Cultured Rat Superior Cervical Ganglion Research Articles

Nerve Growth Factor Sensitizes Superior Cervical Ganglion Neurons to Bradykinin

The sympathetic nervous system plays a role during chronic inflammation. Since levels of nerve growth factor (NGF) are high in inflamed tissues and, in sensory neurons, NGF controls nociception by regulating neuronal sensitivity to noxious stimuli, we hypothesize that NGF also sensitizes sympathetic neurons to bradykinin (BK), a proinflammatory peptide. We cultured rat superior cervical ganglion (SCG) neurons in the presence or absence of NGF. To assess the response of SCG neurons to Bk, we measured cytoplasmic Ca2+ elevations induced by an acute application of Bk. We found Ca2+ elevation to be increased 3-fold in the presence of NGF. These Ca2+ elevations in the presence of NGF depended on external Ca2+ and depolarization of the plasma membrane. Next, we assessed the effect of Bk on the membrane potential by electrophysiological recordings. NGF-treated neurons were significantly depolarized after Bk application, while neurons cultured in the absence of NGF showed no change in membrane potential with Bk. How does NGF render SCG neurons more responsive to a Bk stimulus? We first tested whether NGF application alters BK-induced M current inhibition. Interestingly, we observed full inhibition of M current upon application of BK with and without culture in NGF. Next, we asked whether incubation of SCG neurons with NGF alters activity of other ion channels. One of the ion channels tested, KCa1.1 (Maxi K+ channel), was reduced in current density by 50% in neurons cultured in the presence of NGF. In addition, inhibition of KCa1.1 channels with iberiotoxin in SCG neurons cultured in the absence of NGF depolarized the plasma membrane potential after Bk application. In conclusion, NGF increased Bk-induced electrical excitability of rat SCG neurons by reducing a repolarizing current mediated by KCa1.1 channels. Supported by NIH grant NS008174.

Memantine Inhibits α3β2-nAChRs-Mediated Nitrergic Neurogenic Vasodilation in Porcine Basilar Arteries

Memantine, an NMDA receptor antagonist used for treatment of Alzheimer’s disease (AD), is known to block the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS). In the present study, we examined by wire myography if memantine inhibited α3β2-nAChRs located on cerebral perivascular sympathetic nerve terminals originating in the superior cervical ganglion (SCG), thus, leading to inhibition of nicotine-induced nitrergic neurogenic dilation of isolated porcine basilar arteries. Memantine concentration-dependently blocked nicotine-induced neurogenic dilation of endothelium-denuded basilar arteries without affecting that induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, memantine significantly inhibited nicotine-elicited inward currents in Xenopous oocytes expressing α3β2-, α7- or α4β2-nAChR, and nicotine-induced calcium influx in cultured rat SCG neurons. These results suggest that memantine is a non-specific antagonist for nAChR. By directly inhibiting α3β2-nAChRs located on the sympathetic nerve terminals, memantine blocks nicotine-induced neurogenic vasodilation of the porcine basilar arteries. This effect of memantine is expected to reduce the blood supply to the brain stem and possibly other brain regions, thus, decreasing its clinical efficacy in the treatment of Alzheimer’s disease.

Antihistamine mepyramine directly inhibits KCNQ/M channel and depolarizes rat superior cervical ganglion neurons

The first-generation antihistamines are widely prescribed medications that relieve allergic reactions and urticaria by blocking the peripheral histamine H 1 receptor. Overdose of these drugs often results in serious neuronal toxic effects, including seizures, convulsions and worsening of epileptic symptoms. The KCNQ/M K + channel plays a crucial role in controlling neuron excitability. Here, we demonstrate that mepyramine and diphenhydramine, two structurally related first-generation antihistamines, can act as potent KCNQ/M channel blockers. Extracellular application of these drugs quickly and reversibly reduced KCNQ2/Q3 currents heterologously expressed in HEK293 cells. The current inhibition was concentration and voltage dependent. The estimated IC 50 (12.5 and 48.1 μM, respectively) is within the range of drug concentrations detected in poisoned patients (30–300 μM). Both drugs shifted the I–V curve of KCNQ2/Q3 channel to more depolarized potentials and altered channel gating properties by prolonging activation and shortening deactivation kinetics. Mepyramine also inhibited the individual homomeric KCNQ1–4 and heteromeric KCNQ3/Q5 currents. Moreover, mepyramine inhibited KCNQ2/Q3 current in an outside-out patch excised from HEK293 cells and the inhibitory effect was neither observed when it was applied intracellularly nor affected by blocking phospholipase C (PLC) activity, indicating an extracellular and direct channel blocking mechanism. Finally, in cultured rat superior cervical ganglion (SCG) neurons, mepyramine reduced the M type K + current in a concentration-dependent manner and led to marked membrane potential depolarization. It is likely that these effects may be involved in the adverse neuroexcitatory effects observed in patients experiencing an overdose of antihistamines.

SAD: A Presynaptic Kinase Associated with Synaptic Vesicles and the Active Zone Cytomatrix that Regulates Neurotransmitter Release

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.

Choline induces Ca 2+ entry in cultured sympathetic neurones isolated from rat superior cervical ganglion

Choline has been shown to be a specific agonist at α7 nicotinic acetylcholine receptors, which are the most Ca 2+ permeable of the ionotropic receptor channels. Whole-cell patch recording combined with the measurement of intracellular free Ca 2+ concentration ([Ca 2+] i , using Indo1, in cultured rat superior cervical ganglion neurones demonstrated that application of choline induced a slowly desensitizing inward current and increased [Ca 2+] i . The effect was dose dependent with an EC 50 of 1.6 mM and an n H of 1.19. The relationship between the elevation of [Ca 2+] i (Δ[Ca 2+] i ) and charge transfer analysed under various recording conditions showed that the Δ[Ca 2+] i induced by choline resulted from an influx of Ca 2+ through nicotinic acetylcholine receptors. The effect of choline on the membrane current and Δ[Ca 2+] i was not affected by either short application or pretreatment with α-bungarotoxin (50 nM) and methyllycaconitine (1 nM), two α7 nicotinic receptors antagonists. These results indicate that activation of non-α7 nicotinic acetylcholine receptors by choline significantly increases the Ca 2+ concentration in rat superior cervical ganglion neurones.

Organic cation transporter mRNA and function in the rat superior cervical ganglion.

Reuptake of extracellular noradrenaline (NA) into superior cervical ganglion (SCG) neurones is mediated by means of the noradrenaline transporter (NAT, uptake 1). We now demonstrate by single-cell RT-PCR that mRNA of the organic cation transporter 3 (OCT3, uptake 2) occurs in rat SCG neurones as well. Furthermore, our RT-PCR analyses reveal the presence of mRNA for novel organic cation transporters 1 and 2 (OCTN1 and OCTN2), but not for OCT1 or OCT2 in the ganglion. Making use of the NAT as a powerful, neurone-specific transporter system, we loaded[3H]-N-methyl-4-phenylpyridinium ([3H]-MPP+) into cultured rat SCG neurones. The ensuing radioactive outflow from these cultures was enhanced by desipramine and reserpine, but reduced (in the presence of desipramine) by the OCT3 inhibitors cyanine 863, oestradiol and corticosterone. In contrast, cyanine 863 enhanced the radioactive outflow from cultures preloaded with [3H]-NA. Two observations suggest that a depletion of storage vesicles by cyanine 863 accounts for the latter phenomenon: first, the primary radioactive product isolated from supernatants of cultures loaded with [3H]-NA was the metabolite [3H]-DHPG; and second, inhibition of MAO significantly reduced the radioactive outflow in response to cyanine 863. The outflow of [3H]-MPP+ was significantly enhanced by MPP+, guanidine, choline and amantadine as potential substrates for OCT-related transmembrane transporters. However, desipramine at a low concentration essentially blocked the radioactive outflow induced by all of these substances with the exception of MPP+, indicating the NAT and not an OCT as their primary site of action. The MPP+-induced release of [3H]-MPP+ was fully prevented by a combined application of desipramine and cyanine 863. No trans-stimulation of [3H]-MPP+ outflow was observed by the OCTN1 and OCTN2 substrate carnitine at 100 microM. Our observations indicate an OCT-mediated transmembrane transport of [3H]-MPP+. Amongst the three OCTs expressed in the SCG, OCT3 best fits the profile of substrates and antagonists that cause trans-stimulation and trans-inhibition, respectively, of [3H]-MPP+ release.

SK3 is an important component of K(+) channels mediating the afterhyperpolarization in cultured rat SCG neurones.

1. Our aim was to identify the small-conductance Ca(2+)-activated K(+) channel(s) (SK) underlying the apamin-sensitive afterhyperpolarization (AHP) in rat superior cervical ganglion (SCG) neurones. 2. Degenerate oligonucleotide primers designed to the putative calmodulin-binding domain conserved in all mammalian SK channel sequences were employed to detect SK DNA in a cDNA library from rat SCG. Only a single band, corresponding to a fragment of the rSK3 gene, was amplified. 3. Northern blot analysis employing a PCR-generated rSK3 fragment showed the presence of mRNA coding for SK3 in SCG as well in other rat peripheral tissues including adrenal gland and liver. 4. The same rSK3 fragment enabled the isolation of a full-length rSK3 cDNA from the library. Its sequence was closely similar to, but not identical with, that of the previously reported rSK3 gene. 5. Expression of the rSK3 gene in mammalian cell lines (CHO, HEK cells) caused the appearance of a K(+) conductance with SK channel properties. 6. The application of selective SK blocking agents (including apamin, scyllatoxin and newer non-peptidic compounds) showed these homomeric SK3 channels to have essentially the same pharmacological characteristics as the SCG afterhyperpolarization, but to differ from those of homomeric SK1 and SK2 channels. 7. Immunohistochemistry using a rSK3 antipeptide antibody revealed the presence of SK3 protein in the cell bodies and processes of cultured SCG neurones. 8. Taken together, these results identify SK3 as a major component of the SK channels responsible for the afterhyperpolarization of cultured rat SCG neurones.

Somatic and prejunctional nicotinic receptors in cultured rat sympathetic neurones show different agonist profiles.

1. The release of [3H]-noradrenaline ([3H]-NA) in response to nicotinic acetylcholine receptor (nAChR) agonists was compared with agonist-induced currents in cultured rat superior cervical ganglion (SCG) neurones. 2. [3H]-NA release in response to high concentrations of nicotinic agonists was reduced, but not fully inhibited, by the presence of either tetrodotoxin (TTX) or Cd2+ to block voltage-gated Na+ or Ca2+ channels, respectively. We used the component of transmitter release that remained in the presence of these substances (named TTX- or Cd2+-insensitive release) to pharmacologically characterize nAChRs in proximity to the sites of vesicular exocytosis (prejunctional receptors). Prejunctional nAChRs were activated by nicotinic agonists with a rank order of potency of dimethylphenylpiperazinium iodide (DMPP) > nicotine > cytisine > ACh, and with EC50 values ranging from 22 microM (DMPP) to 110 microM (ACh). 3. [3H]-NA release in response to low concentrations of nAChR agonists was fully inhibited by the presence of either TTX or Cd2+ (named TTX- or Cd2+-sensitive release). TTX-sensitive release was triggered by nicotinic agonists with a rank order of potency of DMPP > cytisine approximately nicotine approximately ACh, which due to its similarity to TTX-insensitive release indicates that it might also be triggered by prejunctional-type nAChRs. The EC50 values for TTX (Cd2+)-sensitive release were less than 10 microM for all four agonists. 4. By contrast to transmitter release, somatic nAChRs as seen by patch clamp recordings were most potently activated by cytisine, with a rank order of potency of cytisine > nicotine approximately DMPP > ACh. EC50 values for the induction of currents exceeded 20 microM for all four agonists. 5. The nicotinic antagonist mecamylamine potently inhibited all transmitter release in response to nicotine. alpha-Bungarotoxin (alpha-BuTX) was, on the other hand, without significant effect on nicotine-induced TTX-insensitive release. The competitive antagonist dihydro-beta-erythroidine (DHbetaE) caused rightward shifts of the dose-response curves for both TTX-sensitive and TTX-insensitive transmitter release as well as for currents in response to nicotine, with pA2 values ranging from 4.03 to 4.58. 6. Due to clear differences in the pharmacology of agonists we propose that nAChRs of distinct subunit composition are differentially targeted to somatic or axonal domains.

G-Protein β-Subunit Specificity in the Fast Membrane-Delimited Inhibition of Ca2+Channels

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gbeta5 subunit produced much weaker effects than Gbeta1 and Gbeta2, whereas Gbeta3 and Gbeta4 were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, Gbeta1 or Gbeta2 coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha1 subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gbeta5 subunit interacted weakly, and Gbeta3 and Gbeta4 failed to interact. Together, these results suggest that Gbeta1 and/or Gbeta2 subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha1 subunit is a principal receptor for this inhibition.

Cyclin Dependent Kinase Inhibitors and Dominant Negative Cyclin Dependent Kinase 4 and 6 Promote Survival of NGF-Deprived Sympathetic Neurons

Neuronal apoptosis plays a critical role in both normal development and disease. However, the precise molecular events controlling neuronal apoptosis are not well understood. Previously, we hypothesized that cell cycle regulatory molecules function in controlling the apoptotic pathways of trophic factor-deprived neurons. To test this hypothesis, we used the RNA alphavirus Sindbis to express three known cyclin dependent kinase inhibitors (CKIs), p16(ink4), p21(waf/cip), and p27(kip1), and dominant negative mutant forms of four known G1 cyclin dependent kinases (CDKs), Cdk2, Cdk3, Cdk4, and Cdk6, in primary cultured rat superior cervical ganglion sympathetic neurons. We demonstrate that expression of each of the CKIs protects the postmitotic cultured neurons from apoptotic death evoked by withdrawal of NGF. In addition, we show that expression of dominant negative forms of Cdk4 or Cdk6, but not Cdk2 or Cdk3, protects NGF-deprived sympathetic neurons from death. Such findings suggest the participation of several CDKs and their cognate cyclins in a neuronal apoptotic pathway.

Transforming growth factor-beta2 selectively alters the developmental expression of the fast transient A-current in cultured rat superior cervical ganglion neurons.

Cultures of neonatal rat superior cervical ganglion (SCG) were utilized to examine the ability of transforming growth factor-beta2 (TGFbeta2) to alter voltage-gated K+ channel development. Whole-cell patch clamp recordings were used to monitor changes in three separate K+ currents: A rapidly inactivating A-current (I(Af)), a slowly inactivating A-current (I(As)), and a non-inactivating current (I(K)). Continuous TGFbeta2 (10 ng/ml) treatment selectively altered the normal developmental decrease in I(Af) expression in SCG neurons, but did not significantly change I(As) or I(K) expression. After 2 weeks of treatment, the mean I(Af) current density in control cultures had decreased 67%, while the I(Af) current density in TGFbeta2 treated cultures remained near initial values (approximately 2.7-fold higher than control). This difference remained even after 4 weeks of exposure. TGFbeta2 did not appear to change the activation kinetics or voltage-dependence of I(Af). These findings indicate that TGFbeta2 may play an important role in modulating the development of neuronal excitability by regulating the expression of voltage-gated K+ channels.

Evaluation of N-type Ca2+ channel currents in cultured rat superior cervical ganglion neurons

Electrophysiological studies of the effects of test compounds on ion channel currents have been useful in the identification of novel therapeutic agents. Here, we examined the use of cultured superior cervical ganglion (SCG) neurons as a model system for the electrophysiological evaluation of N-type Ca2+ channels in vitro. As previously reported, Ca2+ channel currents in acutely dissociated preparations of SCG neurons were mainly N-type, as defined by inhibition of Ca2+ channel current with the specific N-type Ca2+ channel blocker, ω-conopeptides GVIA or MVIIA. However, a cultured preparation that could be used over an extended period of time would be more useful for drug discovery since acutely dissociated preparations require daily dissections. We found that with extended time in culture the amplitudes of Ca2+ channel currents increased with time. While there was a reduction in the percentage of N-type Ca2+ channel component, the majority (70%) of Ca2+ channel currents in long-term cultured SCG neurons remained N-type. A portion of the MVIIA-resistant Ca2+ channel current component was blocked by the ω-conopeptide MVIIC and ω-agatoxin IIIA, but not by ω-agatoxin IVA, a pharmacological profile similar to Q-type Ca2+ channel current. These studies suggest that cultured SCG neurons would be useful for the study of N-type Ca2+ currents even after prolonged time in culture, and may also be used to indicate the selectivity of test compounds for other Ca2+ channel subtypes. Drug Dev. Res. 41:85–90, 1997. © 1997 Wiley-Liss, Inc.

Rapid drug application resolves two types of nicotinic receptors on rat sympathetic ganglion cells.

The properties of nicotinic acetylcholine receptors (AChRs) on cultured rat superior cervical ganglion (SCG) neurons were analysed. AChR agonists [1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), cytisine] were applied to whole cells within 70ms. The desensitization rate of whole-cell currents during constant application of DMPP varied between neurons. The time course of desensitization was fitted by double exponentials with time constants kfast, of between 0.35 and 0.55s, and kslow, of 3-5s. By exchanging intracellular chloride for caesium methanesulphonate, the possibility of interference by a calcium-activated chloride current was excluded. In cells that exhibited a slowly desensitizing current during the application 20 microM DMPP, equimolar cytisine induced a larger peak current compared to the response to DMPP, while in cells with rapidly desensitizing DMPP-induced currents the response to equimolar cytisine was smaller. The differences in desensitization rates and agonist potencies are due to different functional properties of AChR subtypes, as indicated by currents recorded from outside-out patches upon rapid agonist application and removal (2ms each). The results indicate the presence of two distinct AChR subtypes on SCG neurons: one with a fast and one with a slow activation/desensitization rate, but both with similar single-channel conductances. Slow activation/desensitization was found to be associated with a high potency of cytisine/low potency of DMPP. For AChRs with rapid activation/desensitization kinetics the agonist potencies were reversed.

Mammalian myosin Iα is concentrated near the plasma membrane in nerve growth cones

To determine if unconventional myosins play a role in nerve outgrowth, antibodies specific for rat brain derived mammalian myosin Iα (MMIα) were used to label cultured rat superior cervical ganglion nerve cells. Observations were made at both the light and electron microscopic level of resolution using preparative procedures designed to enhance the ability to precisely determine the relationship between antibody label and cellular structures in order to map the distribution and structural association of this myosin. Immunofluorescence showed that MMIα has a punctate distribution throughout the nerve cell body, neurites, and growth cones. In growth cones, MMIα staining is sometimes elevated in thin peripheral regions of high actin content at the leading edge. Immunoelectron microscopy using colloidal gold conjugated antibodies showed that in growth cones MMIα is absent from membranous organelles and is concentrated primarily in the cell cortex adjacent to the cell membrane. The cortical label is equally distributed between upper and lower membranes. The plasma membrane association of the MMIα label persists under conditions in which the actin cytoskeleton is perturbed or removed, suggesting a direct association between a fraction of MMIα and the plasma membrane. MMIα label is also associated with the non-cortical actin cytoskeleton. Partial disruption of the actin cytoskeleton using cytochalasin B causes redistribution of only a subset of MMIα label. These data suggest a complex relationship between MMIα, the actin cytoskeleton, and the plasma membrane in the growth cone. In contrast to its localization in the growth cone, in neuronal cell bodies MMIα is also associated with tubulovesicular structures. This suggests that at this location MMIα may either act as an organelle motor or is passively transported to the plasma membrane on vesicles. © 1996 Wiley-Liss, Inc.

Mammalian myosin Iα is concentrated near the plasma membrane in nerve growth cones

To determine if unconventional myosins play a role in nerve outgrowth, antibodies specific for rat brain derived mammalian myosin I alpha (MMI alpha) were used to label cultured rat superior cervical ganglion nerve cells. Observations were made at both the light and electron microscopic level of resolution using preparative procedures designed to enhance the ability to precisely determine the relationship between antibody label and cellular structures in order to map the distribution and structural association of this myosin. Immunofluorescence showed that MMI alpha has a punctate distribution throughout the nerve cell body, neurites, and growth cones. In growth cones, MMI alpha staining is sometimes elevated in thin peripheral regions of high actin content at the leading edge. Immunoelectron microscopy using colloidal gold conjugated antibodies showed that in growth cones MMI alpha is absent from membranous organelles and is concentrated primarily in the cell cortex adjacent to the cell membrane. The cortical label is equally distributed between upper and lower membranes. The plasma membrane association of the MMI alpha label persists under conditions in which the actin cytoskeleton is perturbed or removed, suggesting a direct association between a fraction of MMI alpha and the plasma membrane. MMI alpha label is also associated with the non-cortical actin cytoskeleton. Partial disruption of the actin cytoskeleton using cytochalasin B causes redistribution of only a subset of MMI alpha label. These data suggest a complex relationship between MMI alpha, the actin cytoskeleton, and the plasma membrane in the growth cone. In contrast to its localization in the growth cone, in neuronal cell bodies MMI alpha is also associated with tubulovesicular structures. This suggests that at this location MMI alpha may either act as an organelle motor or is passively transported to the plasma membrane on vesicles.

Neuropeptide Y inhibits adrenergic transmitter release in cultured rat superior cervical ganglion cells by restricting the availability of calcium through a pertussis toxin-sensitive mechanism

Neuropeptide Y has been reported to inhibit the release of the adrenergic transmitter from sympathetic nerves in many tissues. The purpose of this study was to determine the mechanism of the inhibitory effect of neuropeptide Y on the release of the adrenergic transmitter in cultured superior cervical ganglion cells prelabeled with tritiated norepinephrine. In cultured superior cervical ganglion cells superfused with a HEPES-buffered saline, electrical field stimulation (1 Hz, 30 pulses, 1 ms, 60 V) increased the fractional overflow of tritium. Neuropeptide Y (50 nM) attenuated this depolarization-induced increase in transmitter release. The nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)cyclic AMP (100 μM) and the potassium channel blockers, tetraethylammonium chloride (1 mM) and 4-aminopyridine (300 μM) potentiated the electrically stimulated increase in fractional tritium overflow but failed to alter the inhibitory effect of neuropeptide Y on fractional tritium overflow. Increasing the calcium concentration in the superfusion fluid from 1.8 to 5.4 mM potentiated the electrically stimulated increase in fractional tritium overflow and attenuated the inhibitory effect of neuropeptide Y. Reduction of superfusion fluid calcium concentration to 0.5 mM decreased electrically stimulated fractional tritium overflow and augmented the inhibitory effect of NPY on release of tritium. The fractional release of tritium in response to the calcium ionophore, ionomycin, was not significantly altered by neuropeptide Y. In Fura-2-loaded isolated sympathetic neurites obtained from superior cervical ganglia explants, the depolarization-induced (54 mM KC1) increase in cytosolic calcium was attenuated by neuropeptide Y (50 nM). Pertussis toxin (100 ng/ml for 4 h) pretreatment abolished both the neuropeptide Y-induced decrease in electrically stimulated fractional tritium overflow and the reduction in depolarization-induced increase in cytosolic calcium. These data suggest that NPY inhibits adrenergic transmitter release in cultured superior cervical ganglion cells by decreasing the availability of calcium for the release process by a pertussis toxin-sensitive mechanism.

Vasoactive intestinal peptide suppresses neuronal cell death induced by nerve growth factor deprivation in rat sympathetic ganglion cells in vitro

Neuropeptides were examined for their effects on the survival of cultured rat superior cervical ganglion cells after acute deprivation of nerve growth factor (NGF). Vasoactive intestinal peptide (VIP, 3 μM) delayed the neuronal death about 6 h alone. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM) greatly potentiated its effect, reducing EC 50 from 2.5 μM to 8 nM. The neuronal death was completely suppressed under this condition. On the other hand, substance P (1–100 μM) or enkephalin (1–100 μM) alone did not modify the death, whereas the latter (100 μM) enhanced the survival-promoting effect of membrane depolarization with elevated K +. These results suggest strongly that neuropeptides regulate the NGF-independent survival of sympathetic neurons through a cAMP-dependent mechanism.

Calcium entry through nicotinic receptor channels and calcium channels in cultured rat superior cervical ganglion cells.

1. Patch-clamp techniques in conjunction with indo-1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage-clamp conditions, pressure application of the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/- 26 nM at a clamp potential of -60 mV. This was accompanied by an inward current of -4.53 +/- 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP-induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM-10 microM) but were unaffected by alpha-bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from -80 to -40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine-sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage-dependent calcium current. On average at -60 mV, the M/q ratio of this highly calcium-selective permeability was 1961 mM nC-1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant-field theory, ion-substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.

Zn2+ potentiates ATP-activated currents in rat sympathetic neurons.

The ATP-activated inward current (IATP) in cultured rat superior cervical ganglion neurons and its modulation by extracellular Zn2+ were examined. ATP activated a non-specific cation conductance and caused a transient rise in intracellular Ca2+. The current response was specifically activated by ATP and was blocked by the P2-purinoceptor antagonist, suramin. Low concentrations of extracellular Zn2+ rapidly and reversibly potentiated both IATP and the intracellular Ca2+ rise. The potentiation by 10 microM Zn2+ was dependent on the concentration of agonist; Zn2+ increased the sensitivity of activation without potentiating the maximum response. Higher concentrations of Zn2+ reduced and prolonged the current, consistent with open-channel block. We hypothesize that there exist two sites of action for Zn2+: a positively acting allosteric site that enhances current amplitude and a site, possibly within the pore, that blocks conductance through the channel.

An mRNA homologous to interleukin-1 receptor type I is expressed in cultured rat sympathetic ganglia

Interleukin-1 (IL-1) induces substance P (SP) gene expression in cultured rat superior cervical ganglion (SCG) explants. In order to study the molecular mechanism of this action of IL-1, the presence of an interleukin-1 receptor (IL-1R) activity and the identity of an mRNA homologous to known IL-1R sequence was determined in SCG. The SP increase is blocked by recombinant IL-1 receptor antagonist protein, so IL-1 must be interacting with a specific receptor. We have cloned cDNA homologous to IL-1R Type I from rat SCG using a reverse transcription-polymerase chain reaction (RT-PCR). The resulting cDNA sequence is strongly homologous with mouse and human IL-1R cDNA of the T cell and fibroblast type (type I; encoding an 80-kDa protein). mRNA specific for IL-1R can be readily detected in intact SCG by quantitative RT-PCR and S1 hybridization. However, the level of IL-1R mRNA increases 3–6-fold by 2 days in culture. This increase is independent of the presence of dexamethasone, IL-1β or IL-1 receptor antagonist protein ligands. The increase of IL-1R following explantation, a model of nerve injury, may provide a mechanism linking inflammatory signalling to neuronal phenotypic changes.