Voltage-gated
L-type calcium channel (CaV)
isoforms are well known to play pivotal tissue-specific roles not only in vasoconstriction but also in adrenocortical steroidogenesis including
aldosterone biosynthesis. Alpha-1C subunit
calcium channel (CC) (CaV1.2) is the specific target of
anti-hypertensive CC blockers (CCBs) and its Alpha-1D subunit (CaV1.3) regulates depolarization of cell membrane in
aldosterone-producing cells. Direct effects of CCBs on
aldosterone biosynthesis were previously postulated but their intra-adrenal distribution and effects on
steroid production in primary
aldosteronism (PA) patients have remained virtually unknown. In this study, frozen tissue specimens constituting
tumor, adjacent adrenal gland and peri-adrenal adipose tissues of nine
aldosterone-producing
adenoma (APA) cases were examined for visualization of
amlodipine and
aldosterone themselves using matrix-assisted
laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Liquid chromatography-mass spectrometry (LC-MS) analysis was also performed to quantify
amlodipine and 17 adrenal
steroids in those cases above and compared the findings with immunohistochemical analysis of steroidogenic
enzymes and
calcium channels (CaV1.2 and CaV1.3). Effects of
amlodipine on
mRNA level of
aldosterone biosynthetic
enzymes were also explored using human
adrenocortical carcinoma cell line (H295R).
Amlodipine-specific peak (m/z 407.1 > 318.1) was detected only in
amlodipine treated cases. Accumulation of
amlodipine was marked in adrenal cortex compared to peri-adrenal adipose tissues but not significantly different between APA
tumors and adjacent adrenal glands, which was subsequently confirmed by LC-MS quantification. Intra-adrenal distribution of
amlodipine was generally consistent with that of CCs. In addition, quantitative
steroid profiles using LC-MS and in vitro study demonstrated the lower HSD3B activities in
amlodipine treated cases. Immunoreactivity of CaV1.2 and
HSD3B2 were also correlated. We report the first demonstration of specific visualization of
amlodipine in human adrenal tissues by MALDI-MSI. Marked
amlodipine accumulation in the adrenal glands suggested its direct effects on steroidogenesis in PA patients, possibly targeting on CaV1.2 and suppressing HSD3B activity.