Our aim was to identify which adrenocortical cells produce ouabain and how it is regulated in vitro. With the help of an ouabain radioimmunoassay developed in our laboratory, we found that ouabain is produced both by zona glomerulosa and fasciculata cells. Our results were confirmed by reverse-phase HPLC. ACTH increased ouabain production in both cell types. Angiotensin-II, as well as changes in the potassium concentration of the incubation medium, affected ouabain production only in the zona glomerulosa.
Using histochemical and immunocytochemical methods, cholinergic nerve fibres were demonstrated in the rat adrenal cortex, primarily in the capsule and zona glomerulosa, and in the medulla. Some terminated among the glomerulosa cells or around blood vessels. Occasional fibres were also seen in the fasciculata, ending in islets of chromaffin tissue without ramifications on cortical cells.To clarify the role of cholinergic innervation, a microvolume perifusion system was used to study steroid production by the rat adrenal capsule-glomerulosa. Acetylcholine (ACh) itself had no reproducible effects; however, since variable amounts of endogenous ACh were present, the actions of antagonists were also studied. The M 1 muscarinic receptor antagonist pirenzepine (10 and 100 µM) stimulated aldosterone secretion. This stimulation was abolished by co-incubation with carbachol, the M 1 agonist McN A-343 and by atropine. We found that the action of pirenzepine was blocked by nifedipine (Ca 2+ channel blocker), suggesting that pirenzepine (through release of endogenous ACh) provides an acute stimulus by enhancing Ca 2+ inflow. Hemicholine, a choline uptake blocker, reduced the stimulatory effect of pirenzepine on steroid secretion, confirming that stimulation was of neural origin. Neither the non-selective muscarinic receptor antagonist atropine, the selective M 1 -M 3 muscarinic receptor antagonist 4-DAMP, nor the selective M 2 muscarinic receptor antagonist methoctramine influenced aldosterone output. Receptor-binding studies revealed the existence of M 3 receptors in capsule-glomerulosa homogenates. We conclude that pirenzepine acts on presynaptic M 1 autoreceptors to increase spontaneous ACh release from varicose axon terminals that lie in close proximity to the glomerulosa cells. In turn ACh may thus stimulate steroidogenesis acutely through M 3 receptors. These results support the concept of a direct cholinergic influence on zona glomerulosa function in the rat.
In addition to hypophyseal control, steroid synthesis and secretion in the adrenal cortex is also under direct local neural modulation. We obtained morphological and neurochemical evidence that a substantial proportion of the noradrenergic nerve endings lie in close proximity to zona glomerulosa cells without making synaptic contact, thus providing evidence for a direct local modulatory role of catecholamines in steroid secretion. These noradrenergic neurones, like other noradrenergic neurones in the central nervous system, are able to take up dopamine (DA), convert it partly into noradrenaline (NA) and to release both NA and DA together with the co-transmitter ATP when neuronal activity drives them to do so. These catecholamines and ATP may reach zona glomerulosa cells via diffusion in a paracrine way and modulate the synthesis of aldosterone. The presence of ecto-Ca-ATPases, enzymes that may terminate the effect of ATP, was demonstrated around the nerve profiles indicating that not only ATP but its metabolites (ADP, AMP, adenosine) can also influence the production of aldosterone. These data strongly support the possibility of a paracrine, non-synaptic modulatory role of catecholamines and ATP in the regulation of adrenocortical steroid secretion.
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