David P. Thompson

The Madin Darby Canine Kidney (MDCK) Epithelial Cell Monolayer as a Model Cellular Transport Barrier

Two strains of Madin Darby canine kidney (MDCK) cells were grown on a polycarbonate membrane with 3-µm pores without any extracellular matrix treatment. The membrane, 2.45 cm in diameter, which is part of a commercially obtained presterilized culture insert, provides two chambers when placed in a regular six-well culture plate. This device was found to be convenient for investigating transport of a few selected fluid-phase markers across the MDCK cell monolayer. Both the strain from the American Type Culture Collection (ATCC) and the so-called highly resistant strain I, at a serial passage between 65 and 70, showed a seeding concentration-dependent lag phase followed by a growth phase with a 21-hr doubling time. When seeded at 5 × 104 cells/cm2, cell confluence was achieved in 5 days in a modified Eagles minimum essential medium (MEM) containing 10% fetal bovine serum under a 5% CO2 atmosphere. Similarly, transepithelial electrical resistance (TEER) also reached a plateau value in 5 days. Both light and electron microscopic examinations revealed well-defined junctional structures. Transport of the fluid-phase markers, sucrose, lucifer yellow CH (LY), inulin, and dextran across the MDCK cell monolayers was studied primarily at 37°C following the apical-to-basolateral as well as the basolateral-to-apical direction. Large variations in the steady-state transport rate were observed for a given marker between the cell layer preparations. Thus, the present study proposes an “internal standard” procedure for meaningful comparisons of the transport rate. When normalized to the rate of sucrose, the rate ratio was 1.00:0.80:0.67:0.15 for sucrose:LY:inulin:dextran. This ratio was virtually independent of temperature, cell strain, direction of the marker migration, and TEER value, suggesting a common transport mechanism. The observed rate ratio appears to reflect molecular size and charge. The transport observed in the present study would consist, in theory, of both paracellular shunt and transcellular vesicular transport. Quantitative assessment of each transport mechanism in the overall transport has been difficult. The initial uptake of [3H]dextran estimated for the slowest transport observed in the present study was still 300-fold faster than a literature value. This appears to indicate that the transport observed in the present study is largely through the paracellular shunt pathway.

Frontiers in anthelmintic pharmacology

Research in anthelmintic pharmacology faces a grim future. The parent field of veterinary parasitology has seemingly been devalued by governments, universities and the animal industry in general. Primarily due to the success of the macrocyclic lactone anthelmintics in cattle, problems caused by helminth infections are widely perceived to be unimportant. The market for anthelmintics in other host species that are plagued by resistance, such as sheep and horses, is thought to be too small to sustain a discovery program in the animal health pharmaceutical industry. These attitudes are both alarming and foolish. The recent history of resistance to antibiotics provides more than adequate warning that complacency about the continued efficacy of any class of drugs for the chemotherapy of an infectious disease is folly. Parasitology remains a dominant feature of veterinary medicine and of the animal health industry. Investment into research on the basic and clinical pharmacology of anthelmintics is essential to ensure chemotherapeutic control of these organisms into the 21st century. In this article, we propose a set of questions that should receive priority for research funding in order to bring this field into the modern era. While the specific questions are open for revision, we believe that organized support of a prioritized list of research objectives could stimulate a renaissance in research in veterinary helminthology. To accept the status quo is to surrender.

The structure and function of helminth surfaces

Publisher Summary The external surfaces of parasitic helminths, termed the “cuticle” in nematodes and the “tegument” in trematodes and cestodes, serve many biological roles. The most important is that of a barrier, which shields the organism from external conditions. Other roles include bidirectional transport of inorganic and organic molecules and structural support. The external structures of these organisms exhibit remarkable developmental changes and exhibit a complex biochemistry that is adapted for the parasitic life style. In nematodes and trematodes, the gut surface also plays an important role in digestion, nutrient absorption and transport. This chapter illustrates the structural and functional biology, biochemistry and, where available, molecular biology of these surfaces. The absence of a gut in cestodes has simplified interpretation of functional properties of the external surface. The chapter summarizes that the gastrodermis in trematodes and the intestine in nematodes are composed of cells with microvilli, which amplify the surface area for absorption. However, too little is known about transport across the internal surfaces of trematodes or nematodes to draw definitive conclusions about their importance to the parasites, or the extent to which they resemble analogous processes in other organisms.

Caenorhabditis elegans: how good a model for veterinary parasites?

The organism about which most is known on a molecular level is a nematode, the free-living organism Caenorhabditis elegans. This organism has served as a reasonable model for the discovery of anthelmintic drugs and for research on the mechanism of action of anthelmintics. Useful information on mechanisms of anthelmintic resistance has also been obtained from studies on C. elegans. Unfortunately, there has not been a large-scale extension of genetic techniques developed in C. elegans to research on parasitic species of veterinary (or human) parasites. Much can be learned about the essentials of nematode biology by studying C. elegans, but discovering the basic biology of nematode parasitism can only be gained through comparative studies on multiple parasitic species.

Inhibitory effects of nematode FMRFamide-related peptides (FaRPs) on muscle strips fromAscaris suum

A large number of FMRFamide-related peptides (FaRPs) are found in nematodes, and some of these are known to influence tension and contractility of neuromuscular strips isolated fromAscaris suum body wall. Relaxation of these strips has been noted with five nematode FaRPs. The inhibitory actions of SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) appear to be mediated by nitric oxide, as previously demonstrated with inhibitors of nitric oxide synthase (NOS). This present study showed that the effects of PF1 were also dependent on external Ca++ and were reduced by the Ca++-channel blocker verapamil, observations consistent wirh the finding that nematode NOS is Ca++-dependent. KSAYMRFamide (PF3), KPNFIRFamide (PF4) and KNAFIRFamide (an alanine substituted analog of KNEFIRFamide, AF1, termed A3AF1) also relaxed A.suum muscle strips, but these responses were not affected by NOS inhibitors. PF3 inhibited the activity of strips prepared from the dorsal side of the worm, but contracted ventral strips. Both effects were dependent on the presence of ventral/dorsal nerve cords (unlike PF1/PF2) and were attenuated in medium which contained high K+ or low Ca++. PF4-induced muscle relaxation and hyperpolarization were independent of nerve cords, but were reversed in Cl-free medium, unlike PF1 or PF3. The PF4 effect physiologically desensitized muscle strips to subsequent treatment with PF4 and/or GABA. However, PF4 and GABA were not synergistic in this preparation. The effects of GABA, but not PF4, were reduced in muscle strips treated with the GABA antagonist, NCS 281-93. Following PF4 (or GABA) relaxation, subsequent treatment with higher doses of PF4 caused muscle strip contraction. A3AF1 was found to relax muscle strips and hyperpolarize muscle cells independently of the ventral and dorsal nerve cords, K+, Ca++, and Cl-, and mimicked the inhibitory phase associated with the exposure of these strips to AF1. On the basis of anatomical and ionic dependence, these data have delineated at least four distinct inhibitory activities attributable to nematode FaRPs. Clearly, a remarkably complex set of inhibitory mechanisms operate in the nematode neuromuscular system.

Neuropeptide Signaling Systems - Potential Drug Targets for Parasite and Pest Control.

Current problems of drug resistance in parasites and pests demand the identification of new targets and their exploitation through novel drug design and development programs. Neuropeptide signaling systems in helminths (nematodes and platyhelminths = worms) and arthropods are well developed and complex, play a crucial role in many aspects of their biology, and appear to have significant potential as targets for novel drugs. The best-known neuropeptide family in invertebrates is the FMRFamide-related peptides (FaRPs). Amongst many roles, FaRPs potently influence motor function. The genome sequencing projects of Drosophila melanogaster and Caenorhabditis elegans have revealed unexpected complexity within the FaRPergic systems of arthropods and nematodes, although available evidence for platyhelminths indicates structural and functional simplicity. Regardless of these differences, FaRPs potently modulate motor function in arthropods, nematodes and platyhelminths and there appears to be at least some commonality in the FaRPergic signaling systems therein. Moreover, there is now increasing evidence of cross-phyla activity for individual FaRPs, providing clear signals of opportunities for target selection and the identification and development of broad-spectrum drugs.

Biophysical transport properties of the cuticle of Ascaris suum

The transport properties of isolated cuticle from Ascaris suum were studied using standard two-chamber diffusion cells and a number of radiolabeled permeants which varied in molecular size, lipophilicity and electrical charge. The permeability coefficient of the collagen matrix (lipid-extracted cuticle) vs. molecular radius relationship showed the interdependence of molecular size and electrical charge of the permeants with respect to the aqueous pores of the negatively charged matrix. The permeability of neutral solutes decreased monotonically with size. Protonated amines permeated the aqueous pores faster than neutral solutes of comparable size, while the permeation of anions was slower. The average pore size was estimated to be 1.5 nm in radius. A biophysical model which accounted for diffusion of molecules within a fixed electrostatic field of force and for molecular sieving by the pore channels was used in the mechanistic interpretation of the data. The effective permeability coefficient of the non-lipid-extracted cuticle was delineated into the permeability coefficients of the water-filled collagen matrix and the lipoidal component of the cuticle to determine which layer was the rate-controlling barrier. While each solute was capable of penetrating the water-filled collagen matrix, the rate-determining step for the majority of compounds was passive diffusion across the lipid component, which controlled 75-99% of transport. The exception was water, for which transport kinetics was 75% matrix-controlled. In general, permeation across the lipid-filled tissue was more favorable for small lipophilic compounds because of molecular restriction not only in the aqueous pores, but also in the lipid-filled pores.

Mechanism-based screening: discovery of the next generation of anthelmintics depends upon more basic research.

The therapeutic arsenal for the control of helminth infections contains only a few chemical classes. The development and spread of resistance has eroded the utility of most currently available anthelmintics, at least for some indications, and is a constant threat to further reduce the options for treatment. Discovery and development of novel anthelmintic templates is strategically necessary to preserve the economic and health advantages now gained through chemotherapy. As the costs of development escalate, the question of how best to discover new drugs becomes paramount. Although random screening in infected animals led to the discovery of all currently available anthelmintics, cost constraints and a perception of diminishing returns require new approaches. Taking a cue from drug discovery programmes for human illnesses, we suggest that mechanism-based screening will provide the next generation of anthelmintic molecules. Critical to success in this venture will be the exploitation of the Caenorhabditis elegans genome through bioinformatics and genetic technologies. The greatest obstacle to success in this endeavour is the paucity of information available about the molecular physiology of helminths, making the choice of a discovery target a risky proposition.

Pharmacology of FMRFamide-related Peptides in Helminths

Abstract: Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide‐related peptides (FaRPs), with more yet to be identified. To date, few non‐FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free‐living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand‐receptor recognition features among the Nematoda. Structure‐activity studies on nematode FaRPs have revealed that structure‐activity relationship (SAR) “rules” differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross‐phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross‐phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non‐neuropeptidergic systems, also remains to be elucidated.

THE NERVOUS SYSTEMS OF HELMINTHS AS TARGETS FOR DRUGS

Processes that critically differentiate parasitic helminths and their hosts are obvious candidates for chemotherapeutic intervention. The recognition that neurobiology distinguishes helminths from their vertebrate hosts is due in part to the fact that several efficacious anthelmintics, derived generally from empirical screening, have been found to act selectively on the neuromuscular system of these parasites. In addition, basic physiological and pharmacological research has revealed considerable differences in the ways in which helminths and their hosts transmit information in the nervous system and respond to it in innervated tissues. Unfortunately, most of these differences have yet to be exploited in chemotherapy. The topics for this review include an analysis of mechanistic aspects of the pharmacology of anthelmintics that act on neuromuscular systems and a consideration of the prospects for discovery of novel drugs that act on this system.