Toxin Blockade Research Articles

Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist

We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.

Development of Functionally Selective, Small Molecule Agonists at Kappa Opioid Receptors

The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

Glabellar Contraction Patterns: A Tool to Optimize Botulinum Toxin Treatment

Botulinum toxin is a well-established treatment for dynamic glabellar lines. A previous study evaluated the existence of glabellar contraction "patterns," according to the predominance of eyebrow approximation, depression, or elevation movements, namely "U," "V," "convergent arrows," "omega," and "inverted omega." To confirm contraction patterns in the adult population for a better treatment approach and to verify whether changes occur after repeated treatment. Pairs of photographs-at rest and under contraction-from two groups were retrospectively analyzed: 334 adult volunteers with a predominance of specific movements, being verified and 36 previously treated individuals when they returned for re-injections. The five glabellar contraction patterns were confirmed. Each individual's initial pattern reappeared upon waning of the toxin effect. Interpersonal differences in facial animation are observed. Classifying glabellar wrinkles allows accurate treatment with botulinum toxin, injecting the most commonly recruited muscles with higher doses or into more sites. Muscles not so recruited are spared or injected with lower doses for more-effective and -natural results. Although botulinum toxin blockade causes recruitment of adjacent muscles, the initial muscle contraction pattern is resumed when the effect wanes.

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

Receptor-regulated Interaction of Activator of G-protein Signaling-4 and Gαi

Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind Galpha(i)-GDP subunits free of Gbetagamma. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with Galpha(i1) in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Galpha(i1) reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and Galpha(i1)-YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and alpha(2)-adrenergic receptor-Venus, which were Galpha(i)-dependent and reduced by agonist, indicating that AGS4-Galpha(i) complexes are receptor-proximal. These data suggest that AGS4-Galpha(i) complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.

Milk thistle in liver diseases: past, present, future

Silybum marianum or milk thistle (MT) is the most well-researched plant in the treatment of liver disease. The active complex of MT is a lipophilic extract from the seeds of the plant and is composed of three isomer flavonolignans (silybin, silydianin, and silychristin) collectively known as silymarin. Silybin is a component with the greatest degree of biological activity and makes up 50% to 70% of silymarin. Silymarin is found in the entire plant but it is concentrated in the fruit and seeds. Silymarin acts as an antioxidant by reducing free radical production and lipid peroxidation, has antifibrotic activity and may act as a toxin blockade agent by inhibiting binding of toxins to the hepatocyte cell membrane receptors. In animals, silymarin reduces liver injury caused by acetaminophen, carbon tetrachloride, radiation, iron overload, phenylhydrazine, alcohol, cold ischaemia and Amanita phalloides. Silymarin has been used to treat alcoholic liver disease, acute and chronic viral hepatitis and toxin-induced liver diseases.

Somatic localization of a specific large-conductance calcium-activated potassium channel subtype controls compartmentalized ethanol sensitivity in the nucleus accumbens.

Alcohol is an addictive drug that targets a variety of ion channels and receptors. To address whether the effects of alcohol are compartment specific (soma vs dendrite), we examined the effects of ethanol (EtOH) on large-conductance calcium-activated potassium channels (BK) in cell bodies and dendrites of freshly isolated neurons from the rat nucleus accumbens (NAcc), a region known to be critical for the development of addiction. Compartment-specific drug action was indeed observed. Clinically relevant concentrations of EtOH increased somatic but not dendritic BK channel open probability. Electrophysiological single-channel recordings and pharmacological analysis of the BK channel in excised patches from each region indicated a number of differences, suggestive of a compartment-specific expression of the beta4 subunit of the BK channel, that might explain the differential alcohol sensitivity. These parameters included activation kinetics, calcium dependency, and toxin blockade. Reverse transcription-PCR showed that both BK channel beta1 and beta4 subunit mRNAs are found in the NAcc, although the signal for beta1 is significantly weaker. Immunohistochemistry revealed that beta1 subunits were found in both soma and dendrites, whereas beta4 appeared restricted to the soma. These findings suggest that the beta4 subunit may confer EtOH sensitivity to somatic BK channels, whereas the absence of beta4 in the dendrite results in insensitivity to the drug. Consistent with this idea, acute EtOH potentiated alphabeta4 BK currents in transfected human embryonic kidney cells, whereas it failed to alter alphabeta1 BK channel-mediated currents. Finally, an EtOH concentration (50 mm) that increased BK channel open probability strongly decreased the duration of somatic-generated action potential in NAcc neurons.

Determinants of potency on alpha-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors.

Alpha-conotoxin MII, a peptide toxin isolated from Conus magus, antagonizes a subset of neuronal nicotinic receptors. Rat alpha3beta2 receptors, expressed in Xenopus oocytes, are blocked with an IC(50) of 3.7 +/- 0.3 nM. To identify structural features that determine toxin potency, a series of alanine-substituted toxins were synthesized and tested for the ability to block the function of alpha3beta2 receptors. Circular dichroism and protein modeling were used to assess the structural integrity of the mutant toxins. Three residues were identified as major determinants of toxin potency. Replacement of asparagine 5, proline 6, or histidine 12 with alanine resulted in >2700-fold, 700-fold, and approximately 2700-fold losses in toxin potency, respectively. A decrease in pH improved toxin potency, while an increase in pH eliminated toxin blockade, suggesting that, in the active form of the toxin, histidine 12 is charged. The imidazole ring of histidine 12 protrudes from one side, while asparagine 5 and proline 6 are located at the opposite end of the toxin structure. The side chains of these three residues are exposed on the surface of the toxin, suggesting that they directly interact with the alpha3beta2 receptor.

Angiotensin II phosphorylation of extracellular signal-regulated kinases in rat anterior pituitary cells.

We studied the effects of ANG II on extracellular signal-regulated kinase (ERK)1/2 phosphorylation in rat pituitary cells. ANG II increased ERK phosphorylation in a time- and concentration-dependent way. Maximum effect was obtained at 5 min at a concentration of 10-100 nM. The effect of 100 nM ANG II was blocked by the AT1 antagonist DUP-753, by the phospholipase C (PLC) inhibitor U-73122, and by the MAPK kinase (MEK) antagonist PD-98059. The ANG II-induced increase in phosphorylated (p)ERK was insensitive to pertussis toxin blockade and PKC depletion or inhibition. The effect was also abrogated by chelating intracellular calcium with BAPTA-AM or TMB-8 by depleting intracellular calcium stores with a 30-min pretreatment with EGTA and by pretreatment with herbimycin A and PP1, two c-Src tyrosine kinase inhibitors. It was attenuated by AG-1478, an inhibitor of epidermal growth factor receptor (EGFR) activation. Therefore, in the rat pituitary, the increase of pERK is a Gq- and PLC-dependent process, which involves an increase in intracellular calcium and activation of a c-Src tyrosine kinase, transactivation of the EGFR, and the activation of MEK. Finally, the response of ERK activation by ANG II is altered in hyperplastic pituitary cells, in which calcium mobilization evoked by ANG II is also modified.

Reciprocal Modulation between the α and β4 Subunits of hSlo Calcium-dependent Potassium Channels

Large conductance Ca(2+)-dependent potassium (K(Ca) or maxi K) channels are composed of a pore-forming alpha subunit and an auxiliary beta subunit. We have shown that the brain-specific beta4 subunit modulates the voltage dependence, activation kinetics, and toxin sensitivity of the hSlo channel (Weiger, T. M., Holmqvist, M. H., Levitan, I. B., Clark, F. T., Sprague, S., Huang, W. J., Ge, P., Wang, C., Lawson, D., Jurman, M. E., Glucksmann, M. A., Silos-Santiago, I., DiStefano, P. S., and Curtis, R. (2000) J. Neurosci. 20, 3563-3570). We investigated here the N-linked glycosylation of the beta4 subunit and its effect on the modulation of the hSlo alpha subunit. When expressed alone in HEK293 cells, the beta4 subunit runs as a single molecular weight band on an SDS gel. However, when coexpressed with the hSlo alpha subunit, the beta4 subunit appears as two different molecular weight bands. Enzymatic deglycosylation or mutation of the N-linked glycosylation residues in beta4 converts it to a single lower molecular weight band, even in the presence of the hSlo alpha subunit, suggesting that the beta4 subunit can be present as an immature, core glycosylated form and a mature, highly glycosylated form. Blockage of protein transport from the endoplasmic reticulum to the Golgi compartment with brefeldin A abolishes the mature, highly glycosylated beta4 band. Glycosylation of the beta4 subunit is not required for its binding to the hSlo channel alpha subunit. It also is not necessary for cell membrane targeting of the beta4 subunit, as demonstrated by surface biotinylation experiments. However, the double glycosylation site mutant beta4 (beta4 N53A/N90A) protects the channel less against toxin blockade, as compared with the hSlo channel coexpressed with wild type beta4 subunit. Taken together, these data show that the pore-forming alpha subunit of the hSlo channel promotes N-linked glycosylation of its auxiliary beta4 subunit, and this in turn influences the modulation of the channel by the beta4 subunit.

Mitral cell presynaptic Ca(2+) influx and synaptic transmission in frog amygdala.

Dextran-conjugated Ca(2+) indicators were injected into the accessory olfactory bulb of frogs in vivo to selectively fill presynaptic terminals of mitral cells at their termination in the ipsilateral amygdala. After one to three days of uptake and transport, the forebrain hemisphere anterior to the tectum was removed and maintained in vitro for simultaneous electrophysiological and optical measurements. Ca(2+) influx into these terminals was compared to synaptic transmission between mitral cells and amygdala neurons under conditions of reduced Ca(2+) influx resulting from reduced extracellular [Ca(2+)], blockade of N- and P/Q-type channels, and application of the cholinergic agonist carbachol. Reducing extracellular [Ca(2+)] had a non-linear effect on release; release was proportional to Ca(2+) influx raised to the power of approximately 3.6, as observed at numerous other synapses. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1 microM), blocked 77% of Ca(2+) influx and 88% of the postsynaptic field potential. The P/Q-type Ca(2+) channel blocker, omega-agatoxin-IVA (200 nM), blocked 19% of Ca(2+) influx and 25% of the postsynaptic field, while the two toxins combined to block 92% of Ca(2+) influx and 97% of the postsynaptic field. The relationship between toxin blockade of Ca(2+) influx and synaptic transmission was therefore only slightly non-linear; release was proportional to Ca(2+) influx raised to the power approximately 1.4. Carbachol (100 microM) acting via muscarinic receptors had no effect on the afferent volley, but rapidly and reversibly reduced Ca(2+) influx through both N- and P/Q-type channels by 51% and postsynaptic responses by 78%, i.e. release was proportional to Ca(2+) raised to the power approximately 2.5. The weak dependence of release on changes in Ca(2+) when channel toxins block channels suggests little overlap between Ca(2+) microdomains from channels supporting release or substantial segregation of channel subtypes between terminals. The proportionately greater reduction of transmission by muscarinic receptors compared to Ca(2+) channel toxins suggests that they directly affect the release machinery in addition to reducing Ca(2+) influx.

Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells.

Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell.

Nicotinic Acetylcholine Receptors Containing α7 Subunits Are Required for Reliable Synaptic TransmissionIn Situ

Nicotinic acetylcholine receptors containing alpha7 subunits are widely expressed in the nervous system. The receptors are cation-selective, relatively permeable to calcium, and avid binders of alpha-bungarotoxin. Although the receptors can act both pre- and postsynaptically, their physiological significance is unclear. Using whole-cell patch-clamp analysis of chick ciliary ganglion neurons in situ, we show that the receptors are required for reliable synaptic transmission early in development. Stimulation of the presynaptic nerve root elicited a biphasic synaptic current, including a large rapidly decaying component generated by alpha7-containing receptors. Selective blockade of alpha7-containing receptors by perfusing the ganglion with alpha-bungarotoxin induced failures in synaptic transmission. One-half of the ciliary neurons that were tested failed when stimulated synaptically at 1 Hz, and two-thirds failed at 25 Hz. Failing cells missed, on average, 80% of the trials during a test train of stimuli. The ability to fire synaptically evoked action potentials after toxin treatment was correlated positively with the amplitude of the remaining synaptic current, suggesting that alpha7-containing receptors were needed to augment synaptic responses. Consistent with patch-clamp analysis, toxin blockade reduced the amplitude of the synaptically evoked compound action potential in the postganglionic nerve; it also desynchronized the firing of the remaining units. Methyllycaconitine, another antagonist of alpha7-containing receptors, mimicked alpha-bungarotoxin blockade. Toxin blockade had less impact on transmission in ganglia at the end of embryogenesis. The ability of the receptors to synchronize and sustain population firing, together with their ability to deliver calcium, may influence early developmental events such as target innervation and neuronal survival.

Sea anemone toxins as templates for the design of immunosuppressant drugs

A new class of K channel peptide toxin has been found in sea anemones. So far, four homologous ‘short’ toxins possessing 35–37 residues and 3 disulfide bonds have been isolated and sequenced. The two most thoroughly investigated members of this toxin group are ShK and BgK. The covalent structures of these toxins display no homology with the scorpion K toxins and their disulfide bonding pattern is also different. The NMR-derived solution structures of ShK and BgK toxins are similar in that two short alpha-helices are present, in contrast to the predominantly beta-sheet structures found in scorpion toxins. While BgK blocks Kv1.1,1.2 and 1.3 homomeric channels almost equipotently at 6–15 nM concentrations, ShK blocks Kv1.1 and 1.3 channels at very low (20–100 pM) concentrations and Kv1.2 at a much higher (>10 nM) concentration. The sea anemone peptides also interact with Kv1 heteromers present in rat brain membranes. Additionally, ShK has been shown to block intermediate conductance K(Ca) channels in lymphocytes and erythrocytes. Kv1.3 homomers are localized largely on the surfaces of T-lymphocytes; scorpion toxin blockade of these channels has been shown to inhibit proliferation of these cells in vitro and the development of delayed type hypersensitivity reaction and antibody formation in response to an allogeneic challenge in vivo. ShK toxin also inhibits in vitro lymphocyte proliferation in response to an antibody directed to a lymphocyte membrane antigen. The homomeric lymphocyte Kv1.3 and heteromeric brain Kv1 channel binding domains of ShK and BgK have been located by ‘alanine scans’ of analogs prepared by solid-phase peptide synthesis. Both toxins possess a receptor-binding ‘hot spot’ consisting of a sequentially contiguous Lys-Tyr diad on the second helical segment which spatially resembles a diad consisting of Lys27 and Tyr36 attached to a beta-sheet scaffold in charybdotoxin and related scorpion toxins. From analysis of the structural requirements for blocking Kv1.3 homomers and brain Kv1 heteromers, ShK analogs possessing even greater selectivity for the Kv1.3 channel have been designed. These peptides and smaller peptide-mimetic compounds incorporating the anemone toxin pharmacophore are being developed as potential immunosuppressant agents.

Ca 2+ permeability and Joro spider toxin sensitivity of AMPA and kainate receptors on cerebellar granule cells

We have investigated the Ca 2+ permeability of native kainate- and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate- (AMPA) receptors in cultured rat cerebellar granule cells. Intracellular Ca 2+ ([Ca 2+] i) increases and Mn 2+ quench of fura-2 (a measure of Ca 2+ entry) mediated by kainate receptors were completely dependent on the presence of extracellular Na +. Kainate receptor-mediated [Ca 2+] i rises were reduced 37% by the L-type voltage-gated Ca 2+ channel blocker nifedipine (1 μM). AMPA receptor-mediated [Ca 2+] i rises observed in Na +-free buffer were sensitive to Joro spider toxin (500 nM) blockade showing a 65% reduction, while kainate receptor-mediated [Ca 2+] i responses were largely insensitive. These results suggest that a component of AMPA receptor-mediated [Ca 2+] i increases occurs through Ca 2+ permeable receptors which lack the GluR2 subunit and are Joro spider toxin sensitive. In contrast, kainate receptors do not appear to directly gate significant Ca 2+ but raise [Ca 2+] i through activation of voltage-gated Ca 2+ channels and seem largely insensitive to Joro spider toxin.

Determinants of specificity for alpha-conotoxin MII on alpha3beta2 neuronal nicotinic receptors.

The competitive antagonist alpha-conotoxin-MII (alpha-CTx-MII) is highly selective for the alpha3beta2 neuronal nicotinic receptor. Other receptor subunit combinations (alpha2beta2, alpha4beta2, alpha3beta4) are >200-fold less sensitive to blockade by this toxin. Using chimeric and mutant subunits, we identified amino acid residues of alpha3 and beta2 that participate in determination of alpha-CTx-MII sensitivity. Chimeric alpha subunits, constructed from the alpha3 and alpha4 subunits, as well as from the alpha3 and alpha2 subunits, were expressed in combination with the beta2 subunit in Xenopus laevis oocytes. Chimeric beta subunits, formed from the beta2 and beta4 subunits, were expressed in combination with alpha3. Determinants of alpha-CTx-MII sensitivity on alpha3 were found to be within sequence segments 121-181 and 181-195. The 181-195 segment accounted for approximately half the difference in toxin sensitivity between receptors formed by alpha2 and alpha3. When this sequence of alpha2 was replaced with the corresponding alpha3 sequence, the resulting chimera formed receptors only 26-fold less sensitive to alpha-CTx-MII than alpha3beta2. Site-directed mutagenesis within segment 181-195 demonstrated that Lys185 and Ile188 are critical in determination of sensitivity to toxin blockade. Determinants of alpha-CTx-MII sensitivity on beta2 were mapped to sequence segments 1-54, 54-63, and 63-80. Site-directed mutagenesis within segment 54-63 of beta2 demonstrated that Thr59 is important in determining alpha-CTx-MII sensitivity.

Agonist and toxin sensitivities of ACh-evoked currents on neurons expressing multiple nicotinic ACh receptor subunits.

1. We have investigated the pharmacological properties of functional nicotinic acetylcholine receptors (nAChRs) on neonatal rat sympathetic neurons from the superior cervical ganglion (SCG) to learn more about the subunit composition of these receptors. These neurons express five nAChR transcripts: alpha 3, alpha 5, alpha 7, beta 2, and beta 4; this finding suggests that SCG neurons may express several different, physiologically distinct, subtypes of nAChRs. 2. To identify potential subtypes, we have characterized currents evoked by different nicotinic agonists and determined their sensitivity to blockade by alpha-bungarotoxin (alpha-BTX) and by neuronal bungarotoxin (n-BTX). From dose-response curves, we find that the ED50 for both cytisine and dimethylphenylpiperazinium (DMPP) is 20 microM and for ACh is 52 microM. n-BTX blocks the ACh-gated currents rapidly, but the kinetics for n-BTX removal is dependent on the duration of the application: brief applications were quickly reversible, whereas prolonged applications took orders of magnitude longer to reverse. 3. Using fast (ms) agonist application, we observed no rapidly desensitizing currents despite the high levels of alpha 7 in these neurons, nor did we observe any currents that could be blocked by alpha-BTX. 4. Using Xenopus oocytes expressing alpha 7 receptors, we show that choline evokes a significant current that is blocked by alpha-BTX. In contrast, choline is much less potent on alpha 3 beta 4 receptors expressed in Xenopus oocytes. Choline can also act as a weak agonist for nAChRs on rat SCG neurons, but its evoked current is not blocked by alpha-BTX. 5. Our results indicate that, when measured at the macroscopic level, most functional nAChRs on SCG neurons behave as a uniform population of receptors, at least with respect to agonist activation and toxin blockade. In comparison with known receptors expressed in heterologous systems, the physiological properties of ACh-evoked currents on SCG neurons are most similar to receptors that have coassembled with both beta 2 and beta 4.

A role for cGMP during tetanus toxin blockade of acetylcholine release in the rat pheochromocytoma (PC12) cell line

In order to identify the specific molecular mechanisms involved in neurosecretion, we investigated the mechanism of action of tetanus toxin, a potent presynaptic neurotoxin, in the rat adrenal pheochromocytoma PC12 cell line. It has recently been reported that tetanus toxin is a potent inhibitor of the release of depolarization-evoked 3H-acetylcholine (ACh) from nerve growth factor-differentiated PC12 cells (Sandberg et al., 1989a). In PC12 cells, as in many neural tissue preparations, cGMP accumulation in intact cells increased 6- to 17-fold when stimulated with veratridine (200 microM), carbachol (1 mM), Ba2+ (2 mM), or K+ (30 mM). Preincubation of the cells with tetanus toxin inhibits this accumulation by greater than 95%. The toxin dose-inhibition curves for 3H-ACh release and cGMP accumulation are similar, with half-maximal doses of tetanus toxin seen at approximately 5 nM. The time courses for the development of the effects of tetanus on 3H-ACh release and on cGMP accumulation were also similar. Protocols which elevated intracellular cGMP levels reversed the action of the toxin. For example, evoked ACh release was restored in intoxicated PC12 cells by a 15 min exposure to 100 microM 8-bromo-cGMP. The half-maximal dose was observed at 50 microM nucleotide. Examination of the nucleotide specificity revealed that only cyclic guanine analogs were effective in reversing the effects of tetanus toxin. These results suggested that the inhibition of depolarization-evoked cGMP accumulation is causally related to the action of tetanus toxin on neurosecretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pertussis toxin treatment on metabolism in hamster brown adipocytes

This communication reports the effects of the exotoxin of Bordetella pertussis (pertussis toxin) on hamster brown fat cells. Pertussis toxin significantly increased the lipolytic and respiratory responses to isoproterenol but did not increase the basal rates of either of these processes. In contrast, the stimulation of respiration by the alpha-adrenergic agent phenylephrine was not altered by pertussis toxin. The inhibitory effects of adenosine on stimulated lipolysis, respiration, and adenylate cyclase activity were completely abolished by pertussis toxin, as was the ability of methylxanthines or adenosine deaminase to potentiate isoproterenol stimulation of respiration or lipolysis. These effects of pertussis toxin were associated with an ADP ribosylation of a single membrane protein having a molecular weight of approximately 41. These data demonstrate that pertussis toxin can prevent the inhibitory action of adenosine on brown fat cells and suggest that the effects of the nucleoside on these cells results from inhibition of adenylate cyclase. We further suggest that the enhanced responses to isoproterenol in pertussis-treated adipocytes results from a blockade of the action of endogenous adenosine. In addition to blocking adenosine action, pertussis toxin also abolished the antilipolytic effect of insulin. However, because the antilipolytic effect of insulin was prevented by adenosine deaminase and 3-isobutyl-1-methylxanthine and restored by 2-chloroadenosine, we conclude that insulin action on these cells is dependent on adenosine. Thus pertussis toxin blockade of insulin action appears to be secondary to blockade of adenosine action.

Further evidence that extrinsic acetylcholine acts preferentially on extrajunctional receptors in the chick biventer cervicis muscle

The specificity of action of extrinsic acetylcholine on extrajunctional and junctional receptors in the chick biventer cervicis muscle was studies by determining its ability to protect the responses evoked by acetylcholine and by tetanic nerve stimulation from the blockade by α-bungarotoxin, an irreversible binding agent of acetylcholine receptors. At concentrations of 50–100 μg/ml, acetylcholine caused a desensitization to extrinsic acetylcholine but not to nerve stimulation and protected only the contractile response to extrinsic acetylcholine from the toxin blockade whereas neither the response to tetanic nerve stimulation nor the endplate potentials were protected. For the protection of the latter, higher concentrations of acetylcholine were needed. In the presence of physostigmine, a concentration of acetylcholine as low as 10 μg/ml protected the endplate potentials from the toxin blockade. By contrast, d-tubocurarine protected the tetanic contraction and the endplate potentials induced by nerve stimulation at a concentration which produced the same protection of acetylcholine-induced contraction as that produced by 50–100 μg/ml acetylcholine. These results indicate that in contrast to d-tubocurarine, extrinsic acetylcholine at low concentrations acts preferentially on the extrajunctional receptors in the absence of an anticholinesterase.