Angiotensin II–Induced Ca2+ Influx in Renal Afferent and Efferent Arterioles
Differing Roles of Voltage-Gated and Store-Operated Ca2+ Entry
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.
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
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).
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).
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.
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).
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.
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).
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.
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.
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.
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 pHi 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 factors12 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 contraction9 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.
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.
- © 2000 American Heart Association, Inc.
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