Vascular Myocytes Hypertension, April 1, 2006; 47(4): 735 - 741. [Abstract] [Full Text] [PDF]

				R. Ma, J. Du, S. Sours, and M. Ding Store-Operated Ca2+ Channel in Renal Microcirculation and Glomeruli Experimental Biology and Medicine, February 1, 2006; 231(2): 145 - 153. [Abstract] [Full Text] [PDF]

				A. J. Fuller, B. C. Hauschild, R. Gonzalez-Villalobos, M. S. Awayda, J. D. Imig, E. W. Inscho, and L. G. Navar Calcium and chloride channel activation by angiotensin II-AT1 receptors in preglomerular vascular smooth muscle cells Am J Physiol Renal Physiol, October 1, 2005; 289(4): F760 - F767. [Abstract] [Full Text] [PDF]

				C. S. Facemire and W. J. Arendshorst Calmodulin mediates norepinephrine-induced receptor-operated calcium entry in preglomerular resistance arteries Am J Physiol Renal Physiol, July 1, 2005; 289(1): F127 - F136. [Abstract] [Full Text] [PDF]

				F. H. Hansen, O. B. Vagnes, and B. M. Iversen Enhanced response to AVP in the interlobular artery from the spontaneously hypertensive rat Am J Physiol Renal Physiol, May 1, 2005; 288(5): F1023 - F1031. [Abstract] [Full Text] [PDF]

				Q. Che and P. K. Carmines Src family kinase involvement in rat preglomerular microvascular contractile and [Ca2+]i responses to ANG II Am J Physiol Renal Physiol, April 1, 2005; 288(4): F658 - F664. [Abstract] [Full Text] [PDF]

				R. W. Fallet, H. Ikenaga, J. P. Bast, and P. K. Carmines Relative contributions of Ca2+ mobilization and influx in renal arteriolar contractile responses to arginine vasopressin Am J Physiol Renal Physiol, March 1, 2005; 288(3): F545 - F551. [Abstract] [Full Text] [PDF]

				T. L. Pallone, C. Cao, and Z. Zhang Inhibition of K+ conductance in descending vasa recta pericytes by ANG II Am J Physiol Renal Physiol, December 1, 2004; 287(6): F1213 - F1222. [Abstract] [Full Text] [PDF]

				D. J. Beech, K. Muraki, and R. Flemming Non-selective cationic channels of smooth muscle and the mammalian homologues of Drosophila TRP J. Physiol., September 15, 2004; 559(3): 685 - 706. [Abstract] [Full Text] [PDF]

				B. L. Jensen, U. G. Friis, P. B. Hansen, D. Andreasen, T. Uhrenholt, J. Schjerning, and O. Skott Voltage-dependent calcium channels in the renal microcirculation Nephrol. Dial. Transplant., June 1, 2004; 19(6): 1368 - 1373. [Full Text] [PDF]

				O. B. Vagnes, F. H. Hansen, R. E. F. Christiansen, C. Gjerstad, and B. M. Iversen Age-dependent regulation of vasopressin V1a receptors in preglomerular vessels from the spontaneously hypertensive rat Am J Physiol Renal Physiol, May 1, 2004; 286(5): F997 - F1003. [Abstract] [Full Text] [PDF]

				M.-G. Feng, M. Li, and L. G. Navar T-type calcium channels in the regulation of afferent and efferent arterioles in rats Am J Physiol Renal Physiol, February 1, 2004; 286(2): F331 - F337. [Abstract] [Full Text] [PDF]

				T. L. Pallone, Z. Zhang, and K. Rhinehart Physiology of the renal medullary microcirculation Am J Physiol Renal Physiol, February 1, 2003; 284(2): F253 - F266. [Abstract] [Full Text] [PDF]

				J. G. Murphy, J. N. Herrington, J. P. Granger, and R. A. Khalil Enhanced [Ca2+]i in renal arterial smooth muscle cells of pregnant rats with reduced uterine perfusion pressure Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H393 - H403. [Abstract] [Full Text] [PDF]

				Q. Che and P. K. Carmines Angiotensin II Triggers EGFR Tyrosine Kinase-Dependent Ca2+ Influx in Afferent Arterioles Hypertension, November 1, 2002; 40(5): 700 - 706. [Abstract] [Full Text] [PDF]

				Z. Zhang, K. Rhinehart, and T. L. Pallone Membrane potential controls calcium entry into descending vasa recta pericytes Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2002; 283(4): R949 - R957. [Abstract] [Full Text] [PDF]

				R Flemming, A Cheong, A M Dedman, and D J Beech Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle J. Physiol., September 1, 2002; 543(2): 455 - 464. [Abstract] [Full Text] [PDF]

				L. L. Cribbs Vascular Smooth Muscle Calcium Channels: Could "T" Be a Target? Circ. Res., September 28, 2001; 89(7): 560 - 562. [Full Text] [PDF]

				P. K. Carmines, R. W. Fallet, Q. Che, and K. Fujiwara Tyrosine Kinase Involvement in Renal Arteriolar Constrictor Responses to Angiotensin II Hypertension, February 1, 2001; 37(2): 569 - 573. [Abstract] [Full Text] [PDF]

				E. W. Inscho and A. K. Cook P2 receptor-mediated afferent arteriolar vasoconstriction during calcium blockade Am J Physiol Renal Physiol, February 1, 2002; 282(2): F245 - F255. [Abstract] [Full Text] [PDF]

				X. Wang and R. Loutzenhiser Determinants of renal microvascular response to ACh: afferent and efferent arteriolar actions of EDHF Am J Physiol Renal Physiol, January 1, 2002; 282(1): F124 - F132. [Abstract] [Full Text] [PDF]

				G. Trottier, M. Hollenberg, X. Wang, Y. Gui, K. Loutzenhiser, and R. Loutzenhiser PAR-2 elicits afferent arteriolar vasodilation by NO-dependent and NO-independent actions Am J Physiol Renal Physiol, May 1, 2002; 282(5): F891 - F897. [Abstract] [Full Text] [PDF]

				T. L. Pallone and J. M.-C. Huang Control of descending vasa recta pericyte membrane potential by angiotensin II Am J Physiol Renal Physiol, June 1, 2002; 282(6): F1064 - F1074. [Abstract] [Full Text] [PDF]

				P. B. Hansen, B. L. Jensen, D. Andreasen, and O. Skott Differential Expression of T- and L-Type Voltage-Dependent Calcium Channels in Renal Resistance Vessels Circ. Res., September 28, 2001; 89(7): 630 - 638. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Loutzenhiser, K. Articles by Loutzenhiser, R. Search for Related Content PubMed PubMed Citation Articles by Loutzenhiser, K. Articles by Loutzenhiser, R.