Voltage-gated divalent currents in descending vasa recta pericytes

Abstract

Multiple voltage-gated Ca²⁺ channel (Ca_V) subtypes have been reported to participate in control of the juxtamedullary glomerular arterioles of the kidney. Using the patch-clamp technique, we examined whole cell Ca_V currents of pericytes that contract descending vasa recta (DVR). The dihydropyridine Ca_V agonist FPL64176 (FPL) stimulated inward Ca²⁺ and Ba²⁺ currents that activated with threshold depolarizations to −40 mV and maximized between −20 and −10 mV. These currents were blocked by nifedipine (1 μM) and Ni²⁺ (100 and 1,000 μM), exhibited slow inactivation, and conducted Ba²⁺ > Ca²⁺ at a ratio of 2.3:1, consistent with “long-lasting” L-type Ca_V. In FPL, with 1 mM Ca²⁺ as charge carrier, Boltzmann fits yielded half-maximal activation potential (V_1/2) and slope factors of −57.9 mV and 11.0 for inactivation and −33.3 mV and 4.4 for activation. In the absence of FPL stimulation, higher concentrations of divalent charge carriers were needed to measure basal currents. In 10 mM Ba²⁺, pericyte Ca_V currents activated with threshold depolarizations to −30 mV, were blocked by nifedipine, exhibited voltage-dependent block by diltiazem (10 μM), and conducted Ba²⁺ > Ca²⁺ at a ratio of ∼2:1. In Ca²⁺, Boltzmann fits to the data yielded V_1/2 and slope factors of −39.6 mV and 10.0 for inactivation and 2.8 mV and 7.7 for activation. In Ba²⁺, V_1/2 and slope factors were −29.2 mV and 9.2 for inactivation and −5.6 mV and 6.1 for activation. Neither calciseptine (10 nM), mibefradil (1 μM), nor ω-agatoxin IVA (20 and 100 nM) blocked basal Ba²⁺ currents. Calciseptine (10 nM) and mibefradil (1 μM) also failed to reverse ANG II-induced DVR vasoconstriction, although raising mibefradil concentration to 10 μM was partially effective. We conclude that DVR pericytes predominantly express voltage-gated divalent currents that are carried by L-type channels.

within the outer stripe of the outer medulla, juxtamedullary efferent arterioles divide to form descending vasa recta (DVR), which supply blood flow to the renal medulla. An increasing body of evidence has shown that voltage-gated Ca²⁺ channels (Ca_V) play an important role in the regulation of basal tone and agonist-induced contraction of the juxtamedullary efferent circulation (13–15, 22, 31). Consistent with this evidence, Ca²⁺ channel blockers (CCB) have been observed to enhance medullary perfusion (1, 8, 16, 19, 20, 36, 55). The relative roles of DVR vs. upstream juxtamedullary vascular segments in the actions of CCB cannot be easily discerned, because the overlying renal cortex prevents access in vivo. Cell culture models that replicate the behavior of the pericytes and endothelia that comprise the DVR wall are lacking. To permit ex vivo investigations, DVR have been explanted for microperfusion, microfluorescence, and patch-clamp studies (42, 44, 45), and behavior consistent with Ca_V-mediated regulation has been observed. ANG II, endothelin-1, and vasopressin depolarize DVR pericytes through a variable combination of Ca²⁺-dependent Cl⁻ channel activation and K⁺ channel suppression (4, 43, 44, 59). In the absence of a specific ligand-receptor interaction, depolarization with high extracellular KCl elevates DVR pericyte cytoplasmic Ca²⁺ concentration ([Ca]_CYT) and the L-type CCB diltiazem inhibits KCl-related contraction and [Ca]_CYT elevation (48, 61). Recently, a search for voltage-gated cation entry routes into the pericyte led to surprising identification of a tetrodotoxin (TTX)-sensitive, rapidly inactivating Na⁺ conductance, traced to the voltage-gated Na⁺ channel Na_V1.3, which, combined with Na⁺/Ca²⁺ exchange, might provide a surrogate Ca²⁺ entry mechanism (32, 58).

Motivated by the above-described evidence, functional expression of Ca_V in juxtamedullary efferent arterioles, and immunochemical evidence favoring their presence in DVR pericytes (21, 22, 31), we performed the present study to measure their signature currents. In series 1, we found that the dihydropyridine agonist FPL64176 (FPL) readily stimulates inward Ca²⁺ and Ba²⁺ currents, the characteristics of which are consistent with L-type channels. In the absence of FPL stimulation, otherwise small basal Ca_V currents were readily measured when intracellular Ca²⁺ was chelated and extracellular concentrations of Ca²⁺ or Ba²⁺ were raised to carry charge. Those basal Ca_V currents were blocked by nifedipine, Ni²⁺, and diltiazem, but not calciseptine, ω-agatoxin IVA, or low concentrations of mibefradil. Kinetics, activation and inactivation thresholds, and the ratio of Ba²⁺ to Ca²⁺ conductance are consistent with the predominance of L-type channels. We conclude that the channel architecture of explanted DVR pericytes most closely resembles that described for afferent smooth muscle, where Cl⁻ channel activity and L-type Ca_V control membrane potential and Ca²⁺ entry, respectively (2, 5, 18, 29).

METHODS

Isolation of DVR.

Investigations involving animal use were performed according to protocols approved by the Institutional Animal Use and Care Committee of the University of Maryland. Rats were anesthetized by an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Under full anesthesia, the abdomen was opened and the kidneys were excised, so that euthanasia was induced by concomitant exsanguination. Tissue slices were stored at 4°C in a physiological saline solution (in mM: 155 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4). As described previously (44), for patch-clamp studies, small wedges of renal medulla were dissected and transferred to Blendzyme 1 (Roche) at 0.27 mg/ml in high-glucose DMEM (Invitrogen, without serum), incubated at 37°C for 20 min, transferred to physiological saline solution, and stored at 4°C. Enzymatic tissue digestion was omitted when DVR were explanted for microperfusion (42).

Whole cell patch-clamp recording.

Gigaseals were directly formed on abluminal pericytes of isolated vessels as previously described (44, 48, 57) and studied by ruptured-patch whole cell recording (58). When pericytes showed evidence of open gap junction coupling (prolonged capacitance transients), the cells were abandoned (57); gap junction blockers were not employed in these studies. A two-stage vertical pipette puller (Narshige PP-830) was used to fabricate patch pipettes from borosilicate glass capillaries (1.5 mm OD, 1.0 mm ID; PG52151-4, World Precision Instruments); the patch pipettes were heat polished to a final resistance of 4–8 MΩ. Whole cell recording was performed with a CV201AU headstage and Axopatch 200B amplifier (Axon Instruments, Foster City, CA; 10-kHz sampling) at room temperature. The following buffer, designed to minimize intracellular free Ca²⁺, was used in the electrode (in mM): 115 Cs methanesulfonate, 20 CsCl, 10 NaCl, 2 MgATP, 10 BAPTA or EGTA, and 10 HEPES, with pH adjusted to 7.2 with CsOH. The extracellular buffer, designed to suppress contaminating K⁺ and Cl⁻ currents, consisted of (in mM) 115 NaCl, 30 tetraethylammonium Cl, 1 MgCl₂, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with niflumic acid (100 μM) and glibenclamide (10 μM). CaCl₂ or BaCl₂ was added to the extracellular buffer to carry divalent charge. In those cases where the L-type channel agonist FPL was not used to stimulate currents, TTX (100 nM) was included to inhibit the voltage-gated Na⁺ conductance of DVR pericytes (58). Results were corrected for junction potentials as previously described (37, 44). The cell capacitance observed in these studies [13.6 ± 0.5 pF (SE), n = 78] is similar to that previously observed (12.2 ± 0.5 pF, n = 94) in rat pericytes during study of voltage-gated Na⁺ currents (58).

Vasoactivity measurements.

Vasoactivity was studied by videomicroscopy using isolated, microperfused DVR (42). This preparation requires vessels to be explanted without enzymatic digestion, so that their walls withstand transmural pressure without rupturing. Isolated DVR were mounted on concentric pipettes, cannulated at one end, and held at the other end to permit flow into the collection pipette. The pipettes and vessels were positioned with micromanipulators (Instruments Technology and Machinery, Schertz, TX) near a thermocouple in the narrow entrance region of a custom-built chamber. Temperature was maintained with a feedback system (CN9000A, Omega Engineering, Bridgeport, NJ) at 37°C. The buffer used for dissection, perfusion, and bath was the same as that previously employed for similar experiments: 140 mM NaCl, 10 mM Na acetate, 5 mM KCl, 1.2 mM MgCl₂, 2 mM NaHPO₄/NaH₂PO₄, 1 mM CaCl₂, 5 mM alanine, 0.1 mM l-arginine, 5 mM glucose, 5 mM HEPES, and 0.5 g/dl albumin, pH 7.4 at 37°C (41). DVR were observed through a ×40 objective to yield the final magnification of ×1,300, and images were captured with a Spot camera (Diagnostic Instruments). As in prior studies, constriction was quantified as percent reduction of internal diameter at the point of maximal constriction using National Institutes of Health ImageJ software for offline analysis (4, 41, 59). Diameter changes are expressed as percent constriction: [1 − (D/D₀)] × 100, where D and D₀ are the experimental and baseline diameters, respectively.

Reagents.

FPL and TTX were obtained from Tocris-Cookson (Ellisville, MO); calciseptine and ω-agatoxin IVA from Alomone (Jerusalem, Israel); Blendzyme 1 from Roche Applied Science (Indianapolis, IN); and ANG II, NiCl₂, niflumic acid, mibefradil, nifedipine, diltiazem, and other chemicals from Sigma. Nifedipine, FPL, and niflumic acid were dissolved in DMSO. TTX was dissolved in aqueous 10 mM Na acetate buffer, pH 4.5. ANG II, mibefradil, and NiCl₂ were dissolved in water. Reagents were stored frozen at −20°C, thawed, and diluted 1:1,000 on the day of the experiment. Excess reagents were discarded daily. Blendzyme was stored in 40-μl aliquots of 4.5 mg/ml in water and diluted into high-glucose DMEM on the day of the experiment.

Statistics.

Curve fits were performed with Clampfit 9.2 (Axon Instruments) using Levenberg-Marquardt algorithms. Values are means ± SE. The significance of differences was evaluated with SigmaStat 3.11 (Systat Software, Point Richmond, CA) using parametric or nonparametric tests as appropriate for the data. Comparisons between two groups were performed with Student's t-test (paired or unpaired, as appropriate) or the rank sum test (nonparametric). Comparisons between multiple groups employed one-way ANOVA, repeated-measures ANOVA, or repeated-measures ANOVA on ranks (nonparametric). P < 0.05 was used to reject the null hypothesis.

RESULTS

L-type Ca_V currents stimulated by FPL.

In the first series of experiments, we repeatedly depolarized pericytes from a holding level of −100 to 0 mV at 10-s intervals. Basal and FPL-stimulated currents were measured with EGTA as the Ca²⁺ chelator in the electrode and 1 mM Ca²⁺ as the extracellular charge carrier. As shown in Fig. 1, Aa and Ab, basal (no FPL) inward currents were nearly undetectable under these conditions. In contrast, when FPL was added to the extracellular buffer in sequentially increasing concentrations (0.1, 1.0, and 10 μM), a slowly inactivating inward current emerged when the cells were depolarized.

Fig. 1.

Stimulation of inward Ca²⁺ currents in descending vasa recta (DVR) pericytes by FPL64176 (FPL). Aa: inward currents elicited in 1 mM extracellular Ca²⁺ by sequential depolarizations from −100 to 0 mV in increasing concentrations of FPL. Ab: peak Ca²⁺ current (I_Ca) in DVR pericytes stimulated by 0–10 μM FPL. n = 7. *P < 0.05, **P < 0.01 vs. 0 μM. Ba: voltage dependence of FPL-stimulated inward currents. Bb: peak I_Ca vs. depolarization potential (V_m). n = 9.

Voltage-dependent characteristics of the FPL (10 μM)-stimulated current were further studied using the protocol shown in the inset of Fig. 1Ba and are summarized in Fig. 1Bb. Basal inward current magnitude was <10 pA. In contrast, in FPL they activated at a threshold depolarization of about −40 mV, maximized between −10 and 0 mV, and reached ∼35 pA.

To study characteristics of inactivation of the FPL-induced current, the protocol illustrated in Fig. 2Aa (example in Fig. 2Ab) was executed. Pericytes were held at −110 to −10 mV for 1,000 ms and then depolarized to −10 mV. A summary of the voltage dependence of inactivation (I_Ca/I_Ca,max, where I_Ca is Ca²⁺ current) and activation (g/g_max, where g is conductance) is superimposed in Fig. 2B upon corresponding fits to the Boltzmann equation: y = 1/{1 + exp[s(V_m − V_1/2)/K]}, where V_m is membrane potential, s = −1 or +1 for activation or inactivation, respectively, and V_1/2 and K (slope factor) are constants. Activation was computed from the Ca²⁺ chord conductance (g), where g = I_Ca/(V_m − Ca_rev) and Ca_rev is the reversal potential. Values were normalized to maximum conductance (g_max). Inactivation of the FPL current commenced between −80 and −90 mV with V_1/2 of −57.9 mV and slope factor of 11.0. Activation occurred between −30 and −40 mV with V_1/2 of −33.3 mV and slope factor of 4.4, yielding an overlapping window current between −50 and −10 mV.

Fig. 2.

Voltage dependence of inactivation and activation of FPL-stimulated I_Ca. Aa: protocol used to study inactivation. Pericytes were held at various potentials (V_hold) for 1,000 ms and then depolarized to −10 mV. Ab: effects of various holding potentials on I_Ca. B: inactivation (I_Ca/I_Ca,max, n = 16) and activation [g/g_max (where g is conductance), n = 9] characteristics with 1 mM extracellular Ca²⁺ and 10 mM FPL as agonist. Half-maximal activation poential (V_1/2) and slope factor (K) for Boltzmann fits were −57.9 mV and 11.0 for inactivation and −33.3 mV and 4.4 for activation, respectively.

Blockade of FPL current with nifedipine and Ni²⁺.

The FPL-stimulated inward current was sensitive to nifedipine (1 μM; Fig. 3) and Ni²⁺ (Fig. 4). Representative traces are provided in Fig. 3Aa, where, at baseline, only a rapidly inactivating “fast” inward Na⁺ current is present (58). Subsequently, FPL (10 μM) was introduced to elicit the slowly inactivating component of inward current. This was followed by addition and washout of nifedipine. In similar experiments without FPL, nifedipine failed to affect basal current (Fig. 3Ab). In contrast, it reversibly blocked the FPL-stimulated current (Fig. 3Ac) over the entire range of depolarization potentials (V_m; Fig. 3B). We also tested the effectiveness of Ni²⁺. At baseline, Ni²⁺ (1,000 μM) blocked a very small, depolarization-induced inward current (Fig. 4B). Between 100 and 1,000 μM, Ni²⁺ potently and reversibly inhibited the FPL-stimulated inward currents (Fig. 4C).

Fig. 3.

Nifedipine (Nifd) sensitivity of FPL-stimulated I_Ca. Aa: inward currents elicited in 1 mM extracellular Ca²⁺ by sequential depolarizations from −90 to 0 mV at baseline, in FPL (10 μM), in FPL + nifedipine (1 μM), and after nifedipine washout. Note prominent, rapidly inactivating Na⁺ current (I_Na) followed by slowly inactivating FPL-stimulated I_Ca. Nifedipine blocks I_Ca. Ab: I_Ca at baseline (without FPL), in nifedipine (1 μM), and during nifedipine washout. n = 5. Ac: in experiments similar to example in Aa, FPL increased I_Ca from −0.2 ± 9.0 to −52.6 ± 7.8 pA and nifedipine (1 μM) inhibited I_Ca to −7.3 ± 3.5 pA in a reversible manner. n = 10. *P < 0.05, **P < 0.01. B: voltage dependence of I_Ca in the presence and absence of nifedipine (reproduced from Fig. 1Bb).

Fig. 4.

Inhibition of FPL-stimulated I_Ca by Ni²⁺. A: inward currents elicited by sequential depolarizations from −100 to −10 mV in 1 mM extracellular Ca²⁺ + FPL (10 μM) in increasing concentrations of Ni²⁺ (0, 10, 100, 1,000 μM). B: effects of Ni²⁺ on unstimulated I_Ca. Ni²⁺ had a small, but significant, effect at 1,000 μM. n = 9. *P < 0.05 vs. 0 μM. C: effects of Ni²⁺ on FPL-stimulated I_Ca. Ni²⁺ achieved effective blockade at 100 and 1,000 μM. n = 13. *P < 0.05 vs. 0 μM.

Relative conductance of pericyte Ca_V currents to Ca²⁺ and Ba²⁺.

Conductance of L-type channels to Ba²⁺ generally exceeds that to Ca²⁺ by a factor of ∼2:1, whereas conductance of T-type channels to Ca²⁺ and Ba²⁺ is nearly equal (51, 56). To increase overall inward current to examine that characteristic, we increased the concentration of divalent charge carriers to 10 mM. As shown in Fig. 5A, large FPL-stimulated Ba²⁺ currents (peak −399 pA at −20 mV) exceeded Ca²⁺ currents (peak −251 pA at −10 mV). In a separate series of experiments, we repetitively depolarized individual cells from −110 to −10 mV and exchanged extracellular buffer to contain Ca²⁺ or Ba²⁺ (10 mM) in random order to yield a more rigorous paired comparison of their conductances. Mean currents for Ba²⁺ and Ca²⁺ in FPL were −378 ± 31 and −165 ± 21 pA (P < 0.01), respectively (Fig. 5B).

Fig. 5.

Comparison of relative conductance of FPL-stimulated pericytes to Ba²⁺ and Ca²⁺. A: voltage dependence of peak inward I_Ba and I_Ca in FPL (10 μM) with 10 mM extracellular Ba²⁺ and Ca²⁺. I_Ba exceeded I_Ca, and its activation threshold was shifted toward more negative potentials. n = 9 and 12 for I_Ba and I_Ca, respectively. *P < 0.05, I_Ba vs. I_Ca. B: paired comparison of I_Ba and I_Ca in FPL (10 μM) and extracellular Ca²⁺ or Ba²⁺ (10 mM) exchanged into the bath in random order. n = 10. **P < 0.01, I_Ba vs. I_Ca.

Since unstimulated Ca_V currents in 1 mM Ca²⁺ are too small to study (Fig. 1), we further optimized our conditions for measurement of basal currents by substituting BAPTA (10 mM) for EGTA to rapidly chelate Ca²⁺, thereby reducing any Ca²⁺-dependent inhibition (46). Moreover, we maintained extracellular concentrations of Ca²⁺ and Ba²⁺ at 10 mM (rather than 1 mM) to increase whole cell current and included TTX in the extracellular buffer to prevent the fast voltage-gated Na⁺ current from contaminating any rapidly inactivating T-type currents that might exist. Under those conditions, the basal inward currents were large enough to be measured without use of FPL as an agonist. Where seals were stable, activation and inactivation characteristics for Ba²⁺ and Ca²⁺ (Fig. 6) were examined in random order in the same cells. As shown in Fig. 6A, between test potentials of −10 and 20 mV, basal Ba²⁺ currents consistently exceeded those of Ca²⁺ by >2. Both activated at a threshold of about −30 mV, a somewhat higher potential than observed in FPL (−50 to −40 mV; Fig. 5A). These collective findings are consistent with predominance of L-type Ca_V basal conductance under these experimental conditions. Examination of the voltage dependence of inactivation of basal currents by use of a protocol similar to that used in Fig. 2Aa reinforced this relationship (Fig. 6B) and yielded the Boltzmann fits illustrated in Fig. 6, Ca and Cb. Window currents for both divalent ions are between −40 and +10 mV.

Fig. 6.

Voltage dependence of inactivation and activation in unstimulated DVR pericytes. A: voltage dependence of peak inward I_Ba and I_Ca with 10 mM extracellular Ba²⁺ and Ca²⁺ and BAPTA as electrode chelator. n = 9 and 19 for I_Ca and I_Ba, respectively. *P < 0.05, I_Ba vs. I_Ca. B: voltage dependence of inactivation for Ba²⁺ and Ca²⁺. n = 13 and 21 for I_Ca and I_Ba, respectively. P < 0.05 for all holding potentials less than −10 mV. Ca: Boltzmann fits to activation and inactivation for I_Ca yielded V_1/2 and slope factor of −39.6 mV and 10 for inactivation and 2.8 mV and 7.7 for activation. Cb: Boltzmann fits to activation and inactivation for I_Ba yielded V_1/2 and slope factor of −29.2 mV and 9.2 for inactivation and −5.6 mV and 6.1 for activation.

Sensitivity of pericyte Ba²⁺ currents to L-type channel blockade.

Using the same electrode buffer (BAPTA) and 10 mM Ba²⁺ as charge carrier, we explored the sensitivity of the basal pericyte inward currents to the classical L-type CCB nifedipine and diltiazem. As shown in Fig. 7A, nifedipine (1 μM) was highly effective in eliminating the Ba²⁺ currents. Diltiazem (10 μM) was also effective but exhibited voltage dependence; it was ineffective when cells were held at −90 mV (Fig. 7Ba) but highly effective when the holding potential was raised to −40 mV (Fig. 7Bb). This suggests improvement of access of diltiazem to its binding site with increases in membrane potential (53). The effectiveness of diltiazem in Fig. 7B is consistent with our previous observation that it blocks ANG II-induced vasoconstriction and KCl-induced pericyte [Ca]_CYT elevation (61).

Fig. 7.

Inhibition of I_Ba by nifedipine and diltiazem. A: peak I_Ba in pericytes held at −90 mV and depolarized to test potentials (V_m) in 10 mM Ba²⁺. Inward currents are at baseline, in nifedipine (1 μM), and after washout. n = 8. *P < 0.05, baseline vs. nifedipine. B: peak I_Ba in pericytes held at −90 mV and depolarized to test potentials in 10 mM Ba²⁺ at baseline, in diltiazem (10 μM), and after washout (n = 5). C: peak I_Ba in pericytes held at −40 mV and depolarized to test potentials in 10 mM Ba²⁺ at baseline, in diltiazem, and after washout. n = 5. *P < 0.05, baseline vs. diltiazem.

In view of the exquisite sensitivity of inhibition of rabbit afferent arteriolar KCl constriction to the black mamba toxin calciseptine (EC₅₀ = 80 fM) (22), we also examined its ability to block Ba²⁺ currents in DVR pericytes. Surprisingly, at a 10⁵-fold greater concentration (10 nM), it affected neither basal nor FPL-stimulated Ba²⁺ current (Fig. 8, A and B). Similarly, at the higher concentration of 30 nM, it failed to reverse ANG II (1 nM)-induced contraction of microperfused vessels (Fig. 8C).

Fig. 8.

Lack of effect of calciseptine to reverse I_Ba or ANG II-induced vasoconstriction. A: pericytes were held at −100 mV and depolarized to −10 mV for 300 ms at 10-s intervals. Concatenated responses before (baseline), during (calciseptine), and after (washout) introduction of calciseptine (10 nM) are shown. B: basal (n = 6) and FPL (10 μM, n = 9)-stimulated peak I_Ba using protocol described in A. Calciseptine was without effect. NS, not significant. C: percent reduction of luminal diameter vs. time in microperfused DVR exposed to abluminal ANG II (1 nM) followed by addition and removal of calciseptine (30 nM). Calciseptine failed to reverse ANG II-induced vasoconstriction.

Sensitivity of pericyte Ba²⁺ currents to mibefradil.

Given the growing literature concerning the role of T-type channels in the efferent circulation of the kidney (13, 22, 24, 26, 27, 31), we tested the ability of mibefradil to block DVR pericyte Ba²⁺ currents and vasoconstriction. Perfectly selective T-type Ca_V blockers do not exist; however, it is generally accepted that mibefradil inhibits T-type over L-type Ca_V when it is used at low concentrations, between 10 and 1,000 nM, which blocked efferent arteriolar constriction in other settings (13, 22, 25, 28, 31). In our hands, mibefradil (1 μM) affected neither Ba²⁺ currents nor ANG II-induced DVR vasoconstriction (Fig. 9, A and B). At higher concentration (10 μM), where mibefradil is likely to block L- and T-type Ca_V, it was an effective vasodilator (Fig. 9B).

Fig. 9.

Inhibition of I_Ba- and ANG II-induced vasoconstriction by mibefradil. A: peak I_Ba in pericytes held at −90 mV and depolarized to test potentials (V_m) in 10 mM Ba²⁺. Inward currents are shown at baseline, in mibefradil (1 μM, n = 7), and after washout. Mibefradil was without effect. B: microperfused DVR were exposed to abluminal ANG II followed by addition and removal of mibefradil at 0 μM (sham exchange, n = 6), 1 μM (n = 6), or 10 μM (n = 5). *P < 0.05 vs. sham exchange.

Sensitivity of pericyte Ba²⁺ currents to ω-agatoxin IVA.

Expression of Ca_V2.x in DVR pericytes has been observed by immunostaining, and the P/Q-type Ca_V inhibitor ω-agatoxin IVA (10 nM) is a potent blocker of afferent arteriolar constriction to KCl in the rabbit (2, 21). Since the Ba²⁺ > Ca²⁺ current and slow inactivation observed above might hypothetically contain a component of P/Q-type Ca_V conductance, we tested whether ω-agatoxin IVA provides any blockade. At ≤100 nM, ω-agatoxin IVA was ineffective (Fig. 10).

Fig. 10.

Lack of effect of ω-agatoxin IVA to reverse I_Ba. A: pericytes were held at −100 mV and depolarized to −10 mV for 300 ms at 10-s intervals. Concatenated responses before (vehicle) and during and after introduction of ω-agatoxin IVA are shown. B: peak inward I_Ba using the protocol described in A with ω-agatoxin IVA at 20 nM (n = 9) or 100 nM (n = 10). ω-Agatoxin IVA was without effect.

DISCUSSION

Ca²⁺ influx through Ca_V facilitates elevation of [Ca]_CYT during myocyte contraction. Ca_V are the product of 10 genes, the splice variants of which form the pore-forming α_1a- to α_1l-subunits of the channels. When combined with α₂-, β-, and δ-subunits, they yield an array of properties characterized by variable kinetic behavior, voltage dependence, and sensitivity to toxins and pharmaceuticals (7, 11, 12, 27, 28, 31, 33, 38, 40, 52, 54, 56). Many Ca_V classification schemes exist. One durable approach has been to designate them L-type (Ca_V1.x), T-type (Ca_V2.x), P/Q-type (Ca_V2.1), N-type (Ca_V2.2), and R-type (Ca_V2.3). L-type Ca_V, so designated to describe their long opening and slow rate of inactivation, are common therapeutic targets in the treatment of hypertension, angina, and arrhythmias via dihydropyridine (e.g., nifedipine), phenylalkylamine (verapamil), and benzothiazepine (diltiazem) antagonists. T-type Ca_V, so designated to describe their transient opening and rapid inactivation, are also widely expressed in the cardiovascular system and have become a therapeutic target of less selective antagonists, such as mibefradil, pimozide, and efonidipine, that block L- and T-type channels (27, 28, 38, 40, 56). That therapeutic class may provide additive antihypertensive efficacy, because T-type channels are expressed in smooth muscle of small-resistance vessels (3).

The actions of CCB in the renal circulation have been intensively investigated, and the results point to heterogeneity of expression and action in the cortex and medulla. CCB classes vary in their ability to reverse contraction of and Ca²⁺ entry into vascular smooth muscle of the glomerular afferent and efferent arteriole. Evidence also has shown that their efficacy to block efferent contraction may vary with location of the vessel in the superficial vs. juxtamedullary cortex (21, 22, 27, 31).

Despite reducing the electrochemical driving force for Ca²⁺ entry, membrane depolarization increases [Ca]_CYT of smooth muscle by gating Ca_V conductance. To achieve depolarization without use of vasoactive agonists, a popular maneuver has been to raise the Nernst potential of K⁺ from −90 to ∼0 mV by increasing its extracellular concentration. The ability of CCB to prevent the resultant contraction and rise in [Ca]_CYT is interpreted as evidence for Ca_V expression. In the hydronephrotic kidney preparation, KCl caused nifedipine-sensitive constriction of afferent arterioles that exceeded constriction of the efferent arteriole (35). Similarly, diltiazem preferentially blocked KCl-induced vasoconstriction of isolated perfused afferent arterioles (9). Examination of [Ca]_CYT responses yielded similar results: depolarization increased [Ca]_CYT of afferent, but not efferent, arteriolar smooth muscle (6).

Blockade of responsiveness to agonists such as ANG II has provided an alternate means to assess participation of Ca_V in intrarenal microvessel contraction. When ANG II-stimulated Ca²⁺ entry into interlobular and afferent arteriolar smooth muscle was examined using fura 2, it was found that ANG II-induced increases in [Ca]_CYT were dihydropyridine sensitive (30). Parallel studies found greater sensitivity of ANG II-stimulated [Ca]_CYT increases to nifedipine in the afferent than efferent arteriole (34). L-type signature currents have been elegantly described by electrophysiology in myocytes of the afferent circulation (2, 18). These and other studies have consistently established that L-type voltage-gated channels provide a functionally important route for Ca²⁺ entry into preglomerular smooth muscle.

More recent investigations have expanded the known roles of Ca_V in the kidney by documenting multiple subtype expression and the variable efficacy of pharmacological blockade in the afferent and efferent circulation. In particular, Hansen and colleagues (21, 22) described L-, T-, and P/Q-type channel expression in afferent smooth muscle, as well as both T- and L-type channel expression in efferent arterioles. Efferent Ca_V expression was confined to the juxtamedullary cortex. Those immunochemical observations were reinforced by demonstration of sensitivity of KCl-induced efferent contraction to blockade with calciseptine (an L-type channel antagonist) and mibefradil (a T-type channel blocker) (2, 21, 22, 31). Feng and colleagues (13–15) used the juxtamedullary nephron preparation to examine the roles of arteriolar Ca_V. Pimozide and mibefradil (T-type channel blockers) reduced basal tone of afferent and efferent arterioles. In contrast, successful blockade with diltiazem (L-type channel blocker) occurred only in afferent arterioles, except when generation of nitric oxide was prevented by nitric oxide synthase inhibition. In their hands, T-type channel blockade with pimozide reversed afferent and efferent contraction by ANG II (13–15). Elegant studies in the renal cortex, performed in vivo using a pencil lens camera, also documented that efferent arterioles are dilated by nonselective CCB, such as efonidipine and mibefradil (28). Taken together, a role for Ca_V, particularly T-type channels, in myogenic tone and agonist-induced contraction of juxtamedullary efferent arterioles is well supported, and the ability of combined T- and L-type channel blockade to provide renoprotection through efferent dilation has thus become the subject of clinical investigation (23, 49, 50).

Investigation of the role of Ca_V in the microcirculation of the medulla has lagged behind that of the cortex. Interstitial infusion of diltiazem and intravenous infusion of verapamil selectively raised medullary blood flow (19, 20, 36). Using single-vessel videomicroscopy, Yagil and colleagues (55) observed an increase in papillary vasa recta blood flow by the dihydropyridine blocker CS-905. The effects of CCB on medullary blood flow have also been examined in pathological models. Papillary plasma flow increased when verapamil was infused into the renal artery of dogs subjected to caval constriction (8). Treatment of the spontaneously hypertensive rat with nisoldipine enhanced medullary blood flow and Na⁺ excretion (16). Taken together, the ability of L-type CCB to increase renal medullary blood flow has been a consistent finding. DVR are contractile branches of the juxtamedullary cortical circulation; therefore, it is uncertain whether the effects of L-type channel blockade are attributable to DVR vs. upstream segments.

Because of the relative inaccessibility of outer medullary DVR in vivo, their properties have been studied in isolated vessels (41, 42) or tissue preparations (10). The first evidence favoring participation of Ca_V in vasa recta was the consistency of ANG II-induced pericyte depolarization (44) and the ability of L-type blockade with diltiazem to vasodilate preconstricted vessels and block [Ca]_CYT responses to ANG II, KCl, and PKC activation (60, 61). Robust ability of vasoactive agonists to depolarize pericytes through variable combination of Ca²⁺-dependent Cl⁻ channel activation and K⁺ channel suppression has been a consistent finding (4, 43, 44). Motivated by the associated studies that clearly show Ca_V in juxtamedullary glomerular arterioles (27, 31), we performed the present study of signature Ca_V currents in DVR pericytes. Our initial studies revealed a surprising voltage-gated Na⁺ (Na_V) conductance (32, 58). Ca_V currents, under the conditions used in those studies, were too small to measure. Here, we revised the experimental protocols to enhance the divalent currents, enabling examination of their properties.

In a first series of experiments, we used the dihydropyridine agonist FPL to increase conductance of putative L-type Ca_V. FPL elicited small, but detectable, inward current when 1 mM extracellular Ca²⁺ was the charge carrier (Fig. 1). The slow inactivation, threshold for activation near −40 mV (Fig. 2), and high conductance to Ba²⁺ over Ca²⁺ is consistent with L-type channel behavior (17, 51). Thus the FPL-based studies strongly support Ca_V1.x expression in DVR pericytes but do not rule out conductance by other isoforms.

In an effort to study Ca_V currents in the absence of agonist stimulation, we altered the experimental regimen to increase their magnitude. Since some classes of Ca_V are inhibited by Ca²⁺, we employed BAPTA in place of EGTA to achieve rapid chelation in the vicinity of the channel pore (46). Additionally, we increased the concentration of the extracellular charge carrier from 1 to 10 mM. Those changes yielded the desired effect, such that basal Ba²⁺ and Ca²⁺ currents became readily detectable (Fig. 6). L-type currents peak much later than voltage-gated Na⁺ currents (Fig. 3Aa), so that there is little risk of an error in quantifying Ca_V and Na_V currents when both are present. In contrast, T-type currents typically activate and deactivate more quickly, leading us to use TTX to block Na_V when quantifying basal Ca_V currents of the pericytes. In the absence of FPL, the voltage dependence of the currents shifted by about +10 mV, i.e., stronger depolarizations (to −30 vs. −40 mV in FPL; cf. Fig. 5A with Fig. 6A) were needed to activate the channels. That threshold is more consistent with an L- than a T-type current, which typically activates at lower potentials (56). For specific contrast, the results in Fig. 6, can be compared with the elegant studies of Gordienko et al. (Figs. 7 and 8 in Ref. 18) in which low- and high-threshold T- and L-type currents coexist in isolated preglomerular smooth muscle cells. As discussed above, much other evidence favoring roles for T-type channels has been obtained through pharmacological inhibition of vasoconstriction and immunostaining (13, 15, 22, 26, 28, 31, 47). Evidence that basal DVR pericyte currents are also largely conducted by Ca_V1.x, L-type channels is provided by their near elimination with nifedipine and diltiazem (Fig. 7) and twofold greater conductance to Ba²⁺ over Ca²⁺ (Fig. 6A).

The open probability of Ca_V is determined by a balance of states in which the channels can be opened in response to elevation of membrane potential and inactivation in which the channels cannot respond. Electrophysiologically, that is controlled by holding cells at low potential to remove inactivation and then rapidly shifting to higher potentials to achieve activation; subsequently, closure to the inactivated state occurs spontaneously to limit Ca²⁺ conductance. The range of membrane potentials at which channels can activate without becoming fully inactivated defines the physiological range for function, i.e., the window current. As shown in Figs. 2B and 6C, the window current lies between −40 and +10 mV for basal Ca²⁺ and Ba²⁺ conductance but is shifted toward more negative values (by ∼10 mV) in the presence of FPL. Our prior examination of resting membrane potentials in DVR pericytes and its depolarization in response to agonists (ANG II and endothelin) has shown a shift from −50 to −70 mV to −40 to −20 mV, within the window current range defined here (4, 44).

We extended our studies by testing the effects of other classical Ca_V blockers, calciseptine, mibefradil, and ω-agatoxin IVA. Calciseptine is a toxin derived from the black mamba that purportedly blocks L- over T- or N-type channels (39, 56). Despite the high sensitivity of isolated rabbit arterioles to calciseptine (22, 56), in a separate series of experiments, we could not demonstrate blockade of FPL-stimulated or unstimulated (basal) Ba²⁺ currents with that agent (Fig. 8, A and B). Calciseptine also failed to reverse ANG II-induced vasoconstriction, mitigating against an effect attributable to ruptured-patch methods (Fig. 8C). Given the otherwise strong evidence for L-type channels in this preparation (Figs. 1–7), we speculate that a calciseptine-insensitive Ca_V1.x splice variant exists in the renal medulla of the rat, possibly conferring some resistance to snake venoms. At 1 μM, mibefradil failed to block Ba²⁺ currents or ANG II-induced vasoconstriction (Fig. 9), providing evidence against T-type currents contaminating the results of prior protocols (Fig. 6) and also against participation of T-type channels in ANG II-induced vasoconstriction. At 10 μM, mibefradil dilated microperfused DVR, but that may well be attributable to L-type channel blockade at that concentration. Finally, despite the description of their expression in medullary vascular bundles, P/Q-type channel blockade with ω-agatoxin IVA failed to affect depolarization-induced inward Ba²⁺ currents (Fig. 10). We recognize that the failure to observe signature T- or P/Q-type currents does not rule out participation of such channels in DVR vasoactivity under other physiological conditions. Those channels may be present, but they may be dormant because of posttranslational modifications, internalization, or other inhibitory signaling processes that we have yet to define.

GRANTS

Studies in our laboratory were supported by National Institutes of Health Grants DK-42495, DK-67621, and HL-68686.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.