Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells

1. The action of mibefradil was studied on wild type class A calcium (Ca2+) channels and various class A/L-type channel chimaeras expressed in Xenopus oocytes. The mechanism of Ca2+ channel block by mibefradil was evaluated with two microelectrode voltage clamp. 2. Resting-state dependent block (or initial block) of barium currents (IBa) through class A Ca2+ channels was concentration dependent with an IC50 value of 208+/-23 microM. 3. Mibefradil (50 microM) did not significantly affect the midpoint voltage of the steady-state inactivation curve suggesting that inactivation does not promote Ca2+ channel block. Chimaeric class A/L-type Ca2+ channels inactivating with faster or slower kinetics than wild type class A channels were equally well inhibited by mibefradil as wild type class A channels. 4. Frequent Ca2+ channel activation facilitated IBa inhibition by mibefradil (use-dependent block). Recovery from use-dependent block was voltage-dependent, being slower at depolarized membrane potentials (tau = 75+/-15 s at -70 mV, (n=6) vs tau = 20+/-2 s at -100 mV, (n=6), P<0.05). 5. We suggest that use-dependent block of class A Ca2+ channels by mibefradil occurs because of slow recovery from open channel block (SROB) and not because of drug binding to inactivated channels. 6. Voltage-dependent slow recovery from open state-dependent block provides a molecular basis for understanding the cardiovascular profile of mibefradil such as selectivity for vasculature and relative lack of negative inotropic effects.     

Isolation and characterization of a persistent potassium current in neostriatal neurons

1. Depolarization-activated, calcium-independent potassium (K+) currents were studied with the use of whole cell voltage-clamp recording from neostriatal neurons acutely isolated from adult (> or = 4 wk old) rats. The whole cell K+ current was composed of transient and persistent components. The aims of the experiments were to isolate the persistent component and then to characterize its voltage dependence and kinetics. 2. Application of 10 mM 4-aminopyridine (4-AP) completely blocked the transient currents while reducing the persistent current by approximately 40% [50% inhibitory concentration (IC50), of blockable current = 125 microM]. The persistent K+ current also was reduced by tetraethylammonium (TEA). Two components to the TEA block were present, having IC50s of 125 microM (23% of the blockable current) and 5.9 mM (77% of the blockable current). Collectively, these results suggested that the persistent components of the total K+ current was pharmacologically heterogeneous. The properties of the 4-AP-resistant, persistent K+ current (IKrp) were subsequently studied. 3. The kinetics of activation and deactivation of IKrp were voltage dependent. Examination of the entire activation/deactivation time constant profile showed that it was bell shaped, with time constants being moderately rapid (tau approximately 50 ms) at membrane potentials corresponding to the resting potential of neostriatal cells (approximately -80 mV), becoming considerably longer (tau approximately 100 ms) at potentials near the cells' spike thresholds (approximately -45 mV), and decreasing to a minimum (tau approximately 5 ms) at potentials associated with the peak of the cells' action potentials (approximately +20 mV). The inactivation kinetics of IKrp also were voltage dependent. The time constants of inactivation varied between 1 and 8 s at potentials between -10 and +35 mV. 4. Unlike persistent K+ currents in many other cell types, IKrp activated at relatively hyperpolarized membrane potentials (approximately -70 mV). The Boltzmann function describing activation had a half-activation voltage of -13 mV and a slope factor of 12 mV. In addition, the Boltzmann function describing the voltage dependence of inactivation of IKrp had a relatively depolarized half-inactivation voltage of -55 and a large slope factor of 19 mV, indicating that this current was available over a broad range of membrane potentials (between -100 and -10 mV). 5. Neostriatal neurons recorded in vivo exhibit subthreshold shifts in membrane potential of variable duration (tens of ms to s) from a hyperpolarized resting state to a depolarized state that is limited in amplitude just below spike threshold. The voltage dependence of activation and inactivation of IKrp indicates that it will be available on depolarization from the hyperpolarized state. However, the slow activation rate of this current suggests that it will contribute little either to limiting the amplitude of the initial depolarization associated with entry into the depolarized state or to depolarizing episodes of short duration (e.g., < 50 ms). However, IKrp should limit the amplitude of membrane depolarizations associated with prolonged excursions into the depolarized state.     

Dopaminergic modulation of NMDA-induced whole cell currents in neostriatal neurons in slices: contribution of calcium conductances

The present experiments were designed to examine dopamine (DA) modulation of whole cell currents mediated by activation of N-methyl-D-aspartate (NMDA) receptors in visualized neostriatal neurons in slices. First, we assessed the ability of DA, D1 and D2 receptor agonists to modulate membrane currents induced by activation of NMDA receptors. The results of these experiments demonstrated that DA potentiated NMDA-induced currents in medium-sized neostriatal neurons. Potentiation of NMDA currents occurred at three different holding potentials, although it was more pronounced at -30 mV. It was mediated by D1 receptors, because it was mimicked by D1 agonists and blocked by exposure to a D1 antagonist. Activation of D2 receptors produced inconsistent effects on NMDA-induced membrane currents. Either decreases, increases, or no effects on NMDA currents occurred. Second, we examined the contributions of intrinsic, voltage-dependent conductances to DA potentiation of NMDA currents. Blockade of K+ conductances did not prevent DA enhancement of NMDA currents. However, voltage-activated Ca2+ conductances provided a major contribution to DA modulation. The dihydropyridine L-type Ca2+ channel blockers, nifedipine, and methoxyverapamil (D-600), markedly reduced but did not totally eliminate the ability of DA to modulate NMDA currents. The D1 receptor agonist SKF 38393 also enhanced Ba2+ currents in neostriatal neurons. Together, these findings provide evidence for a complex interplay between DA, NMDA receptor activation and dihydropyridine-sensitive Ca2+ conductances in controlling responsiveness of neostriatal medium-sized neurons.     

Action-potential-like depolarizations relieve opioid inhibition of N-type Ca2+ channels in NG108-15 cells

The ability of action-potential-like waveforms (APWs) to attenuate opioid-induced inhibition of N-type Ca2+ channels was investigated in the neuroblastoma x glioma cell line NG108-15 using whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells, the opioid agonist [d-ala2]-methionine-enkephalin (DAME) reversibly decreased omega-conotoxin-GVIA-sensitive Ba2+ currents (N-type currents). Agonist-mediated inhibition of N-type currents could be transiently relieved by strong unphysiological depolarizing prepulses to +80 mV (facilitation). Significant facilitation was also achieved by conditioning the cell with a train of 15 APWs, which roughly mimicked physiological action potentials (1- to 6-ms-long depolarizations to +30 mV from a holding potential of -40 mV). The APW-induced facilitation depended on both conditioning pulse frequency and duration. Summation of the disinhibition produced by each APW was possible because reinhibition following repolarization to -40 mV was a much slower process (tau=88 ms) than the onset of facilitation at +80 mV (tau=7 ms). These results provide evidence that N-type Ca2+ channel facilitation may be a physiologically relevant process, and suggest that neuronal firing may relieve agonist-induced inhibition of N-type currents to an extent depending on both the shape of action potentials and the frequency of firing.     

Clinical study on five myeloperoxidase specific anti-neutrophil cytoplasmic antibody (MPO-ANCA) positive Churg-Strauss syndrome cases

Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=-80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 microM nifedipine (L-type channels), 21% sensitive to 1 microM omega-conotoxin GVIA (N-type channels), and 60% sensitive to 2 microM omega-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 microM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5-50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.     

Slow closed-state inactivation: a novel mechanism underlying ramp currents in cells expressing the hNE/PN1 sodium channel

To better understand why sensory neurons express voltage-gated Na+ channel isoforms that are different from those expressed in other types of excitable cells, we compared the properties of the hNE sodium channel [a human homolog of PN1, which is selectively expressed in dorsal root ganglion (DRG) neurons] with that of the skeletal muscle Na+ channel (hSkM1) [both expressed in human embryonic kidney (HEK293) cells]. Although the voltage dependence of activation was similar, the inactivation properties were different. The V1/2 for steady-state inactivation was slightly more negative, and the rate of open-state inactivation was approximately 50% slower for hNE. However, the greatest difference was that closed-state inactivation and recovery from inactivation were up to fivefold slower for hNE than for hSkM1 channels. TTX-sensitive (TTX-S) currents in small DRG neurons also have slow closed-state inactivation, suggesting that hNE/PN1 contributes to this TTX-S current. Slow ramp depolarizations (0.25 mV/msec) elicited TTX-S persistent currents in cells expressing hNE channels, and in DRG neurons, but not in cells expressing hSkM1 channels. We propose that slow closed-state inactivation underlies these ramp currents. This conclusion is supported by data showing that divalent cations such as Cd2+ and Zn2+ (50-200 microM) slowed closed-state inactivation and also dramatically increased the ramp currents for DRG TTX-S currents and hNE channels but not for hSkM1 channels. The hNE and DRG TTX-S ramp currents activated near -65 mV and therefore could play an important role in boosting stimulus depolarizations in sensory neurons. These results suggest that differences in the kinetics of closed-state inactivation may confer distinct integrative properties on different Na+ channel isoforms.     

Depolarization-induced facilitation of a plateau-generating current in ventral horn neurons in the turtle spinal cord

Plasticity at the neuronal level commonly involves use-dependent changes in strength of particular synaptic pathways or regulation of postsynaptic properties by modulatory transmitters. Here we analyze a novel form of short-term plasticity mediated by use-dependent facilitation of postsynaptic responsiveness. Using current- and voltage-clamp recordings, we found that all spinal ventral horn neurons able to generate plateau potentials showed depolarization-induced facilitation of the underlying inward current. Facilitation was noticeable when the neurons were depolarized to more than -50 mV at intervals <4 s. When stimulation with fast triangular voltage ramps was used, the inward current activated at a less depolarized potential during the second ramp. The inward current and facilitation was eliminated by nifedipine, a selective antagonist of L-type calcium channels. Depolarization-induced facilitation of low-voltage-activated L-type calcium channels is suggested to be the underlying mechanism. It is noted that facilitation occurs on a time scale compatible with a role in phasic motor activity.     

Inhibition of calcium currents in rat colon sensory neurons by K- but not mu- or delta-opioids

Inhibition of calcium currents in rat colon sensory neurons by kappa- but not mu- or delta-opioids. J. Neurophysiol. 80: 3112-3119, 1998. We previously reported that kappa-, but not mu- or delta-opioid receptor agonists (ORAs) have selective, potentially useful peripheral analgesic effects in visceral pain. To evaluate one potential site and mechanism by which these effects are produced, we studied opioid effects on high-voltage activated (HVA) Ca2+ currents in identified (Di-I) pelvic nerve sensory neurons from the S1 dorsal root ganglion (DRG). Results were compared with opioid effects on cutaneous neurons from L5 or L6 DRG. Di-I-labeled DRG cells were voltage clamped (perforated whole cell patch clamp), and HVA Ca2+ currents were evoked by depolarizing 240-ms test pulses to +10 mV from a holding potential of -60 mV. Neither mu-ORAs (morphine, 10(-6 )M, n = 16; [D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin, 10(-6 )M, n = 12) nor delta-ORAs ([D-Pen2, D-Pen5] enkephalin, 10(-7 )M, n = 16; SNC-80, 10(-7 )M, n = 7) affected HVA Ca2+ currents in colon sensory neurons. In contrast, the kappa-ORAs U50, 488 (10(-6 )M), bremazocine (10(-6)M), and nalBzoH (10(-6 )M) significantly attenuated HVA Ca2+ currents in colon sensory neurons; effects on cutaneous sensory neurons were variable. A nonreceptor selective concentration of naloxone (10(-5 )M) and nor-BNI (10(-6 )M), a selective kappa-opioid receptor antagonist, reversed the inhibitory effect of kappa-ORAs. In the presence of N-, P-, or Q-, but not L-type Ca2+ channel antagonists, the effect of U50,488 on HVA Ca2+ currents was significantly reduced. Pretreatment with pertussis toxin (PTX) prevented the inhibition by U50,488. These results suggest that kappa-opioid receptors are coupled to multiple HVA Ca2+ channels in colon sensory neurons by a PTX-sensitive G protein pathway. We conclude that inhibition of Ca2+ channel function likely contributes in part to the peripheral analgesic action of kappa-ORAs in visceral nociception.     

Electrophysiological characterization of chemosensory neurons from the mouse vomeronasal organ

The mechanism of sensory transduction in chemosensory neurons of the vomeronasal organ (VNO) is not known. Based on molecular data, it is likely to be different from that mediating sensory transduction in the main olfactory system. To begin to understand this system, we have characterized the electrophysiological properties of dissociated mouse VNO neurons with patch-clamp recording. Sensory neurons were distinguished from nonsensory neurons by the presence of a dendrite, by immunoreactivity for olfactory marker protein, and by the firing of action potentials. The resting potential of VNO neurons was approximately -60 mV, and the average input resistance was 3 Gomega. Current injections as small as 1-2 pA elicited steady trains of action potentials that showed no sign of elicited steady trains of action potentials that showed no sign of adaptation during a 2 sec stimulus duration. The voltage-gated conductances in VNO neurons are distinct from those in olfactory neurons. The Na+ current is composed of two components; the major component was TTX-sensitive (Ki = 3.6 nM). The outward K+ current activates at -30 mV with kinetics 10 times slower than for K+ currents in olfactory neurons. The Ca2+ current is composed of at least two components: an L-type current and a T-type current that activates at -60 mV and is not found in olfactory neurons. We find no evidence for cyclic nucleotide-gated channels in VNO neurons under a variety of experimental conditions, including those that produced large responses in mouse olfactory neurons, which is further evidence for a novel transduction pathway.     

Phase I and pharmacologic study of irinotecan in combination with cisplatin for advanced lung cancer

Macroscopic T-type Ca2+ currents, which are often observed in fetal and neonatal cardiac muscle cells, were not found in normal (0 of 17) adult feline ventricular myocytes. However, they were present in most (15 of 21) myocytes isolated from adult feline left ventricles with long-standing pressure-overload-induced hypertrophy. This is the first study to provide evidence in a large mammal, such as the cat, that T-type Ca2+ channels may be reexpressed in adults in association with hypertrophy resulting from slow progressive pressure overload. Importantly, this expression was stable for the duration of the hypertrophy process and was not associated with abrupt pressure overload. T-type Ca2+ currents were separated from L-type Ca2+ currents by exploiting the differences in their voltage dependence of steady-state inactivation. Depolarizations from -80 mV revealed a rapidly activating inward current that peaked in magnitude at -30 mV (-1.8 +/- 0.9 [mean +/- SD] pA/pF) and fully inactivated within 100 milliseconds in 15 of 21 hypertrophied myocytes studied. Further depolarizations activated progressively less T-type Ca2+ current, so that at +10 mV the L-type Ca2+ current predominated. In the hypertrophied myocytes that demonstrated both T-type and L-type Ca2+ currents, two distinct peaks occurred in their current-voltage relations. T-type Ca2+ currents were not evident in any of the 17 normal adult feline left ventricular myocytes studied. The purpose of T-type Ca2+ currents in hypertrophy is unclear. However, their presence may make hypertrophied myocardium more prone to spontaneous action potentials and increase the likelihood for arrhythmias in partially depolarized hypertrophied myocardium.     

Activation of calcium channels by cAMP in STC-1 cells is dependent upon Ca2+ calmodulin-dependent protein kinase II

Activation of L-type calcium channels in the neuroendocrine, cholecytstokinin-secreting cell line, STC-1, is vital for secretion of CCK. In the present study, the regulation of L-type Ca2+ channels by cAMP and Ca2+ calmodulin dependent protein kinase II (CaM-KII) in STC-1 cells was investigated. Exposure to 3-isobutyl-1-methylxanthine (IBMX) increased intracellular cAMP levels, whole cell Ca2+ currents and activated Ca2+ channels in cell-attached membrane patches. Furthermore, in Fura-2AM loaded cells, cytosolic Ca2+ levels increased upon exposure to IBMX. By contrast, pretreatment of cells with the CaM-KII inhibitor KN-62, prevented IBMX activation of Ca2+ channels in cell-attached patches or increases in cytosolic Ca2+ levels. Inclusion of the synthetic peptide fragment 290-309 of CaM-KII, a CaM-KII antagonist, in the pipette solution, blocked the activation of whole cell Ca2+ currents upon addition of IBMX. These results indicate a unique mechanism of L-type Ca2+ channel activation involving two phosphorylation events.     

Characterization of outward currents in neurons of the avian nucleus magnocellularis

Characterization of outward currents in neurons of the avian nucleus magnocellularis. J. Neurophysiol. 80: 2824-2835, 1998. Neurons of the nucleus magnocellularis (NM) preserve the timing of auditory signals through the convergence of a variety of voltage- and ligand-gated ion channels. To understand better how these channels interact, we have characterized the kinetics, voltage sensitivity, and pharmacology of outward currents of NM neurons in brain slices. The reversal potential (Erev) of outward currents varied with potassium concentration as expected for currents carried by potassium. However, Erev was consistently more positive than the Nernst potential for potassium (EK). Deviation of Erev from the calculated EK most likely arose from potassium accumulation in extracellular spaces by potassium conductances active at rest and during depolarizing steps. Three outward potassium currents were studied that varied in voltage and pharmacological sensitivity. A tetraethylammonium (TEA)-sensitive, high-threshold current was activated within 1-5 ms of the onset of depolarization, with a half-maximal activation voltage (V1/2) of -19 mV. It was blocked partially by 4-aminopyridine (4-AP) and was the dominant ionic conductance of NM neurons. A dendrotoxin-I (DTX) and 4-AP-sensitive, low-threshold current had a V1/2 of -58 mV, rapid activation kinetics, and only partial inactivation, with decay time constants between 20 and 100 ms. A rapidly inactivating current was observed that was resistant to TEA and DTX and was blocked by intracellular Cs+. The transient current was inactivated almost completely at the resting potential. The onset of inactivation was fastest at potentials negative to those that caused activation. When intracellular K+ was replaced by Cs+, large inward and outward currents were obtained that corresponded respectively to the above-mentioned DTX- and TEA-sensitive currents. Outward, TEA-sensitive current was carried by Cs+, with a PCs/PK of approximately 0.1. In current-clamped neurons, DTX induced repetitive firing and increased membrane time constant near rest but had little effect on action potential duration. These studies indicate that a low-threshold, DTX-sensitive current plays a key role in making NM neurons highly responsive to the onset and offset of synaptic stimuli.     

A new class of noninactivating K+ channels from aplysia capable of contributing to the resting potential and firing patterns of neurons

From the nervous system of Aplysia, we have cloned a new class of noninactivating K+ channels (aKv5.1) that are activated at low voltage and are capable of contributing to the resting potential and firing patterns of neurons. Expression of aKv5.1 in Aplysia neuron R15 revealed that aKv5.1 exerts an unusual control over cell excitability; it increased the resting potential by more than 20 mV and abolished the spontaneous bursting activity of the cell. In its ability to suppress the endogenous rhythm of R15, aKv5.1 differs in its actions from transient, inactivating K+ channels such as aKv1.1a, an Aplysia homolog of Shaker. aKv1.1a shortens the duration of the spike and increases the afterpotential, but does not suppress bursting. Thus, by expressing different classes of K+ channels, it is possible to redesign, in specific ways, the signaling capabilities of specific, identified neurons.     

Calcium current and inactivation in identified neurons in Hermissenda crassicornis

1. N-type (omega-conotoxin sensitive) calcium currents (ICa) were recorded in identified neurons in Hermissenda crassicornis using low-resistance patch electrodes (0.7 +/- 0.3 M omega; n = 101) under conditions that eliminated inward Na+ currents (choline ions substitution) and suppressed outward K+ currents (Cs+, tetraethylammonium, and 4-AP). Step depolarization from a holding potential of -60 mV to potentials above -30 mV elicited ICa, which peaked approximately 20 mV and declined with increasing depolarizations. 2. Evidence for a low-threshold current was present. Step depolarization from a more hyperpolarizing potentials (e.g., -90 mV) revealed a small shoulder (< 100 pA) at -60 to -40 mV that was sensitive to Co2+ and Ni2+. However, under the conditions examined here (holding potential of -60 mV), the high-voltage-activated current predominated. 3. Barium (Ba2+) and strontium (Sr2+) permeate the Ca2+ channel with similar activation kinetics (ease of permeation; Ba2+ > Ca2+ > Sr2+). Steady-state activation of permeability versus membrane potentials for Ca2+, Ba2+, and Sr2+ as charge carriers could be fitted with the Boltzmann equation, with half-activation voltage and slope factor of 2.9 and 7.7 mV for ICa, -13.1 mV and 7.8 for Ba2+ current (IBa) and -2.3 mV and 7.8 for Sr2+ current (ISr). The time course of activation was monotonic with time constant (tau) for ICa ranging from 2 to 8 ms. 4. The inactivation profile was complex. At negative step potentials (e.g., -20 mV), inactivation of the current was slow. Depolarization steps to relatively positive voltages (e.g., 10 mV) showed more rapid inactivation than those at more positive potentials (e.g., 40 mV). When extracellular Ca2+ was raised from 5 to 10 mM, a biphasic decay (tau fast of 25 +/- 4 ms; and tau slow of 473 +/- 64 ms; mean +/- SD, n = 9) was seen. Such an observation suggested a current-mediated inactivation. 5. With a pulse duration of approximately 350 ms, ISr showed inactivation whereas Ba2+ virtually removed the decay. However, IBa turned off with more prolonged depolarization. 6. A twin-pulse protocol was used to assess the voltage dependence of inactivation: an incomplete U-shaped inactivation curve was observed for ICa, IBa, and ISr. Channels available for inactivation were increased in the presence of Ca2+ ions. 7. Inactivation was further studied with the Ca2+ chelators, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). With 10 mM of BAPTA, in the pipette, inactivation was reduced but not removed.(ABSTRACT TRUNCATED AT 250 WORDS)     

The American Journal of Ophthalmology 1862-1864

A hyperpolarization-activated current (termed I[h]) is believed to provide a pacemaker depolarization in sinoatrial node cells and in some central and peripheral neurons. In the present study, we examined if such an inward cation current exists in primary auditory neurons using the whole-cell patch-clamp technique. A large inward, non-inactivating current was seen during hyperpolarizing steps negative to the resting potential. A depolarizing sag occurred during hyperpolarizing current injection, and upon termination of the current injection there was an overshoot, or a rebound firing. A low concentration of Cs+, but not Ba2+, reversibly blocked the inward current and depolarizing sag. The activation of the current showed voltage dependence with half-activation occurring at -101 +/- 1 mV. The time course of I(h) activation was fitted by double exponential function and was voltage-dependent (time constants: tau1 and tau2 = 480 and 3125 ms at -100 mV, and 66 and 404 ms at -160 mV). The reversal potential of the current was -36 mV measured from tail currents. The conductance of the current was decreased in Na+-free solution, and increased in high K+ solution. Increases in the levels of intracellular cAMP or cGMP enhanced the current. The results suggest that there exists a hyperpolarization-activated inward cation current in mammalian primary auditory neurons. This current may provide a depolarizing current during the membrane hyperpolarization following each firing of the primary auditory nerve.     

Subthalamic nucleus neurons switch from single-spike activity to burst-firing mode

The modification of the discharge pattern of subthalamic nucleus (STN) neurons from single-spike activity to mixed burst-firing mode is one of the characteristics of parkinsonism in rat and primates. However, the mechanism of this process is not yet understood. Intrinsic firing patterns of STN neurons were examined in rat brain slices with intracellular and patch-clamp techniques. Almost half of the STN neurons that spontaneously discharged in the single-spike mode had the intrinsic property of switching to pure or mixed burst-firing mode when the membrane was hyperpolarized from -41.3 +/- 1.0 mV (range, -35 to -50 mV; n = 15) to -51.0 +/- 1.0 mV (range, -42 to -60 mV; n = 20). This switch was greatly facilitated by activation of metabotropic glutamate receptors with 1S,3R-ACPD. Recurrent membrane oscillations underlying burst-firing mode were endogenous and Ca2+-dependent because they were largely reduced by nifedipine (3 microM), Ni2+ (40 microM), and BAPTA-AM (10-50 microM) at any potential tested, whereas TTX (1 microM) had no effect. In contrast, simultaneous application of TEA (1 mM) and apamin (0.2 microM) prolonged burst duration. Moreover, in response to intracellular stimulation at hyperpolarized potentials, a plateau potential with a voltage and ionic basis similar to those of spontaneous bursts was recorded in 82% of the tested STN neurons, all of which displayed a low-threshold Ni2+-sensitive spike. We propose that recurrent membrane oscillations during bursts result from the sequential activation of T/R- and L-type Ca2+ currents, a Ca2+-activated inward current, and Ca2+-activated K+ currents.     

M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons

Modulation of high-voltage-activated Ca2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+ channel current amplitude. The dose-response relationship obtained for ACh-induced inhibition of Ba2+ current (IBa) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of IBa amplitude obtained with 100 microM ACh was approximately 75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (/=30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinic receptor-mediated inhibition. Both dihydropyridine- and omega-conotoxin GVIA-sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes in these neurons. Inhibition of IBa amplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 microM guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-beta-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of IBa by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.     

Inhibition of voltage-dependent calcium channels by prostaglandin E2 in rat melanotrophs

The effects of PGE2 on voltage-dependent Ca2+ channel currents were studied in dissociated rat melanotrophs by the whole-cell configuration of the patch-clamp technique. In about 90% of melanotrophs examined, PGE2 reversibly inhibited voltage-dependent Ba2+ currents elicited by voltage steps from a holding potential of -80 to 0 mV, with an ED50 of 68 nM. The maximum inhibition of Ba2+ currents by 1 microM PGE2 (35.3%) was comparable with that by the maximally effective concentration (100 nM) of dopamine. The EP1/EP3 PGE (EP) agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, mimicked the inhibition by PGE2, whereas the selective EP2 agonist, butaprostol, had little effect. The inhibition by PGE2 was partially, but significantly, reduced by the selective EP1 antagonist, SC-51322. The magnitude of the PGE2-induced inhibition of Ba2+ currents was greatly reduced by pretreatment with pertussis toxin, or by a depolarizing prepulse, to +80 mV, lasting for 50 msec. Although four distinct types (N-, P/Q-, L-, and R-types) of high-threshold Ba2+ currents were observed, PGE2 (1 microM) caused significant inhibition of only P/Q- and L-type currents, which were 17.3 and 10.1%, respectively, of the total Ba2+ currents. These results suggest that PGE2 inhibits P/Q- and L-type Ca2+ channels of rat melanotrophs via EP1 and EP3 receptors, which are coupled to pertussis toxin-sensitive G proteins, and produces both voltage-sensitive and -insensitive inhibition of Ca2+ channels.     

A single non-L-, non-N-type Ca2+ channel in rat insulin-secreting RINm5F cells

Single high-voltage-activated (HVA) Ca2+ channel activity was recorded in rat insulinoma RINm5F cells using cell-attached and outside-out configurations. Single-channel recordings revealed three distinct Ca2+ channel subtypes: one sensitive to dihydropyridines (DHPs)-(L-type), another sensitive to omega -conotoxin (CTx)-GVIA (N-type) and a third type insensitive to DHPs and omega -CTx-GVIA (non-L-, non-N-type). The L-type channel was recorded in most patches between -30 and +30 mV. The channel had pharmacological and biophysical features similar to the L-type channels described in other insulin-secreting cells (mean conductance 21 pS in control conditions and 24 pS in the presence of 5 microM Bay K 8644). The non-L-, non-N-type channel was recorded in cells chronically treated with omega -CTx-GVIA in the presence of nifedipine to avoid the contribution of N- and L-type channels. Channel activity was hardly detectable below -10 mV and was recruited by negative holding potentials (< -90 mV). The channel open probability increased steeply from -10 to + 40 mV. Different unitary current sublevels could be detected and the current voltage relationship was calculated from the higher amplitude level with a slope conductance of 21 pS. Channel activity lasted throughout depolarizations of 300-800ms with little sign of inactivation. Above 0 mV the channel showed a persistent flickering kinetics with brief openings (tau o 0.6 ms) and long bursts (tau burst 60 ms) interrupted by short interburst intervals. The third HVA Ca2+ channel subtype, the N-type, had biophysical properties similar to the non-L-, non-N-type and was best identified in outside-out patches by its sensitivity to omega -CTx-GVIA. The channel was detectable only above -10 mV from a -90 mV holding potential, exhibited a fast flickering behaviour, persisted during prolonged depolarizations and had a slope conductance of about 19 pS. The present data provide direct evidence for a slowly inactivating non-L-, non-N-type channel in insulin-secreting RINm5F cells that activates at more positive voltages than the L-type channel and indicate the possibility of identifying unequivocally single HVA Ca2+ channels in cell-attached and excised membrane patches under controlled pharmacological conditions.