The zona glomerulosa of the adrenal gland is responsible for the synthesis and release of the mineralocorticoid aldosterone. This steroid hormone regulates salt reabsorption in the kidney and blood pressure. The most important stimuli of aldosterone synthesis are the serum concentrations of angiotensin II and potassium. In response to these stimuli, voltage and intracellular calcium levels in the zona glomerulosa oscillate, providing the signal for aldosterone synthesis. It was proposed that the voltage-gated T-type calcium channel CaV3.2 is necessary for the generation of these oscillations. However, Cacna1h knockout mice have normal plasma aldosterone levels, suggesting additional calcium entry pathways. We used a combination of calcium imaging, patch clamp and RNA sequencing to investigate such pathways in the murine zona glomerulosa. Cacna1h-/- glomerulosa cells showed similar calcium levels as wild-type mice in response to stimulation with angiotensin II or potassium. No calcium channels or transporters were upregulated to compensate for the loss of CaV3.2. The calcium oscillations observed in the zona glomerulosa of Cacna1h-/- mice were instead dependent on L-type voltage-gated calcium channels. Furthermore, we found these channels also in the wild-type zona glomerulosa where they can partially compensate for an acute inhibition of CaV3.2. Inhibition of both, T- and L-type calcium channels abolished the increase of intracellular calcium caused by angiotensin II. Our study demonstrates that T-type calcium channels are not required to maintain glomerulosa calcium oscillations and aldosterone production.

Abstract:Precious little is known about useful functionalization strategies of antibodies that will not impair either reactivity or specificity of this valuable reagent. We used an experimental approach to demonstrate our own experience with published protocols. The conjugates obtained were then tested with regard to their performance against commercially available detection kits. Our results are then viewed in light of published precedence to highlight areas where future effort is needed to refine such versatile tools.