Cheryl L. Clark

Drug resistance and neurotransmitter receptors of nematodes : recent studies on the mode of action of levamisole

Here we review recent studies on the mode of action of the cholinergic anthelmintics (levamisole, pyrantel etc.). We also include material from studies on the free living nematode Caenorhabditis elegans. The initial notion that these drugs act on a single receptor population, while attractive, has proven to be an oversimplification. In both free living and parasitic nematodes there are multiple types of nicotinic acetylcholine receptor (nAChR) on the somatic musculature. Each type has different (sometimes subtly so) pharmacological properties. The implications of these findings are: (1) combinations of anthelmintic that preferentially activate a broad range of nAChR types would be predicted to be more effective; (2) in resistant isolates of parasite where a subtype has been lost, other cholinergic anthelmintics may remain effective. Not only are there multiple types of nAChR, but relatively recent research has shown these receptors can be modulated; it is possible to increase the response of a parasite to a fixed concentration of drug by altering the receptor properties (e.g. phosphorylation state). These findings offer a potential means of increasing efficacy of existing compounds as an alternative to the costly and time consuming development of new anthelmintic agents.

Methyridine (2-[2-methoxyethyl]-pyridine]) and levamisole activate different ACh receptor subtypes in nematode parasites: a new lead for levamisole-resistance

The development of resistance to all chemotherapeutic agents increases and needs to be addressed. We are interested in resistance in parasitic nematodes to the anthelmintic levamisole. During studies on methyridine, we found that it gave us a new insight into pharmacological changes associated with levamisole resistance. Initially, electrophysiological investigation using a two‐micropipette current‐clamp recording technique revealed that methyridine acts as a cholinergic agonist on nematode muscle receptors (Ascaris suum). Methyridine (>30 μM) produced reversible concentration‐dependent depolarizations and increases in input conductance. Mecamylamine (30 μM) and paraherquamide (0.3 μM) produced reversible antagonism of the depolarization and conductance responses to methyridine. These observations suggest that methyridine, like acetylcholine and levamisole, gates ion channels on the muscle of parasitic nematodes. The antagonistic effects of dihydro‐β‐erythroidine and paraherquamide on methyridine‐induced contractions of A. suum muscle flaps were then examined to determine if methyridine showed subtype selectivity for N‐subtype (nicotine‐sensitive) or L‐subtype (levamisole‐sensitive) acetylcholine receptors. Dihydro‐β‐erythroidine weakly antagonized the effects of methyridine (but had no effect on levamisole responses). The antagonism of methyridine (pA2, 5.9) and nicotine (pA2, 6.1) by paraherquamide was similar, but was less than the antagonism of levamisole (pA2, 7.0). The antagonist profiles suggested that methyridine has a selective action on the N‐subtype rather than on the L‐subtype. A novel use for a larval inhibition migration assay was made using L3 larvae of Oesophagostomum dentatum. Inhibitory effects of nicotine, levamisole, pyrantel and methyridine on the migration of larvae of levamisole‐sensitive (SENS) and levamisole‐resistant (LEV‐R) isolates were tested at different concentrations. Levamisole and pyrantel (putative L‐subtype‐selective agonists) concentration–response plots were displaced to the right in LEV‐R isolates. Nicotine (an N‐subtype‐selective agonist) and methyridine produced little shift in concentration–response plots in the LEV‐R isolates. Resistance dose ratios were used to calculate the relative selectivity, ρL, for the L‐type receptor (levamisole ρL=1.0; pyrantel ρL=0.93; methyridine ρL=0.17; nicotine ρL=0.06). These observations reveal an N‐subtype‐selective action of methyridine and suggest that levamisole resistance may be associated with a loss of the L‐subtype, but not the N‐subtype receptors. The pharmacology of methyridine suggests an approach for the treatment of levamisole‐resistant parasites.

Levamisole and ryanodine receptors. I: A contraction study in Ascaris suum.

Cholinergic anthelmintics (like levamisole) are important drugs but resistance with reduced responses by the parasite to these compounds is a concern. There is a need to study and understand mechanisms that affect the amplitude of the responses of parasites to these drugs. In this paper, we study interactions of levamisole and ryanodine receptors on contractions of Ascaris suum body muscle flaps. In our second paper, we extend these observations to examine electrophysiological interactions of levamisole, ryanodine receptors (RyRs) and AF2. We report that the maximum force of contraction, g(max), was dependent on the extracellular concentration of calcium but the levamisole EC(50) (0.8 microM) was not. The relationship between maximum force of contraction and extracellular calcium was described by the Michaelis-Menten equation with a K(m) of 1.8mM. Ryanodine inhibited g(max) without effect on EC(50); ryanodine inhibited only 44% of the maximum contraction (K(i) of 40 nM), revealing a ryanodine-insensitive component in the levamisole excitation-contraction pathway. Dantrolene had the same effect as ryanodine but was less potent. The neuropeptide AF2 (1 microM) decreased the levamisole EC(50) to 0.2 microM without effect on g(max); 0.1 microM ryanodine and 100 microM dantrolene, inhibited the g(max) of the AF2-potentiated levamisole response. High concentrations of caffeine, 30 mM, produced weak contraction of the body-flap preparation. Caffeine behaved like ryanodine in that it inhibited the maximum force of contraction, g(max), without effects on the levamisole EC(50). Thus, RyRs play a modulatory role in the levamisole excitation-contraction pathway by affecting the maximum force of contraction without an effect on levamisole EC(50). The levamisole excitation-contraction coupling is graded and has at least two pathways: one sensitive to ryanodine and one not.

Levamisole and ryanodine receptors. II: An electrophysiological study in Ascaris suum.

Resistance to antinematodal drugs like levamisole has increased and there is a need to understand what factors affect the responses to these anthelmintics. In our previous study, we examined the role of ryanodine receptors in muscle contraction pathways. Here we have examined interactions of levamisole receptors, ryanodine receptors (RyRs), the excitatory neuropeptide AF2, and coupling to electrophysiological responses. We examined the effects of a brief application of levamisole on Ascaris suum body muscle under current-clamp. The levamisole responses were characterized as an initial primary depolarization, followed by a slow secondary depolarizing response. We examined the effects of AF2 (KHEYLRFamide), 1 microM applied for 2 min. We found that AF2 potentiated the secondary response to levamisole and had no significant effect on the primary depolarization. Further, the reversal potentials observed during the secondary response suggested that more than one ion was involved in producing this potential. AF2 potentiated the secondary response in the presence of 30 microM mecamylamine suggesting the effect was independent of levamisole sensitive acetylcholine receptors. The secondary response, potentiated by AF2, appeared to be dependent on cytoplasmic events triggered by the primary depolarization. Ion-substitution experiments showed that the AF2 potentiated secondary response was dependent on extracellular calcium and chloride suggesting a role for the calcium-activated anion channel. Caffeine mimicked the AF2 potentiated secondary response and 0.1 microM ryanodine inhibited it. 1.0 microM ryanodine increased spiking showing that it affected membrane excitability. A model is proposed showing ryanodine receptors mediating effects of AF2 on levamisole responses.

Evidence for the Existence of a Luteal Cell Type That Is Steroidogenic and Releases Relaxin

Abstract Secretion of relaxin from cultured luteal cells derived from pregnant sows was detected by a reverse hemolytic plaque assay. In this method, luteal cells are cultured in monolayers together with protein-A-conjugated ovine red blood cells. In the presence of porcine relaxin antiserum and complement, relaxin-releasing cells become surrounded by an area of hemolysis—a plaque—which can be microscopically visualized. After fixation, these same luteal cells in monolayers were stained for the presence of β-hydroxysteroid dehydrogenase, an enzyme marker for steroidogenic cells. Cells could then be classified by their ability to form plaques (relaxin-releasing cells) and/or steroidogenic capability (positive staining). Dual-secretors (large luteal cells that were steroidogenic and released relaxin) could be identified in dispersed luteal cells derived from pigs at all stages of pregnancy examined (Day 22-112 of gestation, n = 9; term is Day 114 ± 2 days). In addition, luteal cells were detected that were either steroidogenic only or released relaxin, and finally, cells that appeared to possess neither endocrine capability. Frequency analysis of functional subtypes indicated approximately equal representation of each in the first half of pregnancy, but an apparent fall in relaxin-releasing cells in the preparturient period. It is suggested that dual-secretors may represent one mechanism that allows the corpus luteum to express multiple endocrine function during pregnancy without the requirement for increased cell numbers.

Lipopolysaccharide and a phorbol ester stimulate secretion of tumor necrosis factor-α from alveolar macrophages through action on overlapping subsets of cells

A cell‐by‐cell analysis of the secretory ability of stimulated, individual alveolar macrophages (AMs) was performed through use of a tumor necrosis factor a (TNF‐α) reverse hemolytic plaque assay. Two functional end points were measured: the percentage of AMs that were TNF‐α secretors and the cumulative amount of TNF‐α secreted by AMs (average plaque area, μm2). Lipopolysaccharide (LPS; 100 μg/ml) increased cumulative TNF‐α release at both 7 and 20 h of incubation. On the other hand, a phorbol ester (phorbol myristate acetate, PMA) stimulated TNF‐α release at 20 h of incubation but not at 7 h. Under nonstimulated culture conditions, 5‐10% of all AMs released detectable TNF‐α. PMA (but not LPS) induced a significant increase in the fraction of AMs capable of releasing TNF‐α (15.1 ± 1.1% vs. 9.0 ± 1.6%, PMA vs. control, P < .05). Differences in the time course of secreted TNF‐α, together with the recruiting effect of PMA, suggest that LPS and PMA target TNF‐α‐ secretory subpopulations of AMs that differ in number and secretory characteristics.

Effects of the muscarinic agonist, 5-methylfurmethiodide, on contraction and electrophysiology of Ascaris suum muscle

Contraction and electrophysiological effects of 5-methylfurmethiodide (MFI), a selective muscarinic agonist in mammals, were tested on Ascaris suum muscle strips. In a contraction assay, MFI produced weak contraction and was less potent than levamisole and acetylcholine. Atropine (3microM) a non-selective muscarinic antagonist in mammalian preparations, did not affect contractions produced by MFI. Mecamylamine (3microM) a nicotinic antagonist in A. suum preparations, blocked the MFI contractions indicating that MFI had weak nicotinic agonist actions. In two-micropipette current-clamp experiments MFI, at concentrations greater than 10microM, produced concentration-dependent depolarizations and small increases in membrane conductance. The depolarizing effects were not abolished by perfusing the preparation in a calcium-free Ascaris Ringer solution to block synaptic transmission, suggesting that MFI effects were mediated by receptors on the muscle and were calcium-independent. A high concentration of mecamylamine, 30microM, only reduced the depolarizing responses by 42%, indicating that MFI also had effects on non-nicotinic receptors. Three non-nicotinic effects in the presence of 30microM mecamylamine were identified using voltage-clamp techniques: (i) MFI produced opening of mecamylamine-resistant non-selective-cation channel currents; (ii) MFI inhibited opening of voltage-activated potassium currents; and (iii) MFI increased the threshold of voltage-activated calcium currents. We suggest that a drug that is more selective for voltage-activated potassium currents, without effects on other channels like MFI, may be exploited pharmacologically as a novel anthelmintic or as an agent to potentiate the action of levamisole. In a larval migration assay we demonstrated that 4-aminopyridine (4-AP: a potassium channel blocker) potentiated the effects of levamisole but MFI did not.

Detection of Relaxin Secretion and Gene Expression in Individual Porcine Luteal Cells

Although large luteal cells of pregnancy contain immunoreactive relaxin (RLX), only about 50% of the cells release RLX in vitro in the reverse hemolytic plaque assay (RHPA) (1). Whether the heterogeneity in RLX secretion by subpopulations of luteal cells can be explained by differences in RLX gene expression is unknown. In situ evidence indicates the RLX gene is expressed by the corpus luteum of pregnancy, but whether a specific subset of luteal cells contains RLX transcript was not determined (2). In this study, RHPA, followed by in situ hybridization histochemistry (ISHH), was used to study the relationship between RLX secretion and gene expression, respectively, by the same luteal cell. These techniques have been used to measure peptide secretion and gene expression in individual pituitary lactotrophs (3). The objective here was to determine whether differences exist between RLX secreting and nonsecreting luteal cells in terms of expression of RLX message.

Relaxin Secretion by Porcine Large Luteal Cells: Effect of Protein Synthesis Inhibitors

Abstract The purpose of the experiments reported herein was to investigate the relative importance of new hormone synthesis to basal and prostaglandin E2-stimulated rates of relaxin release. A relaxin-reverse hemolytic plaque assay was used to monitor relaxin release from individual large luteal cells (LLC) in which new protein synthesis was inhibited by cycloheximide or actinomycin D. These treatments significantly decreased the rate of relaxin release. In addition, cycloheximide reduced the total fraction of LLC possessing the ability to form plaques by about 10%, suggesting complete suppression of relaxin from this subset of cells. Exposure of inhibitor-treated LLC to prostaglandin E2 (a relaxin stimulatory secretagogue) enhanced relaxin release, and restored suppressed LLC back into the secretory population. Taken overall, these results demonstrate that the majority of relaxin-releasing LLC exploit a mixture of newly synthesized and older, stored hormone to achieve basal secretion. A minority of relaxin-releasing LLC, however, appear to depend wholly on newly synthesized hormone for basal secretion. The differential activity (and interaction) of these pathways in individual LLC may provide a potential explanation for the markedly heterogenous manner of hormone release observed in this (and other) cell types, and for the action of relaxin secretagogues.