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(Circulation Research. 2000;87:896.)
© 2000 American Heart Association, Inc.

Cellular Biology

Vascular Smooth Muscle Cells Express the _1A Subunit of a P-/Q-Type Voltage-Dependent Ca²⁺Channel, and It Is Functionally Important in Renal Afferent Arterioles

Pernille B. Hansen, Boye L. Jensen, Ditte Andreasen, Ulla G. Friis, Ole Skøtt

From the Department of Physiology and Pharmacology, University of Southern Denmark–Odense University, Odense, Denmark.

Correspondence to Boye L. Jensen, MD, PhD, Department of Physiology and Pharmacology, University of Southern Denmark–Odense University, Winsloewparken 21,3. DK-5000 Odense C, Denmark. E-mail bljensen{at}health.sdu.dk

	Abstract

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Abstract—In the present study, we tested whether the _1A subunit, which encodes a neuronal isoform of voltage-dependent Ca²⁺ channels (VDCCs) (P-/Q-type), was present and functional in vascular smooth muscle and renal resistance vessels. By reverse transcription–polymerase chain reaction and Southern blotting analysis, mRNA encoding the _1A subunit was detected in microdissected rat preglomerular vessels and vasa recta, in cultures of rat preglomerular vascular smooth muscle cells (VSMCs), and in cultured rat mesangial cells. With immunoblots, _1A subunit protein was demonstrated in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-_1A antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22±4% (n=6) inhibition of inward Ca²⁺ current by -Agatoxin IVA (10^–8 mol/L), which in this concentration is a specific inhibitor of P-type VDCCs. Measurements of intracellular Ca²⁺ in afferent arterioles with fluorescence-imaging microscopy showed 32±9% (n=10) inhibition of the K⁺-induced rise in Ca²⁺ in the presence of 10^–8 mol/L -Agatoxin IVA. In microperfused rabbit afferent arterioles, -Agatoxin IVA inhibited depolarization-mediated contraction with an EC₅₀ of 10^–17 mol/L and complete blockade at 10^–14 mol/L. We conclude that the _1A subunit is expressed in VSMCs from renal preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca²⁺ influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles.

Key Words: smooth muscle • voltage-dependent Ca²⁺ channel • renal • arteriole

	Introduction

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Excitation-contraction coupling in most resistance vessels is highly dependent on Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs), and inhibitors of such channels are in widespread clinical use. Voltage-gated Ca²⁺ influx in vascular smooth muscle cells (VSMCs) is usually ascribed to activation of high voltage–activated (L-type) and in some cases to low voltage–activated (T-type) VDCCs.¹ The VDCC protein complex consists of up to five different subunits. The ₁ subunit forms the pore of the channel, the drug binding site, and the voltage sensor.

In the kidney, L- and T-type Ca²⁺ currents have been identified by patch clamp in smooth muscle cells isolated from renal preglomerular vessels.² Moreover, branching points along the renal resistance vessels are enriched in L-type VDCCs.³ Messenger RNAs encoding ₁ subunits of L- and T-type VDCCs have been demonstrated in the rat kidney by Northern blotting,^{4 5} but in addition to this, mRNA for the _1A subunit, which encodes the neuronal P-/Q-type Ca²⁺ channel, has been shown to be present in kidney cortex.⁴ L- and T-type VDCCs cannot fully account for depolarization-induced Ca²⁺ influx in renal VSMCs.⁶ We investigated, using kidney and aorta, whether P-/Q-type VDCCs are present in vascular smooth muscle, and whether they contribute to Ca²⁺ fluxes and vasoreactivity.

The results demonstrate that _1A subunit mRNA and protein are expressed and that P-type VDCCs contribute to depolarization-mediated Ca²⁺ influx and contraction in VSMCs.

	Materials and Methods

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Cells
Preglomerular VSMCs were prepared as described by Dubey et al.⁷ Renal microvessels were isolated from rat kidneys after iron oxide particle infusion, enzyme-digested, separated by a magnetic field, and suspended in RPMI 1640. When cells were confluent, RNA was isolated (RN'easy kit, Qiagen). The VSMCs were characterized by immunocytochemical staining for smooth muscle -actin (clone 1A4) (Sigma) (Figure 1). Mesangial cells were obtained by outgrowth from isolated glomeruli as previously described.⁸ Only cells from first passage were used for RNA extraction. The aortic cell line A7r5 was from American Type Culture Collection.

View larger version (123K): [in this window] [in a new window]   Figure 1. Figure 1. Immunostaining of renal VSMCs. A, Immunolabeling of a confluent monolayer of VSMCs with anti–-actin antibody. Bar=75 µm. B, Negative control with no primary antibody added.

Microdissection of Rat Preglomerular Vessels and Vasa Recta
Renal vessels for RNA extraction were obtained by dissection of rat kidney tissue from 8 rats according to the protocol of Yang et al.⁹ Two consecutive divisions of preglomerular vessels were isolated. Vasa recta bundles were isolated from outer medulla. Of the preglomerular samples, 40 to 50 "branching points" were pooled. Vasa recta bundle length was measured with a micrometer scale built into the ocular. Total RNA was isolated according to a microadapted protocol of Chomczynski and Sacchi.¹⁰

Reverse Transcription–Polymerase Chain Reaction (RT-PCR) and Cloning
RT-PCR analysis was performed. BamHI or EcoRI restriction sites were added to all oligomers (DNA Technology): _1A: forward: 5'-ATT ACA TCC TGA ACC-3'; reverse: 5'-CTT CAA CTT AGG CAG C-3', covering bases 3564 to 3929, 383 bp (GenBank accession No. M64373). ß-actin: copied from Yu et al.⁴

The cDNA used corresponded to 5 to 10 branching points (preglomerular vessels), 100 ng total RNA (smooth muscle cells and whole kidney) or 1 mm (vasa recta). PCR products were inserted in vector pSP73 (Promega) for heat-shock uptake by competent Escherichia coli (DH5, GIBCO) using standard procedures.¹¹ Plasmid DNA was extracted using the QIAGEN Plasmid Maxi kit. Inserts were sequenced using T7 and SP6 promoter primers on an ABI PRISM 350 sequencer (Perkin Elmer).

Southern Blotting
PCR products were separated by agarose gel electrophoresis and blotted to Zeta Probe GT membranes (Bio-Rad) using standard capillary blotting procedures (transfer buffer: 0.4 mol/L NaOH). Hybridization was allowed overnight to specific probe and in vitro–labeled with -³²P-dCTP, all according to Sambrook et al.¹¹ Autoradiography was performed for 2 to 4 hours on Kodak Biomax MS film.

Western Blotting
Tissues were dissected, snap-frozen, and homogenized in 0.3 mol/L sucrose, 25 mmol/L imidazole, Complete, pH 7.2, and centrifuged 4000g for 15 minutes. Protein concentrations were determined using the Bio-Rad protein assay, with BSA as standard. Cultured cells were rinsed twice in TBS (20 mmol/L Tris-HCl, 137 mmol/L NaCl, pH 7.6), suspended in 100 µL lysis buffer (0.1% Triton-X, 1 tablet/10 mL Complete Mini [Roche Molecular Biochemicals]), and quick-frozen. SDS-PAGE and Western blotting were performed. The primary antibody was anti-_1A subunit (Alomone Labs). Secondary antibody was goat anti-rabbit IgG, HRP-labeled (NEN). Proteins were detected using Renaissance Chemiluminescent Reagent Plus (NEN).

Immunostaining
Renal vascular trees were microdissected from rats after HCl maceration,¹² fixed in 3.7% paraformaldehyde, and permeabilized with methanol plus 0.006% H₂O₂ and immunolabeled. The primary antibodies were rabbit anti-rat _1A antibody and rabbit anti-mouse renin antibody. The secondary antibody was goat anti-rabbit IgG, HRP-labeled. Staining was with diaminobenzidine (DAB⁺ substrate chromogen system, DAKO). Cultures of A7r5 cells were fixed, permeabilized, and immunolabeled with anti-rat _1A antibody in a similar way and then counterstained with hematoxylin.

Patch-Clamp Experiments
Patch-clamp experiments were performed on A7r5 cells at room temperature in the tight-seal whole-cell configuration of the patch-clamp technique¹³ with heat-polished, Sylgard-coated patch pipettes with resistances of 3 to 5.5 M. The pipette solution contained (in mmol/L) CsCl 120, MgCl₂ 3, HEPES-CsOH 5, MgATP 5, and EGTA 10, pH 6.00 (24.1°C). Series resistances were 6 to 20 M and seal resistances were 2 to 15 G. High-resolution membrane currents were recorded with an EPC-9 patch-clamp amplifier (HEKA). Immediately after the whole-cell configuration was obtained, the cells were superfused with a solution that facilitated Ca²⁺ currents (in mmol/L: tetraethylammonium acetate 148, KCl 2.8, MgCl₂ 1.0, BaCl₂ 10.8, and HEPES-CsOH 10 [pH 7.23, 21.4°C]) for 1 to 2 minutes. The cells were then superfused with the same solution supplemented with -Agatoxin IVA (10^–8 mol/L) (Alomone Labs).¹⁴

Measurement of [Ca²⁺]_i by Digital Fluorescence-Imaging Microscopy
Microdissected rabbit afferent arterioles were placed in a perfusion chamber on an inverted microscope, secured by holding pipettes, and loaded with 5 µmol/L fura-2/AM in physiological salt solution (PSS) for 45 minutes at room temperature. Excitation light was provided by a monochromator at 350 and 380 nm, and the output at 510 nm was detected by a charged coupled device camera and light intensifier and analyzed using Metafluor software (Universal Imaging). Measurements of fluorescence intensity were performed at a rate of 1 frame per second, and the ratio of 350:380 was used to calculate [Ca²⁺]_i, applying fura 2/K⁺ calcium standards.¹⁵ In 10 experiments, changes in [Ca²⁺]_i were measured after addition of 100 mmol/L K⁺ (with phentolamine 10^–5 mol/L). After a 5-minute wash in PSS, the vessel was exposed to -Agatoxin IVA (10^–8 mol/L) for 1 minute and the response to K⁺ was measured again in the presence of -Agatoxin IVA.

PSS had the following composition (mmol/L): NaCl 115, NaHCO₃ 25, K₂HPO₄ 2.5, CaCl₂ 1.3, MgSO₄ 1.2, and glucose 5.5. High potassium solution contained (mmol/L) NaHCO₃ 25, NaCl 20, KCl 95, MgSO₄ 1.2, K₂HPO₄ 2.5, CaCl₂ 1.3, and glucose 5.5. Both solutions contained 0.1% BSA at pH 7.4.

Isolation of Rabbit Afferent Arterioles and Microperfusion Protocols
Afferent arterioles were microdissected from 10 rabbits and perfused with PSS.¹⁶ Repetitive depolarizations of each of the perfused afferent arterioles were done in the presence of increasing concentrations of -Agatoxin IVA (10^–20 to 10^–10 mol/L). After preincubation with toxin at each concentration for 1 minute, K⁺ (100 mmol/L) was added for 1 minute. The experiments were recorded on videotape, sequences of interest were digitized, and luminal vessel diameters were assessed using Metamorph imaging software (Universal Imaging).

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Confirmation of Probe Sequences
The _1A PCR amplification product was cloned in vector pSP73, and the insert was sequenced. The insert was 100% identical to the published sequence.¹⁷

Localization of ₁ Subunits in Microdissected Renal Resistance Vessels and VSMCs
We performed RT-PCR analysis and subsequent Southern blotting on RNA extracted from microdissected rat preglomerular vessels and vasa recta bundles and from primary cultures of quiescent renal smooth muscle. Using cDNA from preglomerular microvessels as a template, PCR amplification revealed a significant expression of _1A (Figure 2A). Amplification products for _1A were observed when RNA from the cultured renal smooth muscle cells and mesangial cells was analyzed by RT-PCR and amplified from serial dilutions (Figure 2A). This confirms the expression of the _1A gene in contractile cells. Microdissected vasa recta bundles also expressed _1A subunit mRNA (Figure 2B). To test whether the _1A subunit is expressed in smooth muscle cells from other vascular beds, we examined _1A subunit mRNA expression in aortic smooth muscle cells and confirmed that the aortic cells (A7r5) also expressed _1A (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 2. Figure 2. Expression of 1A subunit mRNA in vascular smooth muscle shown with RT-PCR and Southern blotting. Top, Ethidium bromide–stained agarose gel showing RT-PCR amplification products using primers specific for 1A. Middle, Southern blotting of the above gel, using an 1A-specific probe. Bottom, Ethidium bromide–stained agarose gel showing RT-PCR products for ß-actin. A, Samples were as follows: dilution series of cDNA from VSMCs, cDNA from 5 separate samples of microdissected renal preglomerular resistance vessels, cDNA from cultured mesangial cells (MES), and whole kidney. Negative controls were water and tRNA. indicates molecular weight standard X173HaeIII. B, Demonstration of expression of 1A subunit mRNA in microdissected postglomerular vasa recta bundles (v. recta). For comparison, a sample of microdissected preglomerular vessels (preglom., compare panel A) is shown. Whole kidney is positive control, and tRNA serves as negative control.

Western Blotting
To verify that _1A protein, in addition to mRNA, was present in the VSMCs, we performed Western blots. In immunoblots of protein extracts, cultured renal smooth muscle cells, cultured mesangial cells, and A7r5 cells, as well as aorta and brain, gave rise to bands of the expected size (190 kDa) when labeled with an anti-_1A antibody (Figure 3). By preabsorbing the primary antibody with the peptide CNA1 (used for raising the antibody), the labeling was completely displaced, confirming the specificity.

View larger version (35K): [in this window] [in a new window]   Figure 3. Figure 3. Expression of 1A subunit protein in vascular smooth muscle demonstrated with Western blots. A, Protein isolated from an aortic smooth muscle cell line (A7r5), cultured renal VSMCs, and cultured mesangial cells (MES). Anti-1A+CNA1 indicates negative control; the antibody was preabsorbed with the peptide it was raised against. B, Protein isolated from fresh aorta and brain.

Immunostaining
As a test of the presence of _1A protein in the isolated renal resistance vessels, we performed immunolabeling with anti-_1A antibody of HCl-macerated, microdissected renal microvasculature. This approach revealed expression of the protein throughout the preglomerular vasculature, as well as in the glomeruli (Figure 4A). Preabsorbing the primary antibody with the peptide CNA1 displaced the labeling, and no unspecific labeling was detected in the absence of primary antibody. As a control for the overall specificity of this labeling method on the acid-macerated tissue, we also used an anti-mouse renin antibody. As expected, this antibody gave a very distinct labeling of the endpoints of the afferent arterioles (Figure 4B). We then tested whether the _1A protein was present in the cultured aortic cell line A7r5. Labeling is seen in a granular pattern throughout the cells, verifying expression of the protein in smooth muscle cells (Figure 5A). In the absence of primary antibody, no labeling was seen in A7r5 cells (Figure 5B).

View larger version (45K): [in this window] [in a new window]   Figure 4. Figure 4. Immunolabeling of 1A subunit protein in renal preglomerular vasculature. A, Immunostaining of HCl-treated, microdissected renal vascular trees with anti-1A antibody showing expression of 1A subunit protein throughout the preglomerular vasculature. Bar=500 µm. Negative controls were displacement of antibody by preabsorbing the primary antibody with the peptide CNA1 or without primary antibody. B, Immunostaining with anti-mouse renin antibody of renin-containing cells at the ends of the afferent arterioles. Bar=75 µm.

View larger version (89K): [in this window] [in a new window]   Figure 5. Figure 5. Immunolabeling of 1A subunit protein in cultured VSMCs (A7r5). A, Immunostaining of A7r5 cells with anti-1A antibody showing expression of the protein. Bar=50 µm. B, Negative control without primary antibody.

Patch Clamp
The presence of P-type Ca²⁺ channels was also tested on A7r5 cells with electrophysiology. When these cells were depolarized from a holding potential of -70 to +10 mV, a large negative current was observed (Figure 6A). Significant rundown was observed after 6 to 8 minutes. This current was sensitive to inhibition with the specific P-type VDCC blocker -Agatoxin IVA (10^–8 mol/L) (Figure 6A, upper trace), indicating that part of this current is an inwardly directed Ca²⁺ current through P-type Ca²⁺ channels. -Agatoxin IVA inhibited the current 22.1±4.2% (SE, n=6) (Figure 6B). When Na⁺ currents and K⁺ currents were eliminated by using Cs⁺ and tetraethylammonium in the buffers, a standard voltage-clamp protocol yielded the characteristic I-V relationship for Ca²⁺ currents as shown in Figure 6C. From this relationship, it can be noted that the maximal Ca²⁺ current is observed at +10 mV, which is the reason for the chosen pulse protocol shown in Figure 6A.

View larger version (23K): [in this window] [in a new window]   Figure 6. Figure 6. Inhibition of Ca2+ currents in cultured VSMCs (A7r5) by -Agatoxin IVA as shown by single-cell electrophysiology. A, Immediately after the whole-cell configuration was obtained (cell size 28 pF, seal resistance >2 G, series resistance 16 M) (t=0 seconds), the cell was superfused with a solution that facilitates Ca2+ currents (see online Materials and Methods; data supplement available at http://www.circresaha.org). A repetitive pulse was applied every 10 seconds (from -70 to +10 mV for 40 ms; see pulse protocol above the traces). When the inwardly directed current was large and stable (in this experiment at t=100 seconds, lower trace), the superfusion system was switched to a solution that also contained -Agatoxin IVA (10–8 mol/L). The upper trace shows the effect of -Agatoxin IVA obtained at t=240 seconds in the same cell. This trace has been offset with 15 pA for comparison. B, Histogram showing the inhibitory effect of -Agatoxin IVA on the inwardly directed current. The inhibition amounted to 22.1±4.2% (SE, n=6). C, Steady-state I-V relationship from the cell shown in panel A. The I-V curve was monitored by the response to 12 voltage steps of 10 mV (range, -70 to +50 mV) for 400 ms from a holding potential of -70 mV. The I-V curve was measured at t=278 seconds.

[Ca²⁺]_i Measurements
The effect of the specific P-/Q-type VDCC blocker -Agatoxin IVA on K⁺-induced increase in intracellular Ca²⁺ was tested on afferent arterioles. In the experiment shown, the addition of 100 mmol/L K⁺ rapidly increased the [Ca²⁺]_i from 65 to 205 nmol/L; it then declined slowly until K⁺ was removed. The response to K⁺ after a 5-minute resting period and 1-minute incubation with -Agatoxin IVA was clearly inhibited (205 versus 160 nmol/L) (Figure 7A). No tachyphylaxis was observed to repeated administration of K⁺. In 10 experiments, -Agatoxin IVA inhibited the increase in [Ca²⁺]_i by 32.2±9.4% (SE). This is shown in Figure 7B. The K⁺-induced increases in Ca²⁺ with or without -Agatoxin IVA present were significantly different when tested by Student’s t test (P<0.05).

View larger version (15K): [in this window] [in a new window]   Figure 7. Figure 7. Inhibition of depolarization-induced increase in [Ca2+]i in afferent arterioles by -Agatoxin IVA as shown by fluorescence-imaging microscopy. A, Increase in [Ca2+]i after addition of 100 mmol/L K+ to an afferent arteriole is shown as control (upper trace). After a 5-minute resting period, the vessel was incubated for 1 minute with -Agatoxin IVA (10–8 mol/L) and the response to K+ was measured again (lower trace). B, Histogram showing the inhibitory effect of -Agatoxin IVA on the K+-induced increase in [Ca2+]i. The average inhibition was 32.2±9.4% (SE, n=10).

Isolated, Perfused Arteriole Studies
To test the functional significance of the P-/Q-type Ca²⁺ channel for K⁺-induced contraction in microperfused afferent arterioles, we used -Agatoxin IVA. A test stimulus of 100 mmol/L K⁺ was given initially to ensure viability of the vessel. The addition of -Agatoxin IVA for 1 minute to afferent arterioles perfused at physiological pressures (60 to 80 mm Hg) did not change the basal diameter of the arterioles. The time course of changes in vessel inner diameter from a typical experiment with an afferent arteriole is shown in Figure 8A. In the absence of -Agatoxin IVA, K⁺ (100 mmol/L) closed the arteriolar lumen as previously shown.¹⁸ The K⁺-induced contraction was totally abolished at 10^–12 mol/L of -Agatoxin IVA. After a resting period, the K⁺-induced response was fully restored. All vessels were treated with increasing concentrations of -Agatoxin IVA with a resting period followed by 1 minute of incubation between each addition of K⁺. The dose-response relationship for -Agatoxin IVA obtained in 13 arterioles is shown in Figure 8B. K⁺-induced responses were completely blocked at 10^–14 mol/L of -Agatoxin IVA. The EC₅₀ was 10^–17 mol/L, and the responses to -Agatoxin IVA at different concentrations were significantly different when tested by ANOVA (P<0.01).

View larger version (13K): [in this window] [in a new window]   Figure 8. Figure 8. Inhibition of depolarization-induced contraction in microperfused rabbit afferent arterioles by -Agatoxin IVA. A, Blocking effect of -Agatoxin IVA (-agatx) on K+ action in a single experiment. The sensitivity to K+ was tested before addition of -Agatoxin IVA, in the presence of 10–12 mol/L -Agatoxin IVA (after 1-minute preincubation), and after a resting period without -Agatoxin IVA. B, Dose-response curve for the effect of -Agatoxin IVA on K+-induced contraction (n=13). Basal diameter and response to K+ were tested before exposure to -Agatoxin IVA (squares). In individual arterioles, the sensitivity to repetitive 1-minute depolarizations was tested at increasing concentrations of -Agatoxin IVA (diamonds). After a resting period, the sensitivity to depolarization was tested again (triangle).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we aimed to elucidate the presence of and functional role for P-/Q-type voltage-gated Ca²⁺ channels in resistance vessels. The _1A subunit, which is considered the molecular correlate and minimum component for P-/Q-type Ca²⁺ currents, has previously been localized to the kidney cortex by Northern blotting⁴ and was detected along the nephron⁴ whereas the vessels have not previously been examined. The present set of data demonstrate expression of _1A subunit mRNA and protein in dissected renal microvessels, in aorta, and in cultured renal and nonrenal smooth muscle cells and contractile kidney mesangial cells. Significant immunolabeling for the _1A subunit was found in microdissected preglomerular vessels and cultured smooth muscle cells. Collectively these data suggest a widespread expression of the _1A subunit gene in vascular tissue including smooth muscle cells. The functional studies showed a significant contribution of P-/Q-type Ca²⁺ currents to whole-cell VDCCs in cultured smooth muscle A7r5 cells. In agreement with this, Ca²⁺ influx in response to depolarization was significantly reduced by the specific P-/Q-type Ca²⁺ channel blocker -Agatoxin IVA in isolated rabbit renal afferent arterioles (diameter 20 µm at physiological pressure). This was reflected by an extremely potent inhibition of K⁺-induced contraction of perfused afferent arterioles by -Agatoxin IVA. Together these sets of functional data demonstrate that Ca²⁺ influx and intracellular Ca²⁺ increases mediated by P-/Q-type Ca²⁺ channels are necessary to elicit contraction in response to depolarization, at least in renal resistance vessels. At present, we cannot explain the extreme sensitivity by which -Agatoxin IVA blocks contraction in afferent arterioles. The high sensitivity might reflect voltage-dependent binding of the blocker at depolarized potentials as seen during pressurization or specific modulation of the channel by auxiliary subunits expressed in afferent arterioles.

Alternative splicing of mRNA originating from the _1A subunit gene results in generation of two different gene products considered responsible for P-/Q-type Ca²⁺ currents, respectively.²⁰ The variants are discriminated by their sensitivity to -Agatoxin IVA, which blocks P-type channels at concentrations <10 nmol/L, whereas the Q channels are inhibited at higher concentrations, in the range of 0.1 to 1 µmol/L.²¹ We did not perform a full dose response for the effect of -Agatoxin IVA on Ca²⁺ current and [Ca²⁺]_i. However, the significant effect of -Agatoxin IVA on these parameters at 10 nmol/L, together with the extremely potent inhibition of K⁺-mediated contraction, suggests that the P-type VDCC is the dominant functional _1A subunit gene product in VSMCs of renal resistance vessels.

The demonstration of a P-type VDCC in vascular smooth muscle is compatible with the observation that a significant component of depolarization-induced Ca²⁺ influx in single renal VSMCs is insensitive to typical L-type Ca²⁺ channel blockers.⁶ Similar to renal vascular smooth muscle, it has been shown in contractile glomerular mesangial cells that Ca²⁺ currents are only partially inhibited by antagonists of L-type Ca²⁺ channels.²² Ca²⁺ influx through VDCCs has been suggested to be involved in mesangial cell growth,^{23 24 25} which is important in pathological conditions such as diabetes mellitus and inflammatory glomerular diseases. Our demonstration of the expression of _1A subunit mRNA and protein in mesangial cells raises the possibility that P-/Q-type VDCCs may participate in mesangial cell function. The overall expression of the _1A subunit in vascular tissue was surprising, because this subunit is usually viewed as confined to nervous tissue, being the most abundant ₁ subunit gene product expressed in the brain.²⁶ We cannot rule out that the _1A subunit is present in the perivascular nerves, but our cell culture studies, immunolabeling, and patch-clamp data show that the _1A subunit and functional P-type Ca²⁺ currents are present in smooth muscle cells. In general, we have not found mRNA for other neuronal ₁ subunits (_1D, _1E) in vascular tissue by RT-PCR analysis. Expression of the L-type Ca²⁺ channel _1C subunit in A7r5 smooth muscle cells depends on the state of differentiation of the cells.²⁷ In the present study, _1A subunit mRNA and protein were detected both in vascular cell cultures and in freshly isolated cell and tissue samples. Together, the data imply constitutive expression of P-type Ca²⁺ channels in vascular tissue. Data were recently presented that showed the presence of a nifedipine-insensitive high voltage–activated Ca²⁺ channel in guinea pig mesenteric resistance arterioles (diameter 40 to 100 µm) with some biophysical similarities to the P-type Ca²⁺ channels whereas the pharmacological profile was markedly different from P- or Q-type channels.²⁸ It is conceivable that true arteriolar resistance segments contain as-yet unrecognized subsets of Ca²⁺ channels.

As to the functional role in excitation-contraction coupling, the distinguishing features of P-type Ca²⁺ currents compared with L-type Ca²⁺ currents are their very slow and Ca²⁺-independent rate of inactivation and the strong modulation by G proteins. The last feature is involved in hormonal regulation of channel properties.^{29 30} The presence of P-type currents could significantly expand the time for active Ca²⁺ influx and allow hormonal influence on vascular reactivity. Mutant mice lacking the _1A subunit gene display a phenotype with pronounced neurological deficits with ataxia and dystonia, and the mice die 3 to 4 weeks after birth.³¹ No major abnormalities of cardiovascular control were reported. In humans, mutations in the gene encoding the _1A subunit are associated with neurological disorders such as episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6), as well as familial hemiplegic migraine, which is a disorder with a significant vascular component.^{32 33} Future studies should clarify whether mutations in the _1A subunit gene are involved in the pathogenesis of vascular disorders.

An important observation of the present study is that specific pharmacological inhibition of a single Ca²⁺ channel subtype abolished K⁺-induced contraction. This suggests that several Ca²⁺ channels are present in the smooth muscle and that equal contribution is required for full responsiveness of renal afferent arterioles to depolarization.

The present demonstration of P-type Ca²⁺ channels in the renal vasculature raises the question to what degree previous data on the control of renal blood flow by Ca²⁺ channels reflect interaction of pharmacological blockers with the P-type channel. In this context, it is interesting to note that L-type Ca²⁺ channel blockers, such as the dihydropyridines, verapamil, and diltiazem, have been reported to significantly inhibit P-type Ca²⁺ currents when used in concentrations that are not maximal for the L-type blockade.^{34 35} It is therefore likely that the role of L-type Ca²⁺ channels has been overestimated and that the response to therapeutic concentrations of these compounds includes the sum of inhibiting at least L- and P-type Ca²⁺ channels. We conclude that mRNA and protein encoding an _1A subunit for a P-/Q-type Ca²⁺ current is expressed in VSMCs and mesangial cells, and we provide evidence that P-type VDCCs play a significant functional role in A7r5 cells and in renal afferent arterioles.

	Acknowledgments

 

This work was supported by grants from the Danish Health Science Research Council (9903058, 9601829, and 9902742), the NOVO Nordisk Foundation, The Danish Heart Association (98-1-2-8-22583, 99-2-2-36-22743), the Foundation for the Advancement of Medical Sciences, the Ruth König Petersens Foundation, The Danish Medical Association Research Fund, Alfred Andersens Foundation, the Foundation of 23-9-1909, and Overlægerådets Legatudvalgs Fond. We thank Anthony M. Carter for language revision, Peter D. Ottosen for immunostaining photographs, and Mette Fredenslund and Inge Andersen for skillful technical assistance.

Received June 7, 2000; revision received September 19, 2000; accepted September 19, 2000.

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