Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha

Abstract

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs – which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

PI(4,5)P2-dependent and -independent roles of PI4P in the control of hormone secretion by pituitary cells

“…However, experiments with aT3-1 and LbT2 immortalized mouse gonadotrophs revealed that wortmannin at a concentration that inhibits both PI3K and PI4K has no significant effect on GnRH-induced calcium mobilization, and that PI3K can influence the expression of gonadotroph-specific genes (94). In contrast, we observed no effect of the PI3K inhibitor LY294002 on PRL secretion (14). The observations that (PI(3,4,5) P3 and not PI(4,5)P2 shows a regulatory role on Drosophila's synaptic vesicle exocytosis (95) may also explain the difference between fish and mammalian pituitary cells.…”

“…However, PI(4,5)P2 hydrolysis release IP3 and DAG, whereas PI4P hydrolysis release DAG and inactive IP2, an action that could explain the prolonged production of DAG during sustained GPCR activation (12). The last step in vesicle exocytosis was believed to be solely dependent on PI(4,5)P2 (13), but recent data suggested a possible role for PI4P independent of PI(4,5)P2 in the final step of regulated exocytosis (14). Derived from (9, 10, 14-16).…”

“…There are four catalytic subunits and five regulatory subunits in class I PI3Ks (87). In rat pituitary cells, only the Pik3ca gene reaches the threshold for detection by scRNAseq, as do three regulatory subunit genes: Pik3r1, Pik3r2, and Pik3r3 (14). In general, the RTK-mediated increases in PI3K activity led to the activation of the protein kinase, Akt, which in turn initiates a cascade of cellular responses.…”

Is prolactin receptor signaling a target in dopamine-resistant prolactinomas?

“…Prolactin gene expression is modulated by various signals, stimulatory such as estradiol and inhibitory such as dopamine, that Converge in several signaling pathways such as the AMPc/PKA, PKC, or MAPK pathways ( 19 , 40 , 41 ). The secretion of PRL is another control point, regulated mainly through calcium-dependent- mechanisms ( 42 , 43 ) which can depend on the cell’s electrical activity, e.g., voltage-dependent calcium entry or signaling molecules such as IP3, initiated chiefly by G q/11 -coupled membrane receptors ( 44 ).…”

Evaluation of autoantibody signatures in pituitary adenoma patients using human proteome arrays

“…Acromegaly PAs are primarily caused due to excessive GH secretion, which maintains various glands, bones, cartilages and is involved in carry intracellular signalling in Acromegaly patients [34] and are reported to be ubiquitinated in NFPA patients [35]. Phosphate kinase PI4K2A plays a central role in cell signalling trafficking [36] and maintains calcium balance critical for hormonal release (prolactin secretion) by exocytosis [37]. PI4K2A also regulates VAMP3 involved in vesicular trafficking and GH secretion, which is already reported in rat pituicytes [38].…”