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T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles

2010; Elsevier BV; Volume: 79; Issue: 4 Linguagem: Inglês

10.1038/ki.2010.429

1523-1755

Christian Bjørn Poulsen, Rozh H. Al‐Mashhadi, Leanne L. Cribbs, Ole Skøtt, Pernille Hansen,

Ion Transport and Channel Regulation

Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation–contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium-induced constriction. Further, constriction by the thromboxane analogue U46619 was significantly inhibited by mibefradil at a concentration specific for T-type channels. Using PCR, we found that two channel subtypes, Cav3.1 and Cav3.2, were expressed in microdissected efferent arterioles. Cav3.1 was found by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Cav3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response. Low concentrations of nickel, an agent that blocks Cav3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles. Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation–contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium-induced constriction. Further, constriction by the thromboxane analogue U46619 was significantly inhibited by mibefradil at a concentration specific for T-type channels. Using PCR, we found that two channel subtypes, Cav3.1 and Cav3.2, were expressed in microdissected efferent arterioles. Cav3.1 was found by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Cav3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response. Low concentrations of nickel, an agent that blocks Cav3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles. Changes in pre- and postglomerular vessel diameters are involved in regulation of renal blood flow, glomerular ultrafiltration pressure, and medullary blood flow. These effects influence glomerular filtration rate and salt and water homeostasis and thereby blood pressure. Furthermore, the kidney vascular segments are involved in several pathological conditions such as diabetes and hypertension. Excitation–contraction coupling in renal afferent arteriole involves voltage-gated calcium channels (Cav). In keeping with the central role of Cav, the channels are targets for pharmacological intervention in hypertension. The family of voltage-gated calcium channels are divided into high voltage-activated channels (including L-type Cav) that are activated by a large depolarization, and low voltage-activated channels (to which the T-type Cav belongs) that activate after rather limited depolarization.1.Ertel E.A. Campbell K.P. Harpold M.M. et al.Nomenclature of voltage-gated calcium channels.Neuron. 2000; 25: 533-535Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar Genes encoding mRNAs for Cav α-subunits have been cloned and the L-type channels include Cav 1.1–1.4, and T-type channels comprise three Cav, 3.1–3.3. Vascular smooth muscle cells from preglomerular vessels and freshly microdissected preglomerular vessels express, among others, Cav1.2, Cav3.1, and Cav3.2. Postglomerular vessels from rats showed a differential expression of the voltage-gated calcium channels, with L- (Cav1.2) and T-type (Cav3.1 and 3.2) subunits expressed in efferent arterioles from juxtamedullary glomeruli and in outer medullary vasa recta, whereas no voltage-dependent calcium channels were detected in cortical efferent arterioles.2.Hansen P.B. Jensen B.L. Andreasen D. et al.Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels.Circ Res. 2001; 89: 630-638Crossref PubMed Scopus (179) Google Scholar The activation mechanisms that induce vasoconstriction in afferent and efferent arterioles in the renal cortex are considered different. Thus, depolarization and Cav are involved in the mechanisms that lead to vasoconstriction in the preglomerular vasculature.3.Hayashi K. Wakino S. Sugano N. et al.Ca2+ channel subtypes and pharmacology in the kidney.Circ Res. 2007; 100: 342-353Crossref PubMed Scopus (191) Google Scholar, 4.Navar L.G. Inscho E.W. Majid S.A. et al.Paracrine regulation of the renal microcirculation.Physiol Rev. 1996; 76: 425-536PubMed Google Scholar In efferent arterioles the involvement of Cav is less clear. Calcium influx pathways in efferent vessels have been reported not to be dependent on depolarization5.Loutzenhiser K. Loutzenhiser R. Angiotensin II-induced Ca(2+) influx in renal afferent and efferent arterioles: differing roles of voltage-gated and store-operated Ca(2+) entry.Circ Res. 2000; 87: 551-557Crossref PubMed Scopus (103) Google Scholar and to be resistant to L-type calcium channel antagonists6.Carmines P.K. Fowler B.C. Bell P.D. Segmentally distinct effects of depolarization on intracellular [Ca2+] in renal arterioles.Am J Physiol. 1993; 265: F677-F685PubMed Google Scholar whereas others have shown effects of T-type antagonists.7.Ozawa Y. Hayashi K. Nagahama T. et al.Effect of T-type selective calcium antagonist on renal microcirculation: studies in the isolated perfused hydronephrotic kidney.Hypertension. 2001; 38: 343-347Crossref PubMed Scopus (63) Google Scholar In vivo data confirm the heterogeneity between afferent and efferent arterioles and an important contribution from T-type channels has been suggested for efferent arterioles.8.Honda M. Hayashi K. Matsuda H. et al.Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.J Hypertens. 2001; 19: 2031-2037Crossref PubMed Scopus (54) Google Scholar, 9.Yamamoto T. Hayashi K. Matsuda H. et al.In vivo visualization of angiotensin II- and tubuloglomerular feedback-mediated renal vasoconstriction.Kidney Int. 2001; 60: 364-369Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 10.Furukawa T. Nukada T. Miura R. et al.Differential blocking action of dihydropyridine Ca2+ antagonists on a T-type Ca2+ channel (alpha1G) expressed in Xenopus oocytes.J Cardiovasc Pharmacol. 2005; 45: 241-246Crossref PubMed Scopus (63) Google Scholar Finally, a recent clinical study showed that benidipine (a combined L- and T-type antagonist) caused larger reduction in blood pressure and proteinuria compared with L-type treatment using amlodipine,11.Ohishi M. Takagi T. Ito N. et al.Renal-protective effect of T-and L-type calcium channel blockers in hypertensive patients: an Amlodipine-to-Benidipine Changeover (ABC) study.Hypertens Res. 2007; 30: 797-806Crossref PubMed Scopus (41) Google Scholar suggesting a vasoconstrictor effect of T-type channels on efferent arterioles. The purpose of this study was to determine if voltage-gated calcium channels are present in mice efferent arterioles, and if so, whether they contribute to vascular responses. Furthermore, we investigated the subtype of Cav in postglomerular vessels focusing on L- and T-type channels. Expression and location of Cav were determined by polymerase chain reaction (PCR) and immunostaining. In addition, the functional importance was investigated in isolated mouse perfused efferent arterioles. Resting basal diameter of cortical efferent arterioles vessels was on average 7.9±0.3 μm and application of high potassium solution (HPS, 100 mmol/l potassium) decreased the diameter to 0.1±0.1 μm. After washout, removal of extracellular calcium for 1 min had no effect on basal diameter but blocked the potassium-induced response and the diameter was not significantly different from basal (Figure 1a). To rule out any nerve-mediated effects, we tested the potassium-induced constriction in the presence of an α1-antagonist phentolamine (PHE). Figure 1b shows that administration of PHE (10-4 mol/l) did not change the basal diameter in efferent arterioles and HPS application in the presence of PHE elicited a constriction (from 6.9±0.7 to 0.07±0.05 μm) not different from the HPS response without PHE treatment (from 6.5±0.3 to 0.6±0.6 μm). Eight individual experiments testing the response to HPS in the presence of nifedipine showed that the basal diameter of the vessels was on average 6.6±0.1 μm and HPS treatment decreased the luminal diameter significantly to 0.0±0.0 μm. No significant change in blood vessel diameter could be observed after nifedipine. After incubation with nifedipine HPS decreased the diameter by 36.6±15.5% to 4.2±1.1 μm with a rather large variance between experiments (data not shown). Concentration–response experiments for mibefradil showed a blockade of HPS-elicited vasoconstriction (Figure 2a). The mean basal luminal diameter was 7.2±0.4 μm and HPS led to short complete occlusion of the arterioles (0.0±0.0 μm). Application of mibefradil at increasing concentrations (10-9 to 10-5 mol/l) completely prevented vasoconstriction after HPS. After washout of mibefradil the vessels were fully viable. The thromboxane A2 analogue, U46619, concentration dependently contracted efferent arterioles with an EC50 of 3 × 10-8 mol/l. Mibefradil (10-7 mol/l) significantly inhibited the U46619-induced constriction at 10-7 mol/l (Figure 2b). Another important vasoconstrictor angiotensin II also concentration dependently contracted effect arterioles with maximum response (Emax) at 10-9 mol/l and an EC50 at 1.1 × 10-10 mol/l. Owing to tachyphylaxis the experiment was repeated in the presence of mibefradil (10-7 mol/l) in another set of arterioles. The constriction at 10-9 mol/l of Ang II had a tendency be less in the presence of mibefradil; however it was not significantly inhibited by the T-type antagonist (Figure 2c). Another T-type antagonist, nickel chloride (NiCl2), did not significantly change the resting luminal diameter over 1 min (from 7.3±0.4 to 6.9±0.5 μm). However, NiCl2 had concentration-dependent effects on the response to high potassium (Figure 3). Exposure to HPS alone elicited a transient constriction, which completely closed the vessels for on average 16.2±7.0 s, and which was followed by a dilation. When low concentrations of NiCl2 were applied, the secondary vasodilation was blocked, and a significant increase in the duration of the contraction was observed (Figure 3a). Thus, in the presence of NiCl2 (10-6 mol/l) HPS elicited a sustained constriction lasting on average 48.2±1.1 s. At 10-5 mol/l of NiCl2 the duration of the complete closure of the efferent arteriole lasted 46.7±2.1 s (Figure 3b). At higher concentrations of NiCl2 the initial contraction elicited by HPS was inhibited. Thus, incomplete closure of the blood vessels was observed when HPS was applied in the presence of NiCl2 10-4 mol/l (25.5±7.4) and at 10-3 mol/l no significant response to HPS was observed (Figure 3c and d). NiCl2 caused a concentration-dependent increase in luminal diameter in potassium-constricted vessels, with an EC50 of 1.67 × 10-4 mol/l and a significant inhibition of HPS induced contraction at 10-3 mol/l NiCl2. For the corresponding areas under curve for increasing concentrations of NiCl2, the following data were obtained: 76.6±9.8 s μm at 10-6 mol/l, 84.3±14.2 s μm at 10-5 mol/l, 186.6±22.8 s μm at 10-4 mol/l, 373.8±26.2 s μm at 10-3 mol/l and for HPS alone 261.6±45.3 s μm. Areas under curve for NiCl2 10-6 and 10-5 mol/l were significantly different from HPS alone. Under control condition, high potassium elicits a significant transient constriction in efferent arterioles with complete closure lasting for 11.0±1.5 s. Incubation with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) significantly decreased the basal diameter by 9.3±0.34%, and HPS elicited a constriction significantly faster than in the absence of L-NAME. After L-NAME treatment the duration of K+-elicited contraction increased to 53.4±2.0 s (Figure 4a). In eNOS-/- mice the basal diameter before application of HPS was 8.2±0.6 μm (compared with 8.6±0.7 μm in wild-type (wt) animals) and after application of HPS the diameter decreased to 0.0±0.0 μm (Figure 4b). The duration of the contraction in eNOS-/- mice was not statistically significantly different to that of wt mice treated with L-NAME but significantly different from the duration of the vasoconstriction in wt. Thus, the secondary vasodilation after treatment with HPS is blocked by L-NAME or absence of eNOS. Acetylcholine (Ach)-mediated dilatation was investigated in vessels preconstricted with 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619) (10-6 mol/l) resulted in a luminal diameter of 1.4±1.4 μm and Ach increased the luminal diameter to 7.6±0.2 μm. In the presence of L-NAME and U46619, Ach was unable to change the luminal diameter significantly (Figure 5a). The Ach-induced response was significantly different between the groups with and without L-NAME. In Figure 5b, Ach in the presence of U46619 increased the luminal diameter from 1.1±1.1 to 6.6±0.3 μm. Simultaneous exposure to NiCl2 (10-6 mol/l) led to a similar increase in diameter to 6.6±0.3 μm. To detect whether both Cav3.1 and Cav3.2 were expressed in mouse postglomerular blood vessels, we performed PCR analysis using specific primers on RNA extracted from efferent arterioles. Bands of the expected size were observed for Cav3.1 and Cav3.2 in cortical efferent arterioles and in the positive control using whole brain (Figure 6). The negative controls, water and minus reverse transcriptase, showed no bands. Next, the distribution of Cav3.1 and Cav3.2 was examined in kidney sections. Labeling for Cav3.1 was associated with the entire mouse renal vasculature including glomerular arterioles (Figure 7a). Acid-treated kidney glomeruli with attached efferent arterioles showed Cav3.1 protein in cortical efferent arterioles (Figure 7c). Also human pre- and postglomerular vasculature expressed Cav3.1 protein. Figure 7e shows labeled vasa rectae from outer medulla and staining of a human afferent arteriole is shown in Figure 7f. Finally, labeling for Cav3.2 protein was observed in arteries and arterioles of rat cryosections (Figure 8). Furthermore, separate experiments using immunofluorescence microscopy was performed as this method yielded a higher resolution than DAB staining. In a kidney section, arteries and arterioles positively labeled for Cav3.2, interestingly capillaries and the vessels within the glomerulus were also positive for Cav3.2 (Figure 8c). Using higher magnification, one could easily determined the distribution Cav3.2 in the vessel showing labeling of vascular smooth muscle cells and endothelial cells (Figure 8d).Figure 8Immunohistochemistry for Cav3.2. Rat cryosections (a, c, d) were labeled with anti-Cav3.2 antibody. Omission of primary antibody served as control (b).View Large Image Figure ViewerDownload (PPT) Exposure to a high potassium concentration depolarizes renal preglomerular vessels, activates voltage-gated calcium channels, and stimulates calcium influx and contractions. The involvement of Cav in efferent arteriolar contractility has been unclear but in this study we demonstrate that depolarization of mouse perfused cortical efferent arterioles consistently constricts the blood vessels. Furthermore, our data suggest that T-type Cav potentially of the Cav3.1 subtype are responsible for the potassium-elicited constriction whereas T-type Cav3.2 are involved in the spontaneous dilatation that occurs in the blood vessels after the initial constriction. Depolarizing the efferent arteriole led to a contraction, which was totally dependent on influx of extracellular calcium suggesting involvement of Cav. Activation of T-type and L-type Cav has been shown to mediate α1-adrenoceptor-mediated constriction in guinea pig vas deferens.12.Shishido T. Sakai S. Tosaka T. T- and L-type calcium channels mediate alpha(1)-adrenoceptor-evoked contraction in the guinea-pig vas deferens.Neurourol Urodyn. 2009; 28: 447-454Crossref PubMed Scopus (11) Google Scholar Whether the contraction of efferent arterioles was mediated through activation of the sympathetic nerve terminals and norepinephrine release was tested with PHE as norepinephrine mediates contraction mainly through smooth muscle α1-adrenergic receptors. Because pretreatment with PHE did not change the potassium-elicited response, the response must be independent of norepinephrine and thus probably mediated by a direct smooth muscle cell response. The participation of T-type calcium channels in excitation–contraction coupling was shown by the effects of mibefradil and nickel. Mibefradil selectively inhibits T-type currents at low concentrations with an EC50 about 10 nmol/l13.Klugbauer N. Marais E. Lacinova L. et al.A T-type calcium channel from mouse brain.Pflugers Arch. 1999; 437: 710-715Crossref PubMed Scopus (106) Google Scholar, 14.McDonough S.I. Bean B.P. Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons.Mol Pharmacol. 1998; 54: 1080-1087Crossref PubMed Scopus (131) Google Scholar and the Cav3.1 channel is relatively sensitive to mibefradil.13.Klugbauer N. Marais E. Lacinova L. et al.A T-type calcium channel from mouse brain.Pflugers Arch. 1999; 437: 710-715Crossref PubMed Scopus (106) Google Scholar In this study, mibefradil blocked K+-induced contraction at 10-9 mol/l. Moreover, Ni2+ dose dependently inhibited K+-mediated contraction with an EC50 of 1.67 × 10-4 mol/l. Nickel is a blocker of T-type channels (Cav3.1, Cav3.2, and Cav3.3) but their sensitivity to nickel differs.15.Huguenard J.R. Low-threshold calcium currents in central nervous system neurons.Annu Rev Physiol. 1996; 58: 329-348Crossref PubMed Scopus (635) Google Scholar T-type currents elicited by α13.1 subunits are blocked by Ni2+ in the range 0.5–1 mmol/l,13.Klugbauer N. Marais E. Lacinova L. et al.A T-type calcium channel from mouse brain.Pflugers Arch. 1999; 437: 710-715Crossref PubMed Scopus (106) Google Scholar whereas the Cav3.2 channel is highly sensitive to nickel (EC50 of around 10 μmol/l).16.Williams M.E. Washburn M.S. Hans M. et al.Structure and functional characterization of a novel human low-voltage activated calcium channel.J Neurochem. 1999; 72: 791-799Crossref PubMed Scopus (94) Google Scholar, 17.Lee J.H. Gomora J.C. Cribbs L.L. et al.Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H.Biophys J. 1999; 77: 3034-3042Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar The high concentrations of Ni2+ necessary for blockade of the constriction together with the total inhibition of the response by low-dose mibefradil suggest that Cav3.1 are involved in the excitation contraction mechanism in the efferent arteriole. Nifedipine (L-type antagonist) had variable effects on efferent arterioles. For postglomerular vessels, the microdissection technique allowed us to isolate mRNA from cortical efferent arterioles including both endothelial and vascular smooth muscle cells. In agreement with the functional data, mRNA for Cav3.1 and 3.2 α1-subunits was expressed in isolated cortical efferent arterioles. Immunohistochemistry for Cav3.1 and 3.2 proteins showed the expected labeling of all large vessels and glomerular vessels. The immunostaining for Cav3.1 was repeated by mounting an efferent arteriole with two micropipettes in a tissue bath mounted on an inverted microscope, and making the entire staining procedure on one specimen at the time confirmed that Cav3.1 is expressed in mouse cortical efferent arterioles. Furthermore, Cav3.1 was also observed in human renal vasculature, in both pre- and postglomerular vessels, suggesting that our results could apply to humans. Antibodies specific for the T-type subunit Cav3.2 did not work in the mouse and the experiments were performed on rat sections, in which immunoflourescence microscopy showed the presence of Cav3.2 in rodent renal arterioles in both vascular smooth muscle cells and endothelial cells including cells in the capillaries. A previous study has shown a marked heterogeneity in the expression pattern of calcium channels in renal resistance vessels, with L- (Cav1.2) and T-type (Cav3.1 and 3.2) subunits expressed in efferent arterioles from juxtamedullary glomeruli and in outer medullary vasa recta, but no voltage-dependent calcium channels detected in cortical efferent arterioles. This lack of Cav observed in rat cortical efferent arterioles2.Hansen P.B. Jensen B.L. Andreasen D. et al.Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels.Circ Res. 2001; 89: 630-638Crossref PubMed Scopus (179) Google Scholar suggests a species difference between rat and mouse, which explains the large contractility of mouse efferent arterioles after depolarization. In agreement with this, Feng et al.18.Feng M.G. Li M. Navar L.G. T-type calcium channels in the regulation of afferent and efferent arterioles in rats.Am J Physiol Renal Physiol. 2004; 286: F331-F337Crossref PubMed Scopus (49) Google Scholar showed that an L-type antagonist dilated afferent arterioles in rats whereas it had no effect on efferent arterioles. In contrast, T-type blockade dilated both afferent and efferent arterioles. Furthermore, in vivo studies have shown that T-type activity contributes significantly to efferent arteriolar function8.Honda M. Hayashi K. Matsuda H. et al.Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.J Hypertens. 2001; 19: 2031-2037Crossref PubMed Scopus (54) Google Scholar and that L-type antagonists lead to increased filtration fraction corresponding to an dilatation of afferent but not efferent arterioles, whereas T-type antagonists caused a lower increase in filtration fraction.8.Honda M. Hayashi K. Matsuda H. et al.Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.J Hypertens. 2001; 19: 2031-2037Crossref PubMed Scopus (54) Google Scholar Substances important for efferent arteriolar function in vivo such as angiotensin II and thromboxane were also affected by inhibition of T-type channels. Constriction elicited by the thromboxane analogue U46619 was significantly inhibited whereas a smaller response was observed for the angiotensin II-induced response. The lack of significant inhibition of the angiotensin II-elicited response by mibefradil is in agreement with a previous finding by Loutzenhiser and Loutzenhiser5.Loutzenhiser K. Loutzenhiser R. Angiotensin II-induced Ca(2+) influx in renal afferent and efferent arterioles: differing roles of voltage-gated and store-operated Ca(2+) entry.Circ Res. 2000; 87: 551-557Crossref PubMed Scopus (103) Google Scholar who showed that angiotensin II-induced constriction in efferent arterioles is dependent on activation of store-operated calcium channels. Thus, T-type channels of the Cav3.1 subtype seem to be important in the excitation–contraction response of efferent arterioles and therefore important for the regulation of glomerular filtration rate and filtration fraction. Calcium blockers of the L-type are widely used in conditions such as hypertension but several recent studies have suggested that T-type blockers have additional beneficial effects. In a changeover study, benidipine (L- and T-type antagonist) caused larger reduction in blood pressure and proteinuria compared to L-type treatment using amlodipine.11.Ohishi M. Takagi T. Ito N. et al.Renal-protective effect of T-and L-type calcium channel blockers in hypertensive patients: an Amlodipine-to-Benidipine Changeover (ABC) study.Hypertens Res. 2007; 30: 797-806Crossref PubMed Scopus (41) Google Scholar The observation of superior renoprotective effects with a combined L- and T-type treatment is in agreement with the current demonstration of a vasoconstrictor effect of T-type channels on efferent arterioles. To our initial surprise Ni2+ in low concentrations did not inhibit the constriction of efferent arterioles but rather potentiated the response. Thus, NiCl2 (10-6 mol/l) changed the potassium-elicited constriction from a transient response to a sustained response lasting the total period of potassium administration. As described previously, Cav3.2 is highly sensitive to Ni2+ (EC50 of about 10 μmol/l).16.Williams M.E. Washburn M.S. Hans M. et al.Structure and functional characterization of a novel human low-voltage activated calcium channel.J Neurochem. 1999; 72: 791-799Crossref PubMed Scopus (94) Google Scholar, 17.Lee J.H. Gomora J.C. Cribbs L.L. et al.Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H.Biophys J. 1999; 77: 3034-3042Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar Cav3.2 mRNA was present in mouse efferent arterioles, and all blood vessels observed by immunohistochemistry showed labeling for Cav3.2 in rat smooth muscle cells and endothelial cells. An attempt to confirm the involvement of Cav3.2 with siRNA techniques was unsuccessful because the technical procedures led to loss of endothelial function. On the basis of our results, we propose that inhibition of Cav3.2 blocks the spontaneous dilatation occurring in perfused efferent arterioles after depolarization, although a definitive answer has to await results from Cav3.2-/- mice. The secondary dilatation observed in efferent arterioles after depolarization was likely to be dependent on endothelial NO production, because it was inhibited by L-NAME and it was absent in eNOS-/- mice. In rabbit afferent arterioles, depolarization induces contraction by increasing smooth muscle intracellular calcium concentration, which is followed by calcium increase in the endothelium where it stimulates NO production. This, in turn, curtails vasoconstriction by reducing the calcium sensitivity of the contractile apparatus of the smooth muscle cells.19.Uhrenholt T.R. Schjerning J. Vanhoutte P.M. et al.Intercellular calcium signaling and nitric oxide feedback during constriction of rabbit renal afferent arterioles.Am J Physiol Renal Physiol. 2007; 292: F1124-F1131Crossref PubMed Scopus (16) Google Scholar In the efferent arterioles our new data are consistent with the view that the activation of eNOS involves calcium influx mediated by activation of Cav3.2 located on endothelial cells. A similar role of endothelial T-type channels (Cav3.2) has been suggested in longitudinal conduction of vasodilator responses through an endothelium-dependent mechanism depending on activation of Cav3.2 and calcium-induced eNOS activation.20.Figueroa X.F. Chen C.C. Campbell K.P. et al.Are voltage-dependent ion channels involved in the endothelial cell control of vasomotor tone?.Am J Physiol Heart Circ Physiol. 2007; 293: H1371-H1383Crossref PubMed Scopus (68) Google Scholar The existence of voltage-gated calcium channels in endothelial cells has been somewhat controversial, but Cav3.2 has been shown in endothelial cells of small mesenteric arterioles.21.Braunstein T.H. Inoue R. Cribbs L. et al.The role of L- and T-type calcium channels in local and remote calcium responses in rat mesenteric terminal arterioles.J Vasc Res. 2008; 46: 138-151Crossref PubMed Scopus (39) Google Scholar Lung microvascular endothelial cells express Cav3.122.Wu S. Haynes Jr, J. Taylor J.T. et al.Cav3.1 (alpha1G) T-type Ca2+ channels mediate vaso-occlusion of sickled erythrocytes in lung microcirculation.Circ Res. 2003; 93: 346-353Crossref PubMed Scopus (68) Google Scholar that carries flow-sensitive calcium fluxes which are blocked by mibefradil.23.Wei Z. Manevich Y. Al-Mehdi A.B. et al.Ca2+ flux through voltage-gated channels with flow cessation in pulmonary microvascular endothelial cells.Microcirculation. 2004; 11: 517-526Crossref PubMed Scopus (37) Google Scholar Cav3.2-deficient mice have normal vasoconstrictor responses but they show reduced relaxation of coronary vascular smooth muscle after Ach and nitroprusside. Ni2+ had similar effect in wt animals.24.Chen C.C. Lamping K.G. Nuno D.W. et al.Abnormal coronary function in mice deficient in alpha1H T-type Ca2+ channels.Science. 2003; 302: 1416-1418Crossref PubMed Scopus (284) Google Scholar The authors suggested that Cav3.2 is coupled to the large calcium-activated potassium channel (BKca) and that inhibition/deletion of Cav3.2 led to decreased BKca activity and impaired dilatation. We cannot exclude that a similar interaction between a Cav3.2 and BKca may occur in the vascular smooth muscle cells of the efferent arteriole, but in our experiments the vasodilator effect of Ach was not inhibited by Ni2+. Acetylcholine activates muscarinic receptors on endothelial cells and causes NO production by stimulation of eNOS through release of calcium from intracellular stores. In contrast, the potassium-dependent activation of NO production involves influx of extracellular calcium. The observation that the potassium-dependent dilation was blocked by Ni2+ whereas the ACh-induced dilation was not, suggests that the effect of Ni2+ at low concentrations is neither due to impairment of NO production nor due to impairment of the response to NO in the smooth muscle. This is in agreement with the suggestion that activation of eNOS involves calcium influx mediated by activation of Cav3.2 located on endothelial cells for the potassium-induced response but not for the Ach-induced response. In conclusion, mouse cortical efferent arterioles express T-type calcium channels that are involved in both contraction probably through Cav3.1 and dilatation through Cav3.2. These observations could be relevant for the understanding of regulation of glomerular filtration rate and pressure. The opposite effects of the two T-type channels suggest that antagonists with specific Cav3.1-blocking properties would have clinical advantages over more unspecific T-type antagonists. The experimental protocol was approved by the Danish Animal Experiments Inspectorate under the Danish Ministry of Justice and animal care followed the guidelines of the National Institutes of Health. Male and female C57BL/6 mice (wt; Taconic, Ejby, Denmark), eNOS-/- mice (C57BL/6 background, backcrossed for more than 10 generations; Jackson Laboratory, Bar Harbor, ME), and rats (Sprague–Dawley) had free access to rodent chow and tap water. Mouse cortical efferent arterioles with attached glomeruli were microdissected in Dulbecco's modified Eagle's medium/F12 (4 °C) and perfused retrogradely as described previously.25.Al-Mashhadi R.H. Skott O. Vanhoutte P.M. et al.Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse.Kidney Int. 2009Abstract Full Text Full Text PDF Scopus (36) Google Scholar The efferent arteriole was identified by appearance and its location was compared to the preglomerular vasculature, and was transferred to a thermostated (37 °C) chamber (Warner, Hamden, CT) containing Dulbecco's modified Eagle's medium/F12 (saturated 5% CO2 in air) with 0.1% bovine serum albumin. The chamber was mounted on an inverted microscope (Zeiss Axiovert 10, Oberkochen, Germany). Perfusion occurred by aspiration of the arteriole into a pipette and cannulation with a second pipette followed by increase in driving pressure until the vessel opened, indicating perfusion. The experiments were recorded using a digital camera (DAGE MTI, Michigan City, IN). One frame per second was stored on a computer, using a frame grapper card (Pixel Smart). Images were transferred to custom-made imaging software FluoroFix (produced by Rozh Husain Al-Mashhadi, University of Southern Denmark) and luminal diameter was determined at the most reactive part of the arteriole and at the time with the maximal response. All experimental protocols started with a period of equilibration after the perfusion was established and vessel viability were tested by administration of an HPS (100 mmol/l potassium) added to the bath. All experiments ended by a viability test using HPS or in the absence of contraction ANG II (10-6 mol/l) was applied. Extraluminal calcium was removed for 1 min and the efferent arteriole was challenged with HPS minus calcium for 1 min. To test for potential involvement of nerve-mediated responses, we applied the α-adrenoceptor antagonist PHE 10-4 mol/l to the chamber for 10 min followed by HPS. The effect of nifedipine (5 × 10-6 mol/l; Sigma-Aldrich, St Louis, MO) on HPS-elicited constriction was also tested. Concentration–response curves were obtained for mibefradil (Sigma-Aldrich) in the concentration range 10-9 to 10-5 mol/l. Each concentration of mibefradil was applied for 5 min in the presence of PHE followed by 1 min HPS administration; this was repeated for each mibefradil concentration. The effect mibefradil was tested on U46619 and angiotensin II-induced constrictions. Increasing concentrations of either U46619 (10-8, 3 × 10-8, 6 × 10-8, 10-7 mol/l) or angiotensin II (10-10 to 10-8 mol/l) were applied for 3 min in the presence and absence of mibefradil (10-7 mol/l). Concentration–response kinetics was also determined for another T-type antagonist NiCl2 (Sigma-Aldrich) in the presence of PHE by adding the compound to the bath for 1 min at increasing concentrations (10-6 to 10-3 mol/l) followed by HPS for 1 min. In the next series of experiments, the vessels were incubated with L-NAME (5 × 10-5 mol/l; Sigma-Aldrich) and PHE (10-5 mol/l) for 20 min and the contractility profile to HPS (1 min) was investigated. Furthermore, the time line for the constrictor response to HPS in eNOS-/- mice was determined. Finally, the effect of blocking T-type channels or eNOS for Ach (Sigma-Aldrich)-mediated dilatation was investigated. Efferent arterioles were preconstricted with thromboxane-prostanoid receptor agonist U46619 10-6 mol/l (Sigma-Aldrich) for 3 min and Ach 10-6 mol/l was applied in the presence of U46619 for 2 min. After a resting period, the vessels were incubated with L-NAME 5 × 10-5 mol/l for 20 min and application of U46619 and Ach was repeated. In the last series of experiments, the Ach (10-6 mol/l) induced dilatation in U46619-treated vessels was determined and subsequently the response was assessed in the presence of NiCl2 (10-6 mol/l, 1 min pretreatment). High potassium solution composition in mmol/l: NaHCO3 25, NaCl 20, KCl 95, MgSO4 1.2, K2HPO4 2.5, HEPES 10, CaCl2 1.3, glucose 5. Efferent arterioles were dissected and RNA extracted by acid-guanidinium-phenol-chloroform extraction.26.Hansen P.B. Jensen B.L. Andreasen D. et al.Vascular smooth muscle cells express the alpha(1A) subunit of a P-/Q-type voltage-dependent Ca(2+) channel, and it is functionally important in renal afferent arterioles.Circ Res. 2000; 87: 896-902Crossref PubMed Scopus (82) Google Scholar RT–PCR was performed on 6–7 efferent arterioles with an annealing temperature of 50 °C, 32 cycles. Cav3.1 (forward): 5′-GAACGTGAGGCCAAGAGT-3′, reverse 5′-GCTCGTAAGCGTTCCCCT-3′ coving 221 bp. Cav3.2 (forward): 5′-GCTCTCCCCCGTCTACTTCG-3′, reverse 5′-AGATACTTTGCGCACGACCAGG-3′ covering 247 bp. β-Actin was copied from Yu et al.27.Yu A.S. Hebert S.C. Brenner B.M. et al.Molecular characterization and nephron distribution of a family of transcripts encoding the pore-forming subunit of Ca2+ channels in the kidney.Proc Natl Acad Sci USA. 1992; 89: 10494-10498Crossref PubMed Scopus (67) Google Scholar The use of human material was approved by Danish Ethical Committee and all subjects gave informed consent. The humane renal material comes from patients that have their kidneys removed due to cancer or cysts, the part of the kidney with normal tissue was used. Cav3.1: Paraffin-embedded sections of mouse and human kidneys were treated with 0.2% Triton X followed by 0.5% hydrogen peroxide for 5 min and 3% bovine serum albumin. Next the sections were incubated overnight using primary rabbit anti-Cav3.1 antibody28.Brueggemann L.I. Martin B.L. Barakat J. et al.Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking.Am J Physiol Heart Circ Physiol. 2005; 288: H923-H935Crossref PubMed Scopus (26) Google Scholar diluted 1:500. Cav3.2: Cryosections (5 μm) from rat kidneys were permeabilized by 0.5% Triton X and endogenous peroxidases were blocked by incubation with 0.5% hydrogen peroxide for 10 min followed by 3% goat serum. Next, the sections were incubated with primary rabbit anti-Cav3.2 antibody (Alomone Labs, Jerusalem, Israel) diluted 1:50. Secondary antibody goat anti-rabbit IgG, horseradish-peroxidase-labeled (Dako, Glostrup, Denmark) or Alexa 568-conjugated goat anti-rabbit (Molecular Probes, Eugene, OR) diluted 1:1000 was applied. Horseradish-peroxidase-labeled sections were stained with diaminobenzidine (DAB+ substrate–chromogen system; Dako) and counterstained with hematoxylin. Glomeruli with attached cortical efferent arterioles and preglomerular vascular trees were microdissected from HCl macerated mouse kidney. Staining for Cav3.1 of preglomerular specimens was carried out on a slide while the single efferent arterioles were transferred to a chamber on an inverted microscope and held with glass pipettes.2.Hansen P.B. Jensen B.L. Andreasen D. et al.Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels.Circ Res. 2001; 89: 630-638Crossref PubMed Scopus (179) Google Scholar, 29.Casellas D. Dupont M. Kaskel F.J. et al.Direct visualization of renin-cell distribution in preglomerular vascular trees dissected from rat kidney.Am J Physiol. 1993; 265: F151-F156PubMed Google Scholar The protocol was similar to the Cav3.1 staining described for mouse kidney sections. Data are presented as means±s.e.m. Significance of changes was calculated by one-way analysis of variance with Bonferroni reduction for multiple comparisons and Student's t-test for comparison of two groups. P<0.05 was considered statistically significant. We thank Kristoffer Rosenstand, Lis Teusch, Susanne Hansen, and Kenneth Andersen for technical assistance. This work was supported by grants from the Danish Medical Research Council (271-07-0629 and 271-07-0837), Lundbeck Foundation (R13-A1364), The Danish Heart Foundation (07-10-R60-A1686-B819-22429), and the Danish Kidney Association.