CBD Suppression of EAE Is Correlated with Early Inhibition of Splenic IFN-γ + CD8+ T Cells and Modest Inhibition of Neuroinflammation

Journal of Neuroimmune Pharmacology - In this study cannabidiol (CBD) was administered orally to determine its effects and mechanisms in the experimental autoimmune encephalomyelitis (EAE) model of...     

Is timing everything? Therapeutic potential of modulators of cardiac Na+ transporters

Sodium ion (Na⁺) transporters have roles in the modulation of cardiomyocyte pH and Na⁺ and Ca²⁺ handling. Activation of the cardiac Na⁺-H⁺ exchanger 1 (NHE1) during ischaemia induces arrhythmias, myocardial stunning and irreversible cell injury. As the benefits of NHE1 inhibitors (e.g., amiloride, cariporide) in models of myocardial infarction are usually much greater when used as pretreatment, rather than during or after ischaemia, it is probably not surprising that clinical trials with cariporide in ischaemia have shown little shortterm benefit. NHE1 inhibitors have been shown to be beneficial in animal models of ventricular fibrillation and resuscitation, cardioplegia, hypertrophy and heart failure, and their therapeutic potential in these conditions should be further developed. The Na⁺-HCO₃^- cotransporter (NBC) is also stimulated by intracellular acidification, and part of the benefit of angiotensin-converting enzyme inhibitors after myocardial infarction may be due to inhibition of the NBC. Selective inhibitors of the NBC are required to determine the therapeutic potential of this mechanism. The Na⁺-Ca²⁺ exchanger (NCX) has a major role in cardiac Na⁺ and Ca²⁺ homeostasis and influences cardiac electrical activity. The NCX also has a role in ischaemia/infarction, arrhythmias, hypertrophy and heart failure. NCX inhibitors may have beneficial effects in animal models of ischaemia and reperfusion injury and the therapeutic benefit of these should be further studied in animal models.     

Effect of K+ and Rb+ on the action of verapamil on a voltage-gated K+ channel,hKv1.3: implications for a second open state?

Background and purpose:

Verapamil blocks current through the voltage-gated K⁺ channel K_v1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity.

Experimental“ approach:

We investigated the influence of intra- and extracellular K⁺ and Rb⁺ on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hK_v1.3 channels.

Key results:

Verapamil affinity was highest in high intracellular K⁺ concentrations ([K⁺]_i) and lowest in low [Rb⁺]_i, indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K⁺ and Rb⁺ could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate.

Conclusions and implications:

Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hK_v1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state.     

Inhibitor of PI3Kγ ameliorates TNBS-induced colitis in mice by affecting the functional activity of CD4+CD25+FoxP3+ regulatory T cells

BACKGROUND AND PURPOSE

Phosphoinositide 3-kinase-γ (PI3Kγ) is implicated in many pathophysiological conditions, and recent evidence has suggested its involvement in colitis. In the present study, we investigated the effects of AS605240, a relatively selective PI3Kγ inhibitor, in experimental colitis and its underlying mechanisms.

EXPERIMENTAL APPROACH

Acute colitis was induced in mice by treatment with trinitrobenzene sulphonic acid (TNBS), and the effect of AS605240 on colonic injury was assessed. Pro-inflammatory mediators and cytokines were measured by immunohistochemistry, elisa, real time-polymerase chain reaction and flow cytometry.

KEY RESULTS

Oral administration of AS605240 significantly attenuated TNBS-induced acute colitis and diminished the expression of matrix metalloproteinase-9 and vascular endothelial growth factor. The colonic levels and expression of IL-1β, CXCL-1/KC, MIP-2 and TNF-α were also reduced following therapeutic treatment with AS605240. Moreover, AS605240 reduced MIP-2 levels in a culture of neutrophils stimulated with lipopolysaccharide. The mechanisms underlying these actions of AS605240 are related to nuclear factor-κ (NF-κB) inhibition. Importantly, the PI3Kγ inhibitor also up-regulated IL-10, CD25 and FoxP3 expression. In addition, a significant increase in CD25 and FoxP3 expression was found in isolated lamina propria CD4⁺ T cells of AS605240-treated mice. The effect of AS605240 on Treg induction was further confirmed by showing that concomitant in vivo blockade of IL-10R significantly attenuated its therapeutic activity.

CONCLUSIONS AND IMPLICATIONS

These results suggest that AS605240 protects mice against TNBS-induced colitis by inhibiting multiple inflammatory components through the NF-κB pathway while simultaneously inducing an increase in the functional activity of CD4⁺CD25⁺ Treg. Thus, AS605240 may offer a promising new therapeutic strategy for the treatment of inflammatory bowel diseases.     

An evaluation of memantine ER + donepezil for the treatment of Alzheimer’s disease

ABSTRACT

Introduction: Alzheimer’s disease (AD) is the most common neurodegenerative disease worldwide and carries an immense societal burden. Unfortunately, no curative or disease-modifying treatment has yet been discovered. The currently approved medications are symptomatic. They include two classes: the cholinesterase inhibitors, such as donepezil, and the NMDA receptor antagonist memantine. Most evidence has shown that combining both classes is superior to monotherapy but may complicate the treatment regimen for patients and families. Namzaric®, a fixed dose combination of donepezil and memantine extended-release (ER) (FDC memantine ER/donepezil), was recently approved by the U.S. Food and Drug Administration (FDA) for patients with moderate to severe AD and warrants further consideration as a clinically useful and advantageous pharmacotherapy in AD.

Areas covered: This review discusses the pharmacological properties, efficacy, and safety/tolerability data of this FDC memantine ER/donepezil as well as its benefits and disadvantages for patients and families. A literature search using PubMed was conducted using Namzaric, donepezil, memantine, AD, and medication adherence as keywords.

Expert opinion: Aside from its cost, FDC memantine ER/donepezil improves adherence to medication and reduces caregiver burden. It allows patients to benefit from combination therapy as the disease progresses, especially in those with dysphagia, poor adherence and limited caregiver support.     

Nanoprobing of the Effect of Cu2+ Cations on Misfolding, Interaction and Aggregation of Amyloid β Peptide

Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer’s disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu²⁺. Cu²⁺ cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu²⁺ regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu²⁺ cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu²⁺ cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu²⁺ cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu²⁺ cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu²⁺ cations also stabilize the dimers. No effects of Cu²⁺ cations on Aβ-Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu²⁺ cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu²⁺ cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu²⁺ cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu²⁺ cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu²⁺.     

Levcromakalim- and Isoprenaline-induced Relaxation of Human Isolated Airways—Role of the Epithelium and of K+ Channel Activation

Summary: In this study we have investigated the mechanism of action of levcromakalim and isoprenaline in human isolated airways with respect to the K⁺ channels they activate and the possibility that these smooth muscle relaxants activate K⁺ channels on the airway epithelium. Mechanical removal of the epithelial layer (mean percentage of epithelium present 20±3%, n=20 tissues) did not affect the relaxation responses to levcromakalim or isoprenaline, either in terms of maximal relaxation or sensitivity. Whilst having no effect on isoprenaline-induced relaxation, studied from basal tone, the ATP-sensitive K⁺ channel blocker BRL 31660 (10, 30 and 50 μM) reduced relaxation responses induced (from basal tone) by levcromakalim from 74±6% (of the maximal response to isoprenaline) to 48±12% (n=7), 9±9% (n=4) and 0 (n=4), respectively. Charybdotoxin, a blocker of high conductance Ca²⁺-activated K⁺ channels, at concentrations of 30 and 100 nM, had no effect on either levcromakalim- or isoprenaline-induced relaxation responses and yet charybdotoxin was active at K_Ca channels in outside-out patches of hippocampal granule cells. Moreover, tetraethylammonium (10 mM) inhibited neither isoprenaline- nor levcromakalim-induced relaxation. This study has demonstrated that the relaxation responses elicited in human bronchus to isoprenaline and levcromakalim are likely to be the result of direct effects on the smooth muscle with no contribution from epithelial receptors or K⁺ channels. The actions of levcromakalim appear to be mediated only via activation of K_ATP channels. Further, we have made the important observation that, under the experimental conditions of our study, isoprenaline does not activate the K_Ca channel to produce relaxation in human bronchus.     

Ca2+ regulation of inositol 1,4,5-trisphosphate receptors: can Ca2+ function without calmodulin?

All Ca(2+) channels are regulated by Ca(2+), a feature that allows them to respond to their own activity and to the activities of neighboring Ca(2+) channels. Inhibition by Ca(2+) protects cells from potentially hazardous increases in cytosolic [Ca(2+)], and stimulation can mediate facilitation and regenerative propagation of Ca(2+) signals. Calmodulin is emerging as a key player in regulation of Ca(2+) channels by Ca(2+), but its role is more complex and more beautiful than might have been imagined.     

Effects of Sr2+ and Mg2+ on the phospholipase A and the presynaptic neuromuscular blocking actions of β-bungarotoxin,crotoxin and taipoxin

1.beta-Bungarotoxin, crotoxin and taipoxin, presynaptic neurotoxins of snake venom origin, have about the same phospholipid-splitting activities as a much less toxic cobra phospholipase A2 in the presence of Ca2+ and deoxycholate. 2. Sr2+ was a much less effective activator of the enzymes than is Ca2+, the activation by Sr2+ being only 3-6% for beta-bungarotoxin and crotoxin and 12% for taipoxin. 3. Sr2+ also inhibited the Ca2+ -activated enzymes by 80% in the cases of beta-bungarotoxin and crotoxin, but only 16% in the case of taipoxin. 4. Mg2" had no significant effect on beta-bungarotoxin or crotoxin, but activated taipoxin in the presence or absence of Ca2". 5. In Sr2+ -Tyrode lacking Ca2+ all three toxins exhibited the same immediate depression followed by facilitation in the rat and mouse diaphragms, but the final blocking activity was only 3-10% with beta-bungarotoxin and crotoxin and was 30% with taipoxin. 6. In Sr2+ -Tyrode, increasing in the rate of nerve stimulation had less accelerating effect on the development of neuromuscular block than in Ca2+ -Tyrode for any of the toxins. 7. Removal of Mg2+ from Sr2+ -Tyrode did not diminish the potency of taipoxin in blocking neuromuscular transmission, suggesting that enzyme activity at the outer surface of the axolemma does not contribute to the neuromuscular blocking action. 8. All of the results indicate that there are close correlations between the presynaptic activities of these toxins and their phospholipid-splitting activities in the cationic environment prevailing in the axoplasm. Apparently the final blocking effect of these toxins is due to phospholipase A action within the nerve terminal.     

The H₁-H₂ domain of the α₁ isoform of Na+-K+-ATPase is involved in ouabain toxicity in rat ventricular myocytes

Metals and metalloid species are involved in homeostasis in energy systems such as glucose metabolism. Enlarged adipocytes are one of the most important causes of obesity-associated diseases. In this study, we studied the possibility that various metals, namely, CoCl₂, HgCl₂, NaAsO₂ and MnCl₂ pose risk to or have beneficial effects on white adipose tissue (WAT). Exposure to the four metals resulted in decreases in WAT weight and the size of enlarged adipocytes in mice fed a high-fat diet (HFD) without changes in liver weight, suggesting that the size and function of adipocytes are sensitive to metals. Repeated administration of CoCl₂ significantly increased serum leptin, adiponectin and high-density lipoprotein (HDL) cholesterol levels and normalized glucose level and adipose cell size in mice fed HFD. In contrast, HgCl₂ treatment significantly decreased serum leptin level with the down-regulation of leptin mRNA expression in WAT and a reduction in adipocyte size. Next, we tried to investigate possible factors that affect adipocyte size. Repeated exposure to HgCl₂ significantly decreased the expression levels of factors upon the regulation of energy such as the PPARα and PPARγ mRNA expression levels in adipocytes, whereas CoCl₂ had little effect on those genes expressions compared with that in the case of the mice fed HFD with a vehicle. In addition, repeated administration of CoCl₂ enhanced AMPK activation in a dose-dependent manner in the liver, skeletal muscle and WAT; HgCl₂ treatment also enhanced AMPK activation in the liver. Thus, both Co and Hg reduced WAT weight and the size of enlarged adipocytes, possibly mediated by AMKP activation in the mice fed HFD. However, inorganic cobalt may have a preventive role in obesity-related diseases through increased leptin, adiponectin and HDL-cholesterol levels, whereas inorganic mercury may accelerate the development of such diseases. These results may lead to the development of new approaches to establishing the role of metals in adipose tissue of obesity-related diseases.     

Treatment of colorectal cancer by anticancer and antibacterial effects of hemiprotonic phenanthroline-phenanthroline+ with nanomicelle delivery

Colorectal cancer (CRC) is a common digestive tract tumor worldwide. Specific microorganisms, including Fusobacterium nucleatum (F. nucleatum) and Escherichia coli (E. coli), are abundant in colonic mucosa and can promote the cancer progression and malignancy. Therefore, a therapeutic strategy is proposed to deliver effective drugs to colorectum for both anticancer and antibacteria. Here we used thin-film dispersion method to encapsulate hemiprotonic phenanthroline-phenanthroline⁺ (ph-ph⁺) into nanomicelle. The results showed that the drug-loading nanomicelle had good dispersion, and the particle size was about 28 nm. In vitro assay indicated that the nanomicelle was active against CRC-related obligate and facultative anaerobes. In human CRC cells, the nanomicelle could effectively inhibit cell proliferation and induce apoptosis. In vivo distribution showed that the nanomicelle could release ph-ph⁺ mainly in the colorectum. In CRC model mice, the nanomicelle significantly reduced tumor number and volume, and decreased the bacteria load and colorectal inflammation. Together, the study identifies that the ph-ph⁺nanomicelle has the potential to apply in treating CRC, and also suggests that anticancer combined with antimicrobial therapy would be a feasible way for CRC therapy.     

Forefront of Na+/Ca2+ exchanger studies: stoichiometry of cardiac Na+/Ca2+ exchanger; 3:1 or 4:1?

The stoichiometry of the Na+/Ca2+ exchanger (NCX) had been generally accepted as 3 Na+:1 Ca2+. However, recently a challenging stoichiometry of 4:1 was proposed. Therefore, using guinea pig ventricular cells, we re-examined the stoichiometry by measuring the reversal potential of the NCX current and intracellular Ca2+ concentrations under the whole-cell voltage clamp. We confirmed that the stoichiometry of NCX is 3:1 not 4:1. In addition, we explored the possible reasons for obtaining erroneous results of a 4:1 stoichiometry.     

碳酸饮料成份健康审查：水+糖浆+二氧化碳+色香味添加剂+防腐剂

加入甜水,充入二氧化碳气体——这就是碳酸饮料的制造工艺。这种发展最早、产量最高的软饮料,借“酷爽“之名,已然深入人心。看看这些果汁型、果味型、可乐型、低热量型的碳酸饮料,都是什么在撑腰——你迷恋的碳酸饮料,是这样炼成的——Step 1:调味糖浆=原糖浆+苯甲酸钠+甜味剂+柠檬酸+果汁+香精+色素Step 2:预调甜水=水+调味糖浆(冷却混合)     

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 μM evoked a robust [Ca²⁺]_i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 μM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca²⁺ medium. Removal of extracellular Ca²⁺ reduced the Ca²⁺ signals evoked by 50–100 μM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca²⁺ release and also extracellular Ca²⁺ influx. A concentration of 100 μM SKF 96365 caused significant Mn²⁺ quench of fura-2 fluorescence, which was partly inhibited by La³⁺ (1 mM) or Gd³⁺ (0.1 mM), indicating that the SKF 96365-induced Ca²⁺ influx had two components: one is sensitive to La³⁺ (1 mM) or Gd³⁺ (0.1 mM), the other is not. The internal Ca²⁺ source for the SKF 96365-induced [Ca²⁺]_i transient was the endoplasmic reticulum Ca²⁺ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump nearly abolished the SKF 96365-induced [Ca²⁺]_i increase in Ca²⁺-free medium. In contrast, pretreatment with 100 μM SKF 96365 only partly depleted the thapsigargin-sensitive Ca²⁺ store. Addition of 10 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after prior incubation with 100 μM SKF 96365 in Ca²⁺-free medium, demonstrating that SKF 96365 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was about 40% of that induced by 1 μM thapsigargin. Additional to inducing its own capacitative Ca²⁺ entry, 100 μM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca²⁺ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca²⁺]_i transient. Inhibition of the plasma membrane Ca²⁺ pump with La³⁺ or Gd³⁺, or lowering extracellular Na⁺ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca²⁺]_i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca²⁺-free medium, the thapsigargin-induced [Ca²⁺]_i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca²⁺ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca²⁺ signaling. It induced a considerable increase in [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum store followed by capacitative Ca²⁺ entry. It also caused a direct Ca²⁺ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca²⁺ pump or Na⁺/Ca²⁺ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca²⁺ entry. Received: 21 September 1998 / Accepted: 2 December 1998     

A comparative analysis of the time course of cardiac Ca2+ current response to rapid applications of β-adrenergic and dihydropyridine agonists

Summary A fast perfusion system was used to analyze the kinetics of the response of L-type calcium current (I_Ca) to rapid exposures to -adrenergic or dihydropyridine agonists in whole-cell patch-clamped frog ventricular myocytes. The perfusion system was based on the lateral motion of an array of plastic capillary tubes from which solutions flowed at a velocity of 5 cm/s. Movement from one capillary to the adjacent one occurred in < 20 ms and complete exchange of extracellular solution was achieved in < 50 ms as demonstrated by the block of I_Ca by fastflow application of Cd during a depolarizing pulse. Fastflow applications of increasing concentrations of isoprenaline (Iso) led to a dose-dependent stimulation of I_Ca at [Iso] > 1 nM. The response of I_Ca to Iso always started after a delay of several seconds. The delay duration decreased as [Iso] increased, and was typically 3 s at 10 M Iso. The rising phase of I_Ca increase was monophasic and independent of [Iso] > 100 nM. For short applications of Iso (8.8 s), half maximal and maximal stimulation of I_Ca occurred 20 s and 40 s after the beginning of Iso application, respectively. When Iso was applied during a depolarizing pulse (with Ba as the charge carrier), I_Ba never increased during that pulse. The kinetics of the I_Ca response to Iso were not affected by varying the voltage clamp protocols or the ionic composition of intracellular and extracellular solutions. In comparison with the effects of Iso, the stimulatory effect of the dihydropyridine agonist (–)Bay K 8644 on I_Ca was 15 times faster: delay, half-time to maximal and time to maximal responses were 15 times shorter with (–)Bay K 8644 than with Iso. It is concluded that frog ventricular myocytes respond slowly to a quick application of -adrenergic agonists. Correspondence to: R. Fischmeister at the above address     

Effect of K+ channel blockers on the α2-adrenoceptor-coupled regulation of electrically evoked noradrenaline release in hippocampus

Summary The question whether presynaptic ₂-adrenoceptors regulating noradrenaline release in hippocampus directly couple to tetraethylammonium chloride (TEA) or -dendrotoxin (-DTX)-sensitive K⁺ channels was investigated. Hippocampal slices, prelabelled with [³H] noradrenaline, were superfused in the presence of (+)-oxaprotiline and electrically stimulated with 4 pulses delivered at 100 Hz, in order to avoid autoinhibition due to released noradrenaline.TEA enhanced the evoked [³H]noradrenaline release in rabbit hippocampus in a concentration-dependent manner, yielding an approximately 4-fold increase at 30 mmol/l, whereas the spontaneous outflow of tritium was only slightly affected at this concentration. The ₂-adrenoceptor agonist clonidine, at 10–100 nmol/l inhibited the evoked [³H]noradrenaline release between 77% and 96%. The inhibitory effect of the ₂-agonist was distinctly diminished in the presence of 30 mmol/l TEA but was restored in low Ca²⁺/high Mg²⁺ buffer. Therefore, the diminution of the ₂-agonist effect by TEA observed in experiments with normal Ca²⁺ can be explained by an increase of the Ca²⁺ availability for the release process due to the prolongation of action potentials. In rabbit hippocampus -DTX (10–200 nmol/l) did neither affect the evoked release of [³H]noradrenaline nor its ₂-agonist-induced modulation. However, in rat hippocampus -DTX significantly increased the evoked transmitter release and diminished the effect of clonidine.Taken together, the present data for the rabbit hippocampus exclude the possibility that activation of presynaptic ₂-adrenoceptors inhibits depolarization-evoked [³H]noradrenaline release by inducing an outward K⁺ current through TEA- or -DTX-sensitive K⁺ channels. However, there are species differences between the rabbit and the rat so that in the rat the ₂-adrenoceptors could actually be coupled to K⁺ channels — provided that the release-enhancing properties of -DTX do not account for the ₂-antagonism observed.Correspondence to C. Allgaier at the above address