voltage-gated calcium channels in bovine adrenal glomerulosa cells. All (10 nM) enhances whole-cell T-type calcium channel current and increases the activity ...
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3260-3264, April 1993
Physiology
T-type calcium channels in adrenal glomerulosa cells: GTP-dependent modulation by angiotensin II RICHARD T. MCCARTHY*t, CARLOS ISALESI, AND HOWARD RASMUSSEN*t Departments of *Cell Biology and tMedicine, Yale University School of Medicine, New Haven, CT 06510
Communicated by Gerhard Giebisch, January 4, 1993
adrenal cortex and placed into CaCl2-free Krebs-Ringer bicarbonate (KRB) (120 mM NaCl/25 mM NaHCO3/3.6 mM KCI/1.2 mM MgSO4/1.2 mM NaH2PO4/0.1% dextrose, equilibrated with 5% C02/95% air). Slices were digested with collagenase [10 min at 37°C in KRB containing 0.6 mM CaCl2, 0.1% bovine serum albumin (BSA), and 35 units of collagenase per mg], and cells were dispersed by mechanical agitation. Dispersed cells were filtered through 20-,um mesh (Tetko, Elmsford, NY), collected by centrifugation, and resuspended in KRB containing 1.25 mM CaCl2 and 0.2% BSA, equilibrated with 95% air/5% CO2. Cells were either used within 1-5 hr of the isolation or maintained in culture. Cells for culturing were purified on a 56% Percoll gradient, plated onto uncoated glass coverslips, and grown in 1:1 (vol/vol) Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 10% (vol/vol) horse serum, 2% (vol/vol) fetal bovine serum, 100 ,uM ascorbate, 1.2 ,M a-tocopherol, 0.05 ,uM Na2SeO3, 50 ,uM butylated hydroxyanisole, 5 ,uM metyrapone, 100 units of penicillin per ml, 100 ,ug of streptomycin per ml, 30 j.g of gentamycin per ml, and 3 ug of amphotericin B per ml. After replacement of the serumcontaining medium with serum-free medium (+ 0.2% BSA), the cells were incubated for an additional 24-30 hr before use. All single-channel recordings were performed on primary cultures of glomerulosa cells. Freshly dispersed cells were only used for whole-cell recording. Patch-Clamp Measurements. Whole-cell calcium channel currents were recorded from cells 9-14 ,um in diameter. The bath solution used for recording calcium channel currents contained 117 mM tetraethylammonium chloride, 20 mM BaCl2 or CaCl2, 0.5 mM MgCl2, 5 mM dextrose, 32 mM sucrose, 10 mM Hepes, and 0.2 mM tetrodotoxin (TTX) (pH 7.5; adjusted with CsOH). Bath solutions were oxygenated with 100% 02 and maintained at room temperature. Patch pipettes (2-4 Mfl) were filled either with 108 mM CsCl/10 mM tetrabutylammonium chloride/1l mM bis(2-aminophenoxy)ethane-N,N,N' ,N'-tetraacetate (BAPTA)/0.9 mM CaCl2/6 mM MgCl2/5 mM Na2ATP/0.04 mM GTP/10 mM Hepes, pH 7.2 (adjusted with CsOH) or with 130 mM CsCl/10 mM tetrabutylammonium chloride/1l mM EGTA/0.9 mM CaCl2/2 mM MgCl2/1 mM Na2ATP/0.04 mM GTP/10 mM Hepes, pH 7.2 (adjusted with CsOH). Similar to other cell types, T-type currents were blocked by 100 ,uM cadmium and 50 ,uM nickel (2). Membrane current was sampled at 5, 10, or 20 kHz, having been filtered with an eight-pole low-pass Bessel filter set at a cut-off frequency (-3 decibels) of 1.0, 2.5, or 5.0 kHz, respectively. For analysis, linear-leak and capacitive transient currents were subtracted digitally by appropriately scaling negative test pulses from -90 to -110 mV, which were obtained throughout the experiment. Nonlinear least-
ABSTRACT With the use of whole-cell and single-channel current recordings, we have examined in more detail the site of action of angiotensin II (All) on multiple populations of voltage-gated calcium channels in bovine adrenal glomerulosa cells. All (10 nM) enhances whole-cell T-type calcium channel current and increases the activity of single T-type calcium channels in cell-attached patch recordings. The All-induced enhancement of whole-cell calcium channel currents is dependent on the presence of internal GTP and can be inhibited by the competitive All-receptor antagonist saralasin (1 aiM). These results show that All augments the T-type calcium channel current in bovine adrenal glomerulosa cells.
Voltage-gated calcium channels are an important site of hormone-mediated regulation of calcium influx in a variety of cells. In bovine adrenal glomerulosa cells, two distinct types of voltage-gated calcium channels have been identified (1, 2). These two distinct channel types are designated T-type and L-type. T-type voltage-gated calcium channel currents are distinguished from L-type currents by their more rapid inactivation kinetics and lower voltage threshold of activation (1-4). However, the functional importance of T-type calcium channel currents has been difficult to determine because there are very few pharmacological agents that selectively act on this class of voltage-gated calcium channel current. Angiotensin II (AII) and, more recently, atrial natriuretic peptide have been reported to modulate T-type calcium channel current in bovine adrenal glomerulosa cells (1, 2). These peptides are the first hormones to be identified as modulators of T-type calcium channel current. However, the selectivity of hormonal action for T-type channels has been questioned. While Cohen et al. (1) reported All-induced enhancement of a slowly deactivating T-type calcium channel tail current in adrenal glomerulosa cells, Hescheler et al. (3) reported that AII enhanced L-type calcium channel current in an adrenal carcinoma cell line (Y-1). The resolution of this issue is clearly important since AII represents only one of two hormones to date that have been reported to modulate T-type calcium channel current. To better understand the effect of AII and to determine the messenger pathways involved in channel modulation, we have chosen to reexamine both the site and mechanism of action of AII on calcium channel current in adrenal glomerulosa cells. With the use of whole-cell and single-channel current recordings, we have found that AII modulates T-type calcium channel current in adrenal glomerulosa cells by a readily diffusible second messenger in a GTP-dependent fashion.
METHODS Bovine Adrenal Glomerulosa Cell Isolation and Culture. Bovine adrenal glomerulosa cells were isolated as described (5). The zona glomerulosa layer was dissected from bovine
Abbreviations: AII, angiotensin II; GTP[',.S], guanosine 5'-[y thio]triphosphate; G protein, GTP-binding regulatory protein. tTo whom reprint requests should be addressed at present address: Institute for Preclinical Pharmacology, Miles Inc., 400 Morgan Lane, West Haven, CT 06516.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
3260
Physiology: McCarthy et al.
Proc. Natl. Acad. Sci. USA 90 (1993)
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