Tunneling nanotubes and epithelial bridges are recently discovered new forms of intercellular communication between remote cells allowing their electrical synchronization, transfer of second messengers and even membrane vesicles and organelles. In the present study, we demonstrate for the first time in primary cell cultures prepared from human laryngeal squamous cell carcinoma (LSCC) samples that these cells communicate with each other over long distances (up to 1 mm) through membranous tunneling tubes (TTs), which can be open-ended or contain functional gap junctions formed of connexin 43. We found two types of TTs, containing F-actin alone or F-actin and α-tubulin. In the LSCC cell culture, we identified 5 modes of TT formation and performed quantitative assessment of their electrical properties and permeability to fluorescent dyes of different molecular weight and charge. We show that TTs, containing F-actin and α-tubulin, transport mitochondria and accommodate small DAPI-positive vesicles suggesting possible transfer of genetic material through TTs. We confirmed this possibility by demonstrating that even TTs, containing gap junctions, were capable of transmitting double-stranded small interfering RNA. To support the idea that the phenomenon of TTs is not only typical of cell cultures, we have examined microsections of samples obtained from human LSCC tissues and identified intercellular structures similar to those found in the primary LSCC cell culture.

β₃-adrenergic receptor (β₃-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of β₃-AR in the human atrium. Unexpectedly, β₃-AR activation increased human atrial tissue contractility and stimulated the L-type Ca²⁺ channel current (I_Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I_Ca,L and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective β₃-AR agonists, SR58611 and BRL37344, and a β₃-AR partial agonist, CGP12177, stimulated I_Ca,L in HAMs with nanomolar potency and a 60%–90% efficacy compared with isoprenaline. The β₃-AR agonists also increased contractility but with a much lower efficacy (~10%) than isoprenaline. The β₃-AR antagonist L-748,337, β₁-/β₂-AR antagonist nadolol, and β₁-/β₂-/β₃-AR antagonist bupranolol were used to confirm the involvement of β₃-ARs (and not β₁-/β₂-ARs) in these effects. The β₃-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I_Ca,L stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, β₃-ARs are positively coupled to L-type Ca²⁺ channels and contractility in human atrial tissues through a cAMP-dependent pathway.

Intracellular pH (pH_i) can change during physiological and pathological conditions causing significant changes of electrical and metabolic cell–cell communication through gap junction (GJ) channels. In HeLa cells expressing wild-type connexin45 (Cx45) as well as Cx45 and Cx43 tagged with EGFP, we examined how pH_iaffects junctional conductance (g_j) and g_jdependence on transjunctional voltage (V_j). To characterize V_jgating, we fit the g_j–V_jrelation using a stochastic four-state model containing one V_j-sensitive gate in each apposed hemichannel (aHC); aHC open probability was a Boltzmann function of the fraction of V_jacross it. Using the model, we estimated gating parameters characterizing sensitivity to V_jand number of functional channels. In homotypic Cx45 and heterotypic Cx45/Cx43-EGFP GJs, pH_ichanges from 7.2 to ~8.0 shifted g_j–V_jdependence of Cx45 aHCs along the V_jaxis resulting in increased probability of GJ channels being in the fully open state without change in the slope of g_jdependence on V_j. In contrast, acidification shifted g_j–V_jdependence in the opposite direction, reducing open probability; acidification also reduced the number of functional channels. Correlation between the number of channels in Cx45-EGFP GJs and maximal g_jachieved under alkaline conditions showed that only ~4% of channels were functional. The acid dissociation constant (pK_a) of g_j–pH_idependence of Cx45/Cx45 GJs was ~7. The pK_aof heterotypic Cx45/Cx43-EGFP GJs was lower, ~6.7, between the pK_as of Cx45 and Cx43-EGFP (~6.5) homotypic GJs. In summary, pH_isignificantly modulates junctional conductance of Cx45 by affecting both V_jgating and number of functional channels.

Gap junction (GJ) channels composed of Connexin36 (Cx36) are widely expressed in the mammalian CNS and form electrical synapses between neurons. Here we describe a novel modulatory mechanism of Cx36 GJ channels dependent on intracellular free magnesium ([Mg²⁺]_i). We examined junctional conductance (g_j) and its dependence on transjunctional voltage (V_j) at different [Mg²⁺]_iin cultures of HeLa or N2A cells expressing Cx36. We found that Cx36 GJs are partially inhibited at resting [Mg²⁺]_i. Thus,g_jcan be augmented or reduced by lowering or increasing [Mg²⁺]_i, respectively. Similar changes ing_jandV_j-gating were observed using MgATP or K₂ATP in pipette solutions, which increases or decreases [Mg²⁺]_i, respectively. Changes in phosphorylation of Cx36 or in intracellular free calcium concentration were not involved in the observed Mg²⁺-dependent modulation ofg_j. Magnesium ions permeate the channel and transjunctional asymmetry in [Mg²⁺]_iresulted in asymmetricV_j-gating. Theg_jof GJs formed of Cx26, Cx32, Cx43, Cx45, and Cx47 was also reduced by increasing [Mg²⁺]_i, but was not increased by lowering [Mg²⁺]_i; single-channel conductance did not change. We showed that [Mg²⁺]_iaffects both open probability and the number of functional channels, likely through binding in the channel lumen. Finally, we showed that Cx36-containing electrical synapses between neurons of the trigeminal mesencephalic nucleus in rat brain slices are similarly affected by changes in [Mg²⁺]_i. Thus, this novel modulatory mechanism could underlie changes in neuronal synchronization under conditions in which ATP levels, and consequently [Mg²⁺]_i, are modified.

Both cytosolic fatty acid synthesis (FAS) and mitochondrial fatty acid oxidation (FAO) have been shown to play a role in the survival and proliferation of cancer cells. This study aimed to confirm experimentally whether FAS and FAO coexist in breast cancer cells (BCC). By feeding cells with ¹³C-labeled glutamine and measuring labeling patterns of TCA intermediates, it was possible to show that part of the cytosolic acetyl-CoA used in lipid synthesis is also fed back into the mitochondrion via fatty acid degradation. This results in the transfer of reductive potential from the cytosol (in the form of NADPH) to the mitochondrion (in the form of NADH and FADH₂). The hypothesized mechanism was further confirmed by blocking FAS and FAO with siRNAs. Exposure to staurosporine (which induces ROS production) resulted in the disruption of simultaneous FAS and FAO, which could be explained by NADPH depletion.