Superoxide Enhances Ca²⁺ Entry through L-Type Channels in the Renal Afferent Arteriole

Abstract

Reactive oxygen species regulate cardiovascular and renal function in health and disease. Superoxide participates in acute calcium signaling in afferent arterioles and renal vasoconstriction produced by angiotensin II, endothelin, thromboxane and pressure-induced myogenic tone. Known mechanisms by which superoxide acts include quenching of nitric oxide and increased ADP ribosyl cyclase/ryanodine-mediated calcium mobilization. The effect(s) of superoxide on other calcium signaling pathways in the renal microcirculation is poorly understood. The present experiments examined the acute effect of superoxide generated by paraquat on calcium entry pathways in isolated rat afferent arterioles. The peak increase in cytosolic calcium concentration caused by KCl (40 mM) was 99 ± 14 nM. The response to this membrane depolarization was mediated exclusively by L-type channels as it was abolished by nifedipine but was unaffected by the T-type channel blocker mibefradil. Paraquat increased superoxide production (dihydroethidium fluorescence), tripled the peak response to KCl to 314 ± 68 nM (p<0.001) and doubled the plateau response. These effects were abolished by tempol and nitroblue tetrazolium, but not by catalase, confirming actions of superoxide and not hydrogen peroxide. Unaffected by paraquat and superoxide was calcium entry through store-operated calcium channels activated by thapsigargin-induced calcium depletion of sarcoplasmic reticular stores. Also unresponsive to paraquat was ryanodine receptor-mediated calcium-induced calcium release from the sarcoplasmic reticulum. Our results provide new evidence that superoxide enhances calcium entry through L-type channels activated by membrane depolarization in rat cortical afferent arterioles, without affecting calcium entry through store operated entry or ryanodine receptor-mediated calcium mobilization.

INTRODUCTION

Reactive oxygen species (ROS), such as superoxide $(\cdot {\mathrm{O}}_2^{-})$ and hydrogen peroxide (H₂O₂), influence vascular physiology and pathophysiology ^{1, 2}. $\cdot {\mathrm{O}}_2^{-}$ in the renal vasculature and tubules is an important negative modulator of nitric oxide (NO), a vasodilator and natriuretic agent, by limiting its availability. Together these two opposing radicals provide an important balance in regulating the magnitude of vasoconstriction, sodium excretion and blood pressure (BP)¹. In addition to reducing NO bioavailability, $\cdot {\mathrm{O}}_2^{-}$ acts directly on vascular smooth muscle cells (VSMC) to augment calcium (Ca²⁺) signaling and enhance vasoconstriction ^{1, 3, 4}. Our laboratory has observed that $\cdot {\mathrm{O}}_2^{-}$ participates in and amplifies acute renal vasoconstrictor responses induced by angiotensin II (Ang II) ⁵, endothelin-1 (ET-1) ⁶, and thromboxane (TxA₂) ⁷ and stimulation of cytosolic calcium (Ca²⁺) in the afferent arteriole by these agents ^{8, 9}. $\cdot {\mathrm{O}}_2^{-}$ was implicated as the critical ROS, based on attenuation by dismutation of $\cdot {\mathrm{O}}_2^{-}$ by tempol. Other investigators have reported that $\cdot {\mathrm{O}}_2^{-}$ mediates the acute renal vasoconstriction produced by Ang II in normotensive and hypertensive animals with attenuation by antioxidants ^{5, 10-12}. Moreover, $\cdot {\mathrm{O}}_2^{-}$ potentiates the strength of the myogenic response of cortical afferent arterioles ^13-15.

Excessive vascular and renal $\cdot {\mathrm{O}}_2^{-}$ lead to vascular dysfunction and/or disturbed salt and water homeostasis ^{1, 16, 17}. Oxidative stress caused by increased ROS levels and NO deficiency is associated with renal vasoconstriction and the development of Ang II-induced and salt-sensitive hypertension ^{1, 3, 18-21}. $\cdot {\mathrm{O}}_2^{-}$ activity is enhanced in NO deficient rats, and contributes to abnormal renal function 1, 22. For example, increased $\cdot {\mathrm{O}}_2^{-}$ activity is responsible for inducing salt-sensitive hypertension in endothelial NO synthase knockout mice ^{19, 22}. Administration of superoxide dismutase (SOD) effectively reduces BP in salt-sensitive and salt-independent models of hypertension ^{1, 23}. Knockout mice deficient in extracellular SOD-3 have a higher basal BP than wild-type mice, a phenotype attributed to higher $\cdot {\mathrm{O}}_2^{-}$ and decreased NO levels in the kidney ²⁴. Moreover, chronic Ang II administration produces more pronounced hypertension in SOD-3 deficient mice than in wild-type controls ²⁵.

Ang II-induced hypertension is coupled with oxidative stress in blood vessels ^{26, 27}, and increased renal and non-renal vascular ROS is a common feature in both saltindependent and salt-sensitive hypertension ^{1, 20, 21, 28}. A ROS-dependent rise in renal vascular resistance (RVR) and BP is observed in Ang II-infused hypertensive mice and rats ^{29, 30}. Augmented oxidative stress in the spontaneously hypertensive rat (SHR) involves overexpression of NADPH oxidase and loss of extracellular SOD in the kidney ³¹. The SOD mimetic tempol normalizes elevated basal RVR and BP and restores endothelial function of renal arteries in the SHR and in the 2 kidney, 1 clip Goldblatt model of renovascular hypertension ^{32, 33}, further implicating $\cdot {\mathrm{O}}_2^{-}$ in exaggerated renal vasoconstriction and the potentiation of hypertension during oxidative stress ³⁴. Therefore, increased intrarenal ROS or an abnormal $\cdot {\mathrm{O}}_2^{-}∕\mathrm{NO}$ balance can alter renal hemodynamics and sodium excretion to cause hypertension ¹.

The precise mechanism(s) by which $\cdot {\mathrm{O}}_2^{-}$ affects Ca²⁺ signaling and causes contraction of VSMC in the renal microcirculation is poorly understood. Ca²⁺ signaling studies have linked G-protein coupled receptors (GPCR) for Ang II, ET-1 and catecholamines, and TxA2 to rapid $\cdot {\mathrm{O}}_2^{-}$ production and sensitization of RyR to mobilize Ca²⁺ from sarcoplasmic reticular stores in the renal vasculature ^{5, 7, 9, 35}. In cerebral arteries, ROS generation by hypoxanthine/xanthine oxidase (HX/XO), and by Ang II stimulation activates L-type channels to promote Ca²⁺ entry from the extracellular fluid ³⁶. Many GPCR ligands and perfusion pressure elicit contraction of afferent arterioles by stimulating Ca²⁺ entry through L-type channels ³⁷. Interactions between $\cdot {\mathrm{O}}_2^{-}$ and L-type Ca²⁺ channel activity in the renal microcirculation have not been investigated.

METHODS

See details in Methods in the online-only Data Supplement. All animal studies were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of North Carolina.

Preparation of afferent arterioles

Afferent arterioles (<20 μm in diameter) were isolated from 3 to 6 wk-old (50–120 g) male Sprague-Dawley rats using a magnetized iron oxide-sieving technique as previously described in our laboratory ^{8, 9}. We elected to use young animals because the preparation and purification of single afferent arterioles with little advential tissue from young rats were technically much easier than from mature rats. Pilot studies established that the increase in cytosolic Ca²⁺ concentration produced by 40 mM KCl was similar in young and adult rat afferent arterioles (Online supplement, please see http://hyper.ahajournals.org). All animal research conducted adhered to the NIH Guide for the Care and Use of Laboratory Animals.

Measurement of paraquat-induced cytosolic $\cdot {\mathit{O}}_2^{-}$ production

We used paraquatto generate $\cdot {\mathbf{O}}_2^{-}$ in fresh afferent arterioles. Paraquat (N,N’- dimethyl-4,4’-bipyridinium dichloride) is a classic and well established model for longterm oxidant-initiated toxicity due to its ability to generate $\cdot {\mathbf{O}}_2^{-}$. Paraquat redox cycles with cellular diaphorases and molecular oxygen to generate intracellular $\cdot {\mathbf{O}}_2^{-}$ at levels that do not acutely affect cell viability ^{39, 40}. We selected 1 mM paraquat for testing because it causes marked afferent arteriolar constriction that is reversed by tempol without adversely affecting short-term cell viability ³⁵.

$\cdot {\mathrm{O}}_2^{-}$ production was measured in isolated afferent arterioles using the oxidative-responsive fluorescent dye dihydroethidium (DHE) as previously described ⁷. After a baseline recording of a 90 sec incubation period with paraquat (1mM) or PBS alone as a negative control, a final image was acquired. Some arterioles were incubated with tempol (1 mM) or catalase (250 U/ml) in PBS for 20 min before testing the response to paraquat.

Measurement of cytosolic free calcium concentration

[Ca²⁺]_i was measured in individual afferent arterioles as previously described ^{8, 41, 42}. Fura-2 fluorescence was detected using a CCD camera (Digital Video Camera Co. DVC 1500) after passing through a 510 nm emission filter. [Ca²⁺]_i was determined by ratiometric analysis of Fura-2 emission intensities at two excitation wavelengths (340 nm, 380 nm). Signal intensity was acquired and processed using InCytIm2 software (Intracellular Imaging, Cincinnati, OH).

Protocols

Values for each arteriole were averaged and analyzed to identify the peak and the plateau phases of the response. Peak values were the average of three data points corresponding to the highest measured [Ca²⁺]_i after stimulation. Plateau responses were defined as the average of the 5 data points occurring 45 sec (43-47 sec) after the peak response.

To determine the relative contributions of L-type and T-type channels to Ca²⁺ entry resulting from KCl-induced membrane depolarization, we used selective L-type and Ttype Ca²⁺ channel inhibitors nifedipine (1 μM) and mibefradil (1 μM). Arterioles were incubated for 10 min in HBSS containing either channel blocker prior to stimulation with 40 mM KCl.

To test the effects of $\cdot {\mathrm{O}}_2^{-}$ on Ca²⁺ entry through L-type channels, paraquat was added 90 sec prior to stimulation with 40 mM KCl. The effect of paraquat on Ca²⁺ entry via SOC was determined using a protocol previously described by our laboratory ⁴³. Arterioles were incubated with Ca²⁺-free HBSS containing 10 μM thapsigargin for 20 min and then the thapsigargin-containing HBSS was replaced with a bathing medium composed of Ca²⁺-free PBS with 0.5 mM EGTA and 10 μM thapsigargin. Paraquat (1 mM) was added to the bathing medium 90 sec prior to switching the bath solution to PBS containing Ca²⁺ (1 mM).

To assess the contribution from Ca²⁺-induced Ca²⁺ release (CICR), arterioles were incubated in HBSS containing 50 μM Ry for 20 min to block Ry receptors (RyR) and prevent CICR ⁴¹. After incubation, arterioles were placed in a bathing medium composed of PBS with 1.1 mM Ca²⁺ and 50 μM Ry. In other experiments, paraquat (1 mM) was added 90 sec prior to KCl stimulation.

Statistics

A two-tailed Student's t-test was used to determine significance between groups (Prism Software). P < 0.05 was determined to be significant.

RESULTS

The results for [Ca²⁺]_i are presented as nM ± SEM change from baseline. Baseline [Ca²⁺]i for all afferent arterioles studied averaged 100 ± 4 nM (n=111). Basal values for each group of experiments did not differ statistically from the overall mean (p>0.1).

High KCl stimulates Ca²⁺ entry through L-type channels

Fig. 1 (left panel) shows high KCl (40 mM)-induced depolarization of the plasma membrane of VSMC of afferent arterioles to stimulate Ca²⁺ influx to increase [Ca²⁺]_i.. The initial peak increase in [Ca²⁺]_i averaged 99 ± 14 nM while the sustained plateau phase at 45 s after addition of KCl was 53 ± 10 nM (Fig. 1, right panel). Fig. 1 (right and left panels) shows the complete abolition of high KCl-induced Ca²⁺ entry when L-type channels were blocked by nifedipine (1 μM). Addition of the T-type inhibitor mibefradil (1 μM) did not alter Ca²⁺ responses to high KCl. These observations support previous reports that KCl-induced depolarization exclusively activates voltage-gated L-type Ca²⁺ channels in the rat cortical afferent arteriole ^44-47.

Fig. 1.

Left panel: [Ca²⁺]_i responses to KCl in the presence of the L-type and T-type Ca²⁺ channel inhibitors, nifedipine (n=7) and mibefradil (n=8), respectively, are compared to a control KCl response (n=8). Right Panel: Effects of nifedipine and mibefradil on average peak and plateau [Ca²⁺]_i responses. (*** p<0.005)

Paraquat incresees $\cdot O_2^{-}$ production in afferent arteriolar VSMCs

Fig. 2 shows that paraquat (1 mM) induced a 50% increase in $\cdot {\mathrm{O}}_2^{-}$ generation (DHE fluorescence) in these afferent arteriolar VSMCs. This increase in $\cdot {\mathrm{O}}_2^{-}$ was abolished by tempol (p<0.001) and not affected by catalase.

Fig. 2.

Paraquat elicited a 50% increase in cytosolic $\cdot {\mathrm{O}}_2^{-}$ (DHE fluorescence, n=7, filled bar) as compared to buffer as a negative control (n=7, open bar) and paraquat (n=7, filled bar). The paraquat-induced increase in $\cdot {\mathrm{O}}_2^{-}$ production was abolished by tempol (n=7, p<0.005, gray bar), but was not significantly influenced by catalase (n=7, patterned bar) (*** p<0.005 vs. control, # p<0.005 vs. paraquat).

Paraquat and $\cdot O_2^{-}$ generation increase Ca²⁺ influx induced by membrane depolarization

Fig. 3 (left panel) demonstrates that the addition of paraquat (10⁻³ M) to generate of $\cdot {\mathrm{O}}_2^{-}$ markedly enhanced the Ca²⁺ entry through L-type Ca²⁺ channels stimulated by KCl-induced membrane depolarization but not during basal conditions before addition of KCl. Paraquat increased both the peak and the plateau phases of the response to KCl (Fig. 3, right panel). The peak was enhanced 3-fold to 314 ± 68 nM and the plateau phase was augmented nearly 2-fold to 92 ± 8 nM (p<0.01 for both).

Fig. 3.

Left panel: [Ca²⁺]_i response of freshly isolated afferent arterioles to KCl-induced (40 mM) membrane depolarization before (n=14, open circles) and after paraquat stimulation (n=10, filled circles). Note that paraquat did not affect basal [Ca²⁺]_i prior to stimulation with KCl. Right panel: Average peak and plateau (45 sec after the peak) responses to KCl in the presence and absence of paraquat (*** p<0.005, ** p<0.01).

Fig. 4 (left panel) illustrates the effect of $\cdot {\mathrm{O}}_2^{-}$ dismutation by tempol on the [Ca²⁺]_i response to 40 mM KCl and paraquat stimulation. Tempol (10⁻³ M) had no effect on baseline [Ca²⁺]_i before KCl stimulation, but abolished the enhancement produced by paraquat, yielding results approximating those of KCl alone in the absence of paraquat. Fig. 4 (right panel) shows that the effect of paraquat induced $\cdot {\mathrm{O}}_2^{-}$ production to increase both peak and plateau Ca²⁺ responses to KCl was eliminated by the antioxidant tempol. Similar inhibition of the paraquat stimulation was observed when NBT was used to scavenge $\cdot {\mathrm{O}}_2^{-}$ (NBT results online supplement only, please see http://hyper.ahajournals.org).

Fig. 4.

Left panel: The [Ca²⁺]_i responses to KCl in the presence and absence of paraquat (closed and filled circles, respectively) (same as in Fig. 4), and the effect of tempol dismutation of paraquat-generated $\cdot {\mathrm{O}}_2^{-}$ (n=8, open squares). Right panel: Effect of tempol on the average peak and plateau [Ca²⁺]_i values. (*** p<0.005 vs. control, # p<0.05 vs. paraquat).

Paraquat and $\cdot O_2^{-}$ generation does not increase Ca²⁺ entry through SOC channels

To verify that Ca²⁺ entry took place through SOC and not L-type Ca²⁺ channels, vessels were treated with nifedipine throughout the experiment. Fig. 5 (left panel) displays the magnitude of Ca²⁺ entry through SOC in the presence and absence of $\cdot {\mathrm{O}}_2^{-}$ generated by paraquat. Fig. 5 (right panel) shows the peak and plateau values for [Ca²⁺]_i following activation of Ca²⁺ entry through SOC. Pretreatment with paraquat had no significant effect on Ca²⁺ entry through SOC during either phase (P>0.5).

Fig. 5.

Left panel: The magnitude of Ca²⁺ entry through SOC after thapsigargin-induced depletion of sarcoplasmic reticular Ca²⁺ stores and the lack of effect of paraquat (n=8 for both). Right panel: Peak and plateau values for [Ca²⁺]_i as a result of Ca²⁺entry through SOC before and after addition of paraquat.

Enhancement of Ca²⁺ influx by paraquat does not involve CICR

Increased [Ca²⁺]_i resulting from Ca²⁺ entry through L-type channels is known to increase CICR from the sarcoplasmic reticulum mediated by activation of the ryanodine receptor (RyR) ⁴¹. To test for a possible effect of $\cdot {\mathrm{O}}_2^{-}$ on RyR-mediated CICR, we used Ry (50 μM) to inactivate RyR and prevent CICR secondary to Ca²⁺ entry stimulated by high KCl. As Fig. 6 (left panel) shows, during RyR inactivation to prevent CICR, paraquat enhanced the peak and plateau [Ca²⁺]_i responses initiated by membrane depolarization (p<0.05), again demonstrating the ability of paraquat-derived $\cdot {\mathrm{O}}_2^{-}$ to stimulate L-type channels. Also shown in Fig. 6, Ry decreased the [Ca²⁺]_i response to high KCl, reducing the peak by 50% and the plateau phase by 23%. Thus, RyR-mediated CICR contributes to the [Ca²⁺]_i response when Ca²⁺ entry is stimulated by membrane depolarization. Inactivation of RyR blunted the peak and plateau Ca²⁺ signals after paraquat to approximately the same extent (Fig. 6, right panel). $\cdot {\mathrm{O}}_2^{-}$ enhanced KCl-induced Ca²⁺ influx during paraquat treatment, suggesting $\cdot {\mathrm{O}}_2^{-}$ did not affect RyR-mediated Ca²⁺ mobilization in these experiments. Therefore, we conclude that the paraquat-induced increase in Ca²⁺ entry during membrane polarization is primarily due to L-type channel enhancement, and not by recruitment of SOC or augmented CICR.

Fig. 6.

Left panel: [Ca²⁺]_i responses to KCl during inactivation of RyR-induced CICR in the presence (n=9) and absence (n=8) of paraquat. Right panel: Effect of paraquat on the average peak and plateau [Ca²⁺]_i responses (*** p<0.005, * p<0.05).

DISCUSSION

This study evaluated the effects of $\cdot {\mathrm{O}}_2^{-}$ generated by paraquat on Ca²⁺ signaling in VSMCs of rat renal afferent arterioles. $\cdot {\mathrm{O}}_2^{-}$ was verified as the primary ROS responsible for increased Ca²⁺ influx based on DHE measurement and effective abolition of the paraquat effect on [Ca²⁺]_i by either tempol or NBT (NBT results online supplement only, please see http://hyper.ahajournals.org). Moreover, $\cdot {\mathrm{O}}_2^{-}$ production was negated by tempol but not catalase. Our major new finding is that $\cdot {\mathrm{O}}_2^{-}$ enhanced Ca²⁺ entry in response to membrane depolarization induced by high extracellular KCl. We conclude that $\cdot {\mathrm{O}}_2^{-}$ acted to increase Ca²⁺ entry by increasing the activity of L-type Ca²⁺ channels. The stimulation was rapid, requiring at most 90 sec of exposure to paraquat and was effective upon stimulation with KCl, whereas basal [Ca²⁺]_i before stimulation was unaffected.

Our results extend previous studies showing that membrane depolarization by high KCl exclusively activates L-type Ca²⁺ channels to increase [Ca²⁺]_i and contract the rat cortical afferent arteriole with little to no participation of T-type channels sensitive to mibefradil ^44-46. Both responses were abolished by removal of extracellular Ca²⁺ or by pharmacological inhibition of voltage-gated L-type channels using nifedipine or nitrendipine ^44-46. Based on patch clamp studies of voltage-activated Ca²⁺ currents, freshly isolated VSMCs of the rat afferent arteriole have a high density of L-type channels but do not express functionally active voltage-dependent T-type Ca²⁺ channels ⁴⁷. In rat juxtamedullary afferent arterioles, it is reported that T-type channels are functionally expressed, but do not contribute to constrictor responses to KCl ⁴⁸. On the other hand, another study reports that high KCl-induced Ca²⁺ entry is attenuated in rat juxtamedullary afferent arterioles and rabbit cortical afferent arterioles during inhibition of T-type channels by mibefradil ⁴⁹.

Our results provide important insight into physiological and pathophysiological mechanisms within the kidney as L-type Ca²⁺ channel activity is a critical determinant of contractile tone of the afferent arteriole, whether stimulated by GPCR agonists or increased renal perfusion pressure ³⁷. $\cdot {\mathrm{O}}_2^{-}$ is known to modulate renal hemodynamics 1 and vasoconstrictor responses to GPCR agonists such as Ang II, ET-1 and PE ^{5, 6}. Our finding of a direct action of $\cdot {\mathrm{O}}_2^{-}$ on Ca²⁺ entry provides a mechanism to explain our previous demonstration that NADPH-derived $\cdot {\mathrm{O}}_2^{-}$ mediates the afferent arteriolar [Ca²⁺]_i response to Ang II and ET-1 ^{8, 9}. The immediate increase in [Ca²⁺]_i stimulated by either agonist was attenuated by both tempol and apocynin, indicating participation of NADPH-derived $\cdot {\mathrm{O}}_2^{-}$. Moreover, the actions of Ang II and ET-1 were attenuated by 8-Br-cyclic ADPR and nicotinamide, implicating involvement of ADPR cyclase and RyR in the Ca²⁺ signaling pathway, steps proposed to be downstream of $\cdot {\mathrm{O}}_2^{-}$ production.

ADPR cyclase synthesis of cADPR can sensitize RyR to [Ca²⁺]_i to enhance CICR ⁵⁰. Our earlier studies of afferent arterioles showed that RyR-mediated CICR also contributes to the increased [Ca²⁺]_i following membrane depolarization induced by high KCl ⁴¹. Locking the RyR in the closed position with a high concentration of ryanodine (50-100 μM) attenuated ~50% of the [Ca²⁺]_i response to Ca²⁺ entry through voltagegated L-type channels. Our current studies confirm this finding and extend it in that $\cdot {\mathrm{O}}_2^{-}$ generated by paraquat had essentially no effect on the RyR-mediated CICR response to increased [Ca²⁺]_i secondary to KCl-induced depolarization.

In our studies of freshly isolated afferent arterioles, paraquat did not elicit a change in basal [Ca²⁺]_i, suggesting no acute effect of $\cdot {\mathrm{O}}_2^{-}$ on Ca²⁺ entry in the absence of KCl-induced membrane depolarization when L-type channels are quiescent. This contrasts with other published reports that ROS increased basal [Ca²⁺]_i in VSMC from nonrenal vessels ^{36, 51}. Differences among studies may stem from ROS exposure times and concentrations, intracellular vs extracellular ROS generation, and vascular beds. It is noteworthy that HX/XO primarily generates extracellular ROS, whereas paraquat stimulates intracellular $\cdot {\mathrm{O}}_2^{-}$ production. Our 90 sec exposure time to paraquat / $\cdot {\mathrm{O}}_2^{-}$ was considerably shorter than some previous studies. In cultured rat mesenteric arterial VSMCs, ROS generated by cell membrane permeable LY23583 increased Tempo-9AC florescence after 25 min, a response abolished by tempol, whereas [Ca²⁺]_i was increased after 15 min ⁵⁰. In these nonrenal VSMC of WKY but not SHR, the increase was attributed to augmented Ca²⁺ influx through both L-type and T-type Ca²⁺ channels as it was inhibited by putative selective antagonists verapamil/diltiazem (10 μM) and mibefradil (10 μM), respectively ⁵¹.

Amberg et al. investigated the role of ROS in activating Ca²⁺ entry via L-type Cav1.2 Ca²⁺ channels and the resultant constriction of pressurized, freshly isolated cerebral arteries ³⁶. Total internal reflection fluorescence microscopy revealed that Ang II and endogenous ROS rapidly stimulated Ca²⁺ entry by increasing L-type Ca²⁺ channel sparklet activity. Both the Ang II-induced increase in Ca²⁺ sparklets and arterial tone were abolished by apocynin inhibition of NADPH oxidase, implicating a stimulatory role of ROS, either $\cdot {\mathrm{O}}_2^{-}$ or H₂O₂. Additionally, exogenous ROS generated by HX/XO increased PKCα and L-type Ca²⁺ channel activity and cerebral arterial vasomotor tone as a result of Ca²⁺ entry within 2 min of addition; the latter was abolished by inhibition of L-type channel activity with diltiazem ³⁶. Exogenous ROS increased both Ca²⁺ sparklet activity and sparklet site density in voltage clamped arterial myocytes, further supporting the conclusion that ROS increases Ca²⁺ influx through L-type channels.

L-type channels are sensitive to oxidants, potentially due to direct redox modification of cysteines on the channel, or by redox modification of regulatory proteins involved in channel function ^52-54. $\cdot {\mathrm{O}}_2^{-}$ may amplify Ca²⁺ entry through L-type Ca²⁺ channels by promoting clustering that leads to cooperative gating ⁵⁵. In this manner, increased $\cdot {\mathrm{O}}_2^{-}$ production during ANG II-induced hypertension may explain increased L-type Ca²⁺ channel sparklet activity in arterial VSMC ^{56, 57} and the increased density of L-type channels ⁵⁸. Another new finding of our present studies is that $\cdot {\mathrm{O}}_2^{-}$ does not influence Ca²⁺ entry mediated by SOC in freshly isolated afferent arterioles. In cultured VSMC of porcine coronary artery and bovine pulmonary artery, ROS generated by HX/XO was reported to stimulate [Ca²⁺]_i by inhibiting plasma membrane Ca²⁺-ATPase and SERCA ^59-62. Such inhibition is predicted to deplete intracellular Ca²⁺ stores and thereby indirectly enhance SOC entry. Both TRPC and STIM-Orai channels have been proposed to be the primary SOC channels in other vascular beds ^{63, 64}. AVP stimulation of $\cdot {\mathrm{O}}_2^{-}$ and H₂O₂ production is reported to increase TRPC6 channel activity and Ca²⁺ influx in the A7r5 line of cultured VSMC and cultured mouse aortic VSMC ⁶⁵. Presently, the molecular identity of SOC channels in the renal microcirculation is unknown.

In summary, our major novel finding is that $\cdot {\mathrm{O}}_2^{-}$ enhances Ca²⁺ entry through L-type channels in VSMCs of freshly isolated afferent arterioles. We found no evidence for potentiation by paraquat of either Ca²⁺ entry through SOC or Ca²⁺ mobilization and CICR mediated by RyR.

PERSPECTIVES

It is well established that ROS play a pathophysiological role in the development of hypertension, however the specific mechanisms by which ROS alter renal hemodynamics in health and disease are poorly understood. $\cdot {\mathrm{O}}_2^{-}$ interacts with NO and participates in rapid, acute constriction of the afferent arteriole and increased renal vascular resistance, but the effects on Ca²⁺ signaling pathways in the renal microcirculation are not known. Our studies provide new information that $\cdot {\mathrm{O}}_2^{-}$ acts to enhance Ca²⁺ influx through L-type Ca²⁺ channels in the afferent arteriole, the major preglomerular resistance vessel in the kidney where Ca²⁺ entry through voltage-gated channels is a predominant Ca²⁺ signaling pathway. This stimulatory effect can be reversed both by $\cdot {\mathrm{O}}_2^{-}$ dismutation with tempol and by $\cdot {\mathrm{O}}_2^{-}$ scavenging with NBT. Ca²⁺ entry through store-operated channels resulting from thapsigargin-induced intracellular Ca²⁺ depletion of SR stores is not markedly influenced by cellular $\cdot {\mathrm{O}}_2^{-}$ levels.

During renal autoregulation, changes in tone of the cortical radial arteries and afferent arterioles result from pressure-induced activation of L-type Ca²⁺ channels to maintain RBF, glomerular filtration rate, and buffer pressure-natriuresis. Acute increases in $\cdot {\mathrm{O}}_2^{-}$ modulate the efficiency of renal autoregulation by augmenting the myogenic response of afferent arterioles ^{13, 15}. An enhancement in L-type channel activity by $\cdot {\mathrm{O}}_2^{-}$ may provide a mechanistic explanation for these observations. Increased $\cdot {\mathrm{O}}_2^{-}$ production participates in renal vasoconstriction and sodium retention during the development of hypertension ^{1, 10, 11, 34, 66}. The ability of $\cdot {\mathrm{O}}_2^{-}$ to increase Ca²⁺ entry through L-type channels is likely to play an important role in the vasoconstriction of the preglomerular vasculature often associated with sodium retention and the development and maintenance of hypertension.

NOVELTY AND SIGNIFICANCE.

What is new?

• Our studies provide new information that $\cdot {\mathrm{O}}_2^{-}$ acts to enhance Ca²⁺ influx through L-type Ca²⁺ channels in the afferent arteriole, the major preglomerular resistance vessel in the kidney.

• We also show that superoxide does not participate in other Ca²⁺ signaling mechanisms we investigated, such as store operated calcium entry and ryanodine receptor-mediated calcium mobilization and subsequent calcium-induced-calcium-release.

What is relevant?

• The ability of $\cdot {\mathrm{O}}_2^{-}$ to increase Ca²⁺ entry through L-type channels is likely to play an important role in vasoconstriction the preglomerular vasculature often associated with sodium retention and the development of hypertension.

Summary

• Our studies address a lack of understanding of the mechanisms by which superoxide affects Ca²⁺ signaling and causes contraction of VSMC of renal resistance arterioles.