[3H]dofetilide binding in SHSY5Y and HEK293 cells expressing a HERG-like K+ channel?

The pharmacological characteristics of [3H]dofetilide binding in SHSY5Y, HEK293 and CHO-K1 cells were examined, and in parallel whole cell recordings used to characterise HERG-like K+ currents. Dofetilide affinity was similar in the human cell lines, SHSY5Y (Kd=99.6 nM) and HEK293 (Kd=102.9 nM), but 10 times lower in CHO-K1 cells (Kd=1200 nM). In contrast, clofilium and E4031 had a similar affinity in all three cell lines, whereas WAY 123,398 had no effect. Electrophysiological studies showed that SHSY5Y cells contained a HERG-like K+ current blocked by application of dofetilide to either side of the membrane. Block was faster when dofetilide was applied intracellularly. In contrast, HEK293 and CHO-K1 cells contained no such current, despite the presence of a partial cDNA for HERG in the former. That [3H]dofetilide is specific for I(Kr)/HERG may be questionable, as HEK293 and CHO-K1 cells contain no such functional K+ current.

The action of pyrantel as an agonist and an open channel blocker at acetylcholine receptors in isolated Ascaris suum muscle vesicles.

The anthelmintic pyrantel is believed to act as an agonist at acetylcholine receptors on somatic muscle from the parasite Ascaris suum. This study aimed to confirm this mode of action of pyrantel. Single-channel recordings from muscle vesicles formed from the extrasynaptic region of the bag of somatic muscle cells of Ascaris suum were made using the patch-clamp technique. Pyrantel (0.03-100 microM) activated cation-selective channels with at least 2 conductance levels: main conductance 41 +/- 2.04 pS (mean +/- S.E., n = 28), smaller conductance 22.4 +/- 0.34 pS (mean +/- S.E., n = 8). The current/voltage plots showed a linear relationship. Detailed kinetic analysis revealed that activation of the receptor by pyrantel resulted in at least 2 distinct open and burst states and at least 3 distinct closed states. The mean open time of the channel, with 0.1 microM pyrantel, was 1.53 +/- 0.22 ms (mean +/- S.E., n = 7) at -75 mV. We have previously shown that acetylcholine activated channels with similar properties to the pyrantel-activated channels (Pennington, A.J. and R.J. Martin, 1990, J. Exp. Biol. 154, 201) confirming that pyrantel is an acetylcholine agonist. With high concentrations (100 microM) of pyrantel a sequence of rapid openings and closings of the channel was observed, indicating the presence of an open channel block. Previous experiments have shown that the anthelmintic levamisole, which also acts as an acetylcholine agonist on this preparation, induced channel block at hyperpolarised potentials with high concentrations (Robertson, S.J. and R.J. Martin, 1993, Br. J. Pharmacol. 108, 170). A comparison is made of the actions of the 3 agonists pyrantel, levamisole and acetylcholine at the nicotinic receptor in Ascaris muscle, and implications for the therapeutic use of the compounds are discussed.

Selective enhancement of an A type potassium current by dexamethasone in a corticotroph cell line.

Perforated patch recording was used to examine the effect of the synthetic steroid dexamethasone on the whole cell potassium (K+) current, in the mouse corticotroph tumour cell line AtT20/D16-16. In 15 out of 52 control cells (29%) there was a rapidly-activating, rapidly-inactivating K+ current of the A type, the amplitude of which was strongly dependent on the holding potential in use prior to its activation by depolarising voltage pulses, and which was blocked by 1 mM 4-aminopyridine (4-AP, n = 5). The effect of dexamethasone (100 nM, 2 h, 37 degrees C) was that the A current increased in prevalence (24 out of 31 cells, 77%), lost its dependence on holding potential (over the range studied), and as a result became significantly larger than in controls, for certain voltage steps (peak A current density was 18.5 +/- 2.4 pA/pF (n = 12) for control cells and 26.3 +/- 3.9 pA/pF (n = 18) for dexamethasone treated cells, for a step to +30 mV from -60 mV, values are mean +/- SEM). All cells exhibited a slowly-activating, sustained K+ current, which was unaffected by changes in the holding potential, unaffected by 4-AP and consisted of at least 3 components: one blocked by 30 mM tetraethylammonium(TEA) or 100 nM charybdotoxin (CTX); a second blocked by 100 nM apamin; and a third not blocked by TEA, CTX, apamin, clofilium (100 nM) or niflumic acid (0.1 mM). Dexamethasone produced no change in the slowly-activating, sustained current nor in any of its individual components. The effect of dexamethasone on the A current was completely blocked by 0.1 mM puromycin, a protein synthesis blocker, while puromycin alone did not affect the size or frequency of the A current, nor alter the slowly-activating, sustained current. Secretion studies using 4-AP confirmed that the A current has a role in stimulated adrenocorticotrophic hormone (ACTH) secretion. In summary, AtT20 cells contain at least four types of K+ current: an A current and 3 currents contributing to the slowly-activating current. Selective enhancement of the A current by dexamethasone, shown here to require synthesis of new protein, is one of the mechanisms whereby glucocorticoids exert inhibitory control on ACTH secretion.

The physiology and pharmacology of neuromuscular transmission in the nematode parasite, Ascaris suum.

The organization of Ascaris motoneurones and nervous system is summarized. There is an anterior nerve ring and associated ganglia, main dorsal and ventral nerve cords which run longitudinally, and a small set of posterior ganglia. Cell bodies of motoneurones are found in the ventral nerve cord and occur in 5 repeating 'segments'; each contains 11 motoneurones. Seven morphological types of excitatory or inhibitory motoneurone are recognized. Each Ascaris somatic muscle cell is composed of the contractile spindle; the bag region, containing the nucleus; the arm; and the syncytial region, the location of neuromuscular junctions. The resting membrane potential of muscle is approximately -30 mV and shows regular depolarizing, Ca-dependent 'spike potentials' superimposed on smaller Na(+)- and Ca2(+)-dependent 'slow waves' and even slower 'modulation waves'. The membrane shows high Cl- permeability. Adjacent cells are electrically coupled so that electrical activity in the cells is synchronized. Acetylcholine (ACh) and gamma-aminobutyric acid (GABA) affect the electrical activity. Bath-applied ACh increases membrane cation conductance, depolarizes the cells, alters the frequency and amplitude of spike potentials and produces contraction. Bath-applied GABA increases Cl- conductance, decreases spike activity and causes hyperpolarization and muscle relaxation. The extra-synaptic ACh receptors on the bag region of Ascaris muscle can be regarded as a separate subtype of nicotinic receptor. ACh and anthelmintic agonists (pyrantel, morantel, levamisole) produce a dose-dependent increase in cation conductance and membrane depolarization which is blocked by tubocurarine, mecamylamine but not by hexamethonium. The potency of GABA agonists, with the exception of sulphonic acid derivatives, correlates with the vertebrate GABAa receptor. The potency of antagonists does not. Thus, bicuculline, securinine, pitrazepine, SR95531 and RU5135 are potent vertebrate GABAa antagonists but have little effect on GABA receptors. The potency order of the arylaminopyridazine GABA antagonists: SR95103, SR95132, SR42666, SR95133, SR95531, SR42627 and SR42640 at the Ascaris GABA receptors contrasts with that at vertebrate GABAa receptors. It has been suggested that the receptor is referred to as a GABAn receptor. Patch-clamp studies show that ACh activates a non-selective cation channel which has a main conductance of 40-50pS and apparent mean open time of 1.3 ms; a smaller channel of 20-30 pS with a similar open-time is also activated. Pyrantel and levamisole also produce openings with similar conductances and open-times.(ABSTRACT TRUNCATED AT 400 WORDS)

A patch-clamp study of acetylcholine-activated ion channels in Ascaris suum muscle.

Acetylcholine-activated single-channel currents were recorded from cell-attached and inside-out patches of isolated muscle vesicles from Ascaris suum. Acetylcholine (1-10 mumols l-1) activated cation-selective channels of two amplitudes: 40-50 pS and 25-35 pS. Both channels had linear I/V relationships and mean open durations independent of voltage. The larger conductance was analysed in detail to determine its open-, closed- and burst-time kinetics; the open and burst durations were composed of two components (short and long), while closed durations had at least three components (short, intermediate and long). The data were then corrected to allow for missing short events in order to estimate various parameters including corrected mean open time (1.26 + 0.11 ms, mean +/- S.E.). Values were also derived for the efficacy (beta/alpha = 4.9) and affinity [1/KD = 147 x 10(3) (mol l-1) -1] of acetylcholine at this receptor. Larger concentrations of acetylcholine (25-100 mumols l-1) were shown to produce desensitization and caused single-channel currents to occur in clusters with long closed times (mean 171 s) between clusters. It was concluded that the extrasynaptic muscle of Ascaris suum contains two types of acetylcholine-activated ion channel and these are possible sites of action of various anthelmintic drugs. This paper is the first to describe acetylcholine-activated single-channel currents in invertebrate muscle.

Surface properties of membrane vesicles prepared from muscle cells of Ascaris suum.

To facilitate biochemical, pharmacological, and biophysical studies on the membrane of the body muscle of Ascaris suum, a method for preparing intact vesicles was developed. Vesicles were prepared by incubating a muscle flap preparation with 1 mg/ml collagenase in a saline solution and then washing in saline without enzyme. The vesicles then formed gradually over the next hour as outgrowths of the original surface membrane from the bag region of the muscle. The vesicles were harvested readily by suction using a Pasteur pipette. The structure of the vesicles was examined with the transmission electron microscope. The whole-cell patch-clamp technique showed that the vesicles had a high input resistance and that the membrane was complete. The vesicle membrane was shown to contain Ca-activated Cl channels and gamma-aminobutyric acid-activated Cl channels. The vesicles also were shown to be suitable for fluorescence recovery after photobleaching studies designed to examine lateral and vertical movement of a lipid probe (5-N [octadecanoyl]-aminofluorescein) in the membrane. This probe had a mean lateral diffusion coefficient (DL) of 8.1 x 10(-9) cm2/sec, but only a proportion (68.4%) of the probe was mobile. The latter observation illustrated the nonuniform nature of the membrane. Ivermectin (10(-7) M) had no effect on DL or percent recovery. Trypan blue quenching experiments showed that the lipid probe remained in the outer monolayer of the membrane. These observations illustrate the experimental value of the vesicles; they are potentially useful in discerning anthelmintic mode of action and in drug screening.

A patch-clamp study of effects of dihydroavermectin on Ascaris muscle.

1. Effects of bath-application of the anti-parasitic agent 22,23-dihydroavermectin B1a (DHAVM 20 fM to 0.2 microM) on outside-out and inside-out patches from collagenase-treated muscle membranes of Ascaris suum were examined. 2. DHAVM was tested as a gamma-aminobutyric acid (GABA) agonist by application to outside-out patches. DHAVM failed to open characteristic GABA channels even when high concentrations (20 nM to 0.2 microM) were used. These high concentrations of DHAVM also failed to potentiate opening of channels activated by application of 3-4 microM GABA; instead they acted to depress GABA-activated channel currents by reducing mean conductances and P open. 3. In outside-out patches, low concentrations of DHAVM (1 pM to 100 pM) produced opening of 0-13 channels, the number depending on the patch. The progressive opening of channels gave rise to a 'Staircase' effect. The conductance of these channels was 9-15 pS and open times were long (greater than 100 mS). Ion substitution experiments showed these channels to be permeable to Cl. The channels were not blocked by 20 microM picrotoxin. There was a long delay (greater than 15 s) between DHAVM application and channel opening; this delay and lipophilic nature of DHAVM suggested a site of action in the lipid phase of the membrane. 4. The effect of bath-application of low concentrations of DHAVM on inside-out patches was investigated under low-Ca conditions (to avoid Ca-activated Cl channels). DHAVM (20 fM to 1 nM) did not produce opening of Cl channels but produced opening after a long delay (mean 2.5 minutes) of noisy cation-selective channels which had conductances of 5-30 pS. 5. The actions of DHAVM are discussed.

Energy utilization and gluconeogenesis in isolated leech segmental ganglia: Quantitative studies on the control and cellular localization of endogenous glycogen.

The isolated segmental ganglia of the horse leech Haemopis sanguisuga were used as a model system to study the utilization and control of glycogen stores within nervous tissue. The glycogen in the ganglia was extracted and assayed fluorimentrically and its cellular localization and turnover studied by autoradiography in conjunction with [(3)H]glucose. We measured the glycogen after various periods of electrical stimulation and after incubation with K(+), Ca(2+), ouabain and glucose. The results for each experimental ganglion were compared to a paired control ganglion and the results analysed by paired t-tests. Electrical stimulation caused sequential changes in glycogen levels: a reduction of up to 67% (5-10 min); followed by an increase of up to 124% (between 15-50 min); followed by a reduction of up to 63% (60-90 min). Values were calculated for glucose utilization (e.g. 0.53 ?mol glucose/gm wet weight/min after 90 min) and estimates derived for glucose consumption per action potential per neuron (e.g. 0.12 fmol at 90 min). Glucose (1.5-10 mM) increased the amount of glycogen (1.5 mM by 30% at 60 min) and attenuated the effects of electrical stimulation. Ouabain (1 mM) blocked the effect of 5 min electrical stimulation. Nine millimolar K(+) increased glycogen by 27% after 10 min and decreased glycogen by 34% after 60 min; 3 mM Ca(2+) had no effect after 10 or 20 min and decreased glycogen by 29% after 60 min. Other concentrations of K(+) and Ca(2+) reduced glycogen after 60 min. Autoradiographic analysis demonstrated that the effects of elevated K(+) were principally within the glial cells. We conclude that (i) the glycogen stores in the glial cells of leech segmental ganglia provide an endogenous energy source which can support sustained neuronal activity, (ii) both electrical stimulation and elevated K(+) can induce gluconeogenesis within the ganglia, (iii) that electrical activation of neurons produces changes in the glycogen in the glial cells which are controlled in part by changes in K(+).

Neuropeptides modulate the transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia.

The effects of four neuropeptides (AKH, FMRFamide, proctolin, VIP) on the alterations in glycogen levels induced by other transmitters were studied in isolated leech segmental ganglia. With the exception of FMRFamide, which produced a small increase (14%) in glycogen, the peptides were by themselves without effect on the glycogen levels. Proctolin abolished the glycogenolytic effects of 5-HT, dopamine and octopamine but not histamine. AKH in combination with ACh produced glycogenolysis although each by themselves were ineffective. AKH modified the effects of other transmitters in different ways i.e. by reduction or reversal of effect. VIP and noradrenaline produced an increase in glycogen (cf. noradrenaline alone which decreased glycogen), but did not modify the effects of the other transmitters. FMRFamide produced a complex variety of modulatory effects on the other transmitters. It is concluded that the glycogen stores in leech ganglia, which are localized principally in the glial cells, may be controlled in complex ways by the different combinations of monoamines, amino acids and neuropeptides.

Transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia.

1. The utilization and control of glycogen stores were studied in the isolated segmental ganglia of the horse leech, Haemopis sanguisuga. The glycogen in the ganglia was extracted and assayed fluorimetrically and its cellular localization and turnover studied by autoradiography in conjunction with [3H] glucose. 2. The glycogen levels were measured after incubation with different neurotransmitters for 60 min at 28 degrees C. The results for each experimental ganglion were compared to a paired control ganglion, and the results analysed by paired t-tests. 3. Several transmitter substances (5-HT, octopamine, dopamine, noradrenaline, histamine) produced reductions in glycogen (glycogenolysis); other transmitters (glutamate, GABA) produced increases in glycogen (gluconeogenesis); others (adenosine, glycine) produced reductions or increases, depending on concentration. Acetylcholine had no effect on the glycogen levels. 4. Most of the glycogen in the ganglia is localized in the packet glial cells, which surround the neuron perikarya. Autoradiographic analysis demonstrated that the effects of histamine and dopamine were principally on the glycogen in the glial cells. 5. Adenylate cyclase was demonstrated by electron microscope histochemistry to be localized on the plasma membranes of the glial cells, and to a lesser extent on the neuronal membranes. 6. It is concluded that the changes in glycogen in the glial cells may be party controlled by transmitters via adenylate cyclase. This may provide a sensitive mechanism for coupling neuronal activity with energy metabolism.