Background: Pituitary neuroendocrine tumors (PitNETs) are benign metaplasias that develop in the pituitary gland and due to hormonal overproduction or mass effects have serious adverse effects on the health of the patients. In our previous genomic and transcriptomic studies, we have discovered that ryanodine receptors (RYRs) are one of molecular determinants of pituitary tumor development and functionality, however, however their exact role in PitNET pathogenesis is not clear. Bulk RNA sequencing was performed for PitNET derived pituisphere cultures incubated with 10µm and 10nm concentrations of RYR affecting agents and samples collected at 4, 8, 24, 48, 72 hours from 3 individuals with growth hormone secreting and 5 with non-functional PitNETs. Differential gene expression was conducted using DESeq2 (p.adj < 0.05), functional enrichment was performed with STRING-db. RNA-seq analysis revealed major time dependent alterations in the GH PitNET derived cultures between incubated/non-incubated groups. Most notably, 414 were upregulated while 924 genes were downregulated in the incubated group. However, NF PitNET incubated groups yielded only 11 upregulated and 13 downregulated genes. GH PitNET pituisphere downregulated DEG enrichment revealed their involvement in calcium signaling and neuroactive ligand-receptor interactions. PPI interaction analysis revealed CYSLTR1 and CYSLTR2 as major hubs in the downregulated gene network and BTF3 and CALM1 in the upregulated gene network. Transcriptome profiling revealed distinct alterations between GH- and NF- derived pituispheres incubated with RYR affecting agents, highlighting the specificity of RYR involvement in PitNET pathogenesis.