Two different G-proteins mediate neuropeptide Y and bradykinin-stimulated phospholipid breakdown in cultured rat sensory neurons

We have previously demonstrated that neuropeptide Y (NPY) inhibits voltage sensitive Ca2+ channels in rat dorsal root ganglion neurons and that this effect is mediated by a pertussis toxin-sensitive, guanyl nucleotide-binding protein (G-protein). We now demonstrate that NPY can also stimulate the synthesis of inositol trisphosphate (InsP3) and diacylglycerol in dorsal root ganglion neurons. The effects of NPY were compared with those of bradykinin (BK) which also stimulates phosphoinositide turnover in these cells. NPY-stimulated InsP3 synthesis could be completely blocked by treatment with pertussis toxin and significantly enhanced by cholera toxin although not by other agents which raised cellular concentrations of cyclic AMP. In contrast, the effects of BK were completely unaltered by either toxin. Furthermore the maximal effects of BK and NPY were additive. In spite of the lack of toxin effects, stimulation of InsP3 synthesis produced by BK was clearly mediated by a G-protein. Thus BK stimulated InsP3 production in digitonin-permeabilized neurons, and these effects were enhanced by guanosine 5'-O-(3-thiotriphosphate) and blocked by guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of both NPY and BK were also blocked by treatment of neurons with phorbol esters. Fura-2-based microfluorimetry of single dorsal root ganglion neurons demonstrated that both BK and NPY increased cytoplasmic-free Ca2+ concentration and that both peptides could produce this effect in the same neuron. Both agents could still increase cytoplasmic-free Ca2+ concentration in Ca2+-free medium indicating that the source of the Ca2+ was an intracellular store. Thus, both NPY and BK can activate InsP3 synthesis in the same cell but seem to utilize different G-proteins. NPY utilizes a pertussis toxin-sensitive G-protein and BK a toxin-insensitive one.     

Cloning and functional expression of novel N-type Ca(2+) channel variants

Voltage-dependent Ca(2+) channels are structurally and functionally diverse. As Ca(2+) currents recorded from embryonic chick dorsal root ganglion (DRG) neurons differ significantly from their mammalian counterparts, information on the primary sequence of the chick channels will help define the structural underpinnings of Ca(2+) channel function. Here, we report the cloning and functional expression of full-length Ca(2+) channel alpha(1B) subunit cDNAs derived from chick DRGs. Two variable regions (A and B) have been identified in the cytoplasmic linker between repeats I and II; a third (C) in the carboxyl terminus extends the open reading frame by 525 nucleotides. The A and C inserts are absent, and the B insert is present in all other class B clones reported to date. The unique shorter channels appear to predominate in DRG neurons. Results represent a requisite first step in defining the structural elements that underlie variations in function and modulation of Ca(2+) channels.     

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.     

The Janus-faced atracotoxins are specific blockers of invertebrate K(Ca) channels

The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca(2+)-activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked K(Ca) channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nm, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+)-activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC(50) approximately 10 mum) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.     

17Beta-estradiol inhibits high-voltage-activated calcium channel currents in rat sensory neurons via a non-genomic mechanism

There is increasing evidence that estrogen influences electrical activity of neurons via stimulation of membrane receptors. Although the presence of intracellular estrogen receptors and their responsiveness in dorsal root ganglion (DRG) primary sensory neurons were reported, rapid electrical responses of estrogen in DRG neurons have not been reported yet. Therefore the current study was initiated to examine the rapid effects of estrogen on Ca2+ channels and to determine its detailed mechanism in female rat DRG neurons using whole-cell patch-clamp recordings. Application of 17beta-estradiol (1 microM) caused a rapid inhibition on high-voltage-activated (HVA)-, but not on low-voltage-activated (LVA)-Ca2+ currents. This rapid estrogen-mediated inhibition was reproducible and dose-dependent. This effect was also sex- and stereo-specific; it was greater in cells isolated from intact female rats and was more effective than that of 17alpha-estradiol, the stereoisomer of the endogenous 17alpha-estradiol. In addition, ovariectomy reduced the inhibition significantly but this effect was restored by administration of estrogen in ovariectomized subjects. Occlusion experiments using selective blockers revealed 17beta-estradiol mainly targeted on both L- and N-type Ca2+ currents. Overnight treatment of cells with pertussis toxin profoundly reduced 17beta-estradiol-mediated inhibition of the currents. On the other hand, estradiol conjugated to bovine serum albumin (EST-BSA) produced a similar extent of inhibition as 17beta-estradiol did. These results suggest that 17beta-estradiol can modulate L- and N-type HVA Ca2+ channels in rat DRG neurons via activation of pertussis toxin-sensitive G-protein(s) and non-genomic pathways. It is likely that such effects are important in estrogen-mediated modulation of sensory functions at peripheral level.     

The cross channel activities of spider neurotoxin huwentoxin-I on rat dorsal root ganglion neurons

In this paper, we investigated the action of huwentoxin-I (HWTX-I) purified from the venom of the Chinese bird spider Ornithoctonus huwena on Ca(2+), Na(+) channels of adult rat dorsal root ganglion (DRG) neurons. The results showed that huwentoxin-I could reduce the peak currents of N-type Ca(2+) channels (IC(50) approximately 100 nM) and TTX-S Na(+) channels (IC(50) approximately 55 nM), whereas no effect was detected on TTX-R Na(+) channels. The comparative studies indicated that the selectivity of HWTX-I on Ca(2+) channels was higher that of MVIIA and approximately the same as that of GVIA. HWTX-I is the first discovered toxin with the cross channel activities from the spider O. huwena venom similar to micro O-conotoxins MrVIA and MrVIB.     

Nitric oxide inhibits nociceptive transmission by differentially regulating glutamate and glycine release to spinal dorsal horn neurons

Nitric oxide (NO) is involved in many physiological functions, but its role in pain signaling remains uncertain. Surprisingly, little is known about how endogenous NO affects excitatory and inhibitory synaptic transmission at the spinal level. Here we determined how NO affects excitatory and inhibitory synaptic inputs to dorsal horn neurons using whole-cell recordings in rat spinal cord slices. The NO precursor L-arginine or the NO donor SNAP significantly increased the frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (IPSCs) of lamina II neurons. However, neither L-arginine nor SNAP had any effect on GABAergic IPSCs. L-arginine and SNAP significantly reduced the amplitude of monosynaptic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root with an increase in paired-pulse ratio. Inhibition of the soluble guanylyl cyclase abolished the effect of L-arginine on glycinergic IPSCs but not on evoked monosynaptic EPSCs. Also, inhibition of protein kinase G blocked the increase in glycinergic sIPSCs by the cGMP analog 8-bromo-cGMP. The inhibitory effects of L-arginine on evoked EPSCs and high voltage-activated Ca(2+) channels expressed in HEK293 cells and dorsal root ganglion neurons were abolished by blocking the S-nitrosylation reaction with N-ethylmaleimide. Intrathecal injection of L-arginine and SNAP significantly increased mechanical nociceptive thresholds. Our findings suggest that spinal endogenous NO enhances inhibitory glycinergic input to dorsal horn neurons through sGC-cGMP-protein kinase G. Furthermore, NO reduces glutamate release from primary afferent terminals through S-nitrosylation of voltage-activated Ca(2+) channels. Both of these actions probably contribute to inhibition of nociceptive transmission by NO at the spinal level.     

Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons

Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.     

Fast-deactivating calcium channels in chick sensory neurons

Whole-cell Ca and Ba currents were studied in chick dorsal root ganglion (DRG) cells kept 6-10 in culture. Voltage steps with a 15-microseconds rise time were imposed on the membrane using an improved patch-clamp circuit. Changes in membrane current could be measured 30 microseconds after the initiation of the test pulse. Currents through Ca channels were recorded under conditions that eliminate Na and K currents. Tail currents, associated with Ca channel closing, decayed in two distinct phases that were very well fitted by the sum of two exponentials. The time constants tau f and tau s were near 160 microseconds and 1.5 ms at -80 mV, 20 degrees C. The tail current components, called FD and SD (fast-deactivating and slowly deactivating), are Ca channel currents. They were greatly reduced when Mg2+ replaced all other divalent cations in the bath. The SD component inactivated almost completely as the test pulse duration was increased to 100 ms. It was suppressed when the cell was held at membrane potentials positive to -50 mV and was blocked by 100-200 microM Ni2+. This behavior indicates that the SD component was due to the closing of the low-voltage-activated (LVA) Ca channels previously described in this preparation. The FD component was fully activated with 10-ms test pulses to +20 mV at 20 degrees C, and inactivated to approximately 30% during 500-ms test pulses. It was reduced in amplitude by holding at -40 mV, but was only slightly reduced by micromolar concentrations of Ni2+. Replacement of Ca2+ with Ba2+ increased the FD tail current amplitudes by a factor of approximately 1.5. The deactivation kinetics did not change (a) as channels inactivated during progressively longer pulses or (b) when the degree of activation was varied. Further, tau f was affected neither by changing the holding potential nor by varying the test pulse amplitude. Lowering the temperature from 20 to 10 degrees C decreased tau f by a factor of 2.5. In all cases, the FD component was very well fitted by a single exponential. There was no indication of an additional tail component of significant size. Our findings indicate that the FD component is due to closing of a single class of Ca channels that coexist with the LVA Ca channel type in chick DRG neurons.     

Regulators of G protein signaling proteins as determinants of the rate of desensitization of presynaptic calcium channels.

Norepinephrine inhibits omega-conotoxin GVIA-sensitive presynaptic Ca2+ channels in chick dorsal root ganglion neurons through two pathways, one mediated by Go and the other by Gi. These pathways desensitize at different rates. We have found that recombinant Galpha interacting protein (GAIP) and regulators of G protein signaling (RGS)4 selectively accelerate the rate of desensitization of Go- and Gi-mediated pathways, respectively. Blockade of endogenous RGS proteins using antibodies raised against Galpha interacting protein and RGS4 slows the rate of desensitization of these pathways in a selective manner. These results demonstrate that different RGS proteins may interact with Gi and Go selectively, giving rise to distinct time courses of transmitter-mediated effects.     

Isolation and characterization of two toxins from the Mexican scorpion Centruroides limpidus limpidus Karsch

1. Several toxic polypeptides were found in the venom of the scorpion Centruroides limpidus limpidus. Comparative studies of the potency of the venom in different strains of mice were conducted. 2. A new type of toxin (component II.9), specific for crustaceans (crayfish and isopods), was isolated from this scorpion and was shown to have the following N-terminal amino acid sequence: Lys-Lys-Asp-Gly-Tyr-Leu-Val-Asn-Lys-Tyr-Thr-Gly-Cys-Lys-Val-Asn-Cys- Tyr-Lys-Leu-Gly-Glu-Asn-Lys-Phe-Cys-Asn-Arg-Glu-. 3. A polypeptide toxic to mice (component II.6) from this venom was shown to have the following N-terminal sequence: Lys-Glu-Gly-Tyr-Leu-Val-Asn-His-Ser-Thr-Gly-Cys-Lys-Tyr- Glu-Cys-Tyr-Lys-Leu-Gly-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-. 4. In cultured chick dorsal root ganglion cells, 1 microM of toxin II.6 was shown to reduce the size of sodium currents and to slow-down their activation-inactivation kinetics. The toxin had also a depressive action on the classical Ca2+ current activated at high membrane potentials (greater than 0 mV).     

Differential G protein-mediated coupling of neurotransmitter receptors to Ca2+ channels in rat dorsal root ganglion neurons in vitro

The peptides neuropeptide Y (NPY) and bradykinin (BK) both inhibited Ca2+ currents in rat dorsal root ganglion neurons (DRG) in vitro. The effects of both peptides were completely blocked by treatment of cells with pertussis toxin. Based on antigenic determinants, DRG cells contained at least two pertussis toxin substrates, alpha o (Mr, 39 kd) and alpha i2 (Mr, 40 kd). We examined the ability of three purified bovine alpha subunits (identified with antibodies as alpha o, alpha i1, and alpha i2) to reconstitute the inhibitory effects of NPY and BK. Reconstitution of NPY effects occurred according to the potency series alpha o greater than alpha i1 much greater than alpha i2. However, in the case of BK all three G proteins were approximately equally effective. Whereas complete reconstitution of NPY effects could be obtained with alpha o, no single alpha subunit produced complete reconstitution of BK. Combinations of alpha o and alpha i2, however, were able to completely reconstitute the effects of BK. Thus several G proteins can effect the regulation of Ca2+ channels in these cells. However, neurotransmitters may be selective in the G proteins or combinations of G proteins utilized to achieve this regulation.     

Elevated potassium shortens action potential duration by altering outward currents in chick dorsal root ganglia neurons

Dissociated embryonic chick dorsal root ganglion (DRG) neurons maintained in culture exhibit a mixed Na+/Ca2+ action potential. The characteristic "shoulder" on the repolarizing phase is due to the relatively prolonged inward Ca2+ current. DRG neurons grown in an elevated K+ medium (25 versus. 5 mM) lack the plateau phase of the action potential. Voltage-clamp analysis showed that this plastic change in action potential duration is not due to the loss of the inward Ca2+ current but is partly due to the appearance of a Ca2(+)-dependent, 4-aminopyridine-(4-AP)-sensitive transient outward current. Faster activation of the purely voltage-dependent delayed rectifier outward current also contributes to the rapid repolarization observed in neurons cultured in elevated K+ medium.     

Noncanonical signaling by ionotropic kainate receptors

The potent neurotoxin kainate activates ion channel-forming receptors. However, it can also activate a G protein-coupled signaling pathway to inhibit transmitter release in central neurons. It remains unclear whether the same receptor complex is involved in both signaling activities. Here we show that in a population of dorsal root ganglion cells, exposure to kainate elicits a G protein-dependent increase in intracellular Ca2+. Furthermore, in these cells a brief exposure to kainate inhibited the K+-induced Ca2+ increase, a process that was sensitive to the G protein inhibitor Pertussis toxin and inhibitors of protein kinase C. This metabotropic action did not require ion channel activity and was not observed in neurons prepared from mice deficient for the ion channel-forming subunit GluR5. These results indicate that GluR5, an ion channel-forming subunit, signals through a second messenger cascade, inhibiting voltage-dependent Ca2+ channels. Thus, such a system represents a noncanonical signaling route of ion channel-forming receptors.     

Two high voltage-activated calcium currents are present in isolation in adult rat spinal neurons

In neurons enzymatically isolated from adult rat dorsal root ganglia and used during the following 24 hours, the Ca2+ currents were investigated with the whole-cell patch-clamp technique. In contrast to the neonatal neurons, the salient feature of these adult neurons is the well separated (in the voltage-range) activation and inactivation properties of each recorded current. The low-threshold T-, the high-threshold inactivating N-, and the long-lasting L-currents have a threshold for activation at -60, -45 and -10 mV, and a 50% inactivation at -75, -45 and -5 mV respectively. The N and L currents were poorly affected by 100 microM Ni, a known blocker of T channels and completely blocked by 100 microM Cd2+. Frequently we could find neurons with only one type of current present. We conclude that adult sensory neurons are a better preparation for studying, in isolation, the physiological relevance of the three types of Ca2+ channels.     

Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons.

Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on alpha-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.     

Behavioural and morphological evidence for the involvement of glial cell activation in delta opioid receptor function: implications for the development of opioid tolerance

The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated currents. Blockade and dye uptake is voltage dependent and regulated by external Ca²⁺. The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them.     

Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons

The major component of whole-cell Ca2+ current in differentiated, neuron-like rat pheochromocytoma (PC12) cells and sympathetic neurons is carried by dihydropyridine-insensitive, high-threshold-activated N-type Ca2+ channels. We show that these channels have unitary properties distinct from those of previously described Ca2+ channels and contribute both slowly inactivating and large sustained components of whole-cell current. The N-type Ca2+ currents are modulated by GTP binding proteins. The snail toxin omega-conotoxin reveals two pharmacological components of N-type currents, one blocked irreversibly and one inhibited reversibly. Contrary to previous reports, neuronal L-type channels are insensitive to omega-conotoxin. N-type Ca2+ channels appear to be specific for neuronal cells, since their functional expression is greatly enhanced by nerve growth factor.