Differential Expression of T- and L-Type Voltage-Dependent Calcium Channels in Renal Resistance Vessels
Abstract— The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells showed coexpression of mRNAs for T-type subunits (CaV3.1, CaV3.2) and for an L-type subunit (CaV1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. CaV1.2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed a significant increase of calcium in response to K+ (100 mmol/L) in isolated afferent arterioles (140±25%) and in juxtamedullary efferent arterioles (118±21%). These calcium responses were attenuated by the L-type antagonist calciseptine and by the T-type antagonist mibefradil. Intracellular calcium increased in response to K+ in cortical efferent arterioles (21±9%). Mibefradil and nickel concentration dependently blocked K+-induced contraction of perfused rabbit afferent arterioles. Calciseptine blocked the contraction mediated by K+ (EC50 8×10−14). S-(-)-Bay K 8644 had no effect on vascular diameter in the afferent arteriole. We conclude that voltage-dependent L- and T-type calcium channels are expressed and of functional significance in renal cortical preglomerular vessels, in juxtamedullary efferent arterioles, and in outer medullary vasa recta, but not in cortical efferent arterioles.
The largest drop in intravascular pressure in the kidney occurs in the glomerular arterioles and in the descending vasa recta in the kidney outer medulla. These microvessels are targets for humoral and nervous signals, which interact to determine arteriolar diameters and thereby regulate overall blood flow to the kidney and the distribution of blood flow between kidney regions. There is electrophysiological evidence for the presence of L-type voltage-gated calcium currents and also for T-type calcium currents in single vascular smooth muscle cells (VSMCs) of preglomerular vessels.1,2 Also, excitation-contraction coupling in preglomerular renal vascular segments is highly dependent on calcium entry through voltage-dependent calcium channels.3 For postglomerular vascular segments, conflicting data exist with respect to the involvement of voltage-gated pathways for calcium entry and contraction.3–11 However, with some exceptions,5,6,11 most studies have shown calcium influx pathways in efferent vessels that are not activated by depolarization4,8 and are resistant to L-type calcium channel antagonists.7,9,10 Single-cell calcium currents have not yet been identified in postglomerular vascular smooth muscle. In spite of solid functional and pharmacological evidence for the presence of distinct, segment-specific calcium influx pathways in renal microvessels, the molecular correlates for this reactivity and the sites of their expression have not been defined.
The CaV (α1) subunit of calcium channels constitutes the essential component necessary and sufficient for the expression of voltage-gated calcium currents. Genes encoding mRNAs for a family of CaV subunits have recently been cloned, and several members were expressed in rat kidneys as shown by Northern blotting. These subunits comprise subtypes, which encode L-type calcium currents CaV1.2 (α1C), CaV1.3 (α1D), and CaV1.1 (α1S),12 P-/Q-type current CaV2.1 (α1A),12 and T-type currents CaV3.1 (α1G), CaV3.2 (α1H), and CaV3.3 (α1I).13–15 With the exception of the CaV2.1 subunit,16 there exist no data on the distribution of these crucial subunits in renal resistance vessels. In view of the confusing data on segment-specific activation pathways in renal microvessels and the rather unique presence of a large T-type calcium conductance in afferent vascular myocytes,1 we systematically investigated the molecular and functional distribution of CaV subunits along major vascular resistance segments in the kidney.
Materials and Methods
All animal care and use were according to the guidelines of the National Institutes of Health. Male Sprague-Dawley rats (n=39) (local animal facility) and New Zealand rabbits (n=38) (Harlan, AD Horst, Netherlands) were maintained on standard chow and had free access to tap water.
Preglomerular VSMCs were prepared from rats (180 to 220 g) by outgrowth from preglomerular vessels that had been isolated magnetically after perfusion of the kidney with iron oxide.16,17 Mesangial cells were obtained by outgrowth from isolated glomeruli.16 RNA was isolated by RN′easy mini columns (Qiagen)
Microdissection of Pre- and Postglomerular Vessels
For the preglomerular vascular samples, we dissected from rat kidneys arcuate arteries with divisions into cortical radial arteries and afferent arterioles. Efferent arterioles of juxtamedullary glomeruli were isolated close to the outer stripe and were characterized by a long course into the medulla and by the division into vascular bundles. Vasa recta bundles and single vasa recta were isolated from outer medulla. Because it proved too difficult to obtain sufficient amounts of rat efferent arterioles from superficial glomeruli for RNA isolation, cortical efferent arterioles were microdissected from rabbits (1.8 to 2.5 kg). Of the preglomerular samples, 40 to 50 branching points were pooled, and the length of cortical efferent arterioles, juxtamedullary efferent arterioles, and vasa recta was measured with a scale built into the ocular. Total RNAs were isolated according to a microadapted protocol of Chomczynski and Sacchi18 and by using magnetic beads (Dynal mRNA Direct kit).
Reverse Transcription–Polymerase Chain Reaction (RT-PCR) and Cloning
Published sequences for rat Cav subunit were used to design primers. BamHI or EcoRI restriction sites were added to all oligomers to facilitate cloning (DNA Technology, Denmark).
CaV1.2 (α1C): Forward: 5′-ATCCCCAAGAACCAGCAC-3′; reverse: 5′-GGTGATGGAGATGCGGGAGTT-3′, covering bases 3882 to 4253, 371 bp; set 2: forward: 5′-CCAAGAACCAGCACCAG-3′; reverse: 5′-CCCACAACAATCAAGGC-3′, covering bases 3886 to 4153, 268 bp (GenBank accession No. M59786)
CaV2.3 (α1E): Forward: 5′-CATGCCACAGAACAGGC-3′; reverse: 5′-CTTTATGAGCCGTGCAGC-3′, covering bases 4427 to 4805, 379 bp (GenBank accession No. L15453)
CaV1.3 (α1D): Forward: 5′-AATCCAAGATGTTCAATGACG-3′; reverse: 5′-GTGATGGAGATTCTATTGC-3′, covering bases 4208 to 4470, 262 bp (GenBank accession No. M57682).
CaV3.1 (α1G): Forward: 5′-GAACGTGAGGCCAAGAGT-3′; reverse: 5′-GCTTGTATGCGTTCCCCT-3′, covering bases 3910 to 4130, 221 bp (used for rat); forward: 5′-AGCAACAC-CACCTGTGTC-3′; reverse: 5′-GCAATCACCACCAGGCAC-3′, covering bases 1405 to 1664, 260 bp (used for rabbit) (GenBank accession No. AF027984).
CaV3.2 (α1H): Forward: 5′-GCTCTCACCCGTCTACTTCG-3′; reverse: 5′-AGATACTTTTCGCACGACCAGG-3′, covering bases 5568 to 5795, 256 bp (GenBank accession No. AF290213)
CaV3.3 (α1I): Forward: 5′-CCTTGATACCAGGGAC-3′; reverse: 5′-GGTCATCCATCTTGGTG-3′, covering bases 3316 to 3476, 163 bp (GenBank accession No. AF086827)
β-actin: copied from Yu et al12
The cDNA used for PCR corresponded to 5 to 10 vascular branching points (preglomerular vessels), 100 ng total RNA (smooth muscle cells, mesangial cells, and whole kidney), 0.5 mm cortical efferent arteriole length, or 1 mm juxtamedullary efferent arteriole and vasa recta length.
The RT-PCR amplification products for all investigated CaV subunits were cloned in vector pSP73 (Promega) using standard procedures.19 Plasmid DNA was extracted using the Qiagen Plasmid Maxi Kit according to manufacturer’s instructions. The inserts were sequenced using primers specific for T7 and SP6 promoters on an ABI PRISM 350 sequencer (Perkin Elmer).
Probes were synthesized with [α-32P]dCTP by standard methods. PCR products were separated by agarose gel electrophoresis and blotted to Zeta Probe GT membranes (Bio-Rad, Hercules). Hybridization of specific radioactive probe was allowed overnight.19 The membrane was washed and autoradiography was performed for 2 to 4 hours.
Immunoperoxidase Histochemistry for CaV1.2
Cryosections (5 μm) from rat kidneys were air-dried. Sections incubated with 5% goat serum in TRIS-buffered saline (TBS) for 30 minutes. Next, the sections were incubated with primary rabbit anti-rat CaV1.2 antibody (Alomone Laboratory, Jerusalem, Israel), diluted 1:20 in TBS, for 1 hour. Secondary antibody (goat anti-rabbit IgG, HRP-labeled), diluted 1:500 with TBS, was applied for 45 minutes. The sections were stained with diaminobenzidine (DAB+ substrate-chromogen system, DAKO).
Immunostaining of Isolated Microvessels
The whole preglomerular vasculature was microdissected from rat kidneys after HCl-maceration.16,20 Glomeruli with attached intact vessels were microdissected from superficial and from deep kidney cortex. The staining of large preglomerular specimens was done on a slide, and the single glomerular specimens were transferred to a chamber on an inverted microscope and held with glass pipettes. Samples were fixed and endogenous peroxidases were blocked by incubation with 5% hydrogen peroxide in methanol for 10 minutes, followed by 5% goat serum for 30 minutes. Immunostaining was performed with primary rabbit anti-rat CaV1.2 antibody, diluted 1: 50 in TBS, for 1.5 hours. Secondary antibody (goat anti-rabbit IgG, HRP-labeled), diluted 1:1000 with TBS, was applied for 30 minutes. The vessels were stained with diaminobenzidine (DAB+ substrate-chromogen system).
Measurement of Intracellular Calcium Concentration by Digital Fluorescence- Imaging Microscopy
Intracellular calcium was estimated with fura-2/AM in microdissected glomerular arterioles as described.16 Afferent and cortical efferent arterioles were from rabbits and juxtamedullary efferent arterioles were from male Sprague-Dawley rats. Fluorescence changes were measured in response to K+ (100 mmol/L) for 1 minute. For cortical efferent arterioles, ionomycin (Sigma) served as positive control. The α-adrenoceptor blocker phentolamine (10 μmol/L) was used to exclude nerve-mediated effects on the smooth muscle. The effect of the specific L-type blocker calciseptine (Alomone Labs) and the T-type blocker mibefradil was investigated in separate experiments in juxtamedullary efferent arterioles. K+ was added for 1 minute and the vessel was allowed to rest before calciseptine (10 nmol/L) or mibefradil (100 nmol/L) was added, followed by addition of K+.16
Isolation and Microperfusion of Renal Arterioles
Rabbit afferent arterioles were microdissected, transferred to a thermoregulated chamber on an inverted microscope, and perfused with concentric pipettes.21 A test stimulus of 100 mmol/L K+ was given to ensure viability.
The effect of the T-type calcium channel antagonist mibefradil on K+-induced responses in afferent arterioles was determined. The vessels were incubated for 5 minutes at each of a range of increasing concentrations of mibefradil (10−10 to 10−6 mol/L). After 5 minutes at each concentration, K+ (100 mmol/L) was added for 1 minute. Reversibility was tested by addition of K+ at the end of the experiment. Phentolamine (10−5 mol/L) (Sigma) was added to all solutions.
A second blocker of T-type channels, NiCl2, was tested with 1-minute preincubations at concentrations from 10−6 to 10−3 mol/L.
A specific L-type calcium-channel blocker, calciseptine, was tested with 5-minute preincubations in concentrations from 10−16 to 10−10 mol/L.
To assess, whether activation of L-type calcium channels was sufficient to produce contraction, the L-type calcium channel agonists (±)-Bay K 8644 and S-(-)-Bay K 8644 (Sigma) were tested. Bay-K was added for 1 minute at concentration 10−9 to 10−5 mol/L. This was repeated with 20 mmol/L potassium present.
Rat juxtamedullary efferent arterioles were microdissected and perfused at 30 mm Hg. The response to addition of 100 mmol/L K+ was tested.
Localization of CaV Subunits in Microdissected Renal Resistance Vessels and Smooth Muscle Cells
Cloned PCR-amplification products for CaV1.2, CaV1.3, CaV2.3, CaV3.1, CaV3.2, and CaV3.3 calcium channel subunits were 100% identical to published sequences. By RT-PCR analysis, we observed significant expression of CaV1.2, CaV3.1, and CaV3.2 mRNAs in 5 separate samples of rat preglomerular microvessels and in cultured smooth muscle cells grown from rat preglomerular vessels (Figure 1). No amplification products were observed for CaV1.3, CaV2.3, and CaV3.3 in the rat kidney cortex (Figure 1D). The RT-PCR analysis of postglomerular vascular segments was restricted to CaV1.2, CaV3.1, and CaV3.2. Amplification by PCR for up to 34 cycles with template cDNA from 0.5 mm length of rabbit superficial efferent arteriole did not result in detectable products for CaV1.2, CaV3.1, or CaV3.2 subunits (n=3). Actin was clearly expressed in these vessels (Figure 2A). In contrast, CaV1.2, CaV3.1, and CaV3.2 were expressed in rat juxtamedullary efferent arterioles (cDNA corresponding to 1 mm vessel length was used as template for 32 cycles of PCR, n=4) (Figure 2B). Similarly, CaV3.1, CaV3.2, and CaV1.2 were detected in rat outer medullary vasa recta bundles and in single vasa recta (Figure 2C). Cultured mesangial cells expressed CaV1.2 and CaV3.1 (data not shown). Amplification products were not observed when cDNA or reverse transcriptase was omitted (Figures 2A through 2C).
Immunolocalization of CaV1.2-Subunit Protein in Isolated Vessels and Kidney Sections
Immunohistochemistry for CaV1.2-subunit protein on rat kidney cryosections revealed labeling of all large vessels, glomerular arterioles, and glomeruli (Figure 3, left). Labeling was not seen when the primary antibody was omitted (Figure 3). It was not possible to clearly distinguish between afferent and efferent arterioles in the cryosections. Instead, rat vessels were microdissected from HCl-macerated kidneys and immunostained for CaV1.2 protein. The anti-CaV1.2 antibody strongly labeled all segments of the preglomerular vasculature (and glomeruli) with no difference in intensity at bifurcations or in terminal segments (n=9 separate dissections) (Figure 4A). In 5 single microdissected rat glomeruli, the CaV1.2-subunit antibody did not label cortical efferent arterioles although the parent glomerulus was strongly immunopositive (Figure 4B). In contrast, efferent arterioles from rat juxtamedullary glomeruli were clearly labeled by anti-CaV1.2 antibody (n=3) and vasa recta were also immunopositive (Figure 4C). In parallel with the individual glomeruli, large preglomerular vascular trees served as positive and negative controls. Thus, the immunolabeling data confirm the data on distribution of CaV1.2 mRNA in the rat kidney.
Determination of Intracellular Calcium Concentration
Next, we measured intracellular calcium concentration in microdissected afferent arterioles, in cortical, and in juxtamedullary efferent arterioles in response to K+-induced depolarization (Figure 5A). In rabbit afferent arterioles, the resting calcium concentration was 102±22 nmol/L and K+ elicited an average increase in calcium of 143±25% (n=10; Figure 5B). Positive responses could be repeated several times in the individual specimen. Rabbit cortical efferent arterioles had a resting calcium concentration of 88±13 nmol/L. K+ elicited a small significant change in intracellular calcium (21±9%; n=7; Figure 5B) whereas ionomycin (10 μmol/L) strongly increased calcium in these vessels. In rat juxtamedullary efferent arterioles, resting calcium concentration was 105±11 nmol/L. K+ increased the intracellular calcium concentration by 118±22% (n=7; Figure 5B). The K+-induced increase in calcium in juxtamedullary efferent arterioles was inhibited to 43±17 nmol/L (63±21% inhibition) by 2 minutes of incubation with the specific L-type peptide blocker calciseptine (P<0.01) (Figure 5B). The T-type antagonist mibefradil (100 nmol/L) significantly inhibited the K+-induced increase in calcium both in afferent arterioles (control 72±6 nmol/L, K+ 175±25 nmol/L, K+ and mibefradil 118±23 nmol/L, n=6) and in juxtamedullary efferent arterioles (control 105±20 nmol/L, K+ 242±39, K+ and mibefradil 149±24, n= 8) (Figure 5C). Thus, both L- and T-type calcium channels contribute to depolarization-induced calcium influx in juxtamedullary efferent arterioles.
Isolated Perfused Arterioles
The functional significance of T- and L-type calcium channels for vasoreactivity in response to depolarization was investigated in perfused rabbit afferent arterioles.
In the absence of mibefradil, K+ (100 mmol/L) occluded the arteriolar lumen as previously shown.22 Addition of mibefradil to afferent arterioles did not change the basal diameter of the arterioles. Vasoreactivity to K+ was partially blocked by mibefradil at 10−8 mol/L and reversibly abolished at 10−6 mol/L. The average responses for mibefradil obtained in 11 experiments are shown in Figure 6B. Mibefradil inhibited K+-induced responses with an estimated EC50 of 10−8 mol/L.
Ni2+ did not affect basal diameter of the afferent arterioles. 10−6 mol/L of Ni2+ did not modify K+-induced contraction when the vessel was exposed to Ni2+ for 1 minute. Higher concentrations of Ni2+ blocked the contractile response to K+ concentration dependently and reversibly with an EC50 of 3×10−4 mol/L and total blockade of the vasoreactivity at 10−3 mol/L (n=6) (Figure 6).
Calciseptine did not affect the basal diameter of the afferent arterioles. Calciseptine potently inhibited K+-induced constriction in a concentration-dependent and reversible fashion (Figure 7, n=8) with an EC50 of 8×10−14 mol/L and total blockade at 10−11 mol/L.
The L-type calcium channel agonist Bay-K 8644 was added to perfused afferent arterioles at control levels of K+ (3 mmol/L) and at elevated levels of external K+ (20 mmol/L), which had no effect on vascular diameter by itself. At these concentrations of potassium, there was no response to the mixed (±) type Bay K 8644 (10−9, 10−8, or 10−7 mol/L) (data not shown) (n=5). The more specific enantiomer S-(-)-Bay K 8644 also had no effect on arteriolar diameter in separate experiments (26% reduction at 10−6 and 30% at 10−5 mol/L [not significant], Figure 8). In contrast, potassium (100 mmol/L) occluded the vessel lumen (n=9). In addition, when the vessels were exposed to a constant level of extracellular K+ of 20 mmol/L, S-(-)-Bay K 8644 had no effect on vasoreactivity (23% contraction at 10−5 mol/L, data not shown).
We managed to obtain microperfusion of two rat juxtamedullary efferent arterioles from separate animals. Basal diameters were 18 and 16 μm, respectively. Depolarization of the juxtamedullary efferent arterioles with potassium (100 mmol/L) completely occluded the vessel lumen in both cases.
The present study provides data on the molecular and functional profile of voltage-dependent calcium channels in the renal resistance vasculature. The data show a selective expression of CaV1.2, CaV3.1, and CaV3.2 calcium channel subunits in rat renal microvessels. CaV1.2, CaV3.1, and CaV3.2 mRNAs were found in preglomerular vessels, juxtamedullary efferent arterioles, and vasa recta, whereas no CaV transcripts were found in cortical efferent arterioles. Moreover, in both afferent arterioles and juxtamedullary efferent arterioles, membrane depolarization by K+ evokes calcium increases that are sensitive to specific L- and T-type channel blockers. In contrast, K+ caused a modest increase in intracellular calcium concentration in cortical efferent arterioles. These findings were reflected in a concentration-dependent inhibition of K+-induced contraction of perfused afferent arterioles by blockers of L- and T-type calcium channels. Thus, the functional data demonstrate that, at least in afferent arterioles, the cooperative action of both L- and T-type channels is required to elicit full contraction in response to depolarization. Moreover, the data suggest that T- and L-type calcium channels contribute significantly to excitation-contraction coupling in efferent arterioles of juxtamedullary glomeruli and in vasa recta. All data on the juxtamedullary efferent arterioles were obtained with rat tissue. The cortical efferent arterioles used for molecular study and calcium measurements were from rabbits because these vessels are easier to dissect from rabbits than from rats. The data suggest good correlation across the two species with respect to the absence of calcium channels in cortical efferent arterioles. Thus, these vessels were immunonegative for the L-type calcium channel protein in rats and negative for L-type calcium channel mRNA in the rabbit.
In previous studies, efferent arterioles have been found either to contract in response to K+5,6 or to be insensitive to K+,8 and the intracellular concentration of calcium has been reported to increase11 or to decrease after K+ exposure.4,7 Calcium channel blockers have been shown to have no effect on intracellular calcium7 or vascular diameter8–10 of efferent arterioles or to significantly reduce calcium increases in these vessels.11 Similar to the present findings, Helou and Marchetti11 reported that K+ and angiotensin II raised intracellular calcium concentration in juxtamedullary efferent arterioles in a nifedipine-sensitive way. In preparations like perfused hydronephrotic kidneys8,10 and isolated efferent smooth muscle cells,4 it is not possible to determine the intrarenal origin of the efferent arterioles used for the study. The commonly observed lack of voltage-dependent calcium pathways in these preparations probably reflects a superficial or midcortical origin because the vast majority of efferent arterioles are superficial or midcortical. The efferent arterioles that drain the juxtamedullary glomeruli exclusively supply and regulate blood flow to the renal medulla. Their structure differs markedly from cortical efferent arterioles,23 and the present data show that these vessels are different also at the functional level. It could be expected that L-type and T-type calcium channel blockers would increase blood flow to the medulla. Studies in dogs with intrarenal infusion of the L-type antagonist nicardipine indeed showed a greater increase in flow rate in the juxtamedullary area compared with the cortex.24 Studies with laser-Doppler technique confirm that systemic or local administration of verapamil preferentially increases medullary blood flow.25–27 Thus, the available data on renal blood flow regulation support the notion that L-type calcium channels are involved in the maintenance of tone in vessels supplying the renal medulla. The finding of CaV in juxtamedullary efferent arterioles and vasa recta suggests that the smooth muscle membrane potential is important for control of tone in these vascular segments. In accordance with this, recent data have shown that descending vasa recta depolarize in response to angiotensin II, thus more closely resembling afferent arterioles than cortical efferent arterioles.28
Our data show a widespread and even presence of the classical cardiovascular L-type subunit CaV1.2 along all segments of the rat preglomerular vasculature. With a nonspecific antibody against CaV subunits, a preferential localization in branching points along rat preglomerular resistance vessels has previously been shown.29 This discrepancy might reflect labeling of a different CaV subunit by the nonspecific antibody. In any case, the highly potent blockade of K+-induced vasoreactivity in afferent arterioles by the specific L-type blocker calciseptine, which has no effect on T-type currents,30 underlines the critical contribution of the CaV1.2 subunit to depolarization-induced contraction. These findings confirm and extend a large amount of pharmacological data showing that relatively specific blockers of L-type channels (dihydropyridines, benzothiazepines) potently dilate preglomerular vascular segments constricted by pressure, K+, or agonists.6–8
T-type currents have been shown in rat renal vascular myocytes by patch clamp, but their role in vascular function is poorly understood.1 The membrane potential in pressurized afferent and efferent arterioles is around −40 mV,31 which is presumably more positive than the activation threshold for T-type channels. The participation of T-type calcium channels in excitation-contraction coupling was demonstrated by the inhibitory action of mibefradil on calcium influx in afferent arterioles and juxtamedullary efferent arterioles, and by the inhibition of contraction by nickel and mibefradil in afferent arterioles. The CaV3.1 channel has recently been shown to be relatively sensitive to mibefradil.32 Mibefradil inhibits calcium currents elicited by CaV2.1, CaV2.2, CaV1.2, and CaV2.3 subunits (yielding currents corresponding to P-/Q- type, N-type, L-type, and R-type) in the range 10 to 100 μmol/L,33,34 whereas endogenous or experimentally expressed T-type currents are inhibited with an EC50 around 10 nmol/L.31,33 In our experiments, mibefradil blocked K+-induced contraction in afferent arterioles with an EC50 of 10 nmol/L and significantly inhibited intracellular calcium increases at a concentration of 100 nmol/L, which is still in the specific range. T-type currents elicited by CaV3.1 and CaV3.3 subunits are blocked by Ni2+ in the range 0.5 to 1 mmol/L, whereas only CaV3.2 currents were blocked in the low micromolar range.32,35 The lack of effect of Ni2+ in the low micromolar range suggests that the Cav3 subtype involved in contraction in rabbit afferent arterioles is CaV3.1. It is possible that CaV3.1 is the only T-type subunit expressed in rabbit vessels because the preglomerular expression profile (Figure 1) was obtained with rat vessels. The data suggest a significant contribution of T-type calcium channels to excitation-contraction coupling initiated by K+-depolarization in afferent and juxtamedullary efferent arterioles.
We have previously shown the molecular and functional presence of the CaV2.1 subunit encoding a P-/Q-type current in afferent arterioles and vasa recta.16 Thus, application of either type of specific antagonist of L-, T-, or P-/Q-type channels in relevant concentrations is equally effective in blocking K+-induced contraction of afferent arterioles. Patch-clamp data have clearly shown distinct phenotypes of preglomerular smooth muscle cells; ie, some cells have only L- or T-type calcium currents whereas in others both types are present.1 In cells where the channels are colocalized, it is possible that a cooperative action of all available calcium channels is required to achieve a sufficiently high concentration of intracellular calcium to elicit and maintain contraction. The lack of effect of the calcium channel agonist Bay K would support the notion that activation of a single calcium channel subtype, in this case the L-type, is not sufficient to elicit contraction.
In summary, L-type calcium channels are present at the mRNA, protein, and functional level in afferent and juxtamedullary efferent arterioles, and vasa recta but were not found in cortical efferent arterioles. A similar pattern was found for T-type calcium channels. In the postglomerular vascular segments that regulate and supply blood flow to the kidney medulla, excitation-contraction coupling may depend on calcium influx through voltage-gated pathways.
This work was supported by grants from the Danish Medical Research Council (9601829, 9902742, 9903058), the Novo Nordisk Foundation, The Danish Heart Foundation (99223622743, 01123022896), Ruth T.E. König-Petersen Foundation for Kidney Diseases, the Danish Medical Association Research Fund, the Foundation of 23-9-1909, and the Hartelius Legacy. The authors thank Anthony M. Carter for language revision, Peter D. Ottosen for help with photography of immunostainings, and Mette Fredenslund, Inge Andersen, and Karin Kejling for technical assistance.
Original received March 26, 2001; revision received August 3, 2001; accepted August 3, 2001.
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