Nitric oxide modulates ATP-evoked currents in mouse Leydig cells.

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.

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 æM 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.

Modulation of gap junction mediated intercellular communication in TM3 Leydig cells.

Long-term modulation of intercellular communication via gap junctions was investigated in TM3 Leydig cells, under low and high confluence states, and upon treatment of the cells for different times with activators of protein kinase A (PKA) and protein kinase C (PKC). Cells in low confluence were readily coupled, as determined by transfer of the dye Lucifer Yellow; on reaching confluence, the cells uncoupled. Western blots and RT-PCR revealed that connexin 43 (Cx43) was abundantly expressed in TM3 Leydig cells and its expression was decreased after the cells achieved confluence. Stimulation of PKA or PKC induced a decrease in cell-cell communication. Staurosporin, an inhibitor of protein kinases, increased coupling and was able to prevent and reverse the uncoupling actions of dibutyryl cAMP and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Under modulation by confluence, Cx43 was localized to the appositional membranes when cells were coupled and was mainly in the cytoplasm when they were uncoupled. In addition, cAMP and TPA reduced the surface membrane labeling for Cx43, whereas staurosporin increased it. These data show a strong correlation between functional coupling and the membrane distribution of Cx43, implying that this connexin has an important role in intercellular communication between TM3 cells. Furthermore, increased testosterone secretion in response to luteinizing hormone was accompanied by a decrease in intercellular communication, suggesting that gap junction mediated coupling may be a modulator of hormone secretion in TM3 cells.

The resting potential of mouse Leydig cells: role of an electrogenic Na+/K+ pump.

Resting potentials (Vm) were measured in mouse Leydig cells, using the whole-cell patch-clamp technique. In contrast to conventional microelectrode measurements, where a biphasic potential was observed, we recorded a stable Vm around -32.2 +/- 1.2 mV (mean +/- SEM, n = 159), at 25 degrees C, and an input resistance larger than 2.7 x 109 W. Although Vm is sensitive to changes in the extracellular concentrations of potassium and chloride, the relationship between Vm and these ions' concentrations cannot be described by either the Goldman-Hodgkin-Katz or the Nernst equation. Perifusing cells with potassium-free solution or 10?3 M ouabain induced a marked depolarization averaging 20.1 +/- 3.2 mV (n = 9) and 23.1 +/- 2.8 mV, (n = 7), respectively. Removal of potassium or addition of ouabain with the cell voltage-clamped at its Vm, resulted in an inwardly directed current, due to inhibition of the Na+K+ATPase. The pump current increased with temperature with a Q10 coefficient of 2.3 and had an average value of -6.5 +/- 0.4 pA (n = 21) at 25 degrees C. Vm also varied strongly with temperature, reaching values as low as -9.2 +/- 1.2 mV (n = 22) at 15 degrees C. Taking the pump current at 25 degrees C and a minimum estimate for the membrane input resistance, we can see that the Na+K+ATPase could directly contribute with 17.7 mV to the Vm of Leydig cells, which is a major fraction of the ?32.2 +/- 1.2 mV (n = 159) observed.

Short term regulation of cell-cell communication in TM3 Leydig cells. A perforated patch study.

Determination of the junctional conductance (g(j)) in TM3 Leydig cells by the dual whole cell patch clamp technique (DWCPC) shows that coupling undergoes a rapid and irreversible run down. Addition of ATP or cAMP derivatives to the pipette solution has been shown to prevent this phenomenon in several tissues, but this same treatment is unable to inhibit run down in Leydig cells. Because the run down in junctional conductance may pose serious problems to the interpretation of results, we also measured g(j) by using the double perforated patch clamp technique (DPPT). Access to the cell interior was achieved by adding 200 microgram/ml of nystatin to the pipette solution. With this method, run down in g(j) was greatly reduced, amounting to no more than 5% of the initial value. Exposure of the cells, under DWCPC or DPPT, to dibutyryl cAMP or to tumor promoting agent (TPA) led to a decrease in cell to cell communication. Staurosporine, a PKC inhibitor, increased g(j) and was able to prevent and reverse the uncoupling action of cAMP or TPA. Our results indicate that cell-cell communication in Leydig cells is down regulated by both protein kinases A and C, interacting in a complex manner.

TsTX-IV, a short chain four-disulfide-bridged neurotoxin from Tityus serrulatus venom which acts on Ca2+-activated K+ channels.

The primary structure of TsTX-IV, a neurotoxin isolated from Tityrus serrulatus scorpion venom, is reported. Its amino acid sequence was determined by automated Edman sequential degradation of the reduced and carboxymethylated toxin and of relevant peptides obtained by digestion with Staphylococcus aureus strain V8 protease or trypsin and cleavage by CNBr. The complete sequence showed 41 amino acid residues, which account for an estimated molecular weight of 4520, and eight half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518. Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K+ channels. TsTx-IV was able to block Ca2+-activated K+ channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced.

Patch-clamp detection of macromolecular translation 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 nucleus.

Calcium-activated potassium channels are involved in the response of mouse Leydig cells to human chorionic gonadotropin.

The patch-clamp technique was used to investigate the involvement of ion channels in the response of Leydig cells to gonadotropic hormones (viz. hCG). Recordings in the cell-attached configuration (pipette containing 140 mM KCl) showed unitary events with conductance of 187.9 +/- 5.2 pS (N = 24 patches) in about 70% of the cells. These channels were potassium selective and the open channel probability (Po) was always about 1% for displacement of potential from the resting value in the range of -20 to +60 mV. Treatment of the cells with hCG (2 ng/ml) led to a large increase in the frequency of openings, concomitant with a reduction in the mean closed time and there was essentially no effect on the mean open time of the channel. Dibutyryl cAMP (100 microM) produced an effect similar to that of hCG and both required external calcium for their action. No direct effect of either dibutyryl cAMP or hCG were observed in inside-out patches. Reversal potential measurements on excised inside-out patches demonstrated that the channels were highly potassium selective with unitary conductance of about 206.8 +/- 6.36 pS (mean +/- SEM of 6 measurements), and an estimated permeability of 3.6 x 10(-13) +/- 0.2 x 10(-13) cm3/s (mean +/- SEM for 6 measurements), in symmetrical 140 mM KCl. The activity of the channel in excised patches was very sensitive to the free-calcium concentration on the intracellular surface of the channel. Po evaluated at +60 mV increased from 3% at 10 nM to 47% at 100 nM free calcium. The Hill coefficient under these conditions was 1.1. These results demonstrate that Leydig cells have a Ca2(+)-activated K+ channel of large unitary conductance, which can be activated upon the binding of hCG to receptors in the cell membrane.

Calcium-activated potassium channels ar involved in the response of mouse Leydig cells to human chorionic gonadotropin

The patch-clamp technique was used to investigate the involvement of ion channels in the response of Leydig cells to gonadotropic hormones (viz.hCG). Recordings in the cell-attached configuration (pipette containing 140 mM KCl) showed unitary events with conductance of 187.9 + or - 5.2 pS(N = 24 patches) in about 70 percent of the cells. These channels were potassium selective and the open channel probability (Po) was always about 1 percent for displacemtne of potential from the resting value in the range of -20 to +60 mV. Treatment of the cells with hCG (2 ng/ml) led to a large increase in the frequency of openings, concomitant with a reduction in the mean closed time and there was essentially no effect on the mean open time of the channels. Dibutyryl cAMP (100 µM) produced an effect similar to that of hCG and both required external calcium for their action. No direct effect of either dibutyryl cAMP or hCG were observed in inside-out patches. Reversal potential measurements on excised inside-out patches demonstrated that the channels were highly potassium selective with unitary conductance of about 206.8 + or - 6.36 pS(mean + or - SEM of 6 measurements), and an estimated permeability of 3.6 x 10-13 + or - 0.2 x 10--13 cm3/s (mean + or - SEM for 6 measurements), in symmetrical 140 mM KCl. The activity of the channel in excised paches was very sensitive to the free-calcium concentration on the intracellular surface of the free-calcium concentration on the intracellular surface of the channel. Po evaluated at + 60mV increased from 3 percent at 10 nM to 47 percent at 100 nM free calcium. The Hill coefficient under these conditions was 1.1. These results demonstrate that Leydig cells have a Ca2+ -activated K+ channel of large unitary conductance, which can be activated upon the binding of hCG to receptors in the cell membrane

Hurst analysis in the study of ion channel kinetics

Ion channels are protein molecules which can assume distinct open and closed conformational states. The transitions between these states can be controlled by the electrical field, ions and/or drugs. Records of unitary current events show that short open-time intervals are frequently adjacent to much longer closed-time intervals, and vice-versa, suggesting that the kinetic process has memory, i.e., the intervals are correlated in time. here the rescaled range analysis (R/S Hurst analysis) is proposed as a method to test for correlation. Simulations were performed with a two-state Markovian model, which has no memory. The calculated Hurst coefficients (H) presented a mean + or - SD value of 0.493 + or - 0.025 (N = 100). For the Ca2+ -activated K+ channels of Leydig cells, H wass equal to 0.75, statistically different (1 percent level) from that calculated for the memoryless proces. Randomly shuffling the experimental data resulted in an H = 0.55, not significantly different (1 percent level) from that found for the two-state Markovian model. For a linear three-state Markovian model, H was equal to 0.548 + or - 0.017 (N = 15), agin not significantly different (1 percent level) from that of the memoryless proces. Although the tree-state Markovian model adequately describes the open-and closed-time distributions, it does not account for the correlation found in this Ca2+ -activatedK+ channel. Our results ilustrate the efficacy of the R/S analysis in determining whether successive opening and closing events are correlated in time and can be of help in deciding which odel should be used to describe the kinetics of ion channels

Glomerular permeability to macromolecules in gentamicin-treated rats

To determine the effect of gentamicin on the functional properties of the glomerular barrier, 44 Wistar rats received daily doses of 80 mg/kg body weight for 6 days. Glomerular permeability to neural dextrans and albumin was evaluated by day 6 and albuminuria was determined on the 1st, 3rd and 5th days of treatment. Treatment induced an intense increase in albuminuria from 74 ug/24 h to 11.5 mg/24 h on the 5th day of treatment (N=11). This increase was associated with the presence of large amounts of albumin in elements of the glomerular filter and in the apical region of the proximal tubular cells (N=4). Fractional clearances of neutral dextrans having molecular radii in the range of 18-41 A were not significantly different in control (N=5) and gentamicin-treated rats (N=7). These results show that gentamicin, a polycation at pH 7.4, produces an increase in the glomerular permeability to negatively charged macromolecules in rats, probably due to interaction of the polycation with negative charges in the glomerular filter

Ca2+ induced down-regulation of epithelial amiloride-sensitive channels reconstituted into planar lipid bilayers

Multichannel experiments were carried out to investigate the Ca2+ - induced down-regulation of epithelial Na+ channels reconstituted into planar lipid bilayer membranes. Reconstitution was achueved by fusion of vesiculated apical membrane fragments to solvent-free planar lipid bilayers. We found that the presence of micromolar concentrations of Ca2+ on the side to which the vesicles were added substantially lowered the channel-mediated current. The inhibition was strongly influenced by pH. At pH 8.0 all the current was blocked by 1 mM calcium, whereas at pH 7.1 the inhibition was about 80%. The blocking kinetics was clearly voltage-dependent. The mechanism of blocking cannot be explained either in terms of interations with a single site, or by a model in which two blocking sites are assumed.