Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleu

Effects of nitric oxide on magnocellular neurons of the supraoptic nucleus involve multiple mechanisms

Physiological evidence indicates that the supraoptic nucleus (SON) is an important region for integrating information related to homeostasis of body fluids. Located bilaterally to the optic chiasm, this nucleus is composed of magnocellular neurosecretory cells (MNCs) responsible for the synthesis and release of vasopressin and oxytocin to the neurohypophysis. At the cellular level, the control of vasopressin and oxytocin release is directly linked to the firing frequency of MNCs. In general, we can say that the excitability of these cells can be controlled via two distinct mechanisms: 1) the intrinsic membrane properties of the MNCs themselves and 2) synaptic input from circumventricular organs that contain osmosensitive neurons. It has also been demonstrated that MNCs are sensitive to osmotic stimuli in the physiological range. Therefore, the study of their intrinsic membrane properties became imperative to explain the osmosensitivity of MNCs. In addition to this, the discovery that several neurotransmitters and neuropeptides can modulate their electrical activity greatly increased our knowledge about the role played by the MNCs in fluid homeostasis. In particular, nitric oxide (NO) may be an important player in fluid balance homeostasis, because it has been demonstrated that the enzyme responsible for its production has an increased activity following a hypertonic stimulation of the system. At the cellular level, NO has been shown to change the electrical excitability of MNCs. Therefore, in this review, we focus on some important points concerning nitrergic modulation of the neuroendocrine system, particularly the effects of NO on the SON

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration

Nitric oxide modulates ATP-evoked currents in mouse Leydig cells

<div><p>Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.</p></div