Voltage-dependent calcium channels in the renal microcirculation

Boye L. Jensen, Ulla G. Friis, Pernille B. Hansen, Ditte Andreasen, Torben Uhrenholt, Jeppe Schjerning and Ole Skøtt

Physiology and Pharmacology, University of Southern Denmark, Odense C, Denmark

Correspondence and offprint requests to: Boye L. Jensen, Physiology and Pharmacology, University of Southern Denmark, Winsløwparken 21, DK-5000 Odense C, Denmark. Email: bljensen{at}health.sdu.dk

Keywords: kidney; flour; current; arteriole

Introduction

Voltage-dependent calcium channels (Cav) play an important role in the excitation–contraction coupling in vascular smooth muscle by linking hormone-induced depolarization to calcium influx and contraction. In keeping with this central role of Cav, the channels are targets for pharmacological intervention in conditions such as hypertension.

Cav are heteromeric multisubunit proteins that are composed of an {alpha}1-subunit, and auxilliary ß- and {alpha}2{delta}-subunits. The Cav {alpha}1-subunit contains the essential components necessary and sufficient for the expression of voltage-gated calcium currents, i.e. the calcium pore, the voltage sensor and the drug-binding sites. Up to now, 10 genes encoding mRNAs for Cav {alpha}1-subunits have been cloned. Based on their electrophysiological characteristics, these gene products are divided into high voltage-activated (HVA) channels that need a large depolarization to be activated, and low voltage-activated (LVA) channels that are activated after rather limited depolarization. The HVA channels comprise L-type channels (Cav 1.1–1.4) and neuronal channels (Cav 2.1–2.3), and the LVA channels comprise three different T-type channels (Cav 3.1–3.3) [1]. There exist splice variants of several of the Cav. Thus, P- and Q-type currents are carried by gene products of the same gene that has been alternatively spliced [2].

In the kidney, the glomerular arterioles are centrally involved in the control of renal blood flow and glomerular filtration, as well as medullary blood flow and thereby salt and water reabsorption. For a number of years, there has existed functional evidence for the presence of Cav in pre-glomerular vessels [3]. Electrophysiological measurements on vascular smooth muscle cells derived from renal vessels have shown both L- and T-type Cav [4], but a more detailed molecular characterization of the different types of Cav in the kidney vasculature was lacking, and prompted us to study this issue [5,6] with a combination of molecular and functional techniques.

Cav mRNA in renal pre-glomerular microcirculation

As a basis for isolation of mRNA, we have used two sources: (i) freshly microdissected renal afferent arterioles and cortical radial arteries; and (ii) renal vascular smooth muscle cells that have been cultured by outgrowth from renal pre-glomerular vessels isolated by the iron oxide perfusion method. Mesangial cells were obtained by outgrowth from isolated glomeruli.

By using primers based on published sequences, we showed, by reverse transcription–polymerase chain reaction (RT–PCR) and Southern blotting, that cultured vascular smooth muscle cells from pre-glomerular vessels and freshly microdissected pre-glomerular vessels express mRNA of Cav 1.2 (L-type, {alpha}1C), Cav 2.1 (P/Q-type, {alpha}1A), Cav 3.1 (T-type, {alpha}1G) and Cav 3.2 (T-type, {alpha}1H). In contrast, no expression was detected for Cav 1.3 (L-type, {alpha}1D), Cav 2.2 (N-type, {alpha}1B), Cav 2.3 (R-type, {alpha}1E) or Cav 3.3 (T-type, {alpha}1I) [5,6]. It was expected to find expression of Cav 1.2, because this is the classical L-type calcium channel found in the cardiovascular system, in vascular smooth muscle and in the heart. Also, we had expected to find expression of T-type Cav, but now we were in the position to determine the molecular subtype. The real surprise in these findings was the observation of Cav 2.1, which encodes a classical neuronal Cav—the P/Q-type voltage-dependent calcium channel [7] which had not been described in vascular tissue before.

L-type Cav [Cav 1.2 ({alpha}1C)] in the renal pre-glomerular microcirculation

The finding of Cav 1.2 mRNA was confirmed at the protein level by using a commercial antibody (Alomone, Jerusalem, Israel). Cav 1.2 protein was present in whole vascular trees that had been dissected out of the kidneys after acid maceration. Cav 1.2 was also demonstrated in all pre-glomerular vascular segments and glomeruli when using cryosections of unfixed tissue. However, if we used perfusion fixation, labelling was no longer seen in the blood vessels, but became apparent in several tubular segments. We therefore refrained from using this antibody on fixed tissue. Immunopositive staining was also observed in single fresh myocytes isolated from renal pre-glomerular vessels. Consistent with the finding of Cav 1.2 mRNA in glomerular mesangial cells, positive glomerular immunolabelling was observed both in the acid-macerated preparations and in the cryosections.



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Fig. 1. Structure of Cav. The channel is a heteromeric protein consisting of three subunits. The {alpha}1-subunit is the most important subunit and contains the calcium pore, the voltage sensor and the drug-binding sites. The ß-subunit and {alpha}2{delta}-subunit modify the electrical properties of the channel, and may assist in correct targeting of the channel complex to the cell membrane.

 


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Fig. 2. Overview of the family of voltage-dependent calcium channels, which presently has 10 members divided into three main groups: the Cav 1 family, which comprises L-type channels; the Cav 2 family with neuronal calcium channels; and the Cav 3 family, which comprises T-type calcium channels. In renal pre-glomerular vasculature, we found evidence for mRNA expression of Cav 1.2, Cav 2.1, Cav 3.1 and Cav 3.2. We found no expression of Cav 1.3, Cav 2.2, Cav 2.3 or Cav 3.3.

 
As a functional test system, we have used microperfused afferent arterioles that have been dissected out from rabbit kidney, transferred to a thermoregulated chamber and perfused with concentric pipettes mounted in a motorized movable track system. A video recorder continously records the arteriolar responses to exchanges of bath solution. The standard procedure includes activation of Cav with a high K concentration for 1 min, which induces a complete occlusion of the vascular lumen. This is followed by a step-up procedure with increasing concentrations of inhibitors, and with addition of a high K concentration after 5 min pre-incubation at each concentration of toxin. In these experiments, we have used toxin blockers, because traditional pharmacological blockers are not entirely specific. Calciseptine, which is a toxin from the Black Mamba [8], is an efficient and specific blocker of L-type calcium channels, and a complete block was observed after exposure of the vessels to calciseptine. The block was complete at 10–11 mol/l. We therefore conclude that the pre-glomerular vessels express Cav 1.2 at the mRNA and protein level, and that these channels have functional significance for constriction in renal afferent arterioles.

P/Q-type Cav [Cav 2.1 ({alpha}1A)] in the renal pre-glomerular microcirculation

The finding of Cav 2.1 mRNA in freshly microdissected renal vessels and in cultured renal vascular smooth muscle cells was surprising, because this Cav has not been demonstrated previously in vascular smooth muscle. We therefore isolated protein from cultures of renal vascular smooth muscle cells, cultured mesangial cells, cells from the aortic smooth muscle cell line, A7r5, and from fresh aorta, and obtained labelling with the correct size in western blots. The existence of Cav 2.1 protein in the kidney vessels was substantiated further by labelling of renal vascular trees obtained with the acid maceration procedure, and by immunohistochemistry on cryosections. Further evidence that the Cav 2.1 protein was present in the renal myocytes came from immunolabellings of single isolated myocytes from kidney vessels. Immunocytochemistry on cultures of A7r5 cells also showed labelling with the Cav 2.1 protein antibody, and these cells were therefore used to demonstrate calcium currents with whole-cell patch-clamp.We used a pulse protocol with a step from a holding potential of –70 mV to + 10 mV, which produced maximal HVA currents. After the initial measurement, the bath solution was changed to a solution containing 10 nmol/l {omega}-agatoxin IVA, which is a specific blocker of P- and Q-type calcium currents, and which blocked ~22% of the calcium current. The toxin is derived from the Funnel Web Spider, and is specific for P-type currents in concentrations <=10 nmol/l [9], while >100 nmol/l is necessary for block of Q-type calcium currents. From this experiment, it was concluded that P-type calcium channels contribute to calcium influx in vascular smooth muscle cells.

As a further functional control experiment, we tested the ability of {omega}-agatoxin IVA to interfere with the increase in intracellular calcium concentration [Ca2+]i that occurs after depolarization of afferent arterioles with a high K+ concentration. [Ca2+]i was measured with the conventional fura-2 method, using ratiometric imaging with a monochromator. The resting calcium concentration was 70 nmol/l and increased to 200 nmol/l after depolarization. Treatment with {omega}-agatoxin IVA inhibited 30% of the calcium increase after depolarization, showing that P-type calcium channels contribute significantly to the increase in [Ca2+]i after depolarization.

As the final functional test, we assessed the ability of {omega}-agatoxin IVA to block constriction in microperfused afferent arterioles. The afferent arterioles turned out to be extremely sensitive to the toxin. A total block of K-induced vasoconstriction was observed already at 10–14 mol/l. The inhibition was completely reversible.

We conclude from these experiments that vascular smooth muscle cells in the renal pre-glomerular microcirculation express Cav 2.1 at the mRNA and protein level, and that these channels contribute to calcium influx and are important for vascular constriction after depolarization. The observation of Cav 2.1 protein in cultures from A7r5 cells and fresh aorta, and the demonstration of calcium currents carried by Cav 2.1 channels in the aortic cell line, suggest that this neuronal calcium channel is widely distributed in vascular tissue.

T-type Cav [Cav 3.1 ({alpha}1G) and Cav 3.2 ({alpha}1H)] in the renal pre-glomerular microcirculation

Based on the data on mRNA expression, we concluded that the Cav 3.1 and 3.2 were expressed in the pre-glomerular microcirculation. However, for some reason, it seems that nobody has been able to generate good antibodies for the T-type calcium channels, and we have to wait for these before we can carry out the western blots and immunostainings that could confirm the expression of T-type calcium channels at the protein level. The functional description of T-type calcium channels is hampered by the lack of the specific toxins that exist for the other Cav. According to the literature, there exists one toxin, kurtoxin, which is said to have specific T-type-blocking properties, but it is not available from commercial sources and the originators do not supply it. It can be found in the US patent directory, and may show that the commercialization which has begun to haunt the basic sciences is an obstacle to development rather than an aid. We are therefore left with pharmacological blockers that are not completely specific. The most widely used of the pharmacological blockers with T-type-blocking properties is mibefradil, which does not block L-type channels at low concentrations. We observed an inhibition of the K-induced increase in [Ca2+]i in afferent arterioles when mibefradil was added in a concentration of 10–7 mol/l. In the microperfused afferent arterioles, mibefradil was also able to block K-induced contraction. The dose–response curve was rather flat, covering 5 logs, which is consistent with the view that, at high concentrations, mibefradil blocks more than one Cav [10]. On the other hand, the EC50 value was 10–8 mol/l and corresponds closely to the EC50 value of 1.4 x 10–8 mol/l published for the Cav 3.1 channel when studied with electrophysiology in expression systems. A further indication that T-type channels may be of functional use is the observation that nickel blocks K-induced contraction in the afferent arterioles with an EC50 value of 3 x 10–4 mol/l [11].



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Fig. 3. Immunostainings with anti-CaV1.2 antibody (L-type calcium channel). Upper panel: cortical glomerulus. Left: strong labelling was associated with the afferent arteriole and the glomerulus, while the efferent arteriole was unlabelled. Right: microphotograph in black and white of the same specimen. Note the division into peritubular capillaries. Lower panel: juxtamedullary glomerulus. Left: glomerulus, efferent arteriole and vasa rectae were immunopositive. In the right-hand corner, the arteriole divides into descending vasa rectae. Right: microphotograph in black and white of the same specimen. From [6] with permission from the American Heart Association.

 
In conclusion, we have provided specific evidence at the mRNA level that Cav 3.1 and Cav 3.2 are expressed in the pre-glomerular vessels, and we have circumstantial evidence that these channels may have functional significance for depolarization-induced calcium increase and vasoconstriction, but further insight into the biology of the T-type channels, probably has to await the availability of specific antibodies and toxins.

Heterogeneity of Cav in the renal microcirculation

The renal microcirculation is organized differently in the renal cortex and medulla. In the cortex, the blood passes from the glomeruli into the efferent arterioles, which are rather thin-walled structures that break up into peritubular capillaries. In contrast, juxtamedullary efferent arterioles that supply the renal medulla have a different structure. They are longer and thicker, and often have a several layers of vascular smooth muscle cells in the wall. After a course of several hundred microns, they break up into the descending vasa rectae. There is rather good functional evidence from the literature that the activation mechanisms that induce vasoconstriction in afferent and efferent arterioles in the renal cortex are different. Thus most of the evidence suggests that depolarization and Cav are involved in the mechanisms that lead to vasoconstriction in the pre-glomerular vasculature [3,1214]. This is in good agreement with the molecular and functional data presented above. On the other hand, efferent arterioles seem to be less dependent of Cav, even when looking at the same hormone. Thus, angiotensin II induces vasoconstriction in afferent arterioles by a mechanism that involves depolarization and activation of Cav, while angiotensin II induces vasoconstriction in efferent arterioles independenly of depolarization and Cav. Receptor-operated channels and store-operated channels with release from intracellular stores seem to be involved in the regulatory pathways in the efferent arterioles [15].

We decided to look more into this question and compared the expression and function of Cav in the efferent microcirculation in renal cortex and renal medulla. The microdissection technique allowed us to isolate mRNA from cortical efferent arterioles, from juxtamedullary efferent arterioles and from vasa rectae. In these experiments, we looked for expression of L- and T-type channels (Cav 1.2, Cav 3.1 and Cav 3.2). In cortical efferent arterioles, there was no Cav 1.2 mRNA expression, and this was confirmed by immunostainings of acid-macerated microdissected glomerular complexes that contained both arterioles. These immunostainings were carried out by mounting the specimens with two micropipettes in a tissue bath mounted on an inverted microscope, and performing the entire staining procedure on only one specimen at a time. In contrast to the result with cortical efferent arterioles, it turned out that juxtamedullary efferent arterioles and vasa rectae (both bundles and individual vasa rectae) expressed Cav 1.2 mRNA and clearly showed immunopositive labelling for Cav 1.2 protein [6].

As a functional correlate to these experiments, we tested the ability of a high potassium concentration to induce an increase in the [Ca2+]i in cortical and juxtamedullary efferent arterioles and compare them with afferent arterioles. The results fully confirmed the molecular data, since depolarization with high K+ induced an increase in the [Ca2+]i in juxtamedullary efferent arterioles and afferent arterioles, while the [Ca2+]i in cortical efferent arterioles was largely insensitive to depolarization. Similarly to the present findings, Helou and Marchetti [16] reported that K+ and angiotensin II raised the [Ca2+]i in juxtamedullary efferent arterioles in a nifedipine-sensitive way. In addition, very recent data have shown that angiotensin II initiates vasoconstriciton in descending vasa rectae by a mechanism that depends on depolarization and activation of Cav [17].

We conclude from these experiments that in contrast to cortical efferent arterioles, the juxtamedulllary efferent arterioles and vasa rectae contain Cav. This observation provides a molecular explanation for the observation that calcium channel blockers cause a preferential increase in medullary blood flow [18]. Furthermore, a preferential effect of calcium channel blockers on the renal medullary blood flow may lead to a relative wash-out of medullary osmotic gradients of urea and thereby decrease the absorption of NaCl in the thin ascending limb of Henle in the inner medulla. The resulting increase in NaCl excretion may explain the natriuretic effect of calcium channel blockers, which may be central for their ability to reduce arterial blood pressure in long-term treatment.

This finding suggests that the cellular activation mechanisms that lead to vasoconstriction in the microcirculation of the renal medulla has much in common with the activation mechanisms observed in the pre-glomerular vasculature.

In conclusion, our results from the kidney microcirculation have shown that vascular smooth muscle cells contain a number of different Cav, which all contribute to the function of the small blood vessels. This ensemble of Cav that have differing activation and inactivation kinetics, are influenced by different intracellular signalling molecules, and allows for a much more subtle regulation of calcium influx than we had anticipated. In addition, the results show that there is a highly heterogeneous distribution of calcium channel subtypes in various vascular segments permitting differences in hormonal activation mechanisms.

Acknowledgments

Work from the authors’ laboratory was supported by grants from the Danish Medical Research Council (9601829 9902742, 9903058), the Novo Nordisk Foundation, The Danish Heart Foundation (98-1-2-8-22583, 99223622743, 01123022896), the Ms 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 and Alfred Andersens Foundation Overlægerådets Legatudvalgs Fond, the Foundation for the Advancement of Medical Sciences.

Conflict of interest statement. None declared.

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