Mary D. Coyne

Purification and properties of calmodulin from adrenal cortex

Calmodulin (CaM), a multifunctional calcium binding protein with no known enzymatic activity, has been purified to homogeneity from bovine adrenal cortex. The purification included anion exchange on DE-52 cellulose, ammonium sulfate precipitation, and separation by molecular sieving on Sephadex G-150. The yield of CaM from 900 g of whole adrenal was 150 mg. Adrenocortical CaM showed a molecular weight of 18,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, an isoelectric point of 4.1, and demonstrated a characteristic shift in mobility on polyacrylamide gels in the presence of calcium. The spectral properties of adrenocortical CaM differed slightly from those of CaM isolated from bovine brain. Minor differences were observed in peptide maps and amino acid composition between adrenocortical and brain CaM, but adrenocortical CaM contained a single trimethyl-lysine residue characteristic of all mammalian forms of CaM isolated to date. Adrenocortical CaM is biologically active in the stimulation of activator-deficient phosphodiesterase, and showed a half-maximal effective concentration (EC50) of 3 nM for stimulation of adenylate cyclase from Bordetella pertussis.

Calcium channels do not play a role in the steroid response to ACTH IN Y1 adrenocortical cells.

Y1 cells derived from mouse tumor zona fasciculata cells (ZF) were used to assess the importance of extracellular (EC) calcium availability via voltage-dependent calcium channels (VDCC) on steroidogenesis. The steroidogenic response to ACTH was investigated in the presence of blockers known to affect both calcium and potassium channels in Y1 cells. Y1 cells respond to either ACTH (100 pM) or cAMP (300 microM) at low EC Ca2+ (1 microM) suggesting that EC Ca2+ is not absolutely necessary for a steroidogenic response. However, increases in Ca2+ from 0.05-2.2 mM induced a small but significant biphasic response, first stimulating then inhibiting steroidogenesis. Nickel and amiloride, blockers of T-type Ca2+ channels in Y1 cells, did not depress ACTH-induced steroidogenesis. The dihydropyridine, nifedipine, which is an L-type channel antagonist did not affect ACTH-induced steroidogenesis while the agonist, Bay K 8644 was consistently inhibitory. Neither pimozide nor omega-conotoxin which suppressed Ca2+ currents in Y1 cells inhibited ACTH-induced steroidogenesis. Depolarization of the membrane which would activate VDCCs was inhibitory rather than stimulatory. The present studies using blockers of both voltage-dependent Ca2+ and K+ channels suggest that EC Ca2+ plays a modulatory role in ACTH-induced steroidogenesis in Y1 cells but the data do not support the concept that activation of voltage dependent calcium channels are an important mechanism for steroidogenesis.

Voltage dependent calcium and potassium currents in Y-1 adrenocortical cells are unresponsive to ACTH

In this report we use both whole cell and perforated patch clamp recording techniques to characterize calcium and potassium channels in Y-1 adrenocortical cells in order to assess their responsiveness to ACTH. Both transient and long-lasting components of an inward calcium current were identified which were similar to T and L-type Ca2+ currents. With Ba2+ as the charge carrier, the transient current activated at voltages more hyperpolarized than -50 mV with V1/2 for activation at -78.1 mV, and for steady state inactivation at -52.3 mV. The L-type current activated at -20 mV, with a V1/2 for activation at -29.9 mV and steady state inactivation at -44.2 mV. Under perforated patch conditions the response was shifted to more depolarized voltages. Both currents were responsive to agents which usually affect T- or L-type Ca2+ currents. The transient current was completely blocked by 50 microM lanthanum or 200 microM nickel and partially blocked by 300 mM amiloride. Cadmium (100 microM) and nifedipine (300 nM) completely blocked the long-lasting current while omega-conotoxin GVIA (1992 nM) inhibited the current by only 20-25%. The agonist, Bay K 8644 was stimulatory at 50 nM. Both transient and sustained outward potassium currents similar to A-type and delayed rectifier currents, respectively, were present. The transient current demonstrated fast activation at voltages more positive than -10 mV, inactivation with continued depolarization and steady state inactivation at V1/2 = -50 mV. The sustained current activated rapidly and had minimal inactivation with continued depolarization. The transient current was blocked by 5 mM 4AP and the sustained by 25 mM TEA. While Y-1 cells contain both calcium and potassium currents similar to those found in other adrenocortical cells, none of the currents were affected by ACTH or AII, secretagogues which stimulate steroidogenesis. These data, combined with the inability of both Ca2+ and K+ channel blockers to alter ACTH-induced steroidogenesis as reported earlier, suggests that neither calcium nor potassium currents are responsive to ACTH in Y-1 cells.

Effect of the growth hormone-secreting tumor StW5 on pituitary and adrenal gland function in rats.

A growth hormone-secreting tumor (StW5 was implanted into male rats and resulted in a tripling of adrenal weight concomitant with a 30% decrement in pituitary weight. Plasma concentrations of corticosterone in tumor-bearing (TB) rats were significantly elevated at rest or after ACTH injections or the stress of either anesthesia. The rise in plasma concentrations of corticosterone was due mainly to the large increment in adrenal size although a significant increase in adrenal responsiveness to ACTH was demonstrated in vitro. In addition, plasma corticosterone concentrations were higher in TB rats despite both a doubling of the blood volume and a 50% increase in liver capacity to metabolize corticosterone. Pituitary ACTH content was significantly lower in TB rats, but these pituitary glands could still release near-normal quantities of ACTH as shown both by in vitro incubations and adrenal corticosterone output following ether stress.