Background/Objectives: Copper (Cu²⁺) is an essential trace element that participates as a cofactor in key metabolic enzymes such as cytochrome c oxidase and superoxide dismutase. However, excessive copper exposure can be toxic and disturbances in copper homeostasis have been associated with neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. Despite growing evidence linking copper to neuronal dysfunction, the cellular mechanisms by which Cu²⁺ affects neuronal excitability and neurotransmission remain poorly understood. The aim of this study was to investigate the effects of acute Cu²⁺ exposure on ionic currents involved in cellular excitability and neurotransmitter release in bovine chromaffin cells. Methods: Primary cultures of bovine chromaffin cells were used as a neuroendocrine model to study cellular excitability. Voltage-dependent ionic currents were recorded using the whole-cell patch-clamp technique in voltage-clamp configuration. Catecholamine secretion was monitored by amperometry, and cytosolic Ca²⁺ dynamics were measured in fluo-4-loaded cells during depolarization induced by high K⁺ stimulation. Results: Acute Cu²⁺ exposure produced a concentration-dependent enhancement of depolarization-evoked catecholamine release. In parallel, Cu²⁺ inhibited voltage-dependent calcium (ICa), sodium (INa), potassium (IKv), and calcium/voltage-dependent potassium (IKCa-v) currents in a concentration-dependent and partially reversible manner. In addition, Cu²⁺ increased basal cytosolic Ca²⁺ levels while reducing the amplitude of depolarization-evoked Ca²⁺ transients. Conclusions: Acute Cu²⁺ exposure exerts a dual effect in bovine chromaffin cells, inhibiting the ionic currents that support cellular excitability while potentiating catecholamine secretion. This apparent paradox is consistent with a disruption of intracellular Ca²⁺ homeostasis, in which elevated basal cytosolic Ca²⁺ may facilitate exocytosis despite reduced depolarization-evoked Ca²⁺ entry. These findings provide new insight into the mechanisms by which copper may alter neuronal signaling and contribute to neurotoxicity.

Background/Objectives: Origanum majorana L. has reported antioxidant and other bioactive properties, but its effects on excitability and intracellular Ca²⁺ handling in excitable cells remain poorly defined. We investigated how a hydroalcoholic marjoram extract modulates membrane ion currents, cytosolic Ca²⁺ signaling, and MTT-reducing activity in bovine chromaffin cells and primary rat cortical neurons. Methods: A pressurized liquid O. majorana extract (ethanol:water, 70:30) was chemically characterized by HPLC-PAD. Whole-cell patch-clamp and current-clamp recordings were used to assess voltage-dependent Ca²⁺, Na⁺, and K⁺ currents and membrane excitability in bovine chromaffin cells. Fluo-4-AM measurements were used to evaluate Ca²⁺ responses to K⁺, caffeine, and histamine. MTT assays assessed changes in MTT-reducing activity under veratridine-induced Ca²⁺ overload and oligomycin/rotenone-induced mitochondrial stress in bovine chromaffin cells and cortical neurons prepared from embryonic day 18 Sprague-Dawley rats. For inferential statistics, replicate cells or wells from the same primary culture were averaged and the independent culture was used as the biological unit. Results: Culture-level analysis confirmed significant reductions in voltage-dependent Ca²⁺ and Na⁺ currents at 1.5 µg/mL (1 µL/mL) marjoram extract, together with significant overall effects on the putative Ca²⁺-dependent and voltage-dependent K⁺ current components. The extract depolarized the resting membrane potential and markedly suppressed repetitive action-potential firing. Mean K⁺-evoked Fluo-4 responses were reduced by approximately 20%, although this effect did not reach statistical significance at the culture level. Caffeine-evoked Ca²⁺ responses were higher in the presence of 0.015–1.5 µg/mL (0.01–1 µL/mL) marjoram extract, but these differences did not remain significant after correction for multiple comparisons. Histamine-evoked responses were unchanged. In MTT assays, significant preservation of MTT-reducing activity was limited to selected conditions, mainly at lower extract concentrations in bovine chromaffin cells, whereas the neuronal comparisons did not remain significant after multiplicity correction. Because extract-only, concentration-matched vehicle, cell-free extract/MTT interference controls, and an independent orthogonal assay of cell number or membrane integrity were not included, the MTT findings are reported strictly as changes in MTT-reducing activity and not as direct evidence of cytoprotection or neuroprotection. Conclusions:O. majorana extract modulates membrane excitability through effects on multiple plasma-membrane ion currents. The higher mean caffeine-evoked Ca²⁺ responses are compatible with altered Ca²⁺ mobilization from ryanodine-sensitive intracellular stores, but they do not demonstrate direct activation or sensitization of RyR receptors. The MTT findings do not establish cytoprotection or neuroprotection. These findings are correlative, and a causal contribution of ryanodine-sensitive intracellular stores to the preservation of MTT-reducing cellular metabolic activity remains to be established.