Upregulation of dorsal root ganglion (alpha)2(delta) calcium channel subunit and its correlation with allodynia in spinal nerve-injured rats.

Peripheral nerve injury can lead to a persistent neuropathic pain state in which innocuous tactile stimulation elicits pain behavior (tactile allodynia). Spinal administration of the anticonvulsant gabapentin suppresses allodynia by an unknown mechanism. In vitro studies indicate that gabapentin binds to the alpha(2)delta-1 (hereafter referred to as alpha(2)delta) subunit of voltage-gated calcium channels. We hypothesized that nerve injury may result in altered alpha(2)delta subunit expression in spinal cord and dorsal root ganglia (DRGs) and that this change may play a role in neuropathic pain processing. Using a rat neuropathic pain model in which gabapentin-sensitive tactile allodynia develops after tight ligation of the left fifth and sixth lumbar spinal nerves, we found a >17-fold, time-dependent increase in alpha(2)delta subunit expression in DRGs ipsilateral to the nerve injury. Marked alpha(2)delta subunit upregulation was also evident in rats with unilateral sciatic nerve crush, but not dorsal rhizotomy, indicating a peripheral origin of the expression regulation. The increased alpha(2)delta subunit expression preceded the allodynia onset and diminished in rats recovering from tactile allodynia. RNase protection experiments indicated that the DRG alpha(2)delta regulation was at the mRNA level. In contrast, calcium channel alpha(1B) and beta(3) subunit expression was not co-upregulated with the alpha(2)delta subunit after nerve injury. These data suggest that DRG alpha(2)delta regulation may play an unique role in neuroplasticity after peripheral nerve injury that may contribute to allodynia development.

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells.

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.

The time and space machine: continuous measurement of drug-induced behavior patterns in the rat.

Because of the increasing demand for refined techniques to record drug-induced motoric changes, we designed and evaluated a computer monitoring system with continuous measurement of different parameters of rat motor activity. This system is particularly useful for chronic drug studies because it can characterize patterns of behavior and combines the residential and experimental environments, thus enabling automated behavioral measurement without experimenter intervention. Behavioral responses are detected by a capacitance-sensing device that generates bipolar analog voltages representing the location of the rat in its home cage. These voltages are first transduced, then amplified, and finally converted to digital signals in a computer that processes the input using algorithms to define specific responses. The technique pinpoints the exact location of the rat and identifies many kinds of responses simultaneously (e.g., rearing, circling) by tracking the path of movement or setting threshold limits. Some threshold values (representing rearing, gross movements, fine movements) were validated against stereotypy rating scales for amphetamine, apomorphine, and beta-phenylethylamine. Among these drugs, quantitatively distinct response profiles were obtained. The system has wide applications for studies of biological rhythms, sleep, aging, and drug toxicology.

Effect of ?-conotoxin GVIA on Ca(2+) influx and endogenous acetylcholine release from chicken brain preparations.

?-Conotoxin GVIA (?-conotoxin) is an inhibitor of neuronal voltage sensitive calcium channels (VSCCs). We have examined the effects of various manipulations aimed at elucidating the effects of ?-conotoxin particularly on K(+) stimulated (45)Ca(2+) influx and endogenous acetylcholine (ACh) release. The initial phase of K(+) stimulated (45)Ca(2+) influx was accompanied by a large rapid release of ACh measured by a modification of the chemiluminescent technique of Israël and Lesbats (J. Neurochem.39, 248-250, 1982). ACh release was greatly reduced (?90% ) in Ca(2+) free solution or by Cd(2+) (100 ?M) period ?-conotoxin (1 ?M) completely blocked the initial phase of (45)Ca(2+) influx (1-3 s) from synaptosomes and reduced K(+) stimulated ACh release from tissue slices. ?-Conotoxin (0.1 ?M) reduced ACh release which was reversed in the presence of high Ca(2+) concentrations. The dihydropyridine VSCC inhibitor (?)202-791 or agonist (+)202-791 had no effect on ACh release or (45)Ca(2+) influx. The pattern of responses on (45)Ca(2+) influx and endogenous ACh release was consistent with the suggestion that ?-conotoxin inhibits N-type VSCCs and that these channels are intimately related to ACh release.

Characterization of sodium-dependent, high-affinity serotonin uptake in rat spinal cord synaptosomes.

Synaptosomal accumulation of [3H]serotonin was used to determine if the rat spinal cord possesses a high-affinity neuronal uptake system for serotonin. Two temperature-dependent accumulation processes were found, one sodium-dependent, the second sodium-independent. Sodium-dependent [3H]serotonin accumulation was linear with sodium concentrations up to 143 mM, was associated with the purified synaptosomal fraction (P2B), and decreased 76% by osmotic lysis, 88% by sonication, and 96% by 0.1% Triton X-100. Drug inhibition studies demonstrated fluoxetine to be the most potent inhibitor of this system (IC50 0.075 microM) while desipramine (IC50 0.43 microM) and nomifensine (IC50 0.95 microM) were less potent. Kinetic analysis revealed that sodium-dependent accumulation in purified synaptosomes was saturable at low [3H]serotonin concentrations (Ku = 50 nM, Vmax = 4 pmol/mg protein/min). Sodium-independent [3H]5-HT accumulation was substantially less sensitive to fluoxetine, desipramine and nomifensine. While sodium-independent accumulation was not significantly affected by osmotic lysis, it was markedly increased by prior sonication of tissue. Also, in contrast to sodium-dependent accumulation, sodium-independent accumulation was evenly distributed in all tissue fractions, and was not saturable at low [3H]serotonin concentrations. It is concluded that sodium-dependent [3H]serotonin accumulation reflects uptake into spinal serotonergic nerve terminals while sodium-independent accumulation probably reflects a temperature-sensitive binding to membrane fragments. Comparison to brain uptake of serotonin and the necessity for using 37 degrees C sodium-free blanks rather than 0 degree C blanks in spinal cord homogenates is discussed.

Presynaptic serotonin receptors regulate [3H]serotonin release from rat spinal cord synaptosomes.

Superfused rat spinal cord synaptosomes were studied to determine if inhibitory serotonin (5-HT) receptors (autoreceptors) exist on spinal serotonergic nerve terminals. Exogenous 5-HT (1-50 nM) produced a concentration-dependent inhibition of K+-induced [3H]5-HT release but did not affect basal [3H]5-HT release. A 32-44% inhibition was produced by 30 nM 5-HT. The inhibitory effect of 30 nM 5-HT was effectively antagonized by 100 nM metitepine, a 5-HT autoreceptor antagonist. The results provide evidence for the existence of 5-HT autoreceptors in rat spinal cord tissue.

Relative contributions of G protein, channel, and receptor to voltage-dependent inhibition of neuronal N-type and P/Q-type calcium channels in HEK 293 cell lines.

The voltage-dependent modulation of neuronal voltage-gated calcium channels by heterotrimeric G protein-coupled receptors potentially provides a means for activity-dependent modulation of synaptic efficacy. Recent attention has focused upon the molecular mechanisms by which such G proteins influence the biophysical properties of calcium channels. We have used an HEK 293-based heterologous system which stably expresses human neuronal calcium channels to address the relative contributions of receptor, G protein, and channel to voltage-dependent inhibition. We find that the receptor and channel subtype only insignificantly influence the time it takes to re-establish modulation following voltage-dependent relief of inhibition. In contrast, the G protein subtype mediating inhibition appears to play a significant part in this process. These results emphasize the importance of G protein subtype in the modulation of neuronal calcium channels.

Fluoxetine-induced inhibition of synaptosomal [3H]5-HT release: possible Ca(2+)-channel inhibition.

Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K(+)-induced [3H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations greater than 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K+ used to depolarize the synaptosomes and the concentration of external Ca2+. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [3H]5-HT release induced by the Ca(2+)-ionophore A 23187 or Ca(2+)-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K(+)-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca2+ channels and Ca2+ entry. Whereas fluoxetine and paroxetine inhibited binding of [3H]nitrendipine to the dihydropyridine-sensitive L-type Ca2+ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K(+)-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K(+)-induced increase in intracellular free Ca2+ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K(+)-induced [3H]5-HT release by antagonizing voltage-dependent Ca2+ entry into nerve terminals.

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.

An assessment of spectral analysis of amphetamine-induced behavior.

Testing of a new radio frequency capacitance field type transducer and power spectrum analysis system for assessment of rat behavior is described. Power spectrum estimates of amphetamine-induced behavior had an orderly relationship with behavior ratings ranging from inactive to intense stereotypy. The effects of thorazine dose-response blocking on amphetamine-induced behavior were linear. Separation between adjacent doses could not be accomplished with a single frequency, but required differential frequency-time period information.

The inositol 1,4,5-trisphosphate-forming agonist histamine activates a ryanodine-sensitive Ca2+ release mechanism in bovine adrenal chromaffin cells.

The role of a Ca(2+)-induced Ca2+ release (CICR) mechanism in the generation of agonist-induced increases of intracellular free Ca2+ concentration ([Ca2+]i) was studied in bovine adrenal chromaffin cells. In single cells, repetitive stimulations with caffeine at 200-s intervals evoked reproducible spikes of [Ca2+]i. Ryanodine, an agent that interacts with the CICR channel of muscle, inhibited the caffeine-induced spikes of [Ca2+]i in a "use-dependent" way. High affinity binding sites for [3H]ryanodine (Kd 3.3 nM, Bmax 26 fmol/mg protein) were also detected in membranes from chromaffin cells, supporting the presence of a caffeine- and ryanodine-sensitive CICR channel. Pretreatment of single cells with caffeine + ryanodine to reduce the size of the caffeine-sensitive Ca2+ compartment inhibited a subsequent spike of [Ca2+]i evoked by histamine, a D-myo-inositol 1,4,5-trisphosphate-forming agonist. This demonstrates that a significant portion of the Ca2+ released by histamine comes from a caffeine- and ryanodine-sensitive pool. Ryanodine inhibited by 50% the size of [Ca2+]i spikes evoked by repetitive stimulation with histamine and did so in a use-dependent manner. These data suggest that, in addition to D-myoinositol 1,4,5-trisphosphate, activation of a caffeine- and ryanodine-sensitive CICR channel participates in the generation of histamine-induced release of intracellular Ca2+.

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells.

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.

Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment.

Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.