L-type Ca2+ channels predominantly influence preglomerular arterioles, but there is less information regarding the role of T-type Ca2+ channels in regulating the renal microvasculature. We compared the effects of T- and L-type channel blockade on afferent and efferent arterioles using the in vitro blood-perfused juxtamedullary nephron preparation. Single afferent or efferent arterioles of Sprague-Dawley rats were visualized and superfused with solutions containing Ca2+ channel blockers. We confirmed that L-type channel blockade with diltiazem dilates afferent arterioles but has no significant effects on efferent arterioles. In contrast, T-type channel blockade with pimozide (10 μmol/l) or mibefradil (1 μmol/l) dilated both afferent (26.8 ± 3.4 and 24.6 ± 1.9%) and efferent (19.2 ± 2.9 and 19.1 ± 4.8%) arterioles. Adding diltiazem did not significantly augment the dilation of afferent arterioles elicited by pimozide and mibefradil, and adding pimozide after diltiazem likewise did not elicit further vasodilation. Diltiazem blocked the depolarization-induced afferent arteriolar constriction elicited by 55 mM KCl; however, the constrictor response to KCl remained intact during treatment with 10 μM pimozide. Pimozide also prevented the afferent arterioles from exhibiting autoregulatory-mediated constrictor responses to increases in perfusion pressure. We conclude that T-type channel blockers dilate efferent arterioles as well as afferent arterioles and diminish afferent arteriolar autoregulatory responses to changes in perfusion pressure. To the extent that these agents exert their effects primarily on T-type Ca2+ channels in our experimental setting, these results indicate that T-type channels are functionally expressed in juxtamedullary afferent and efferent arterioles and may act cooperatively with L-type channels to regulate afferent arteriolar resistance. Because L-type channels are not functionally expressed in efferent arterioles, T-type channels may be particularly significant in the regulation of efferent arteriolar function.
- renal autoregulation
voltage-dependent Ca2+ channels are divided into two classes: high-voltage-activated (HVA) Ca2+ channels including the L-type channels (CaV1), the neuronal N-, P/Q-, and R-type channels (CaV2), and low-voltage-activated (LVA) Ca2+ channels, the T-type channels (CaV3) (18, 48). The T-type Ca2+ channel is characterized by low-threshold activation, fast inactivation, slow deactivation kinetics, and small unitary conductance (4, 48). T-type Ca2+ currents are involved in many important cellular processes of the cardiovascular system, such as in the electromechanical coupling in smooth muscle cells and in the sinoatrial node pacemaker activity (34, 57). Both HVA and LVA Ca2+ currents have been observed previously in vascular smooth muscle (VSM) cells from preparations including mesenteric artery, coronary artery, rabbit ear artery, and renal arteries (1, 5, 6, 20, 23, 25). There is electrophysiological evidence for the presence of both L-type Ca2+ currents and T-type Ca2+ currents in single VSM cells of preglomerular vessels (23, 52). L-type Ca2+ channel blockers have been shown to exert their vasodilatory effects predominantly on preglomerular arterioles and have only modest or nonsignificant effects on efferent arterioles (10, 19, 31, 38, 54, 55). In addition, treatment with L-type Ca2+ channel blockers prevents or diminishes autoregulatory responses of afferent arteriolar diameter and renal blood flow (24, 26, 46, 54).
More recently, increased attention has been focused on T-type Ca2+ channels and their possible physiological and pathophysiological roles. Three genes encoding the T-channel pore subunits were identified and designated α1G (CaV3.1), α1H (CaV3.2), and α1I (CaV3.3) (13, 37, 43, 48, 62). T-type Ca2+ channels CaV-3.1 and CaV-3.2 have been shown to be expressed in the kidney including juxtamedullary efferent arterioles of rat kidney (25). Administration of mibefradil, a T-type Ca2+ channel blocker, was shown to reduce afferent and efferent arteriolar resistances in spontaneously hypertensive rats (45), increase renal blood flow in vivo in dogs (28), and block the ANG II-induced efferent arteriolar constriction observed in the isolated, perfused hydronephrotic rat kidney (47). However, the role of T-type Ca2+ channels in renal autoregulation and its effect on the in vitro juxtamedullary-perfused nephron model have not been determined.
We tested the hypothesis that T-type Ca2+ channels are functionally expressed in both juxtamedullary afferent and efferent arteries and contribute to the basal regulation of the pre- and postglomerular arteriolar resistances. We used the in vitro blood-perfused juxtamedullary nephron technique and a potent T-type Ca2+ channel blocker pimozide (3, 7, 12, 15, 21, 27, 48) to compare the effects of T-type and L-type Ca2+ channel blockade on afferent and efferent arteriolar diameters of juxtamedullary nephrons and on the interaction between T-type and L-type Ca2+ channels. However, it is recognized that the T-type blockers available are not completely selective. As a means of assessing the effects of pimozide on L-type Ca2+ channels in afferent arteries, its ability to block the effects of 55 mM KCl on depolarization-induced constriction was tested. We further determined the effects of T-type channel blockade on the afferent arteriolar autoregulatory responses to increases in renal perfusion pressure.
MATERIALS AND METHODS
The experiments were performed in accordance with the guidelines and practices established by the Tulane University Animal Care and Use Committee.
Afferent and efferent arteriolar diameters were assessed in vitro using the isolated blood-perfused juxtamedullary nephron technique combined with videomicroscopy, as previously described (10, 26, 54). Each experiment used one male Sprague-Dawley rat (Charles River Laboratories, Wilmington, MA), weighing 370-410 g, serving as blood donor and kidney donor. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a cannula was inserted in the left carotid artery for blood collection. Donor blood was collected in a heparinized (500 U) syringe via the carotid arterial cannula and centrifuged to separate the plasma and cellular fractions. The buffy coat was removed and discarded. After sequential passage of the plasma through 5- and 0.22-μm filters (Gelman Sciences, Ann Arbor, MI), erythrocytes were added to achieve a hematocrit of 33%. This reconstituted blood was passed through a 5-μm nylon mesh and thereafter stirred continuously in a closed reservoir that was pressurized with a 95% O2-5% CO2 gas mixture.
The right kidney was perfused through a cannula inserted in the superior mesenteric artery and advanced into the right renal artery. The perfusate was a Tyrode's solution (pH 7.4) containing 5.1% BSA and a mixture of L-amino acids thereafter stirred continuously in a closed reservoir that was pressurized with a 95% O2-5% CO2 gas mixture. The kidney was excised and sectioned longitudinally, retaining the papilla intact with the perfused dorsal two-thirds of the organ. The papilla was reflected to expose the pelvic mucosa and tissue covering the inner cortical surface. Overlying tissue was removed to expose the tubules, glomeruli, and related vasculature of the juxtamedullary nephrons. The arterial supply of the exposed microvasculature was isolated by ligating the larger branches of the renal artery.
After the dissection was completed, the Tyrode's perfusate was replaced with the reconstituted blood. Perfusion pressure was monitored by a pressure catheter centered in the tip of the perfusion cannula. Renal perfusion pressure was regulated by adjusting the rate of gas inflow into the blood reservoir and set at 100 mmHg. The inner cortical surface of the kidney was continuously superfused with a warmed (37°C) Tyrode's solution containing 1% BSA. The tissue was transilluminated on the fixed stage of a microscope (Nikon) equipped with a water-immersion objective (×40). Video images of the microvessels were transferred by a Newvicon camera (model NC-67M; Dage-MTI, Michigan City, IN) through an image enhancer (model MFJ-1452; MFJ Enterprises, Starkville, MS) to a video monitor (Conrac Display Systems, Covina, CA). The video signal was recorded on videotape for later analysis. Afferent and efferent arteriolar inside diameters were measured at 30-s intervals using a calibrated digital image-shearing monitor (Instrumentation for Physiology and Medicine, San Diego, CA). Single afferent or efferent arterioles were visualized. Treatments were administered by superfusing the tissue with a Tyrode's solution containing the agent to be tested or vehicle. Pimozide and diltiazem were obtained from Sigma Chemical (St. Louis, MO) and mibefradil was kindly provided by Dr. J.-P. Clozel (Hoffmann La Roche, Basel, Switzerland).
Experimental protocols. A single afferent or efferent arteriole that showed adequate blood flow was selected for study. After a 10-min equilibration period, an experimental protocol was initiated consisting of consecutive 10-min treatment periods. Steady-state diameter determinations were calculated from the average of measurements obtained during the final 5 min of each 10-min treatment period.
The first experimental protocol was performed to determine the effects of the T-type Ca2+ channel blockers, pimozide and mibefradil (3, 7, 12, 15, 21, 27, 48), and compare them with those of the L-type Ca2+ channel blocker, diltiazem, on afferent and efferent arterioles. Arteriolar inside diameter was measured at a renal arterial pressure of 100 mmHg during control conditions and after sequential exposure of the vessel to superfusate solutions of various compositions. The dose-response relationship of pimozide on afferent and efferent arteriolar diameters using pimozide at a concentration of 0.1, 1, 10, and 100 μmol/l was established. The effects of pimozide (10 μmol/l), mibefradil (1 μmol/l), and diltiazem (10 μmol/l) on afferent and efferent arteriolar diameters were compared.
A second series of experiments was performed to determine the synergy or overlap between the effects of T-type and L-type Ca2+ channel blockers. Afferent arteriolar inside diameter was measured during sequential exposure of the kidney to vehicle, 5 μmol/l pimozide and 5 μmol/l pimozide plus 10 μmol/l diltiazem; or 1 μmol/l mibefradil and 1 μmol/l mibefradil plus 10 μmol/l diltiazem. Alternately, afferent arterioles were first treated with diltiazem followed by diltiazem plus pimozide.
A third series of experiments was performed to determine the effects of the Ca2+ channel blockers in inhibiting voltage-dependent afferent arteriolar vasoconstriction elicited by depolarizing concentrations of KCl. Experiments involved a control period followed by a 10-min exposure to an isotonic solution containing 55 mM KCl or a 5-min exposure to 10 μmol/l pimozide or 10 μmol/l diltiazem followed by superfusion with a solution containing 10 μmol/l pimozide or 10 μmol/l diltiazem plus 55 mM KCl (30, 35, 44). The afferent arteriolar response to KCl was reassessed. The superfusion solution was modified by replacing part of the NaCl with KCl but maintaining the original isoosmolality and all the other constituents in the normal Tyrode's solution.
A fourth series of experiments was conducted to determine the role of T-type Ca2+ channels in mediating autoregulatory responses. Autoregulatory behavior was assessed by increasing renal arterial pressure in a step-wise manner from 100 to 125 and 150 mmHg. Renal arterial pressure was kept constant at each pressure step for at least 3 min before subsequent changes in pressure. After the control studies, the tissue was superfused with Tyrode's solution containing 5 or 10 μmol/l pimozide, and the process as described above testing the responses to increases in renal arterial pressure was repeated.
Statistical analysis. All data are reported as means ± SE. Data were analyzed by two-way ANOVA or one-way ANOVA, followed by a Bonferroni's multiple-comparison post hoc test. Values of P < 0.05 were considered statistically significant.
Effects of pimozide, mibefradil, and diltiazem on afferent and efferent arteriolar diameters. As illustrated in Fig. 1, superfusion with 0.1, 1, 10, and 100 μmol/l pimozide caused dilation of afferent arterioles with diameters increasing from 17.0 ± 0.4 to 17.4 ± 0.5, 19.8 ± 0.5, 21.7 ± 0.3, and 22.0 ± 0.4 μm, respectively (2.1 ± 0.4, 16.2 ± 1.7, 27.5 ± 3.5, and 29.5 ± 3.1%, n = 5). Pimozide also caused dilation of efferent arterioles with diameters increasing by 4.4 ± 0.6, 15.8 ± 1.7, 21.3 ± 2.2, and 22.8 ± 2.7%, n = 5, respectively. Although the lower concentrations of pimozide caused significant dilation of afferent and efferent arterioles, the most effective concentration used was 10 μmol/l. Figure 2 illustrates the afferent and efferent arteriolar responses to pimozide, mibefradil, and diltiazem. As previously reported (10, 54), superfusion with solutions containing diltiazem (10 μmol/l) caused a dilation of afferent arterioles, with average diameters increasing from 17.2 ± 1.2 to 22.3 ± 1.7 μm, for an increase of 29.6 ± 3.5% (n = 7, P < 0.01). Diltiazem did not exert significant actions on efferent arterioles, which varied slightly from 20.5 ± 0.3 to 21.0 ± 0.3 μm (n = 6, P > 0.05). Pimozide (10 μmol/l) and mibefradil (1 μmol/l) caused dilation of afferent arterioles with diameters increasing from 16.5 ± 0.4 to 20.8 ± 0.6 μm (26.8 ± 3.4%, n = 12, P < 0.01) and from 16.7 ± 0.5 to 20.8 ± 0.7 μm (24.6 ± 1.9%, n = 5, P < 0.01), respectively. Pimozide at a lower concentration (5 μmol/l) also caused a similar dilation of afferent arterioles with diameter increasing 25.0 ± 3.2% (n = 7, P < 0.01). In contrast to the predominant preglomerular actions of diltiazem, pimozide and mibefradil elicited significant dilation of efferent arterioles with diameters increasing 20.3 ± 0.3 to 24.2 ± 0.5 μm (19.2 ± 2.9%, n = 12, P < 0.01) and from 18.8 ± 0.4 to 22.4 ± 1.1 μm (19.1 ± 4.8%, n = 5, P < 0.01), respectively.
As shown in Fig. 3, adding diltiazem (10 μmol/l) to the pimozide (5 μmol/l)- or mibefradil (1 μmol/l)-containing solutions did not elicit further afferent arteriolar dilation than that caused by pimozide or mibefradil alone with diameter changing from 19.7 ± 1.2 to 20.1 ± 1.2 μm, 1.5 ± 0.5% (n = 7, P > 0.05) and from 20.8 ± 0.7 to 21.0 ± 0.5 μm, 1.2 ± 0.9% (n = 5, P > 0.05), respectively. Thus diltiazem superimposed on pimozide and mibefradil did not elicit further dilation of afferent arterioles. To test if the pimozide effects would be additive or overlapping with diltiazem, the order was reversed with pimozide (10 μmol/l) added to the diltiazem (10 μmol/l). Renal perfusion pressure was raised to 150 mmHg to increase vascular tone further. Pimozide (10 μmol/l) added to diltiazem (10 μmol/l)-treated afferent arterioles did not elicit further dilation with diameter increasing only slightly from 17.7 ± 1.8 to 18.2 ± 1.7 μm, 3.4 ± 1.1% (n = 4).
Effects of pimozide and diltiazem on afferent arteriolar response to KCl. Figure 4 shows the effects of T- and L-type Ca2+ channel blockers in inhibiting voltage-dependent afferent arteriolar vasoconstriction elicited by superfusion with a solution containing 55 mM KCl, which has been shown to directly depolarize the membrane and open L-type Ca2+ channels (30, 35, 44). KCl elicited a marked constriction of afferent arterioles, with average diameter decreasing from 17.2 ± 0.5 to 9.8 ± 0.5 μm for a decrease of 43.1 ± 2.6% over a 10-min period (n = 6, P < 0.01). Pretreatment with pimozide (10 μmol/l) caused initial vasodilation but failed to prevent the vasoconstriction induced by high KCl. Afferent arteriolar diameter decreased from 20.1 ± 0.9 to 12.9 ± 1.2 μm for a decrease of 35.5 ± 6.4% (n = 5, P < 0.01). In contrast, pretreatment with diltiazem (10 μmol/l) dilated the afferent arterioles and prevented the sustained vasoconstriction with afferent arteriolar diameter increasing from 20.6 ± 1.1 to 21.3 ± 1.3 μm over a 10-min period for a slight increase of 3.2 ± 1.3% (n = 5, P < 0.01 vs. KCl alone group and KCl plus pimozide group).
Effects of pimozide on autoregulation of afferent arteriolar diameters in response to increased renal perfusion pressure. The effects of pimozide at concentrations of 5 and 10 μmol/l on afferent arteriolar diameter responses to elevations of renal perfusion pressure are shown in Fig. 5. Afferent arteriolar diameter averaged 19.2 ± 0.7 μm at 100 mmHg and decreased significantly to 16.2 ± 1.0 μm (16.1 ± 2.3%, n = 6, P < 0.01) and 14.0 ± 1.3 μm (27.6 ± 4.0%, n = 6, P < 0.01) with elevations in renal perfusion pressure to 125 and 150 mmHg, respectively. In contrast, afferent arteriolar diameter actually increased slightly from 21.2 ± 0.6 to 21.7 ± 0.6 μm (2.3 ± 0.8%, n = 5, P < 0.01 vs. control group) and to 22.1 ± 0.6 μm (4.0 ± 1.2%, n = 5, P < 0.01 vs. control group) and 21.5 ± 1.1 to 22.4 ± 1.0 μm (4.2 ± 1.3%, n = 6, P < 0.01 vs. control group) and to 23.4 ± 1.1 μm (9.1 ± 2.9%, n = 6, P < 0.01 vs. control group) in the presence of 5 or 10 μmol/l pimozide, respectively. Thus 5 μmol/l as well as 10 μmol/l pimozide prevented the autoregulatory-mediated afferent arteriolar vasoconstriction in response to increases in renal perfusion pressure.
It is well recognized that voltage-gated Ca2+ channels are important for regulating entry of extracellular Ca2+ in VSM cells. The influx of Ca2+ is essential for the development of muscle tension, which mediates arterial vasoconstriction, and ultimately influences systemic blood pressure. Renal resistance arteries rely on Ca2+ influx pathways to maintain their normal tone and can undergo either dilation or constriction, depending on the contractile state of the VSM cells. Ca2+ channels have been shown to exert substantial influences on the renal microvasculature and on renal hemodynamic function (10, 23, 25, 26, 38, 45, 47, 52, 54). In particular, the ability of the renal microvasculature to exhibit autoregulatory-mediated increases in renal vascular resistance in response to increases in renal perfusion pressure is dependent on the integrity of Ca2+ entry via L-type Ca2+ channels (26, 46, 54).
The present study confirmed that diltiazem, a selective L-type Ca2+ channel antagonist, elicits marked dilation of afferent arterioles but does not have significant effects on efferent arterioles and further demonstrates that the T-type Ca2+ channel blockers pimozide and mibefradil elicit dilation of both preglomerular arterioles and postglomerular arterioles in the juxtamedullary nephron preparation. To the extent that these agents exert their effects primarily on T-type channels in our experimental setting, these findings are consistent with an important role of T-type Ca2+ channels in regulating afferent and efferent arteriolar tone. These data help explain results from in vivo dog experiments, using the T-type Ca2+ channel blocker mibefradil in which it was shown that both nifedipine and mibefradil increase renal blood flow but only nifedipine increased glomerular filtration rate (28). Our observations are supported further by studies of Hansen et al. (25) that T-type Ca2+ channels (CaV3.1 and CaV3.2) are expressed on both afferent and efferent arterioles of juxtamedullary nephrons. However, we recognize that there is no absolutely specific T-type Ca2+ channel blocker available. Our choice of agents was based on studies showing that mibefradil has a 10-fold greater selectivity for T-compared with L-type Ca2+ channels (42, 56) and was 79 times less effective than lacidipine in reducing the vasoconstriction induced by 60 mM KCl (41). Furthermore, pimozide has been shown to be an even more potent relatively selective T-type Ca2+ channel blocker (3, 7, 12, 15-17, 21, 27, 48, 51). Santi et al. (51) tested serial neuroleptics, finding that pimozide is the most potent T-type channel blocker, and other investigators showed that mibefradil was less potent than pimozide at blocking various T-type Ca2+ channels (4, 22). Nevertheless, it is possible that other uncharacterized mibefradil- and pimozide-sensitive channels, other than the well-characterized T-type channels, could play a role in the responses observed. Although pimozide can also block dopamine D2 receptors, it has been demonstrated that endogenous dopamine and low-dose exogenous dopamine cause renal arteriolar dilation, increase renal blood flow, and decrease renal vascular resistance due to activation of dopamine D1 and D2 receptors (2, 33). In the isolated, perfused hydronephrotic rat kidney, Steinhausen et al. (53) observed that superfusion with dopamine dilated both the afferent and efferent arterioles and this effect was inhibited by a dopamine receptor antagonist. Thus blocking dopamine receptors would be expected to cause vasoconstriction, not vasodilation, making it unlikely that the dilation of afferent and efferent arterioles caused by pimozide is associated with blockade of dopamine receptors.
Similar to other T-type Ca2+ channel blockers, pimozide possesses modest L-type channel blocking action, but the present study demonstrates that the efferent arterioles that are not dilated by L-type channel blocker were markedly dilated following treatment with T-type channel blockers. In addition, it has been demonstrated that the localization of L-type Ca2+ channels is predominantly on VSM cells of preglomerular arterioles (22), Ca2+ influx pathways in efferent vessels are not activated by depolarization (38), and L-type channels are not functionally expressed in efferent arterioles (9, 10, 19, 55). Thus the mibefradil- and pimozide-induced efferent vasodilation cannot be due to an effect to block L-type Ca2+ channels.
Although it is recognized that the effects on afferent arterioles could be due to blockade of L-type Ca2+ channels, it is widely accepted that L-type HVA currents require a strong depolarization to evoke opening. In contrast, T-type LVA currents are evoked by weak depolarizations. Membrane depolarization by high KCl is known to stimulate Ca2+ entry through activation of HVA Ca2+ channels (35, 36). In the study of Murphy et al. (44), KCl (51 mM) was shown to cause a 31 ± 1.8% contraction and an increase in cytosolic Ca2+ to 288 ± 12 nM in renal arterial smooth muscle cells of rats. L-type Ca2+ channel blockade can block the high KCl-induced current, but T-type Ca2+ channel blockade cannot. In our study, we confirmed that the L-type Ca2+ channel blocker diltiazem prevented the afferent arteriolar vasoconstriction elicited by high-KCl solution. In contrast, we found that T-type Ca2+ channel blockade with pimozide did not prevent the vasoconstriction elicited by high KCl. These results are consistent with previous reports that diltiazem blocks the KCl-induced vasoconstriction in afferent arterioles of juxtamedullary nephrons (30) and that in isolated afferent arterioles, the increased [Ca2+]i elicited by depolarization with medium containing 50 mM KCl was totally blocked by nifedipine (49) and further supported by recent reports that the high KCl-induced cell contraction and increase in [Ca2+]i are prevented by L-type Ca2+ channel blockade in renal arterial smooth muscle cells (44). Also, the [Ca2+]i transient evoked by high-KCl depolarization of two-cell embryo was inhibited by diltiazem and verapamil but was not blocked by pimozide at a concentration of 25 μmol/l (40). We observed that pimozide at a concentration of 10 μmol/l did not block KCl-induced vasoconstriction, indicating that even at this concentration pimozide does not exert substantive effects on L-type channels. Even at a lower concentration (1 μmol/l), thought to have minimal effect on L-type Ca2+ channels, pimozide caused significant afferent and efferent vasodilation indicating that the vasorelaxation caused by pimozide is not likely to be due to a direct blockade of L-type Ca2+ channels in afferent arterioles. These findings are also consistent with a recent finding showing that nifedipine blocked the conducted Ca2+ response to electrical stimulation of isolated renal interlobular arteries but mibefradil in concentrations up to 1 μM did not (50).
To determine the degree of interaction in afferent arterioles, we found that while the dilation elicited by a T-type Ca2+ channel blocker was similar to that elicited by an L-type channel blocker, superimposing L-type Ca2+ channel blocker on pimozide-treated vessels did not enhance the dilation already elicited by T-type Ca2+ channel blockade. We also observed that pimozide added to diltiazem-treated afferent arterioles did not elicit further dilation. These data suggest that the T-type and L-type Ca2+ channels act in a sequential or cooperative manner. It is known that HVA Ca2+ channels require substantial depolarization of the membrane for activation, whereas LVA Ca2+ channels open at a relatively low membrane potential (8). The activation threshold for L-type is less negative than that for T-type Ca2+ channel in VSM cells; examples include canine bronchial smooth muscle: -35 mV for L-type and -60 mV for T-type (32); rat colonic smooth muscle: -40 mV for L-type and -70 to -60 mV for T-type (61); rat tail artery: -20 mV for L-type and -50 mV for T-type (58). At physiological pressures, VSM cell resting potentials range from -40 to -60 mV (14). In these tissues, T-type Ca2+ channel activation threshold is very close to the resting membrane potential and is much closer to the resting membrane potential than L-type Ca2+ channel activation threshold. Experiments using the in vitro perfused hydronephrotic rat kidney perfused at 80 mmHg reported VSM cell potentials of -40 mV for afferent arterioles and -38 mV for efferent arterioles, indicating values very close to the inactivation range of T-type Ca2+ channels (39). As recently suggested, however, it is likely that when the resting membrane potentials are relatively more depolarized, the function of T-type Ca2+ channels is speculated to be carried on by a relatively small and sustained Ca2+ window current. Thus small depolarizations shift the membrane potential into the T-type window-current range of voltages, which allow a sustained physiologically relevant Ca2+ entry. (11, 60). This Ca2+ influx may further elevate the membrane potential into the L-type Ca2+ channel activation range of voltages to facilitate the activities of HVA L-type Ca2+ channels. Blockade of the LVA T-type Ca2+ channels may result in membrane hyperpolarization, which may lead to inactivation of the HVA Ca2+ channels. Although T-type Ca2+ currents cause a small depolarization, it may be sufficient to gate the activity of the HVA L-type Ca2+ channels (29, 48, 60) because the L-type Ca2+ channel activation threshold is also close to the resting membrane potential in VSM cells (14). Thus the T-type Ca2+ current seems to be “amplified” due to the cooperative action with L-type Ca2+ channels. Ca2+ influx through both T-type and L-type Ca2+ channels determines the contractile status of smooth muscle and T-type Ca2+ channel activity may be more important at membrane potentials near the resting level.
The finding that pimozide and mibefradil masked the effects of diltiazem suggests that there is a cooperativity between LVA Ca2+ channels and HVA Ca2+ channels to control the influx of Ca2+ in afferent arteriolar VSM cells.
In the present study, we found that similar to L-type Ca2+ channel blockade, T-type Ca2+ channel blockade also completely prevented autoregulatory-mediated responses in afferent arteriole diameter to increases in renal perfusion pressure. In the kidney, intact voltage-gated Ca2+ channels are crucial to the maintenance of afferent arteriolar autoregulatory capability, which is believed to provide the primary protection against hypertensive glomerular injury (24). Both L-type and T-type blockers reduce renal autoregulatory capability (24). Elevations of intravascular pressure stimulate stretch-activated Ca2+ channels leading to depolarization and constriction of precapillary arterioles contributing to blood flow regulation (46, 59). T-type Ca2+ channel blockade may restrain the depolarization caused by elevation of intravascular pressure, and in this manner, prevent the Ca2+ influx via L-type channels needed for renal autoregulatory responses.
In summary, we found that in contrast to L-type channel blockade with diltiazem, which only dilates afferent arterioles, T-type Ca2+ channel blockade with pimozide and mibefradil caused dilation of efferent arterioles as well as of afferent arterioles. No significant difference was observed between the dilation of afferent arterioles caused by T-type Ca2+ channel blockade alone and that caused by the combination of T-type Ca2+ channel blockade plus L-type Ca2+ channel blockade. As a test of selectivity, we observed that the L-type Ca2+ channel blocker prevented the high-KCl-induced afferent arteriolar vasoconstriction but that T-type Ca2+ channel blockade did not prevent high KCl-induced afferent arteriolar vasoconstriction. Nevertheless, pimozide blocked the ability of the afferent arterioles to exhibit autoregulation in responses to increases in perfusion pressure. To the extent that these agents exert their effects primarily on T-type Ca2+ channels in our experimental setting, these data are consistent with the premise that LVA T-type Ca2+ channels cooperate with HVA L-type Ca2+ channels to control the influx of Ca2+ in afferent arteriolar VSM and that T-type Ca2+ channels are functionally expressed in both afferent and efferent arteries and contribute to the regulation of both afferent and efferent arteriolar resistance. Because L-type Ca2+ channels are not functionally expressed in efferent arterioles of normal rats, T-type Ca2+ channels may be particularly significant in the regulation of efferent arteriolar resistance.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-18426 and by a Health Excellence Fund grant from the Louisiana Board of Regents.
The authors thank Dr. K. Elmslie and Dr. E. Inscho (Medical College of Georgia, Augusta, GA) for helpful discussions. We also thank Dr. L. M. Harrison-Bernard and Dr. A. Nishiyama for technical advice and D. Olavarrieta for assistance in preparing the manuscript.
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