Characterisation of the muscarinic receptor subtype M3 in the bovine zona fasciculata-reticularis cells by receptor binding, mRNA and functional studies

Abstract

In the present work we have investigated which muscarinic (M) receptor subtype is responsible for the steroidogenic effect of muscarinic agonists in bovine zona fasciculata-reticularis (ZFR) cells in culture. Radioligand binding studies using the muscarinic antagonist [ Reverse transcriptase (RT)-PCR analysis of bovine ZFR mRNAs using specific primers for M 2 , M 3 and M 4 receptors revealed the expression of only M 3 mRNA. Moreover, carbachol significantly stimulated inositol phosphate accumulation, but had no inhibitory effect on basal or ACTH-induced cAMP production. Indeed, carbachol potentiated ACTH-induced cAMP production and this effect was, in part, mediated through protein kinase C. Lastly, neomycin, an inhibitor of phosphoinositide turnover, significantly attenuated carbachol-evoked cortisol production. Thus, pharmacological, biochemical and mRNA studies indicate that the M 3 receptor subtype is responsible for the biological effects of muscarinic agonists in bovine ZFR cells.

M3-subtype muscarinic receptor activation stimulates intracellular calcium oscillations and aldosterone production in human adrenocortical HAC15 cells

“…Intriguingly, our RT-PCR data found M 2 mRNA present at levels on par with M 3 . This stands in contrast to one of the aforementioned bovine studies which found mRNA for M 3 , but not for M 2 or M 4 (Jánossy et al, 1999). (Because the sequences were unknown at the time, it was not possible for the authors to test for bovine M 1 or M 5 .)…”

“…This activity was attributed to muscarinic receptors that stimulated phosphoinositide turnover and Ca 2+ mobilization but did not alter the cyclic nucleotide pool. Subsequent reports using muscarinic agonists in bovine cells suggested that ACh-mediated phosphoinositide turnover and cortisol production was most consistent with M 3 receptors coupled to phospholipase C (Clyne et al, 1994; Jánossy et al, 1999; Walker et al, 1990). Other work explained the weak steroidogenic effect of carbachol as a consequence of fast internalization of the M 3 receptor (Jánossy et al, 2001).…”

Pituitary–Adrenal Responses to Oxotremorine and Acute Stress in Male and Female M1 Muscarinic Receptor Knockout Mice: Comparisons to M2 Muscarinic Receptor Knockout Mice

“…The M 3 receptor appears to predominate in the adrenals (36, 37); therefore, some of the corticosterone responses in the present study may have resulted from adrenal M 3 receptor activation, with oxotremorine being a nonselective muscarinic agonist. That the female corticosterone responses to oxotremorine were of much greater magnitude relative to ACTH responses than were the male responses suggests a greater number and/or sensitivity of M 3 receptors in the female adrenal cortex.…”

Pituitary‐Adrenal Responses to Cholinergic Stimulation and Acute Mild Stress are Differentially Elevated in Male and Female M2 Muscarinic Receptor Knockout Mice

“…In addition to M 2 receptors, other muscarinic receptors appear to regulate corticosterone responses differentially in males versus females. The M 3 receptor appears to predominate in the adrenals (42, 43); therefore, some of the corticosterone response may have resulted from adrenal M 3 receptor activation. The finding that female corticosterone responses to cholinergic stimulation were of much greater magnitude relative to ACTH responses than were male responses, suggests a greater number and/or sensitivity of M 3 receptors in the female adrenal cortex.…”