KSAYMRFamide (PF3/AF8) is present in the free-living nematode, Caenorhabditis elegans

The effect of cAMP on a ryanodine-sensitive Ca2+ release from microsomal vesicles of rat parotid acinar cells was studied. After a steady state of ATP-dependent 45Ca2+ uptake into the vesicles, cAMP was added to the medium with thapsigargin (TG) to block a reuptake of 45Ca2+. The addition of cAMP (1.0 mM) with TG released about 10% of the 45Ca2+ that had been taken up. The cAMP-induced 45Ca2+ release was strongly inhibited by pretreatment of the vesicles with 500 microMM ryanodine. Preincubation with cAMP (1 mM) abolished ryanodine (10 microM)-induced 45Ca2+ release. The presence of a specific inhibitor of cAMP-dependent protein kinase (PKA) H-89 (10 microMM) inhibited the cAMP-induced 45Ca2+ release. These results indicate that in rat parotid acinar cells, cAMP can activate a ryanodine-sensitive Ca2+ release mechanism in the endoplasmic reticulum and that this activation is via a PKA-dependent process.     

Ca2+ permeation in cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.     

Clinical challenge. Osteomyelitis of the distal half of the metacarpus with associated involucrum and bone sequestrum formation

Treatment of cultured carrot cells with dibutyryl cAMP or forskolin resulted in the appreciable decrease in extracellular K+ concentration. This decrease was found to be transient and the concentration of the ion in the culture medium restored to the original level within few minutes. The cAMP-induced decrease in K+ level in the medium was almost completely inhibited when carrot cells were incubated in the presence of K+ channel blockers, CsCl and tetraethylammonium chloride. Appreciable amounts of 45Ca2+ were discharged from 45Ca2+-loaded inside-out vesicles of carrot plasma membrane by the stimulation with cAMP, however, the release of the ion was significantly inhibited in the presence of the K+ channel blockers. The release of 45Ca2+ from the vesicles was also observed when K+ current was evoked with an ionophore, valinomycin, even in the absence of cAMP. These results suggest that the gating of some of the inward K+ channels located at plasma membrane of cultured carrot cells is controlled by cytoplasmic concentration of cAMP and the inward K+ current across the plasma membrane induced by the nucleotide elicits Ca2+ influx into the cells possibly by the activation of voltage-dependent Ca2+ channels.     

Ionic mechanisms involved in the spontaneous firing of tegmental pedunculopontine nucleus neurons of the rat

We have previously defined three types of tegmental pedunculopontine nuclei neurons based on their electrophysiological characteristics: Type I neurons characterized by low-threshold Ca2+ spikes, Type II neurons which displayed a transient outward current (A-current), and Type III neurons having neither low-threshold spikes nor A-current [Kang Y. and Kitai S. T. (1990) Brain Res. 535, 79-95]. In this report, ionic mechanisms underlying repetitive firing of Type I (n=15) and Type II (n=69) neurons were studied in in vitro slice preparations. Type I neurons did not fire rhythmically but their spontaneous firing frequency ranged from 0 to 19.5 spikes/s (mean 9.7 spikes/s). The spontaneous firing of Type II neurons was rhythmic, with a mean frequency of 9.6 spikes/s (range 3.5-16.0 spikes/s). Choline acetyltransferase immunohistochemistry combined with biocytin labeling indicated that none of the Type I neurons were immunopositive to choline acetyltransferase, while 60% (42 of 69) of Type II neurons were immunopositive. There was no apparent difference in the electrophysiological membrane properties of immunopositive and immunonegative Type II neurons. At membrane potentials subthreshold for Na+ spikes (-50 mV), spontaneous membrane oscillations (11.6 Hz) were observed: these underlie the spontaneous repetitive firing of Type I neurons. The subthreshold membrane oscillation was tetrodotoxin sensitive but was not affected by Ca2+-free medium. A similar tetrodotoxin-sensitive subthreshold membrane oscillation (10.5 Hz) was also observed in Type II neurons. However, in Type II neurons a membrane oscillation was also observed at higher membrane potentials (-50 mV). This high-threshold oscillation was insensitive to tetrodotoxin and Na+-free medium, but was eliminated in Ca2+-free conditions. The amplitude and frequency of the high-threshold oscillation was increased upon membrane depolarization. At the most prominent oscillatory level (around -40 mV), the high-threshold oscillation had a mean frequency of 8.8 Hz. The high-threshold Ca2+ spike was triggered from the peak potential (-35 to -30mV) of the high-threshold oscillation. Application of tetraethylammonium chloride (< 5 mM) increased the amplitude of the high-threshold oscillation, while nifedipine greatly attenuated the high-threshold oscillation without changing the shape of the high-threshold Ca2+ spike. Application of Cd2+ eliminated both the high-threshold oscillation and the high-threshold Ca2+ spike, and omega-conotoxin reduced the size of the high-threshold Ca2+ spike without affecting the frequency of the high-threshold oscillation. Nickel did not have any effect on either the high-threshold oscillation or the high-threshold Ca2+ spike. These data suggest an involvement of N- and L-type Ca2+ channels in the generation of the high-threshold oscillation and the high-threshold Ca2+ spike, respectively. The results indicate that a persistent Na+ conductance plays a crucial role in the subthreshold membrane oscillation, which underlies spontaneous repetitive firing in Type I neurons. On the other hand, in addition to a persistent Na+ conductance for subthreshold membrane oscillation, a voltage-dependent Ca2+ conductance with Ca2+-dependent K+ conductance (for the high-threshold oscillation) may be responsible for rhythmic firing of Type II neurons.     

Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs

Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs.     

Evidence for contribution by increased cytoplasmic Na+ to the insulinotropic action of PACAP38 in HIT-T15 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized to pancreatic nerve terminals and stimulates insulin secretion. The insulinotropic effect of PACAP38 in insulin-producing HIT-T15 cells is accompanied by increases in cellular cAMP and cytoplasmic Ca2+ ([Ca2+]cyt). As also intracellular Na+ is important for insulin secretion after glucose and other cAMP forming peptides, we examined the Na+ dependence of the insulinotropic effect of PACAP38 in HIT-T15 cells. We found that PACAP38 (100 nM)-induced insulin secretion was diminished by approximately 50% by removal of extracellular Na+ (replaced by equimolar N-methyl-D-glucamine). In contrast, removal of Na+ did not diminish the formation of cellular cAMP (measured by radioimmunoassay) or the increase in [Ca2+]cyt (measured in FURA-2AM-loaded cell suspensions) induced by PACAP38. Furthermore, PACAP-38 increased the cytoplasmic Na+ ([Na+]cyt) in single HIT-T15 cells as measured by the fluorophore sodium-binding benzofran isophthalate. This increase was reduced by removal of extracellular Na+ and by inhibition of protein kinase A by H-89. We conclude that the insulinotropic action of PACAP38 is Na+-dependent. We propose that PACAP38 opens plasma membrane Na+ channels by an action partially mediated by cAMP and protein kinase A, and the subsequent raise in [Na+]cyt elicits insulin secretion by an as yet unsolved mechanism.     

Calmodulin is involved in membrane depolarization-mediated survival of motoneurons by phosphatidylinositol-3 kinase- and MAPK-independent pathways

In the present work, we find that the elevation of extracellular K+ concentration promotes the survival of chick spinal cord motoneurons in vitro deprived of any neurotrophic support. This treatment induces chronic depolarization of the neuronal plasma membrane, which activates L-type voltage-dependent Ca2+ channels, resulting in Ca2+ influx and elevation of the cytosolic free Ca2+ concentration. Pharmacological reduction of intracellular free Ca2+ or withdrawal of extracellular Ca2+ reversed the effects of depolarization on survival. The intracellular Ca2+ response to membrane depolarization developed as an initial peak followed by a sustained increase in intracellular Ca2+ concentration. The depolarizing treatment caused tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) without involving tyrosine kinase receptor activation. The calmodulin antagonist W13 inhibited the survival-promoting effect induced by membrane depolarization but not the tyrosine phosphorylation of MAPK. Moreover, depolarization did not induce phosphatidylinositol-3 kinase (PI-3K) phosphorylation in our cells, and the PI-3K inhibitor wortmannin did not suppress the survival-promoting effect of K+ treatment. These results suggest that calmodulin is involved in calcium-mediated survival of motoneurons through the activation of PI-3K- and MAPK-independent pathways.     

Evidence for the involvement of inositol trisphosphate but not cyclic nucleotides in visual transduction in Hermissenda eye

Although several second messengers are known to be involved in invertebrate photoresponses, the mechanism underlying invertebrate phototransduction remains unclear. In the present study, brief injection of inositol trisphosphate into Hermissenda photoreceptors induced a transient Na+ current followed by burst activity, which accurately reproduced the native photoresponse. Injection of Ca2+ did not induce a significant change in the membrane potential but potentiated the native photoresponse. Injection of a Ca2+ chelator decreased the response amplitude and increased the response latency. Injection of cGMP induced a Ca2+-dependent, transient depolarization with a short latency. cAMP injection evoked Na+-dependent action potentials without a rise in membrane potential. Taken together, these results suggest that phototransduction in Hermissenda is mediated by Na+ channels that are directly activated by inositol trisphosphate without mobilization of cytosolic Ca2+.     

A test of how well the repeatability of courtship predicts its heritability

In mossy fiber synapses of the hippocampal CA3 region, LTP is induced by cAMP and requires the synaptic vesicle protein rab3A. In contrast, CA1-region synapses do not exhibit this type of LTP. We now show that cAMP enhances glutamate release from CA3 but not CA1 synaptosomes by (1) increasing the readily releasable pool as tested by hypertonic sucrose; (2) potentiating release evoked by KCl depolarization, which opens voltage-gated Ca2+ channels; and (3) by enhancing Ca2+ action on the secretory apparatus as monitored by the Ca2+-ionophore ionomycin. In rab3A-deficient synaptosomes, forskolin still enhances KCl- and sucrose-induced glutamate release but not ionomycin-induced release. Our results show that cAMP has multiple actions in mossy fiber synapses, of which only the direct activation of the secretory apparatus requires rab3A and functions in mfLTP.     

Interaction between adrenaline and epidermal growth factor in the control of liver glycogenolysis in mouse

Epidermal growth factor (EGF) stimulates glycogenolysis in mouse liver, but the effect requires concentrations that are only achieved in plasma upon adrenergic stimulation of EGF release from submandibular salivary glands. Thus, we studied the interaction between adrenaline and EGF in liver glycogen metabolism, both in whole animals and in isolated hepatocytes. Adrenaline administered to anesthetized mice stimulated both the endocrine secretion of EGF from submandibular salivary glands and the degradation of glycogen in the liver. In sialoadenalectomized mice, adrenaline administration did not increase plasma EGF concentration. In these animals, the glycogenolytic response to adrenaline was enhanced. The sensitivity of hepatocytes to adrenaline was similar in cells from sialoadenalectomized and sham-operated mice. EGF, added to isolated hepatocytes, reduced the glycogenolytic effect of adrenaline (the maximal effect but not the ED50). Adrenaline stimulated glycogen degradation through both an alpha1-adrenergic mediated Ca2+ increase and a beta-adrenergic-mediated cAMP increase. EGF did not interfere with the rise of cytosolic Ca2+ but decreased the cAMP signal. EGF did not decrease the glycogenolytic effect of phenylephrine or VP (which increased cytosolic Ca2+ but not cAMP), but EGF decreased both the glycogenolytic effect and the cAMP signal generated by glucagon or forskolin. EGF did not interfere with the glycogenolytic effect of CPT-cAMP or bt2-cAMP. The effect of EGF on cAMP was blocked by 3-isobutyl-1-methylxanthine. These results demonstrate that the effect of EGF on the glycogenolytic action of adrenaline involves interference with the generation of the cAMP signal. We suggest that EGF induces such an effect through the activation of a phosphodiesterase.     

Differential effect of halothane and forskolin on platelet cytosolic Ca2+ mobilization and aggregation

BACKGROUND: Previous works have suggested that the impairment of platelet aggregation by halothane was partly related to a stimulation of cyclic adenosine monophosphate (cAMP) production, to an inhibitory effect on Ca2+ signaling, or both. Intracellular Ca2+ measurements therefore were undertaken, first to determine the critical steps in the platelet CaZ+ signaling cascade most likely to be affected by halothane or by an increase in cAMP production, and second to establish if the effect of halothane involves aggregation-related biochemical pathways triggered by an increase in internal Ca2+. METHODS: Human washed platelets were treated with halothane or forskolin for 5 min before application of either platelet-activating factor, thrombin, U46619, or thapsigargin. The cytosolic Ca2+ concentration ([Ca2+]i) was measured with the fluorescent Ca2+ indicator fura-2. Nephelometric measurements were also performed to assay the aggregation process. RESULTS: Our results indicate that pretreating platelets with halothane leads to a partial impairment of the [Ca2+]i increase induced either by U46619, thrombin, or platelet-activating factor, but this had no significant effect on the [Ca2+]i response triggered by thapsigargin. In addition, our results show that halothane inhibits platelet aggregation triggered by U46619, but not by thapsigargin. Conversely, forskolin completely inhibited the [Ca2+]i response to U46619 and thapsigargin and prevented platelet aggregation induced by both agonists. CONCLUSIONS: These results suggest that halothane and cAMP exert their effects on platelet aggregation and Ca2+ signaling through different mechanisms, and that halothane cannot impair platelet aggregation independently of phospholipase C stimulation.     

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.     

Membrane potential regulates inositol 1,4,5-trisphosphate-controlled cytoplasmic Ca2+ oscillations in pituitary gonadotrophs

The influence of membrane potential (Vm) on cytoplasmic calcium ([Ca2+]i) oscillations during the sustained extracellular Ca(2+)-dependent phase of the Ca2+ signaling response to gonadotropin-releasing hormone (GnRH) was analyzed in cultured pituitary gonadotrophs. In agonist- and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-stimulated cells, sustained [Ca2+]i oscillations were extinguished by hyperpolarization after 3-15 min despite the availability of Ca2+ in the extracellular medium. Single depolarizing pulses transiently restored the amplitude of the sustained spiking in a dihydropyridine- and extracellular Ca(2+)-sensitive manner. The responses to depolarization showed a marked dependence on Vm that was correlated with the steady-state inward Ca2+ current. In addition, repetitive application of brief depolarizing pulses modulated the frequency of agonist- and Ins(1,4,5)P3-controlled spiking; depolarization pulses at frequencies lower than the intrinsic rate of episodic Ca2+ release triggered large transients between the autonomous spikes, whereas higher frequencies of depolarizing pulses overcame the original Ca2+ spiking frequency. These extrinsically driven and extracellular Ca(2+)-dependent oscillations were sensitive to the Ca(2+)-ATPase blocker, thapsigargin, but not to ryanodine. On the other hand, spontaneous firing and application of depolarizing pulses to nonstimulated cells failed to induce thapsigargin-sensitive oscillations. These findings demonstrate that the pattern of Ca2+ signaling in gonadotrophs does not depend exclusively on the Ins(1,4,5)P3 concentration, but also on the excitable status of the cell. Such modulation of the Ins(1,4,5)P3-controlled Ca2+ signaling system by changes in Vm could provide a mechanism for the integration of multiple inputs that utilize diverse signal transduction pathways.     

Endoscopic features of the columnar-lined esophagus

Electrophysiological characterization of neurons within the rat subiculum was carried out with intracellular recordings in an in vitro slice preparation. Subicular neurons responded to threshold pulses of depolarizing current delivered at a resting membrane potential (RMP) of 45.7+/-5.8 mV (mean+/-SD, n=85) with an initial burst of three to five fast action potentials that rode on a depolarizing envelope and was terminated by an afterhyperpolarization (burst AHP) (duration 113+/-35 ms; peak amplitude 2.7+/-0.6 mV, n=10). Tonic firing replaced the bursting mode at membrane potential less negative than -55 mV. Suprathreshold depolarizing pulses evoked at RMP both an initial burst and successive tonic firing. Intracellular staining with biocytin showed morphological features typical of pyramidal cells (n=8). The relationship between frequency of repetitive firing and injected current (f-I) revealed that the burst firing frequency (250-300 Hz) was only slightly influenced by the amount of injected current. By contrast, the f-I curve of the tonic firing phase depended upon current intensity: it displayed an initial segment that increased at first linearly and then turned into a plateau for both the early and the late inter-spike intervals. The frequency of the tonic firing declined only slightly with time, thus suggesting a lack of adaptation. During tonic firing, each single action potential was followed by a fast AHP and a depolarizing afterpotential. Termination of repetitive firing was followed by an AHP (spike-train AHP; duration 223+/-101 ms, peak amplitude 5.6+/-2.4 mV, n=17). Fast spike-train and burst AHPs were reduced by bath application of the Ca2+-channel blockers Co2+ (2 mM) and Cd2+ (1 mM) (n=8), thus suggesting the participation of Ca2+-dependent K+ conductances in these AHPs. Subicular bursting neurons generated persistent, subthreshold voltage oscillations at 5.3+/-1 Hz (n=20) during steady depolarization positive to -60 mV; at values positive to -55 mV, the oscillatory activity could trigger clusters of single action potentials with a periodicity of 0.9-2 Hz. Oscillations were not prevented by application of excitatory amino acid receptor and GABA(A) receptor antagonists (n=5), Ca2+-channel blockers (n=5), or Cs+ (3 mM; n=4), but were abolished by the Na+-channel blocker tetrodotoxin (1 microM; n=6). Our findings demonstrate that pyramidal-like subicular neurons generate both bursting and non-adapting tonic firing, depending upon their membrane potential. These neurons also display oscillatory activity in the range of theta frequency that depends on the activation of a voltage-gated Na+ conductance. These electrophysiological properties may play a role in the process of signals arising from the hippocampal formation before being funnelled towards other limbic structures.     

Differential regulation of N- and Q-type Ca2+ channels by cyclic nucleotides and G-proteins

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway delimited to the membrane. In addition, modulation of Ca2+ channel activity by protein kinases also contributes to the dynamic regulation of neuronal physiology. Recently, differences in these modulations between Ca2+ channel subtypes have been shown in several neuronal preparations. Here we show that two types of presynaptic Ca2+ channel (N-type and Q-type) are differentially regulated by cAMP and G-proteins using a Xenopus oocyte expression system. Treatment to increase cytosolic cAMP concentration with forskolin and 3-isobutyl-1-methylxanthine (IBMX) markedly potentiated Q-type channel current, and the enhancement was reversed by protein kinase A inhibitors. Much smaller enhancement was observed in N-type channel current after the cAMP elevation. When large depolarizing prepulse was applied to the oocytes for evaluation of the tonic inhibition of Ca2+ channels by intrinsic G-protein activity, N-type channel current elicited a large prepulse facilitation but Q-type channels did not. The tonic inhibition of N-type channels was abolished by an intracellular perfusion with a 'cut-open' recording configuration, or by co-expression with G(alpha o). When kappa opioid receptors were co-expressed and stimulated with agonists, depolarization-resistant inhibition was more apparent in Q-type channels than in N-type channels. These results suggest that Q-type channels are more susceptible to the protein kinase A-mediated facilitation than N-type channels, and that activity of N-type channels can be more highly regulated in a voltage-dependent manner by G(betagamma) than that of Q-type channels. These differences may account for the selective regulation of neurotransmitter release by these Ca2+ channels.     

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.     

Modulation of bursts and high-threshold calcium spikes in neurons of rat auditory thalamus

Neurons in the ventral partition of the medial geniculate body are able to fire high-threshold Ca2+-spikes. The neurons normally discharge such spikes on low-threshold Ca2+-spikes after the action potentials of a burst. We studied membrane mechanisms that regulate the discharge of high-threshold Ca2+-spikes, using whole-cell recording techniques in a slice preparation of rat thalamus. A subthreshold (persistent) Na+-conductance amplified depolarizing inputs, enhancing membrane excitability in the tonic firing mode and amplifying the low-threshold Ca2+-spike in the burst firing mode. Application of tetrodotoxin blocked the amplification and high-threshold Ca2+-spike firing. A slowly inactivating K+ conductance, sensitive to blockade with 4-aminopyridine (50-100 microM), but not tetraethylammonium (2-10 mM), appeared to suppress excitability and high-threshold Ca2+-spike firing. Application of 4-aminopyridine increased the low-threshold Ca2+-spike and the number of action potentials in the burst, and led to a conversion of the superimposed high-threshold Ca2+-spike into a plateau potential. Application of the Ca2+-channel blocker Cd2+ (50 microM), reduced or eliminated this plateau potential. The tetrodotoxin sensitive, persistent Na+-conductance also sustained plateau potentials, triggered after 4-aminopyridine application on depolarization by current pulses. Our results suggest that high-threshold Ca2+-spike firing, and a short-term influx of Ca2+, are regulated by a balance of voltage-dependent conductances. Normally, a slowly inactivating A-type K+-conductance may reduce high-threshold Ca2+-spike firing and shorten high-threshold Ca2+-spike duration. A persistent Na+-conductance promotes coupling of the low-threshold Ca2+-spike to a high-threshold Ca2+-spike. Thus, the activation of both voltage-dependent conductances would affect Ca2+ influx into ventral medial geniculate neurons. This would alter the quality of the different signals transmitted in the thalamocortical system during wakefulness, sleep and pathological states.     

Functional Ca2+ and Na+ channels on mouse Schwann cells cultured in serum-free medium: regulation by a diffusible factor from neurons and by cAMP

Regulation of expression of functional voltage-gated ion channels for inward currents was studied in Schwann cells in organotypic cultures of dorsal root ganglia from E19 mouse embryos maintained in serum-free medium. Of the Schwann cells that did not contact axons, 46.5% expressed T-type Ca2+ conductances (ICaT). Two days or more after excision of the ganglia, and consequent disappearance of neurites, ICaT were detectable in only 10.9% of the cells, and the marker 04 disappeared. On Schwann cells deprived of neurons, T- (but not L-) type Ca2+ conductances were re-induced by weakly hydrolysable analogues of cAMP, and by forskolin (an activator of adenylyl cyclase) after long-term treatment (4 days). With CPT cAMP (0.1-2 mM), 8Br cAMP, db cAMP or forskolin (0.01 or 0.1 mM), the proportion of cells with ICaT was not significantly different from the proportion in the cultures with neurons. These agents also induced expression in some cells of tetrodotoxin-resistant Na+ currents, which were rarely induced by neurons, but 04 was not re-induced by cAMP analogue treatments that re-induced ICaT. Inward currents (Ba2+ or Na+) were partly restored (P < 0.05) on Schwann cells cultured for 6-7 days beneath a filter bearing cultured neurons. In contrast, addition of neuron-conditioned medium was ineffective. The results suggest that neurons activate, via diffusible and degradable factors, a subset of Schwann cell cAMP pathways leading to expression of IcaT, and activate additional non-cAMP pathways that lead to expression of 04.     

Synaptically activated low-threshold muscarinic inward current sustains tonic firing in rabbit prevertebral sympathetic neurons

Whole-cell patch-clamp experiments were performed on non-dissociated rabbit coeliac sympathetic neurons in the presence of nicotinic blockers. Coeliac neurons were classified as either silent or spontaneously active (pacemaker) cells. Under voltage-clamp conditions, pacemaker cells exhibited a steady-state N-shaped current-voltage relationship due to the presence of a persistent voltage-dependent inward current in the potential range of -100 to approximately -20 mV. This inward current sustained the regular firing activity of pacemaker cells and was absent from quiescent neurons. It disappeared in the presence of tetrodotoxin and in low Ca(2+)-high Mg2+ external solutions and was enhanced by eserine. Splanchnic nerve stimulation induced slow regenerative depolarizations and firing discharges in silent neurons by activating a low-threshold voltage-sensitive inward current. The synaptic current had a U-shaped voltage-dependence from -96 to approximately -20 mV and exhibited the dynamic properties of the muscarinic voltage-dependent inward current INa,M. It gave the current-voltage relationship an N shape similar to that observed in spontaneously active cells. The muscarinic antagonists atropine and pirenzepine abolished the inward current present in pacemaker cells and that induced by nerve stimulation in silent neurons. These data provide evidence that both spontaneous firing activity and nerve-evoked depolarizing responses in coeliac neurons are sustained by the activation of the muscarinic Na,M current. The tonic activation of INa,M in spontaneously firing cells results from a sustained Ca(2+)-dependent tetrodotoxin-sensitive release of acetylcholine. This study provides evidence that the role of the muscarinic receptors is not purely a neuromodulatory one, but that these receptors are directly involved in ganglionic neurotransmission.