Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain.

The large diversity of peptides from venomous creatures with high affinity for molecules involved in the development and maintenance of neuropathic pain has led to a surge in venom-derived analgesic research. Some members of the α-conotoxin family from Conus snails which specifically target subtypes of nicotinic acetylcholine receptors (nAChR) have been shown to be effective at reducing mechanical allodynia in neuropathic pain models. We sought to determine if three such peptides, Vc1.1, AuIB and MII were effective following intrathecal administration in a rat neuropathic pain model because they exhibit different affinities for the major putative pain relieving targets of α-conotoxins. Intrathecal administration of α-conotoxins, Vc1.1, AuIB and MII into neuropathic rats reduced mechanical allodynia for up to 6 h without significant side effects. In vitro patch-clamp electrophysiology of primary afferent synaptic transmission revealed the mode of action of these toxins was not via a GABA(B)-dependent mechanism, and is more likely related to their action at nAChRs containing combinations of α3, α7 or other subunits. Intrathecal nAChR subunit-selective conotoxins are therefore promising tools for the effective treatment of neuropathic pain.

Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Spatial distribution and developmental appearance of postjunctional P2X1 receptors on smooth muscle cells of the mouse vas deferens.

P2X1-type purinoceptors have been shown to mediate fast transmission between sympathetic varicosities and smooth muscle cells in the mouse vas deferens but the spatial organization of these receptors on the smooth muscle cells remains inconclusive. Voltage clamp techniques were used to estimate the amplitudes of spontaneous excitatory junction currents (SEJCs) in cells of the vas deferens longitudinal smooth muscle layer. These currents involved the activation of about 6% of the P2X-type channels present on the cell, as compared to whole cell currents produced when isolated smooth muscle cells were exposed to maximal concentrations of either ATP or alpha,beta-MeATP. Immunofluorescence staining of the vas deferens with antibodies against P2X1 receptor showed a diffuse, grainy distribution over the entire membrane of each smooth muscle cell. Anti-P2X1 staining was not markedly clustered beneath anti-SV2-stained sympathetic varicosities. Similar results were obtained for cells in the urinary bladder. During development, P2X1 mRNA was detected as early as embryonic day 15 (E15). Increasing intensities of diffuse immunostaining for P2X1 were observed in the walls of the bladder, tail artery, and aorta from E15 until 6 weeks postnatal. The vas deferens showed increasing intensities of diffuse staining of its smooth muscle layers between 2 and 6 weeks postnatal, consistent with the time-course of development of fast purinergic transmission described previously. Together, the results suggest that the response of smooth muscle of the vas deferens to ATP released from sympathetic varicosities relies on rapidly desensitizing P2X1 receptors, distributed diffusely across the smooth muscle cell surface.

Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia.

Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists omega-conotoxin GVIA (N type), nifedipine and nimodipine (L type), omega-conotoxin MVIIC and omega-agatoxin IVA (P/Q type), and Ni2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals.

P2 purinoceptor blocker suramin antagonises NMDA receptors and protects against excitatory behaviour caused by NMDA receptor agonist (RS)-(tetrazol-5-yl)-glycine in rats.

It has been reported that suramin, an anthelminthic, trypanocidal agent and an inhibitor of P2 receptors, may antagonise N-methyl-D-aspartate (NMDA) subtype of the excitatory amino acid receptors. Both NMDA receptors and P2X subclass of P2 receptors are ligand-gated Ca2+-selective channels and, since the increased influx of Ca2+ into neurons has been linked to neurotoxicity, simultaneous inhibition of P2X and NMDA receptors in vivo by suramin could represent an effective neuroprotective treatment. We have found that suramin inhibited the binding of [3H]CGP 39653 to NMDA receptor binding sites in vitro and reduced the frequency of NMDA channel openings in patch-clamp studies. Suramin (1 mM) had no effect on [3H]kainate binding in vitro. In vivo, intracerebroventricular (I.C.V.) injections of suramin (70 nmol/brain) antagonised convulsive effects of the NMDA agonist (RS)-(tetrazol-5-yl)-glycine (TZG, LY 285265). Suramin, however, did not prevent neurotoxic lesions in the hippocampus caused by I.C.V. administration of TZG. Increasing the dose of suramin resulted in death from severe respiratory depression.

Effect of P2-purinoceptor antagonists on glutamatergic transmission in the rat hippocampus.

1. A study has been made of the effects of P2-purinoceptor antagonists on the evoked excitatory postsynaptic currents (e.p.s.cs) generated in CA1 pyramidal cells on stimulation of Schaffer collaterals and in CA3 pyramidal cells on stimulation of mossy fibres. The effects of these antagonists on currents generated in the cells on application of glutamate has also been determined. 2. Suramin blocked the evoked e.p.s.cs with an 50% inhibition (ID50) of 62 +/- 8 microM (mean +/- s.e.mean, n = 17), spontaneous miniature e.p.s.cs and the currents induced by application of 100 microM glutamate with an ID50 = 121 +/- 36 microM (n = 15) in all the cells studied. 3. Reactive Blue 2 (RB-2) in a concentration of 200 microM decreased the e.p.s.cs by 80 +/- 10% (n = 6) and the glutamate-activated currents by 83 +/- 3% (n = 6). 4. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) in the concentration-range of 40-500 microM decreased the amplitude of the e.p.s.cs in 12 out of 13 cells studied. PPADS at 200 microM reduced the amplitude of the e.p.s.cs by 60 +/- 10% (n = 3). PPADS did not affect the glutamate-induced currents in 4 cells and produced potentiation of the current amplitude by 60 +/- 10% in 4 other cells. 5. These results suggest that both presynaptic and postsynaptic P2-purinoceptors in the hippocampus can modulate the release and action of endogenous glutamate.

Opioids acting on delta-receptors modulate Ca2+ currents in cultured postganglionic neurones of avian ciliary ganglia.

Opioid agonists inhibited Ba2+ currents in cultured postganglionic neurons of 3-15-day-old embryonic avian ciliary ganglia. Leucine-enkephalin (LENK, 10-20 microM) inhibited transient Ba2+ currents by 50% and sustained currents by 21%. The delta-opioid receptor agonist, DPDPE (0.02-10 microM), also inhibited Ba2+ currents, but kappa- (U50488H) and mu- (DAMGO) receptor agonists had no effect. Inhibition of Ba2+ currents showed profound desensitisation during prolonged application of agonists, being reduced to approximately 25% of the original response after 4 min superfusion with leucine-enkephalin (10-20 microM).

Existence of two fast inactivation states in cardiac Na channels confirmed by two-stage action of proteolytic enzymes.

Fast inactivation of Na channels in neonatal cardiac cells was removed by the action of proteolytic enzymes trypsin or papain. Two stages were apparent in the time course of this process. During the first one, both number of channel reopenings and the mean open time increased markedly even though fast inactivation remained complete. The second stage was manifested by the disappearance of all signs of fast inactivation without further noticeable changes in channel mean open time. At the same time the nonrandom clustering of blank response (response without channel openings) trials became prominent. The data obtained support the interpretation of two separate fast inactivation states in cardiac Na channels as suggested in our previous papers (Zilberter et al. (1989) in Neuromuscular Junction (Sellin, L.C., Libelius, R. and Thesleff, S., eds.), pp. 43-50, Elsevier, Amsterdam, and Zilberter et al. (1991) J. Mol. Cell. Cardiol. 23, (Suppl.) 61-72).

Ca-sensitive slow inactivation and lidocaine-induced block of sodium channels in rat cardiac cells.

Macroscopic and single Na channel currents were studied using the patch-clamp technique in rat cardiac cells. Slow inactivation (SI) characterized by 100 ms order kinetics was investigated. This inactivation was incomplete, as no more than 50% of Na channels were able to enter the SI state during 1-2 s of membrane depolarization. The maximal fraction of Na channels in the SI state decreased with increasing external Ca concentration. Single-channel analysis led us to conclude that Na channels undergo transition from the resting (R) state to the SI state, bypassing both open (O) and ordinary fast inactivation (I) states. A new fast inactivation state (Y) was postulated between the R and SI states. Exposure to the local anesthetic, lidocaine, decreased the probability of Na channel opening and dramatically slowed reactivation. The latter effect was due to lidocaine interaction with the Y state rather than the I state. Increasing external Ca concentration in the presence of lidocaine diminished the fraction of Na channels capable of making the transition to a slowly reactivatable state (lidocaine-bound Y and/or SI states).

Potentiation of glutamate-activated currents in isolated hippocampal neurons.

"Fast chemical stimulation" was shown to induce potentiation of glutamate-activated currents in neurons isolated from rat hippocampus. A fast application system allowed solution changes up to a rate of 20 Hz. In Mg2(+)-free solution, the response to glutamate application immediately after repetitive stimulation with glutamate plus glycine was increased by 25%-88%, returning to control levels over 10-15 min. Enhancement of glutamate-induced currents was also seen after stimulation with solutions containing aspartate or NMDA plus glycine. Aspartate-induced currents were not potentiated. These and other observations demonstrate that in a purely "postsynaptic" system, short-term potentiation can be induced and is mediated via NMDA receptors whereas the potentiated current is carried via non-NMDA glutamate receptor channels.