Addressing the use of PDIF-CN2 molecules in the development of n-type organic field-effect transistors for biosensing applications.

BACKGROUND: There is no doubt that future discoveries in the field of biochemistry will depend on the implementation of novel biosensing techniques, able to record biophysiological events with minimal biological interference. In this respect, organic electronics may represent an important new tool for the analysis of structures ranging from single molecules up to cellular events. Specifically, organic field-effect transistors (OFET) are potentially powerful devices for the real-time detection/transduction of bio-signals. Despite this interest, up to date, the experimental data useful to support the development of OFET-based biosensors are still few and, in particular, n-type (electron-transporting) devices, being fundamental to develop highly-performing circuits, have been scarcely investigated. METHODS: Here, films of N,N'-1H,1H-perfluorobutyldicyanoperylene-carboxydi-imide (PDIF-CN2) molecules, a recently-introduced and very promising n-type semiconductor, have been evaporated on glass and silicon dioxide substrates to test the biocompatibility of this compound and its capability to stay electrically-active even in liquid environments. RESULTS: We found that PDIF-CN2 transistors can work steadily in water for several hours. Biocompatibility tests, based on in-vitro cell cultivation, remark the need to functionalize the PDIF-CN2 hydrophobic surface by extra-coating layers (i.e. poly-l-lysine) to favor the growth of confluent cellular populations. CONCLUSIONS: Our experimental data demonstrate that PDIF-CN2 compound is an interesting organic semiconductor to develop electronic devices to be used in the biological field. GENERAL SIGNIFICANCE: This work contributes to define a possible strategy for the fabrication of low-cost and flexible biosensors, based on complex organic complementary metal-oxide-semiconductor (CMOS) circuitry including both p- (hole-transporting) and n-type transistors. This article is part of a Special Issue entitled Organic Bioelectronics-Novel Applications in Biomedicine.

Vasorelaxation by hydrogen sulphide involves activation of Kv7 potassium channels.

Hydrogen sulphide (H2S) has been recently hypothesized to be an endogenous adipocyte-derived relaxing factor, evoking vasorelaxation of conductance and resistance vessels. Although the activation of ATP-sensitive potassium channels is known to play a central role in H2S-induced vasorelaxation, activation of vascular Kv7 voltage-gated potassium channels has also been suggested. To investigate this possibility, the ability of selective activators and blockers of distinct classes of potassium channels to affect vasodilation induced by the H2S-donor NaHS, as well as NaHS-induced Rb(+) efflux in endothelium-denuded rat aortic rings, was investigated. NaHS-induced changes of membrane potential were fluorimetrically assessed on human vascular smooth muscle (VSM) cells. Modulation of Kv7.4 channels by NaHS was assessed by electrophysiological studies, upon their heterologous expression in CHO cells. In isolated aortic rings, NaHS evoked vasorelaxing responses associated with an increase of Rb(+)-efflux. NaHS promoted membrane hyperpolarization of human VSM cells. These effects were antagonized by selective blockers of Kv7 channels. The H2S-donor caused a left-shift of current activation threshold of Kv7.4 channels expressed in CHO cells. Altogether, these results suggest that the activation of Kv7.4 channels is a key mechanism in the vascular effects of H2S. Given the relevant roles played by Kv7.4 channels in VSM contractility and by H2S in circulatory homeostasis regulation, these findings provide interesting insights to improve our understanding of H2S pathophysiology and to focus on Kv7.4 channels as novel targets for therapeutic approaches via the "H2S-system".

Towards the realization of label-free biosensors through impedance spectroscopy integrated with IDES technology.

Impedance spectroscopy (IS) is a powerful technique for analysis of the complex electrical impedance of a large variety of biological systems, because it is sensitive both to surface phenomena and to changes of bulk properties. A simple and convenient method of analysis of cell properties by IS is described. An interdigitated electrodes configuration was used for the measurements; human epithelial cells were grown on the device to investigate the complex dielectric response as a function of frequency, in order to test the suitability of the device for use as a label-free biosensor. To test the ability of the device to detect channels in the cell membrane, the effect of drugs known to affect membrane integrity was also investigated. The frequency response of the admittance (i.e. the reciprocal of the impedance) can be well fitted by a model based on very simple assumptions about the cells coating the device surface and the current flow; from the calculations, membrane-specific capacitance and information about cell adhesion can be inferred. These preliminary efforts have shown that our configuration could lead to a label-free non-invasive technique for biosensing and cellular behavior monitoring which might prove useful in investigation of the basic properties of cells and the effect of drugs by estimation of some fundamental properties and modification of the electrical characteristics of the device.

KV7 channels regulate muscle tone and nonadrenergic noncholinergic relaxation of the rat gastric fundus.

Voltage-dependent type 7 K+ (KV7) channels play important physiological roles in neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators in the rat gastric fundus and the expression of KV7 channels in this tissue. Muscle tone and electrical field stimulation (EFS)-evoked relaxations of precontracted longitudinal muscle strips of the rat gastric fundus were investigated under nonadrenergic noncholinergic conditions by organ bath studies. Gene expression was studied by real-time PCR and tissue localization of channels was investigated by immunohistochemistry. The KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean pD2 and Emax of 5.4 and 48% of the maximal U46619-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed U46619-precontracted strips, with pD2s of 4.7 and 4.4 and Emax of 93% and 91% of the maximal relaxation induced by papaverine, respectively. XE-991 concentration-dependently inhibited retigabine-induced relaxation with a pIC50 of 6.2. XE-991 and DMP-543, another KV7 channel blocker, increased by 13-25% or reduced by 11-21% the relaxations evoked by low- or high-frequency EFS, respectively. XE-991 also reduced the relaxation induced by vasoactive intestinal polypeptide (VIP) by 33% of controls. Transcripts encoded by all KV7 genes were detected in the fundus, with 7.4 and 7.5 showing the highest expression levels. KV7.4 and 7.5 channels were visualized by confocal immunofluorescence in both circular and longitudinal muscle layers. In conclusion, in the rat proximal stomach, KV7 channels appear to contribute to the resting muscle tone and to VIP- and high-frequency EFS-induced relaxation. KV7 channel activators could be useful relaxant agents of the gastric smooth muscle.

Specification of pore properties by the carboxyl terminus of inwardly rectifying K+ channels.

Inwardly rectifying potassium (K+) channels (IRKs) maintain the resting membrane potential of cells and permit prolonged depolarization, such as during the cardiac action potential. Inward rectification may result from block of the ion conduction pore by intracellular magnesium (Mgi2+). Two members of this family, IRK1 and ROMK1, which share 40 percent amino acid identity, differ markedly in single-channel K+ conductance and sensitivity to block by Mgi2+. The conserved H5 regions were hypothesized to determine these pore properties because they have this function in voltage-dependent K+ channels and in cyclic nucleotide-gated channels. However, exchange of the H5 region between IRK1 and ROMK1 had no effect on rectification and little or no effect on K+ conductance. By contrast, exchange of the amino- and carboxyl-terminal regions together transferred Mg2+ blockade and K+ conductance of IRK1 to ROMK1. Exchange of the carboxyl but not the amino terminus had a similar effect. Therefore, the carboxyl terminus appears to have a major role in specifying the pore properties of IRKs.

Spermine and spermidine as gating molecules for inward rectifier K+ channels.

Inward rectifier K+ channels pass prominent inward currents, while outward currents are largely blocked. The inward rectification is due to block by intracellular Mg2+ and a Mg(2+)-independent process described as intrinsic gating. The rapid loss of gating upon patch excision suggests that cytoplasmic factors participate in gating. "Intrinsic" gating can be restored in excised patches by nanomolar concentrations of two naturally occurring polyamines, spermine and spermidine. Spermine and spermidine may function as physiological blockers of inward rectifier K+ channels and "intrinsic" gating may largely reflect voltage-dependent block by these cations.

Exchange of conduction pathways between two related K+ channels.

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.

Differences between the deep pores of K+ channels determined by an interacting pair of nonpolar amino acids.

The pore of a chimeric K+ channel, CHM, differed from its parental host channel, Kv2.1, by 9 amino acids. Four were located in a putative deep region and 5 in a nearby outer mouth. Point reversions were without restorative effects, and reversions V369I or L374V in the deep pore produced novel phenotypes. Among double mutations, only V369I and L374V were effective in restoring the Kv2.1 pore phenotype. Adding a change in charge at Q382K in the outer pore fully restored the parental phenotype. Thus, the pore appears to have an inner, deep region where ions such as K+ and TEA+ may be regulated by nonpolar residues and an outer region where ions may be regulated by charged residues.

herg encodes a K+ current highly conserved in tumors of different histogenesis: a selective advantage for cancer cells?

The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) that plays a fundamental role in heart excitability by regulating the action potential repolarization (IKr); mutations of this gene are responsible for the chromosome 7-linked long QT syndrome (LQT2). In this report, we show that in a variety (n = 17) of tumor cell lines of different species (human and murine) and distinct histogenesis (neuroblastoma, rhabdomyosarcoma, adenocarcinoma, lung microcytoma, pituitary tumors, insulinoma beta-cells, and monoblastic leukemia), a novel K+ inward-rectifier current (IIR), which is biophysically and pharmacologically similar to IHERG, can be recorded with the patch-clamp technique. Northern blot experiments with a human herg cDNA probe revealed that both in human and murine clones the very high expression of herg transcripts can be quantified in at least three clearly identifiable bands, suggesting an alternative splicing of HERG mRNA. Moreover, we cloned a cDNA encoding for IIR from the SH-SY5Y human neuroblastoma. The sequence of this cDNA result was practically identical to that already reported for herg, indicating a high conservation of this gene in tumors. Consistently, the expression of this clone in Xenopus oocytes showed that the encoded K+ channel had substantially all of the biophysical and pharmacological properties of the native IIR described for tumor cells. In addition, in the tumor clones studied, IIR governs the resting potential, whereas it could not be detected either by the patch clamp or the Northern blot techniques in cells obtained from primary cell cultures of parental tissues (sensory neurons and myotubes), whose resting potential is controlled by the classical K+ anomalous rectifier current. This current substitution had a profound impact on the resting potential, which was markedly depolarized in tumors as compared with normal cells. These results suggest that IIR is normally only expressed during the early stages of cell differentiation frozen by neoplastic transformation, playing an important pathophysiological role in the regulatory mechanisms of neoplastic cell survival. In fact, because of its biophysical features, IIR, besides keeping the resting potential within the depolarized values required for unlimited tumor growth, could also appear suitable to afford a selective advantage in an ischemic environment.

Do glia have heart? Expression and functional role for ether-a-go-go currents in hippocampal astrocytes.

Potassium homeostasis plays an important role in the control of neuronal excitability, and diminished buffering of extracellular K results in neuronal Hyperexcitability and abnormal synchronization. Astrocytes are the cellular elements primarily involved in this process. Potassium uptake into astrocytes occurs, at least in part, through voltage-dependent channels, but the exact mechanisms involved are not fully understood. Although most glial recordings reveal expression of inward rectifier currents (K(IR)), it is not clear how spatial buffering consisting of accumulation and release of potassium may be mediated by exclusively inward potassium fluxes. We hypothesized that a combination of inward and outward rectifiers cooperate in the process of spatial buffering. Given the pharmacological properties of potassium homeostasis (sensitivity to Cs(+)), members of the ether-a-go-go (ERG) channel family widely expressed in the nervous system could underlie part of the process. We used electrophysiological recordings and pharmacological manipulations to demonstrate the expression of ERG-type currents in cultured and in situ hippocampal astrocytes. Specific ERG blockers (dofetilide and E 4031) inhibited hyperpolarization- and depolarization-activated glial currents, and ERG blockade impaired clearance of extracellular potassium with little direct effect on hippocampal neuron excitability. Immunocytochemical analysis revealed ERG protein mostly confined to astrocytes; ERG immunoreactivity was absent in presynaptic and postsynaptic elements, but pronounced in glia surrounding the synaptic cleft. Oligodendroglia did not reveal ERG immunoreactivity. Intense immunoreactivity was also found in perivascular astrocytic end feet at the blood-brain barrier. cDNA amplification showed that cortical astrocytes selectively express HERG1, but not HERG2-3 genes. This study provides insight into a possible physiological role of hippocampal ERG channels and links activation of ERG to control of potassium homeostasis.

Effect of maitotoxin on cytosolic Ca2+ levels and membrane potential in purified rat brain synaptosomes.

In this study, the effects of the marine toxin maitotoxin on cytosolic Ca2+ levels and membrane potential in rat brain synaptosomes were evaluated. Maitotoxin (10 ng/ml) caused a remarkable increase of intrasynaptosomal Ca2+ levels monitored by the fluorescent probe fura-2. This increase was prevented by the removal of external Ca2+ ions. Tetrodotoxin, as well as the removal of extracellular Na+ ions, failed to affect maitotoxin-induced increase of intrasynaptosomal Ca2+ levels. Also the complete removal of all monovalent and divalent cations, except Ca2+ ions, from the incubation medium (0.32 M sucrose substitution), was unable to prevent the effect of maitotoxin on intrasynaptosomal Ca2+ levels. Maitotoxin (0.3-10 ng/ml), produced a dose-dependent depolarization of synaptosomal membranes, which required the presence of extracellular Ca2+ ions. The substitution of extracellular Na+ with choline or the removal of all cations from the incubation medium and their replacement with an isotonic concentration of sucrose (0.32 M), did not prevent the depolarizing effect exerted by maitotoxin. Also under these two ionic conditions, the effect of maitotoxin on membrane potential was critically dependent on the presence of 1 mM extracellular Ca2+. The depolarizing effect exerted by maitotoxin on synaptosomal membrane potential was also observed when extracellular Ca2+ ions were substituted with an equimolar concentration of Ba2+ or Sr2+ ions. In summary, these results appear to suggest that, in presence of 1 mM extracellular Ca2+ ions, maitotoxin depolarizes synaptosomal plasmamembrane by promoting the influx of extracellular Ca2+ ions. This enhanced influx of Ca2+ causes an increase of intrasynaptosomal Ca2+ levels.

The Na(+)-Ca2+ exchanger activity in cerebrocortical nerve endings is reduced in old compared to young and mature rats when it operates as a Ca2+ influx or efflux pathway.

The activity of the Na(+)-Ca2+ exchanger, which regulates the entry and the extrusion of Ca2+ ions from nerve endings was investigated in Percoll-purified cerebrocortical synaptosomes of aged rats. 45Ca2+ uptake in a Na(+)-free medium and 45Ca2+ efflux in a 145 mM Na+ medium were significantly reduced in cerebrocortical synaptosomes from aged rats (24 months) as compared to those occurring in young (4 months) and mature (14 months) rats. 45Ca2+ influx induced by 55 mM K+, a concentration of K+ ions which selectively promotes Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC), was significantly reduced in mature and aged rats as compared to that occurring in young rats. The impairment of these mechanisms in aged rats is not accompanied by any variation of fura-2 monitored Ca2+ levels under resting and depolarizing conditions.

Effect of different organic and inorganic blockers of calcium entry on the release of endogenous dopamine from tuberoinfundibular neurones.

In the present study the effect of different blockers of calcium entry belonging to different chemical classes on basal and K+-elicited release of endogenous dopamine (DA) from tuberoinfundibular dopaminergic neurones was studied in vitro. For this purpose fragments of hypothalamus containing arcuate-periventricular nuclei and median eminence were incubated in vitro and endogenous DA released into the medium was assayed by radioenzymatic assay. The organic blockers of calcium entry, nitrendipine, nimodipine, nifedipine, diltiazem and flunarizine did not modify basal or K+-evoked release of endogenous DA, unless very large concentrations (100 microM) of nifedipine or diltiazem were used. The phenylalkylamine methoxyverapamil (D-600) consistently inhibited K+-stimulated release of endogenous DA in concentrations of 50 and 100 microM. Cobalt and lanthanum, two ions with an ionic radius similar to that of calcium and which are known to inhibit calcium fluxes through nerve membranes, significantly blocked release of endogenous DA elicited by 35 mM K+. In summary, the results of the present study showed that calcium channels in the tuberoinfundibular dopaminergic system displayed a different sensitivity to various classes of blockers of calcium entry. Inorganic blockers of calcium entry, like lanthanum and cobalt, appeared to be the most effective in blocking Ca2+-dependent release of endogenous DA, whereas, among the organic calcium antagonists, phenylalkylamines seemed to possess a certain degree of effectiveness.

Inhibition of HERG1 K(+) channels by the novel second-generation antihistamine mizolastine.

1. Ventricular arrhythmias are rare but life-threatening side effects of therapy with the second-generation H(1) receptor antagonists terfenadine and astemizole. Blockade of the K(+) channels encoded by the Human Ether-à-go-go-Related Gene 1 (HERG1) K(+) channels, which is the molecular basis of the cardiac repolarizing current I(Kr), by prolonging cardiac repolarization, has been recognized as the mechanism underlying the cardiac toxicity of these compounds. 2. In the present study, the potential blocking ability of the novel second-generation H(1) receptor antagonist mizolastine of the HERG1 K(+) channels heterologously expressed in Xenopus oocytes and in HEK 293 cells or constitutively present in SH-SY5Y human neuroblastoma cells has been examined and compared to that of astemizole. 3. Mizolastine blocked HERG1 K(+) channels expressed in Xenopus oocytes with an estimated IC(50) of 3.4 microM. Mizolastine blockade was characterized by a fast dissociation rate when compared to that of astemizole; when fitted to a monoexponential function, the time constants for drug dissociation from the K(+) channel were 72.4+/-11.9 s for 3 microM mizolastine, and 1361+/-306 s for 1 microM astemizole. 4. In human embryonic kidney 293 cells (HEK 293 cells) stably transfected with HERG1 cDNA, extracellular application of mizolastine exerted a dose-related inhibitory action on I(HERG1), with an IC(50) of 350+/-76 nM. Furthermore, mizolastine dose-dependently inhibited HERG1 K(+) channels constitutively expressed in SH-SY5Y human neuroblastoma clonal cells. 5. The results of the present study suggest that the novel second-generation H(1) receptor antagonist mizolastine, in concentrations higher than those achieved in vivo during standard therapy, is able to block in some degree both constitutively and heterologously expressed HERG1 K(+) channels, and confirm the heterogeneity of molecules belonging to this therapeutical class with respect to their HERG1-inhibitory action.

New insights into the second generation antihistamines.

Second generation antihistamines are recognised as being highly effective treatments for allergy-based disease and are among the most frequently prescribed and safest drugs in the world. However, consideration of the therapeutic index or the benefit/risk ratio of the H1 receptor antagonists is of paramount importance when prescribing this class of compounds as they are used to treat non-life threatening conditions. There are many second generation antihistamines available and at first examination these appear to be comparable in terms of safety and efficacy. However, the newer antihistamines in fact represent a heterogeneous group of compounds, having markedly differing chemical structures, adverse effects, half-life, tissue distribution and metabolism, spectrum of antihistaminic properties, and varying degrees of anti-inflammatory effects. With regard to the latter, there is growing awareness that some of these compounds might represent useful adjunct medications in asthma therapy. In terms of safety issues, the current second generation grouping includes compounds with proven cardiotoxic effects and others with the potential for adverse drug interactions. Moreover, some of the second generation H1 antagonists have given cause for concern regarding their potential to cause a degree of somnolence in some individuals. It can be argued, therefore, that the present second generation grouping is too large and indistinct since this was based primarily on the concept of separating the first generation sedating compounds from nonsedating H1 antagonists. Although it is too early to talk about a third generation grouping of antihistamines, future membership of such a classification could be based on a low volume of distribution coupled with a lack of sedating effects, drug interactions and cardiotoxicity.

Human ether-a-gogo related gene (HERG) K+ channels as pharmacological targets: present and future implications.

Electrophysiological and molecular biology techniques have widely expanded our knowledge of the diverse functions where K+ channels are implicated as potential and proven pharmacological targets. The aim of the present commentary is to review the recent progress in the understanding of the functional role of the K+ channels encoded by the human ether-a-gogo related gene (HERG), with particular emphasis on their direct pharmacological modulation by drugs, or on their regulation by pharmacologically relevant phenomena. About 3 years have passed since the cloning, expression, and description of the pathophysiological role of HERG K+ channels in human cardiac repolarization. Despite this short lapse of time, these K+ channels have already gained considerable attention as pharmacological targets. In fact, interference with HERG K+ channels seems to be the main mechanism explaining both the therapeutic actions of the class III antiarrhythmics and the potential cardiotoxicity of second-generation H1 receptor antagonists such as terfenadine and astemizole, as well as of psychotropic drugs such as some antidepressants and neuroleptics. It seems possible to anticipate that the main tasks for future investigation will be, on the one side, the better understanding of the intimate mechanism of action of HERG K+ channel-blocking drugs in order to elucidate the conditions regulating the delicate balance between antiarrhythmic and proarrhythmic potential and, on the other, to unravel the pathophysiological role of this K+ channel in the function of the brain and of other excitable tissues.

Cytoplasmic alkalinization induced by insulin through an activation of Na(+)-H+ antiporter inhibits tyrosine hydroxylase activity in striatal synaptosomes.

Insulin dose-dependently inhibited tyrosine hydroxylase (TH) activity and increased intrasynaposomal pH (pHi) in rat striatal nerve endings. Both these effects of insulin on TH and pHi were prevented by the 5-(N-methyl-N-(guanidinocarbonylmethyl) amiloride (MGCMA), a putative selective inhibitor of the Na(+)-H+ antiporter. Interestingly when, by changing the extracellular pH (pHo), the pHi was increased, from 7.1 up to 7.5, an equivalent inhibition of TH activity occurred. The inhibitory action exerted from insulin on TH activity disappeared when the hormone was added to synaptosomes whose pHi was lowered to 6.83. Collectively, the results of the present study showed that insulin inhibited TH activity in striatal synaptosomes. This effect seems to involve the activation of the Na(+)-H+ antiporter. This exchange system once activated, may induce an intrasynaptosomal alkalinization, a condition in which TH activity is inhibited.

Inhibition of depolarization-induced [3H]noradrenaline release from SH-SY5Y human neuroblastoma cells by some second-generation H(1) receptor antagonists through blockade of store-operated Ca(2+) channels (SOCs).

In the present study, the effect of the blockade of membrane calcium channels activated by intracellular Ca(2+) store depletion on basal and depolarization-induced [3H]norepinephrine ([3H]NE) release from SH-SY5Y human neuroblastoma cells was examined. The second-generation H(1) receptor blockers astemizole, terfenadine, and loratadine, as well as the first-generation compound hydroxyzine, inhibited [3H]NE release induced by high extracellular K(+) concentration ([K(+)](e)) depolarization in a concentration-dependent manner (the IC(50)s were 2.3, 1.7, 4.8, and 9.4 microM, respectively). In contrast, the more hydrophilic second-generation H(1) receptor blocker cetirizine was completely ineffective (0.1-30 microM). The inhibition of high [K(+)](e)-induced [3H]NE release by H(1) receptor blockers seems to be related to their ability to inhibit Ca(2+) channels activated by Ca(i)(2+) store depletion (SOCs). In fact, astemizole, terfenadine, loratadine, and hydroxyzine, but not cetirizine, displayed a dose-dependent inhibitory action on the increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)) obtained with extracellular Ca(2+) reintroduction after Ca(i)(2+) store depletion with thapsigargin (1 microM), an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump. The rank order of potency for SOC inhibition by these compounds closely correlated with their inhibitory properties on depolarization-induced [3H]NE release from SH-SY5Y human neuroblastoma cells. Nimodipine (1 microM) plus omega-conotoxin (100 nM) did not interfere with the present model for SOC activation. In addition, the inhibition of depolarization-induced [3H]NE release does not seem to be attributable to the blockade of the K(+) currents carried by the K(+) channels encoded by the human Ether-a-Gogo Related Gene (I(HERG)) by these antihistamines. In fact, whole-cell voltage-clamp experiments revealed that the IC(50) for astemizole-induced hERG blockade is about 300-fold lower than that for the inhibition of high K(+)-induced [3H]NE release. Furthermore, current-clamp experiments in SH-SY5Y cells showed that concentrations of astemizole (3 microM) which were effective in preventing depolarization-induced [3H]NE release were unable to interfere with the cell membrane potential under depolarizing conditions (100 mM [K(+)](e)), suggesting that hERG K(+) channels do not contribute to membrane potential control during exposure to elevated [K(+)](e). Collectively, the results of the present study suggest that, in SH-SY5Y human neuroblastoma cells, the inhibition of SOCs by some second-generation antihistamines can prevent depolarization-induced neurotransmitter release.

The Na(+)-Ca++ exchanger in central nerve endings: the relationship between its pharmacological blockade and dopamine release from tuberoinfundibular hypothalamic neurons.

2', 4'-Dimethylbenzamiloride (DMB), an inhibitor of Na(+)-Ca++ antiporter dose-dependently (10-100 microM) inhibited Na(+)-dependent 45Ca++ efflux from brain synaptosomes. This compound was also able to stimulate basal release of [3H]DA from superfused TIDA neurons. Another amiloride analogue, 5-N-methyl-N-guanidinocarbonylmethylamiloride (MGCMA, 100-300 microM), which lacks of inhibitory properties on the Na(+)-Ca++ antiporter, failed to modify basal [3H]DA release from TIDA neurons. In addition, when the antiporter operates as a Ca(++)-influx pathway, DMB dose-dependently inhibited Na(+)-dependent 45Ca++ uptake in brain synaptosomes, whereas it did not prevent K(+)-induced 45Ca++ uptake, which reflets the activation of voltage-operated Ca++ channels. Finally DMB inhibited ouabain-induced [3H]DA release, which depends on the activation of the Na(+)-Ca++ exchanger due to the inhibition of the Na+/K(+)-ATPase pump.

Maitotoxin and Bay-K-8644: two putative calcium channel activators with different effects on endogenous dopamine release from tuberoinfundibular neurons.

In the present study we report on the effects of two putative calcium channel activators, maitotoxin and the dihydropyridine BAY-K-8644, on endogenous dopamine release from tuberoinfundibular dopaminergic neurons. Maitotoxin stimulated basal dopamine release and this effect was calcium-dependent. By contrast BAY-K-8644 failed to produce any modification of basal or high potassium-induced dopamine release. These results suggest that maitotoxin, unlike BAY-K-8644, represents a suitable tool to investigate the functional role of calcium channels in central dopaminergic neurons.