It is known that estrogen receptor (ER) has extranuclear signaling functions in addition to classical genomic pathway, and estrogenic actions have been reported in ER-negative breast carcinoma cells. However, significance of cytoplasmic-ER immunoreactivity has not been reported in ER-negative breast carcinoma tissues. We immunolocalized cytoplasmic ER in 155 ER-negative breast carcinoma tissues and evaluated its clinicopathological significance including the prognosis. As a comparative cohort set, we also used 142 ER-positive breast carcinomas. Cytoplasmic-ER immunoreactivity was detected in the carcinoma cells, but not in the non-neoplastic mammary epithelium. Cytoplasmic-ER immunoreactivity was positive in the 35 out of 155 (23%) ER-negative breast carcinoma cases, whereas it was detected only in 2 out of 142 (1.4%) ER-positive cases. Cytoplasmic ER status was positively associated with cytoplasmic-PR status, but inversely associated with Ki67 labeling index or distant free-relapse survival rate. Moreover, cytoplasmic-ER status turned out to be an independent good prognostic factor for both distant relapse-free survival and breast cancer specific survival. These findings suggested that cytoplasmic ER plays important roles in the ER-negative breast carcinoma, and cytoplasmic ER is a potent good prognostic factor. Among the ER-negative breast cancer patients, clinical benefit of chemotherapy may be limited in the cytoplasmic-ER positive cases.

Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types have many common properties in membrane excitability and signaling. However, AMC cells function as endocrine cells while sympathetic ganglion cells are neurons. In rat sympathetic ganglion cells, muscarinic M₁ and M₄ receptors mediate excitation and inhibition via suppression of M-type K⁺ channels and suppression of voltage-dependent Ca²⁺ channels, respectively. On the other hand, M₁ receptor stimulation in rat AMC cells also produces excitation by suppressing TWIK-related acid sensitive K⁺ (TASK) channels. However, whether M₄ receptors are coupled with voltage-dependent Ca²⁺ channel suppression is unclear. We explore this issue electrophysiologically and biochemically. Electrical stimulation of nerve fibers in rat adrenal glands trans-synaptically increased the Ca²⁺ signal in AMC cells. This electrically evoked increased Ca²⁺ signal was not altered during muscarine-induced increase in Ca²⁺ signal, whereas it decreased significantly during a GABA-induced increase, due to a shunt effect of increased Cl⁻ conductance. The whole-cell current recordings revealed that voltage-dependent Ca²⁺ currents in AMC cells were suppressed by adenosine triphosphate, but not by muscarinic agonists. The fractionation analysis and immunocytochemistry indicated that Ca_V1.2 Ca²⁺ channels and M₄ receptors are located in the raft and non-raft membrane domains, respectively. We concluded that muscarinic stimulation in rat AMC cells does not produce voltage-dependent Ca²⁺ channel inhibition. This lack of muscarinic inhibition is at least partly due to physical separation of voltage-dependent Ca²⁺ channels and M₄ receptors in the plasma membrane.