alpha 1H T-type Ca2+ channel is the predominant subtype expressed in bovine and rat zona glomerulosa

Andrew D. Schrier, Hongge Wang, Edmund M. Talley, Edward Perez-Reyes, and Paula Q. Barrett

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The low voltage-activated (T-type) Ca2+ channel has been implicated in the regulation of aldosterone secretion from the adrenal zona glomerulosa by extracellular K+ levels, angiotensin II, and ACTH. However, the identity of the specific subtype mediating this regulation has not been determined. We utilized in situ hybridization to examine the distribution of three newly cloned members of the T-type Ca2+ channel family, alpha 1G, alpha 1H, and alpha 1I, in the rat and bovine adrenal gland. Substantial expression of only the mRNA transcript for the alpha 1H-subunit was detected in the zona glomerulosa of both rat and bovine. A much weaker expression signal was detected for the alpha 1H transcript in the zona fasciculata of bovine. Whole cell recordings of isolated bovine adrenal zona glomerulosa cells showed the native low voltage-activated current to be inhibited by NiCl2 with an IC50 of 6.4 ± 0.2 µM. Because the alpha 1H subtype exhibits similar NiCl2 sensitivity, we propose that the alpha 1H subtype is the predominant T-type Ca2+ channel present in the adrenal zona glomerulosa.

in situ hybridization; adrenal cortex; low voltage-activated calcium channels; nickel inhibition


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE THREE CONCENTRIC ZONES within the cortex of the mammalian adrenal gland were first described as the zona glomerulosa, zona fasciculata, and zona reticularis in 1866 by Arnold (2). These three zones have different functional roles in steroid hormone production: mineralocorticoids are produced in the zona glomerulosa, glucocorticoids in the zona fasciculata, and C19 steroids in the zona reticularis (32). In the zona glomerulosa, aldosterone secretion is regulated by adrenocorticotropin (ACTH), angiotensin II (ANG II), and K+ in a Ca2+-dependent manner (1, 11).

Agonist-stimulated and plasma [K+] regulation of aldosterone secretion in the zona glomerulosa is dependent on changes in membrane potential (24, 25, 29, 30) and the influx of extracellular Ca2+ (5, 14, 15). Because small elevations in extracellular [K+] would be expected to increase low voltage-activated (LVA), T-type Ca2+ channel currents in rat and bovine adrenal glomerulosa cells preferentially (6, 31, 33), LVA Ca2+ channels may provide the link between small depolarizations in membrane potential and the influx of extracellular Ca2+. The low voltage of activation and slow deactivation kinetics of the LVA Ca2+ channel make it ideally suited to maximize the amount of Ca2+ entry at depolarized membrane potentials, where channel inactivation may be incomplete, and thus provide a sensor for coupling minimal changes in membrane potential to prolonged influxes of Ca2+. Such biophysical properties of LVA Ca2+ channels would allow them to facilitate a prolonged Ca2+ influx to sustain the secretion of aldosterone from the zona glomerulosa.

Recently, three separate members of the T-type Ca2+ channel family have been cloned from human and rat tissues (8, 17, 26, 27) . The alpha 1G, alpha 1H, and alpha 1I subtypes all exhibit currents characteristic of native T-type channels when expressed in HEK-293 cells or oocytes. However, the alpha 1H subtype has been shown to be inhibited by low micromolar concentrations of NiCl2 (<10 µM), distinguishing it from the alpha 1G and alpha 1I subtypes, which typically require >100 µM NiCl2 for inhibition (18). Furthermore, recent work using in situ hybridization indicated that the mRNA expression for the three subtypes each exhibited unique and complementary distribution patterns throughout the rat central and peripheral nervous systems (36). The subtype that contributes to LVA activity in the zona glomerulosa is not known.

We have set out to identify which of the three T-type Ca2+ channel subtypes are present throughout the rat and bovine adrenal gland. In situ hybridization studies in rat and bovine adrenal tissue indicated predominant expression of mRNA for the alpha 1H subtype. In the rat adrenal, detectable levels of alpha 1H mRNA were localized exclusively to the outer cortical regions that circumscribed the zona glomerulosa, while alpha 1H mRNA was detected in both the zona glomerulosa and zona fasciculata of the bovine adrenal gland. In addition, whole cell voltage-clamp recordings of T-type Ca2+ channel currents from isolated bovine zona glomerulosa cells displayed a high sensitivity to low micromolar concentrations of NiCl2. Together, these data indicate that the alpha 1H subtype is the predominant LVA Ca2+ channel expressed in the zona glomerulosa of the rat and bovine adrenal.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. Male Sprague-Dawley rats (250-350 g) were anesthetized with ketamine/xylazine before surgical removal of the adrenal gland or decapitation and removal of the brain. Adrenal glands and brains were immediately frozen on dry ice, cut into 10-µm-thick sections, thaw mounted onto charged slides (Superfrost Plus; Fisher Scientific, Houston, TX), and stored at -80°C for later use. Two- to four-day-old calf adrenal glands (Florida Biologicals) were obtained immediately after slaughter, cleaned of adherent fat, frozen on dry ice, and cut into 10-µm-thick sections. The sections were thaw mounted onto charged slides and stored at -80°C for later use. Pretreatment of the slides before hybridization was described previously (36). In short, slides were allowed to equilibrate for ~5 min before they were fixed in a fresh 4% paraformaldehyde solution. The slides were then subjected to a series of washes in 0.1 M PBS (pH 7.4), a 5-min treatment with glycine (0.2% in 0.1 M PBS), and a 10-min treatment with acetic anhydride (0.25% in 0.1 M triethanolamine, 0.9% NaCl, pH 8) before undergoing dehydration and delipidation effected by a series of increasing ethanol washes. The hybridization reaction was carried out overnight at 37°C in the presence of a hybridization buffer containing 50% formamide, 600 mM NaCl, and 60 mM standard sodium citrate (SSC), 1× Denhardt's solution (Sigma), 10% dextran sulfate, 100 mM dithiothreitol, 250 µg/ml yeast tRNA, and 1 mg/ml salmon sperm DNA. The slides were then subjected to a series of four 15-min washes in SSC at 55°C, followed by a 1-h wash in SSC at room temperature.

Oligonucleotide probes. The rationale and design of the rat-specific oligonucleotide probes were described previously (36). In short, 33-bp antisense probes were designed to detect the I-II cytoplasmic loop for each of the three T-type Ca2+ channel gene products. The I-II cytoplasmic loop contains a much lower degree of sequence similarity than the transmembrane region and enables the design of probes specific to each gene transcript (8). Multiple probes recognizing different regions in the I-II loop were designed for each subtype to confirm individual probe effectiveness. The probes listed in Table 1 demonstrated specific binding and were used either alone (see Fig. 1) or in combination (see Fig. 3). For the bovine study, 33-mer bovine-specific probes were made containing 1- or 2-bp mismatches with the corresponding region of the rat sequence. The mismatches provide an internal control for the effectiveness of each probe by examining their distribution under described conditions, specifically the rat brain (36). Additional probes were designed specific to the bovine tyrosine hydroxylase gene and the bovine CYP11B gene. These controls provided a positive indication of successful hybridization along with morphological indication of cellular phenotype in the tissue preparations. Competition experiments utilizing an ~1,000-fold excess of unlabeled probe were used to assay the nonspecific interaction associated with each probe. Probe sequences were checked against sequences in the GenBank to ensure no cross-reactivity with other Ca2+ channel gene products or other sequences in the database. Probes were labeled with [33P]dATP (NEN) by using terminal deoxyribonucleotidyl transferase (Life Technologies, Gaithersburg, MD) and were purified by ethanol precipitation. The sequences for the probes used in this study are listed in Table 1.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Selected oligonucleotide sequences used for in situ hybridization and PCR experiments



View larger version (89K):
[in this window]
[in a new window]
 
Fig. 1.   Distribution of 3 mRNA transcripts of the T-type Ca2+ channel family in the rat adrenal gland. Levels of mRNA were detected by in situ hybridization with 33P-labeled oligonucleotide probes specific to the 3 members of the low voltage-activated (LVA) channel family: alpha 1G (top), alpha 1H (middle), and alpha 1I (bottom). Right: control autoradiographs in which a competition assay using 1,000-fold excess of unlabeled probe (cold comp) was used to measure nonspecific binding. Left: specific binding of the labeled oligonucleotide probe (antisense) to the alpha 1H mRNA transcript is readily detectable in the outer layers of the adrenal cortex. In contrast, very little to no signal was detected for the alpha 1G and alpha 1I mRNA transcripts. The multiple probes for each subtype listed in Table 1 were combined and utilized as a cocktail mixture. Scale bar, 2.5 mm.

Sequencing of I-II loop. Total RNA was isolated and reverse transcribed to cDNAs from bovine adrenal glomerulosa cells and bovine brain cerebellum. A set of degenerate PCR primers for all subtypes was designed to specifically recognize the highly conserved transmembrane regions flanking the I-II loop (Table 1). PCR products were then subcloned into the TA cloning vector pCR2.1 (Invitrogen, Carlsbad, CA) before transformation into competent cells (INValpha Ft, Invitrogen). Positive clones were detected by color and identified through restriction enzyme digest before sequencing. I-II loop sequences were then compared with their human isoforms for identification purposes.

Cell isolation. Neonatal bovine adrenal glomerulosa cells were isolated by collagenase digestion, as described previously (12, 19, 20). The glomerulosa layer was carefully cut away from the adrenal cortex into thin sections and stored in a Ca2+/BSA-free standard Krebs-Ringer bicarbonate (KRB) buffer containing (in mM) 120 NaCl, 25 NaHCO3, 3.5 KCl, 1.2 MgSO4, 1.2 NaH2PO4, and 0.1% dextrose, equilibrated with 95% air-5% CO2 (pH 7.4). The slices were digested twice at 37°C for 10 min with collagenase (160 U/mg; Worthington, Freehold, NJ) before being mechanically dispersed, purified through a 20-µm nylon mesh (Tetko, Elmsford, NY), recovered by centrifugation, and resuspended in KRB containing 1.25 mM CaCl2 and 0.2% BSA. A discontinuous (30%/56%) Percoll gradient (Pharmacia, Piscataway, NJ) was used to further purify the cells before they were resuspended in the Ca2+/BSA KRB. Isolated cells were then stored overnight in a 50:50 DMEM/F-12:KRB (2 mM K+) solution. For recording, cells were plated directly onto collagen-coated slides (50 µg/ml in 0.2 N acetic acid; Becton Dickinson, Bedford, MA) in 1:1 DMEM/F-12 medium with 100 U/ml penicillin and 100 µg/ml streptomycin ~3 h before experimentation.

Patch-clamp experiments. Electrophysiology experiments were carried out as described previously (19, 20). Briefly, Ca2+ currents were recorded from single adrenal glomerulosa cells (12-18 µm) adhered to collagen-coated slides. The slides were placed into a 500-µl microincubator chamber perfused by gravity at a rate of 0.5 ml/min with an external bath solution containing (in mM) 134 tetraethylammonium chloride, 10 CaCl2, 0.5 MgCl2, 5 dextrose, 32 sucrose, and 10 HEPES-Cs (filtered, pH 7.4). Patch pipettes (2-4 MOmega ) were made from 0010 glass capillaries (World Precision Instruments, Sarasota, FL) by using an L/M-3P-A vertical puller (List Medical Electronic, Darmstadt, Germany). The standard internal pipette solution contained (in mM) 115 CsCl, 1 tetrabutylammonium chloride, 11 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 0.9 CaCl2, 1 MgCl2, 20 HEPES, 5 MgATP, and 1 LiGTP (pH 7.2). Whole cell currents were recorded with the use of an Axopatch 200A amplifier and pCLAMP 6.0 software (Axon Instruments) by applying a 10.4-ms test pulse to -15 mV from a holding potential of -90 mV. Tail currents were elicited by a 28-ms repolarization step to -70 mV and recorded. Currents were sampled at 12.5 kHz and filtered with an eight-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA) set to a cutoff frequency of 2 kHz. Ni2+ inhibition experiments were performed by exchange of the bath solution with standard external solution containing increasing concentrations of NiCl2 (stock 100 mM). Currents were allowed to stabilize before the Ni2+ concentration was incremented in the perfusate.

Data analysis. In situ hybridization slides were exposed to autoradiographic film (Hyperfilm beta -max or Hyperfilm H3; Amersham, Arlington Heights, IL) for either 5 or 10 days. Autoradiographs were digitally captured with a DAGE-16E microscope mounted to a light box. Images were manipulated collectively as a group in Adobe Photoshop (Adobe Systems).

Exponential fits to the Ca2+ channel tail currents were determined by using Clampfit software (Axon Instruments). These exponential fits were used to assess the amplitude of the tail current at the fit start time (1.2 ms) and to calculate the inhibition of the Ca2+ current as a function of Ni2+. A sigmoidal dose-response analysis was performed by using Prism (GraphPad, San Diego, CA). The data are means ± SE for percent inhibition of each concentration of Ni2+ and were fitted to a four-component logistic equation
Y=D+(A−D)/{1+10∧<SUP>[(log<IT> B</IT>−<IT>X</IT>)<IT>∗C</IT>]</SUP>}
where X represents log[Ni2+], Y is the response, A is the maximum value (100%), B is the IC50, C is the Hill slope, and D is the minimum value.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha 1H subtype of the LVA Ca2+ channel family is expressed in the zona glomerulosa of rat adrenal tissue. Prominent LVA currents have been recorded in rat adrenal cortical cell preparations that contain a mixture of aldosterone-producing glomerulosa cells and corticosterone-producing fasciculata cells (9, 21, 25, 29, 30). In situ hybridization studies were carried out to determine the subtype-specific expression and localization of members of the LVA Ca2+ channel family in rat adrenal tissue. High levels of alpha 1H mRNA expression were readily detectable in the rat adrenal in contrast to extremely low levels of mRNA expression for the alpha 1G subtype and a virtual absence of mRNA expression for the alpha 1I subtype (Fig. 1, left). The distribution of the alpha 1H message was limited to the outer region of the cortical layer of the tissue; however, exact zonal distribution could not be determined. Cold competition experiments utilizing a 1,000-fold excess of unlabeled probe reduced the intensity of the strong alpha 1H signal and the weak alpha 1G signal equivalently, indicating that the interactions were specific for each mRNA transcript (Fig. 1, right).

Sequencing of the putative I-II loop of the three members of the bovine LVA Ca2+ channel family. The detected dominant expression of alpha 1H LVA channel mRNA in the zona glomerulosa of the rat prompted us to determine the expression pattern in the bovine adrenal gland, where LVA currents have been characterized in cell preparations containing exclusively cells from the zona glomerulosa (6, 22, 28, 34). Preliminary experiments were conducted with rat-specific oligonucleotides because sequence information for the bovine family of LVA Ca2+ channels was not available from GenBank. However, these rat probes could not identify message for any member of the LVA channel family in bovine adrenal tissue, prompting us to sequence the I-II loop for each paralog of the bovine channel family. cDNA was prepared by reverse transcription of total RNA from isolated bovine adrenal zona glomerulosa cells and bovine cerebellum. A set of degenerate PCR primers (Table 1) was designed to recognize highly conserved sequences in transmembrane regions flanking the I-II loop. These primers hybridized with sequences across LVA family members as well as across species. PCR products were subcloned into TA cloning vector, and restriction enzyme digests identified positive clones. Thirteen positive clones, identified by color, were sequenced and identified as the bovine variant of the alpha 1H. Only PCR product for the alpha 1H channel was detected in bovine adrenal glomerulosa cells (data not shown). The I-II loop sequences for alpha 1G and alpha 1I were obtained from bovine cerebellum RNA, which has been shown by previous work to express these two family members in the rat (36). Clearly, the identification of mRNA for all three subtypes with the use of our degenerate primers attests to the efficacy of the primer design strategy. The sequences for the putative I-II loop of the three members of the T-type Ca2+ channel family in bovine are listed in Fig. 2A. A dendrogram indicating sequence homology between the bovine family members and their respective human or rat counterparts within the I-II loop is shown in Fig. 2B. The relatively low degree of homology within this loop among the members of the LVA Ca2+ channel family provided regions from which to design probes specific to each subtype.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 2.   cDNA sequence information for the putative I-II loop of the 3 members of the bovine LVA Ca2+ channel family. A: mRNA from bovine adrenal glomerulosa cells and bovine brain cerebellum was reverse transcribed to produce cDNA for PCR reactions. After PCR, the products were subcloned, subjected to restriction enzyme digestion, and sequenced. Bold regions indicate the location of the I-II loop where specific probes were designed to identify the different T-type channel subtypes. The sequences have been deposited in GenBank with the following accession numbers: alpha 1H, AF236638; alpha 1G, AF236639; and alpha 1I, AF236640. Bold regions in the cloned loop sequences for alpha 1H, alpha 1G, and alpha 1I indicate hybridization sequences for bovine probes that are named below the start of the complementary sequence (alpha 1I 1, alpha 1G 1, alpha 1H 1, alpha 1H 2, alpha 1G 2, and alpha 1I 2). B: dendrogram indicating the sequence homology within the I-II loop between the 3 members of the bovine LVA family with their rat or human counterpart (GenBank accession nos.: alpha 1G, AF027984; alpha 1H, AF073931; and alpha 1I, AF086827).

Bovine adrenal tissue expresses the alpha 1H subtype of the T-type Ca2+ channel family. Antisense oligonucleotide probes specific to the three members of the bovine LVA Ca2+ channel family were designed from the sequences of their putative I-II loops, listed in Fig. 2. Figure 3 displays the autoradiographs from an in situ hybridization study designed to detect and localize the channel-specific expression of the three members of the LVA Ca2+ channel family. Figure 3, left, depicts alpha 1G, alpha 1H, and alpha 1I mRNA expression in addition to mRNA expression for the cellular phenotype markers tyrosine hydroxylase and CYP11B. Tyrosine hydroxylase is present in dopamine-producing cells in the medulla of the bovine adrenal gland, while CYP11B is a multifunctional P-450 enzyme that catalyzes either the formation of aldosterone or cortisol from corticosterone in either the zona glomerulosa or zona fasciculata of the bovine adrenal cortex, respectively (32). These markers allowed us to localize the mRNA signal to a specific zonal region within the bovine adrenal gland. As in the rat adrenal gland, the predominant subtype-specific expression was limited to the alpha 1H transcript. alpha 1H probes identified two regions with markedly different levels of message abundance in the bovine adrenal gland (Fig. 3). The predominant expression signal was present near the outer cortical region of the adrenal tissue, while a much weaker signal was present within the cortex just proximal to this area of expression. On the basis of the distribution signal of the marker enzyme CYP11B, the alpha 1H transcript is expressed predominantly in the zona glomerulosa with lower levels of expression confined to the zona fasciculata. The detection of alpha 1H message in the zona fasciculata is consistent with previously reported electrophysiological data indicating T-type Ca2+ channel activity in both the zona glomerulosa and the zona fasciculata in the bovine adrenal gland (6, 10, 22, 23, 28, 37).


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 3.   In situ hybridization characterization of the bovine adrenal gland indicates that alpha 1H is the predominant T-type Ca2+ channel transcript. Oligonucleotide probes were designed specific to sequence information obtained for each bovine T-type Ca2+ channel isoform and 2 cellular phenotype markers: tyrosine hydroxylase (TH), which identifies the medulla, and 11beta -hydroxylase (CYP11B), which is present in both the zona glomerulosa and zona fasciculata. Autoradiographs indicate predominant expression of the alpha 1H mRNA message vs. alpha 1G or alpha 1I (left). Competition control experiments indicate the level of nonspecific binding of each probe to the tissue (right). The alpha 1H mRNA message was localized to the outer cortical regions with significant mRNA expression in the zona glomerulosa and much weaker mRNA expression in the zona fasciculata. Little to no mRNA expression of the alpha 1G- or alpha 1I-subunit was detected throughout the adrenal. The results were generated from the following bovine probes listed in Table 1: alpha 1G2, alpha 1H1, alpha 1I1, TH, and CYP11B.

Figure 3, right, displays the competition control experiments used to assess nonspecific binding for each of the labeled probes. These controls provide two critical pieces of information. First, in the case of the alpha 1H transcript, competition with 1,000-fold excess unlabeled probe eliminated the specific distribution signal identified in Fig. 3, left. Second, the controls indicate that, in the case of alpha 1G and alpha 1I, a specific expression pattern was not evident because competition with 1,000-fold excess of unlabeled probe exhibited the same general background expression as observed with the specific probes (Fig. 3). Together, these data indicate that the alpha 1H subtype is the predominant T-type Ca2+ channel transcript in the zona glomerulosa of the adrenal gland.

T-type Ca2+ currents recorded from isolated bovine adrenal zona glomerulosa cells exhibit Ni2+ sensitivity. To determine whether these data indicating high levels of alpha 1H mRNA correlate with the expression of functional channel protein, we took advantage of the differential Ni2+ sensitivity of the LVA Ca2+ currents from expressed T-type channel clones. After heterologous expression, the alpha 1H subtype exhibits a much greater sensitivity to inhibition by NiCl2 (IC50 ~10 µM of blockable current component) than either the alpha 1G or alpha 1I subtype (IC50 ~200-300 µM) (18). Thus we performed whole cell voltage-clamp recordings of zona glomerulosa cells in the presence of increasing concentrations of NiCl2. T-type Ca2+ currents were elicited with a test pulse from -90 mV to -15 mV upon repolarization to -70 mV. Exponential fits to the tail currents elicited upon repolarization were used to measure the effect of Ni2+ on LVA channel activity. Figure 4A displays representative current tracings from a single cell recorded in the presence of increasing [Ni2+]. The LVA current was almost completely abolished by as little as 100 µM Ni2+. The dose dependence of Ni2+ inhibition is shown in Fig. 4B. Clearly, the LVA current present in the zona glomerulosa cells was blocked by low micromolar concentrations of Ni2+. The IC50 for NiCl2 inhibition was calculated to be 6.4 ± 0.2 µM with a Hill slope of -1.14 ± 0.05 (n = 7-11). This sensitivity to low micromolar concentrations compares with the IC50 for alpha 1H currents heterologously expressed in HEK-293 cells (12 µM) and indicates a functional role for the alpha 1H subtype in the adrenal zona glomerulosa. However, at 100 µM [Ni2+], only ~80% of the measured current was inhibited by Ni2+. Incomplete block by Ni2+ of alpha 1H current at a low IC50 is consistent with cloned Ca2+ channel data of our own (unpublished observations) and others (17) showing that a component of alpha 1H current remains uninhibited. At higher concentrations block of Ca2+ current by Ni2+ is no longer diagnostic of LVA Ca2+ channel subtypes because high voltage-activated Ca2+ channel subtypes are inhibited with IC50 values similar to those reported for alpha 1G and alpha 1I LVA channels (39). Thus, when Ni2+ is used, it is not possible to assign the residual component of current in the bovine glomerulosa cell to a specific channel subtype. Nevertheless, the in situ hybridization results coupled with the electrophysiological data from isolated zona glomerulosa cells indicate that the alpha 1H subtype is the predominant T-type Ca2+ channel active in the zona glomerulosa of the bovine adrenal.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4.   Ni2+ inhibition of T-type Ca2+ currents in isolated bovine adrenal zona glomerulosa cells. A: T-type Ca2+ channel currents recorded from isolated bovine adrenal zona glomerulosa cells in the presence of increasing [Ni2+] by using a test pulse (10 ms) from -90 mV to -15 mV, followed by a repolarization step (28 ms) to -70 mV to elicit tail currents. The tracings are representative of an average of 5 episodes for each of the following [Ni2+]: 0, 3, 10, 30, and 100 µM. The records were blanked over a 0.3-ms time range after depolarization to remove artifact. B: dose-response analysis of the inhibition of these currents indicates high sensitivity to low micromolar [Ni2+]. Each data point represents the mean ± SE (n = 7-11) of the percent inhibition as measured from exponential fits to the tail currents. The IC50 of 6.4 ± 0.2 µM is consistent with data previously reported for the alpha 1H subtype (18).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The T-type Ca2+ channel plays an important role in adrenal zona glomerulosa physiology. Molecular identification of the members of the T-type Ca2+ channel family has facilitated our study of the identity of the subtypes expressed in the adrenal zona glomerulosa. Using in situ hybridization, we demonstrate that the alpha 1H subtype is the predominant LVA Ca2+ channel present in the rat adrenal cortex, presumably within the zona glomerulosa. Furthermore, electrophysiological data coupled with in situ hybridization studies indicate that the alpha 1H subtype is also the predominant LVA channel in the bovine zona glomerulosa. In both species, substantially lower levels of alpha 1G mRNA transcript were detected, indicating region-specific expression of the different LVA Ca2+ channel subtypes. This finding is consistent with a report describing differential distributions of the LVA family members in the central nervous system as well (36).

A role for the influx of Ca2+ during steroidogenesis and aldosterone secretion from the zona glomerulosa has been purported for several years (1, 4, 11, 35). In fact, the presence of LVA channel activity in the zona glomerulosa of the rat and bovine is well documented (6, 9, 21, 22, 25, 29, 30, 34). These currents are similar to T-type currents described in neuronal systems, which can be characterized by their distinct half-activation potential, rapid inactivation, slow deactivation, and small single-channel conductance (13). Our demonstration of the predominant expression of alpha 1H mRNA in the zona glomerulosa of both the rat and bovine provides a molecular identity to the LVA current active in mediating aldosterone secretion. However, the role of the LVA Ca2+ channel in cortisol secretion from the zona fasciculata is less clear. Several reports have identified voltage-dependent Ca2+ currents in isolated bovine fasciculata cells (10, 23, 37), in contrast to rat fasciculata cells, where their presence has been excluded (37, 38). One exception, however, is a study demonstrating small LVA currents in freshly isolated cells that are lost within 1 day of cell culture (3). Nonetheless, these currents displayed the distinct half-activation potential, rapid inactivation, slow deactivation, and small single-channel conductance described for T-type currents in other systems. Our in situ hybridization results coincide with the majority of previous observations in that mRNA for the alpha 1H subtype was detected in the zona fasciculata of the bovine but not in that of the rat. However, we cannot conclusively rule out T-type Ca2+ channel activity in the rat fasciculata because morphological characterization of the zonation of the rat adrenal was not carried out.

Even though we have shown that the alpha 1H subtype is the predominant LVA channel present in the zona glomerulosa, it is also probable that the alpha 1G subtype is present as well, because low levels of the alpha 1G subtype were detected in our in situ hybridization studies. The biophysical properties of the cloned alpha 1G and alpha 1H channels appear to be remarkably similar. Both channels, when expressed in oocytes or HEK-293 cells, respectively, open near resting membrane potential, inactivate rapidly, exhibit slow deactivation, and have small unitary conductances (7, 8, 27). However, recently Kozlov et al. (16) demonstrated that the cloned subtypes exhibit distinct kinetics of Ca2+ entry in response to different action potential frequencies and duration. Even though action potentials do not play a role in aldosterone secretion from the zona glomerulosa, the differences in Ca2+ entry through each subtype may induce different signaling patterns that uniquely alter cell physiology. Furthermore, while the alpha 1G and alpha 1H subtypes contain ~90% sequence identity across their transmembrane-spanning regions, the intracellular loops and amino and carboxy termini exhibit a much wider divergence in sequence similarity, making these regions potential targets for differential regulation (7).

This disproportionate expression pattern, distinct kinetics of Ca2+ entry during mock action potentials, and highly divergent intracellular loops and termini for the alpha 1G and alpha 1H LVA Ca2+ channels could allow the channels to either subserve different functional roles in various systems or mediate the same function under different modes of regulation. Our laboratory has demonstrated that LVA Ca2+ channel current in bovine zona glomerulosa cells is, in fact, regulated by calmodulin-dependent protein kinase II activity (20). This kinase increases the frequency of channel opening at negative membrane potentials, which results in a shift in the half-activation potential of the channel to more hyperpolarized potentials. Such a shift would increase the ability of the cell to respond with an increase in aldosterone secretion to even smaller changes in membrane potential and could account for the high sensitivity of the adrenal zona glomerulosa cell to relatively small fluctuations in external [K+] or physiological concentrations of hormonal agonists. If mechanisms for LVA Ca2+ channel regulation are indeed subtype specific, then one would ultimately expect differences in aldosterone secretion based on specific Ca2+ channel subtype expression, i.e., differences in cellular physiology could be accounted for by differences in T-type Ca2+ channel expression. The validation of these predictions awaits future experimentation.


    ACKNOWLEDGEMENTS

We thank Dr. Douglas A. Bayliss for experimental guidance and input during the development of this project.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-36977 (to P. Q. Barrett).

Address for reprint requests and other correspondence: P. Q. Barrett, Dept. of Pharmacology, Univ. of Virginia Health System, PO Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735 (E-mail: pqb4b{at}virginia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 March 2000; accepted in final form 12 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aguilera, G, and Catt KJ. Participation of voltage-dependent calcium channels in the regulation of adrenal glomerulosa function by angiotensin II and potassium. Endocrinology 118: 112-118, 1986[Abstract].

2.   Arnold, J. Ein Betrag zu der feiner Struktur und dem Chemismus der Nebennieren. Arch Pathol Anat Physiol Klin Med 35: 64-107, 1866.

3.   Barbara, JG, and Takeda K. Voltage-dependent currents and modulation of calcium channel expression in zona fasciculata cells from rat adrenal gland. J Physiol (Lond) 488: 609-622, 1995[Abstract].

4.   Barrett, PQ, Bollag WB, Isales CM, McCarthy RT, and Rasmussen H. Role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev 10: 496-518, 1989[ISI][Medline].

5.   Capponi, AM, Lew PD, Jornot L, and Vallotton MB. Correlation between cytosolic free Ca2+ and aldosterone production in bovine adrenal glomerulosa cells. Evidence for a difference in the mode of action of angiotensin II and potassium. J Biol Chem 259: 8863-8869, 1984[Abstract/Free Full Text].

6.   Cohen, CJ, McCarthy RT, Barrett PQ, and Rasmussen H. Ca channels in adrenal glomerulosa cells: K+ and angiotensin II increase T-type Ca channel current. Proc Natl Acad Sci USA 85: 2412-2416, 1988[Abstract].

7.   Cribbs, LL, Gomora JC, Daud AN, Lee J, and Perez-Reyes E. Molecular cloning and functional expression of ca(v)31c, a T-type calcium channel from human brain. FEBS Lett 466: 54-58, 2000[ISI][Medline].

8.   Cribbs, LL, Lee JH, Yang J, Satin J, Zhang Y, Daud A, Barclay J, Williamson MP, Fox M, Rees M, and Perez-Reyes E. Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family. Circ Res 83: 103-109, 1998[Abstract/Free Full Text].

9.   Durroux, T, Gallo-Payet N, and Payet MD. Three components of the calcium current in cultured glomerulosa cells from rat adrenal gland. J Physiol (Lond) 404: 713-729, 1988[Abstract].

10.   Enyeart, JJ, Mlinar B, and Enyeart JA. T-type Ca2+ channels are required for adrenocorticotropin-stimulated cortisol production by bovine adrenal zona fasciculata cells. Mol Endocrinol 7: 1031-1040, 1993[Abstract].

11.   Fakunding, JL, Chow R, and Catt KJ. The role of calcium in the stimulation of aldosterone production by adrenocorticotropin, angiotensin II, and potassium in isolated glomerulosa cells. Endocrinology 105: 327-333, 1979[ISI][Medline].

12.   Fern, RJ, Hahm MS, Lu HK, Liu LP, Gorelick FS, and Barrett PQ. Ca2+/calmodulin-dependent protein kinase II activation and regulation of adrenal glomerulosa Ca2+ signaling. Am J Physiol Renal Fluid Electrolyte Physiol 269: F751-F760, 1995[Abstract/Free Full Text].

13.   Huguenard, JR. Low-threshold calcium currents in central nervous system neurons. Annu Rev Physiol 58: 329-348, 1996[ISI][Medline].

14.   Kojima, I, Kojima K, and Rasmussen H. Role of calcium fluxes in the sustained phase of angiotensin II- mediated aldosterone secretion from adrenal glomerulosa cells. J Biol Chem 260: 9177-9184, 1985[Abstract/Free Full Text].

15.   Kojima, I, and Ogata E. Direct demonstration of adrenocorticotropin-induced changes in cytoplasmic free calcium with aequorin in adrenal glomerulosa cell. J Biol Chem 261: 9832-9838, 1986[Abstract/Free Full Text].

16.   Kozlov, AS, McKenna F, Lee JH, Cribbs LL, Perez-Reyes E, Feltz A, and Lambert RC. Distinct kinetics of cloned T-type Ca2+ channels lead to differential Ca2+ entry and frequency-dependence during mock action potentials. Eur J Neurosci 11: 4149-4158, 1999[ISI][Medline].

17.   Lee, JH, Daud AN, Cribbs LL, Lacerda AE, Pereverzev A, Klockner U, Schneider T, and Perez-Reyes E. Cloning and expression of a novel member of the low voltage-activated T- type calcium channel family. J Neurosci 19: 1912-1921, 1999[Abstract/Free Full Text].

18.   Lee, JH, Gomora JC, Cribbs LL, and Perez-Reyes E. Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys J 77: 3034-3042, 1999[Abstract/Free Full Text].

19.   Lu, HK, Fern RJ, Luthin D, Linden J, Liu LP, Cohen CJ, and Barrett PQ. Angiotensin II stimulates T-type Ca2+ channel currents via activation of a G protein, Gi. Am J Physiol Cell Physiol 271: C1340-C1349, 1996[Abstract/Free Full Text].

20.   Lu, HK, Fern RJ, Nee JJ, and Barrett PQ. Ca2+-dependent activation of T-type Ca2+ channels by calmodulin-dependent protein kinase II. Am J Physiol Renal Fluid Electrolyte Physiol 267: F183-F189, 1994[Abstract/Free Full Text].

21.   Matsunaga, H, Maruyama Y, Kojima I, and Hoshi T. Transient Ca2+-channel current characterized by a low-threshold voltage in zona glomerulosa cells of rat adrenal cortex. Pflügers Arch 408: 351-355, 1987[ISI][Medline].

22.   Matsunaga, H, Yamashita N, Maruyama Y, Kojima I, and Kurokawa K. Evidence for two distinct voltage-gated calcium channel currents in bovine adrenal glomerulosa cells. Biochem Biophys Res Commun 149: 1049-1054, 1987[ISI][Medline].

23.   Mlinar, B, Biagi BA, and Enyeart JJ. Voltage-gated transient currents in bovine adrenal fasciculata cells I T-type Ca2+ current. J Gen Physiol 102: 217-237, 1993[Abstract].

24.   Natke, E, Jr, and Kabela E. Electrical responses in cat adrenal cortex: possible relation to aldosterone secretion. Am J Physiol Endocrinol Metab Gastrointest Physiol 237: E158-E162, 1979[Abstract/Free Full Text].

25.   Payet, MD, Benabderrazik M, and Gallo-Payet N. Excitation-secretion coupling: ionic currents in glomerulosa cells: effects of adrenocorticotropin and K+ channel blockers. Endocrinology 121: 875-882, 1987[Abstract].

26.   Perez-Reyes, E. Three for T: molecular analysis of the low voltage-activated calcium channel family. Cell Mol Life Sci 56: 660-669, 1999[ISI][Medline].

27.   Perez-Reyes, E, Cribbs LL, Daud A, Lacerda AE, Barclay J, Williamson MP, Fox M, Rees M, and Lee JH. Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature 391: 896-900, 1998[ISI][Medline].

28.   Quinn, SJ, Brauneis U, Tillotson DL, Cornwall MC, and Williams GH. Calcium channels and control of cytosolic calcium in rat and bovine zona glomerulosa cells. Am J Physiol Cell Physiol 262: C598-C606, 1992[Abstract/Free Full Text].

29.   Quinn, SJ, Cornwall MC, and Williams GH. Electrical properties of isolated rat adrenal glomerulosa and fasciculata cells. Endocrinology 120: 903-914, 1987[Abstract].

30.   Quinn, SJ, Cornwall MC, and Williams GH. Electrophysiological responses to angiotensin II of isolated rat adrenal glomerulosa cells. Endocrinology 120: 1581-1589, 1987[Abstract].

31.   Quinn, SJ, and Williams GH. Regulation of aldosterone secretion. Annu Rev Physiol 50: 409-426, 1988[ISI][Medline].

32.   Rainey, WE. Adrenal zonation: clues from 11beta -hydroxylase and aldosterone synthase. Mol Cell Endocrinol 151: 151-160, 1999[ISI][Medline].

33.   Rossier, MF, Burnay MM, Maturana A, and Capponi AM. Duality of the voltage-dependent calcium influx in adrenal glomerulosa cells. Endocr Res 24: 443-447, 1998[ISI][Medline].

34.   Rossier, MF, Python CP, Capponi AM, Schlegel W, Kwan CY, and Vallotton MB. Blocking T-type calcium channels with tetrandrine inhibits steroidogenesis in bovine adrenal glomerulosa cells. Endocrinology 132: 1035-1043, 1993[Abstract].

35.   Spat, A, Enyedi P, Hajnoczky G, and Hunyady L. Generation and role of calcium signal in adrenal glomerulosa cells. Exp Physiol 76: 859-885, 1991[ISI][Medline].

36.   Talley, EM, Cribbs LL, Lee JH, Daud A, Perez-Reyes E, and Bayliss DA. Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19: 1895-1911, 1999[Abstract/Free Full Text].

37.   Yanagibashi, K, Kawamura M, and Hall PF. Voltage-dependent Ca2+ channels are involved in regulation of steroid synthesis by bovine but not rat fasciculata cells. Endocrinology 127: 311-318, 1990[Abstract].

38.   Yanagibashi, K, Papadopoulos V, Masaki E, Iwaki T, Kawamura M, and Hall PF. Forskolin activates voltage-dependent Ca2+ channels in bovine but not in rat fasciculata cells. Endocrinology 124: 2383-2391, 1989[Abstract].

39.   Zamponi, GW, Bourinet E, and Snutch TP. Nickel block of a family of neuronal calcium channels: subtype and subunit-dependent action at multiple sites. J Membr Biol 151: 77-90, 1996[ISI][Medline].


Am J Physiol Cell Physiol 280(2):C265-C272
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society