Angiotensin II (ANG II) is the most potent and the most physiologically important stimulator of aldosterone synthesis and secretion from the adrenal zona glomerulosa. Because steroidogenesis by adrenal glomerulosa (AG) cells is mediated in part by Ca2+ influx through T- and L-type Ca2+ channels, we evaluated whether T-type Ca2+ channels are regulated by ANG II. We observe that ANG II enhances T-type Ca2+ current by shifting the voltage dependence of channel activation to more negative potentials. This shift is transduced by the ANG II type 1 receptor. The effect of the hormone is not mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) as it is not prevented by CaMKII(281-302), a peptide inhibitor of the catalytic region of the kinase. Rather, this shift is mediated by the activation of a G protein, Gi, because it is abolished by cell pretreatment with pertussis toxin and by cell dialysis with a monoclonal antibody generated against recombinant Gi alpha. This effect of ANG II on T-type Ca2+ channels should increase Ca2+ entry in AG cells at physiologically relevant voltages and result in a sustained increase in aldosterone secretion.

The effect of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca(2+) channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of approximately 9 pS in 110 mM BaCl(2) or CaCl(2). In the cell-attached patch, elevation of bath Ca(2+) from 150 nM to 1 microM raised intracellular Ca(2+) in K(+)-depolarized (140 mM) cells and evoked an increase in the LVA Ca(2+) channel probability of opening (NP(o)) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP(o) elicited by elevated Ca(2+) concentration. Furthermore, administration of a mutant recombinant CaMKIIalpha exhibiting cofactor-independent activity in the absence of elevated Ca(2+) produced a threefold elevation in LVA channel NP(o). These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca(2+).

The T-type Ca2+ channel is unique among voltage-dependent Ca2+ channels in its low threshold for opening and its slow kinetics of deactivation. Here, we evaluate the importance of intracellular Ca2+ (Cai2+) in promoting low-threshold gating of T-type channels in adrenal glomerulosa cells. We observe that 390 nM to 1.27 microM Cai2+ enhances T-type current by shifting the voltage dependence of channel activation to more negative potentials. This Ca(2+)-induced shift is mediated by calmodulin-dependent protein kinase II (CaMKII), because it is abolished by inhibitors of CaMKII but not of protein kinase C and is subsequently restored by exogenous calmodulin. This Ca(2+)-induced reduction in gating threshold would render T-type Ca2+ channels uniquely suited to transduce depolarizing stimuli of low amplitude into a Ca2+ signal sufficient to support a physiological response.

We recently reported that elevations in the intracellular Ca2+ concentration ([Ca2+]i) enhance low-voltage-activated, T-type, Ca2+ channel activity via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Here, we document CaMKII activity in bovine adrenal glomerulosa (AG) cells and assess the importance of CaMKII in depolarization-induced Ca2+ signaling. AG cell extracts exhibited kinase activity toward a CaMKII-selective peptide substrate that was dependent on both Ca2+ [half-maximal concentration for Ca2+ activation (K0.5) = 1.5 microM] and calmodulin (K0.5 = 46 nM) and was sensitive to a calmodulin antagonist and a CaMKII peptide inhibitor. On cell treatment with elevated extracellular potassium (10-60 mM) or angiotensin II, Ca(2+)-independent CaMKII activity increased to 133-205% of basal activity. Ca(2+)-independent kinase activity in agonist-stimulated extracts was inhibited by the CaMKII inhibitor peptide, 1(-)[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a cell-permeable inhibitor of CaMKII, reduced the agonist-induced stimulation of Ca(2+)-independent CaMKII activity. KN-62 also diminished depolarization-induced increases in [Ca2+]i without affecting the membrane potential. These observations suggest that CaMKII is activated in situ by aldosterone secretagogues and augments Ca2+ signaling through voltage-gated Ca2+ channels.

1. Changes in extracellular K+ concentration (delta[K+]o) evoked by electrical current were measured with K+-selective microelectrodes (K-ISMs) in the retina of the frog eyecup. 2. In the superfusate at 20 microns above the inner limiting membrane (ILM), current-evoked delta[K+] was a function of current polarity and strength; its amplitude decreased as the K-ISM was moved higher above the ILM. Responses were similar whether measured with K-ISMs containing the Corning exchanger or a valinomycin-based liquid membrane. No current-evoked delta[Ca2+] could be detected with Ca-selective microelectrodes (Ca-ISMs). 3. Within the retina, a complex spatiotemporal profile of current-evoked delta[K+]o was observed. Strophanthidin abolished responses in the proximal retina, but had little effect on the response in the superfusate. A blocker of K+ channels (Ba2+) depressed responses in the superfusate, but not in the proximal retina. 4. Quantitative analysis of these responses indicates a transport number for K+ of 0.18 at onset of current, and that decreases over a few seconds. In contrast, a transport number of approximately 0.01 is predicted from the expected ionic concentrations within extracellular space. 5. These findings are compatible with the delta[K+] above the ILM being due to transcellular movement of K+ through Müller cells. The results suggest that K+ spatial buffering may be particularly potent in the retina. Furthermore, determinations of tissue characteristics by passage of electrical current must take into account that at least 17% of the current does not travel through extracellular space.