Regulation of Aldosterone Secretion by Substance P and the Neurokinin Type 1 Receptor in Aldosterone-Producing Adenomas

Abstract

Background: Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism, the most frequent form of secondary hypertension. Although somatic mutations in ion channels within APA have been shown to activate Ca²⁺ signaling and drive aldosterone production, the pathophysiology of primary aldosteronism remains partially understood. SP (Substance P), encoded by the TAC1 gene, is a neuropeptide of the tachykinin family, known for its role in stimulating aldosterone production through activation of the neurokinin 1 receptor (NK1R) in the human adrenal cortex. The aim of our work was to investigate the presence of SP nerve fibers and the NK1R in a large series of APA to assess the potential role of tachykinins in the pathophysiology of primary aldosteronism.

Methods: Using molecular, immunohistochemical, and functional techniques, 56 APA tissues were analyzed to assess the expression of SP and NK1R and their impact on aldosterone secretion.

Results: SP-positive nerve fibers were detected in 90% of the APA tissues, localized both within and around the adenomas, which also showed strong NK1R expression. Functional studies revealed that SP stimulated aldosterone secretion in 6 of 10 APA cultures. The NK1R antagonist aprepitant inhibited SP-induced aldosterone secretion in 3 of the 4 SP-responsive APA cultures on which the antagonist was tested. Additionally, in perifused APA explants, SP influenced aldosterone pulsatility, resulting in enhanced mineralocorticoid secretion.

Conclusions: These findings suggest that the SP-NK1R signaling pathway may contribute to APA pathophysiology and represent a novel potential target for the pharmacological treatment of PA in a subset of patients.

Keywords: adenoma; adrenal cortex; aldosterone; neurokinin 1 receptor; primary aldosteronism; substance P.

Figure 1. Expression of tachykinins in APA.

A, Quantitative RT‐PCR analysis of TAC1, TAC3, and TAC4 mRNAs in 42 independent APAs. Each dot indicates 1 adenoma. Data are presented as mean±SEM. P<0.0001. B, Comparison of the expression levels of the TAC1 gene between APA subgroups: C, classical APAs and nonclassical APAs (left panel); NKD and KCNJ5 (middle panel), and correlation of the expression levels of the TAC1 gene with APA diameter (right panel). C through G. Immunohistochemical analysis of substance P in adenoma and adjacent adrenal tissue representative of n=56 independent APAs. C through F, Microphotographs showing a classical APA. C, SP‐positive longitudinal nerve fibers (arrow) and granulation in the immediate vicinity of steroidogenic cells in an APA region positive for CYP11B2. D, High magnification view showing granular SP staining at the periphery (arrow) and within steroidogenic cells (arrowhead). E, CYP11B2 immunostaining. The boxed areas highlight the zones of interest shown in (C) and (F) panels, exhibiting positive SP staining in both the adenoma (C) and adjacent tissues (F), respectively. F, SP‐positive fibers (arrows) surrounding a blood vessel in the adjacent adrenal tissue. G, SP‐positive fibers (arrows) in close contact with steroidogenic cells in the zona glomerulosa and zona fasciculata in the adrenal tissue adjacent to another classical APA. H, SP‐staining in the adjacent tissue to the adenoma detected in nerve fibers (arrows) and ganglia (G) in a nonclassical APA. APA indicates aldosterone‐producing adenoma; C, classical; KCNJ5, KCNJ5 mutation detected; NC, nonclassical; NKD, no KCNJ5 mutation detected; RT‐PCR, reverse transcription polymerase chain reaction; SP, Substance P; V, vessel; ZF, zona fasciculata; and ZG, zona glomerulosa.

Figure 2. Expression of tachykinin receptors in APA.

A, Quantitative RT‐PCR analysis of TACR1, TACR2, and TACR3 mRNAs in 42 independent APAs. Each dot indicates 1 adenoma. Data are presented as mean±SEM. P=0.012; P<0.0001. B, Comparison of the expression levels of the TACR1 gene between different APA subgroups. classical APAs; nonclassical APAs (left panel) P=0.045; NKD and KCNJ5 (middle panel); CYP11B2‐positive nodules in nonclassical APAs (<2 nodules vs ≥2 nodules, right panel). C through F, Distribution of NK1R immunostaining in APA and adjacent adrenal tissue representative of n=56 independent APAs. C, E, F, NK1R staining in a classical APA and its adjacent adrenal cortex. C, Overall NK1R staining pattern in an APA and the adjacent adrenal cortex. D, Closer view of the NK1R staining in a different classical APA, highlighting areas of staining present in the zona glomerulosa or organized in micronodules within the adrenal tissue adjacent to the adenoma (arrows). E, Close‐up view of NK1R‐positive staining in the ZG and nerve ganglia in the adrenal cortex adjacent to the adenoma). F, High magnification view illustrating the distribution of NK1R staining in a group of adenoma cells. Arrow and arrowhead show membrane and cytoplasmic NK1R staining, respectively. APA indicates aldosterone‐producing adenoma; C, classical; Ca, capsule; G, nerve ganglia; KCNJ5, KCNJ5 mutation detected; NC, nonclassical; NKD, no KCNJ5 mutation detected; NK1R, neurokinin type 1 receptor; RT‐PCR, reverse transcription polymerase chain reaction; and ZG, zona glomerulosa.

Figure 3. Comparison of NK1R and CYP11B2 expressions in APA by immunohistochemistry.

A‐F, Comparison of NK1R and CYP11B2 immunostainings in the adenoma and adjacent adrenal tissue, illustrated by microphotographs representative of n=33 independent APAs. All panels depict a classical APA. A and B, Low magnification views of NK1R (A) and CYP11B2 (B) immunostainings in the same APA tissue. C and D: Closer view of NK1R (C) and CYP11B2 (D) staining patterns in adenoma and adrenal cortex adjacent to adenoma (Adjacent). E‐F: Higher magnification views showing NK1R (E) and CYP11B2 (F) staining in the zona glomerulosa and adrenal capsule in the adjacent tissue. APA indicates aldosterone‐producing adenoma; Ca, capsule; NK1R, neurokinin type 1 receptor; and ZG, zona glomerulosa.

Figure 4. Effect of tachykinins on aldosterone production by APA cells in primary culture.

A, Effect of SP on aldosterone production by cultured APA cells (n=10 independent APA cell cultures, each performed in quadruplicate) (P=0.003). SP (10⁻⁷ M) significantly increased aldosterone production in 6 APA samples. B, Effect of increasing concentrations of SP (10⁻¹⁰ M to 10⁻⁶ M) on aldosterone production in 1 of the 6 SP‐sensitive APAs (from patient 1 [P1]), showing a dose‐dependent increase in aldosterone secretion (EC₅₀=1.71±0.3 nM, Emax 228.9±14.3% of basal level). C, Effect of SP (10⁻⁷ M) alone or in the presence of the NK1R antagonist aprepitant (10⁻⁹ M) on aldosterone production by cultured APA cells (n=3 independent cultures). The stimulatory effect of SP was blunted by AP (P=0.008). D, Effect of neurokinin A (10⁻⁷ M) on aldosterone production by cultured APA cells (n=2 independent cultures from patients 7 [P7] and 8 [P8]). NKA significantly increased aldosterone production in the APA cell culture derived from P7 (P=0.008) but did not significantly modify aldosterone release in the cell culture derived from P8 (P=0.084). Data represent the mean±SEM of the values obtained in independent experiments and are expressed as % of basal level. In all culture experiments, aldosterone secretion was normalized to basal levels. AP indicates aprepitant; APA, aldosterone‐producing adenoma; EC₅₀, half maximal effective concentration; NKA, neurokinin A; NK1R, neurokinin type 1 receptor; and SP, Substance P.

Figure 5. Effect of SP on aldosterone production by perifused APA tissues.

Aldosterone production kinetics in basal state and in response to SP (10⁻⁶ M, administered for 20 minutes) in 5 independent perifused APA tissues. Perifusion experiments were conducted over a total duration of 250 minutes. A, Integrated aldosterone response using the area under the curve. B, Mean aldosterone levels calculated as the average of all values during each condition. C, Nadir aldosterone levels, representing the lowest nonpeak values in each condition. D, Aldosterone pulse frequency expressed as the number of pulses per h. E, Mean aldosterone pulse interval, defined as the average time (in min) between consecutive pulse peaks. F, Pulse amplitude, calculated as the difference between each peak and its preceding nadir. For each tissue, the data were normalized to basal secretion levels to allow evaluation of the aldosterone response to SP irrespective of the variability of the spontaneous aldosterone production among APAs. Error bars represent the SEM. Data are expressed as % basal level. APA indicates aldosterone‐producing adenoma; and SP, Substance P.