Fasciola hepatica: motility response to metabolic inhibitors in vitro

The effects of metabolic inhibitors on the in vitro motility of Fasciola hepatica have been determined by means of an isometric transducer system. Sodium fluoride, an inhibitor of glycolysis, causes a long-term suppression of motility; this is also the effect of sodium iodoacetate (another glycolysis inhibitor) at low concentrations (1 X 10(-5) M and below). However, higher concentrations of iodoacetate induce a rapid inhibition of activity leading to a spastic paralysis. Both rotenone and oligomycin, which act as inhibitors of oxidative phosphorylation, produce a long-term suppression of movement. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and carbonylcyanide-m-chlorophenylhydrazone, which are uncouplers of oxidative phosphorylation, induce a spastic paralysis of the fluke; this is rapid at high concentrations (1 X 10(-4) and 1 X 10(-5) M). A brief stimulation of activity is evident at 1 X 10(-5) M and lasts longer at 1 X 10(-6) and 1 X 10(-7) M, before inhibition sets in. There is no stimulation at low concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (1 X 10(-8) and 1 X 10(-9) M), only inhibition leading to a medium-term spastic paralysis. In contrast, a third uncoupler, 2,4-dinitrophenol, causes a flaccid paralysis and the effect is rapid only at high concentrations, being accompanied by an initial increase in muscle tone at 1 X 10(-2) M and a brief stimulation of motility at 1 X 10(-3) M. Stimulation lasts longer at 1 X 10(-4) and 1 X 10(-5) M, but is not evident at concentrations below this. The effects on motility at these lower concentrations are essentially long term in nature. That the rapid effects of the uncouplers on muscle tone and motility are not due primarily to uncoupling is shown by 2,4,6-trinitrophenol and hydroquinone, compounds structurally related to 2,4-dinitrophenol. 2,4,6-Trinitrophenol is a membrane-impermeable compound devoid of uncoupling activity; at 1 X 10(-3) M, it causes an immediate inhibition of activity and increase in muscle tone. The antioxidant hydroquinone produces an initial stimulation of motility with some increase in tone, but this is followed by a sharp decline and a short-term flaccid paralysis. The results are discussed in relation to the postulated effects of certain fasciolicides on the energy metabolism of the liver fluke.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Efficacy of various chemotherapeutic agents on the growth of Spironucleus vortens, an intestinal parasite of the freshwater angelfish

Seven chemotherapeutic agents (dimetridazole, metronidazole, pyrimethamine, albendazole, fenbendazole, mebendazole and magnesium sulfate) were examined for growth inhibition on the cultivation of Spironucleus vortens. Dimetridazole and metronidazole were effective in inhibiting the parasite's growth. At concentrations of 1 microgram ml-1 or higher, both dramatically decreased numbers of parasites. At 24 h exposure, 33% of parasites were inhibited when exposed to dimetridazole or metronidazole at concentrations of 2 and 4 micrograms ml-1, respectively. Dimetridazole at 4 micrograms ml-1 or higher concentrations decreased the number of organisms to 50% or less after 48 h exposure. During the same period of time, the numbers of parasites decreased to 50% or less when exposed to metronidazole at 6 micrograms ml-1 or higher. Pyrimethamine at concentrations of 1 to 10 micrograms ml-1 was not effective in inhibiting the parasite's growth. Albendazole and fenbendazole at concentrations of 0.1 and 0.5 microgram ml-1 were similar in inhibiting the growth of the organism. Both compounds suppressed parasite growth at concentrations of 1.0 microgram ml-1 or higher after 24 h exposure. Mebendazole inhibited the parasite's growth at concentrations of 0.5 microgram ml-1 or higher. At 72 h exposure, 45 to 50% of the parasites were inhibited when exposed to mebendazole at concentrations higher than 0.5 microgram ml-1. Magnesium sulfate at concentrations of 70 mg ml-1 or higher also suppressed the growth of parasites after 24 h exposure. These results indicate that dimetridazole, metronidazole and mebendazole are the most effective chemotherapeutic agents in vitro at inhibiting the growth of S. vortens.     

Low efficacy of single-dose albendazole and mebendazole against hookworm and effect on concomitant helminth infection in Lao PDR

Background

Albendazole and mebendazole are increasingly deployed for preventive chemotherapy targeting soil-transmitted helminth (STH) infections. We assessed the efficacy of single oral doses of albendazole (400 mg) and mebendazole (500 mg) for the treatment of hookworm infection in school-aged children in Lao PDR. Since Opisthorchis viverrini is co-endemic in our study setting, the effect of the two drugs could also be determined against this liver fluke.

Methodology

We conducted a randomized, open-label, two-arm trial. In total, 200 children infected with hookworm (determined by quadruplicate Kato-Katz thick smears derived from two stool samples) were randomly assigned to albendazole (n = 100) and mebendazole (n = 100). Cure rate (CR; percentage of children who became egg-negative after treatment), and egg reduction rate (ERR; reduction in the geometric mean fecal egg count at treatment follow-up compared to baseline) at 21–23 days posttreatment were used as primary outcome measures. Adverse events were monitored 3 hours post treatment.

Principal Findings

Single-dose albendazole and mebendazole resulted in CRs of 36.0% and 17.6% (odds ratio: 0.4; 95% confidence interval: 0.2–0.8; P = 0.01), and ERRs of 86.7% and 76.3%, respectively. In children co-infected with O. viverrini, albendazole and mebendazole showed low CRs (33.3% and 24.2%, respectively) and moderate ERRs (82.1% and 78.2%, respectively).

Conclusions/Significance

Both albendazole and mebendazole showed disappointing CRs against hookworm, but albendazole cured infection and reduced intensity of infection with a higher efficacy than mebendazole. Single-dose administrations showed an effect against O. viverrini, and hence it will be interesting to monitor potential ancillary benefits of a preventive chemotherapy strategy that targets STHs in areas where opisthorchiasis is co-endemic.

Clinical Trial Registration

Current Controlled Trials ISRCTN29126001     

Chinese medicinal herbs Muh-Shiang Bin-Lang-Wan increases the motility of sphincter of Oddi in anesthetized rabbits through activation of M1 muscarinic receptors

The sphincter of Oddi (SO) plays an important role in regulating the bile flow into the duodenum. This study was designed to investigate the effect of Chinese Medicinal Herbs Muh-Shiang-Bin-Lang-Wan (MSBLW) and their mechanism of action on regulating the motility of SO in rabbits. The activity of SO in anesthetized rabbits was measured by using a continuously perfused open-tip manometric method. The rabbits were administered with different doses of MSBLW through naso-gastric tubes. The SO motility before and after the administration of MSBLW were recorded, and analyzed with a computer equipped with an off line analysis software. The results showed that the SO activity, in terms of tonic pressure and phasic contraction pressure, were significantly changed. A significant lower tonic pressure and a higher phasic contraction pressure were noticed 40-60 min after administration of MSBLW with a peak response at 0.5-1.0 gm range. The responses were blocked by pretreatment of muscarinic receptors (M1) antagonist, pirenzepine (10 mg/kg, orally). We conclude that MSBLW is effective in increasing the SO motility in rabbits through activation of M1 muscarinic receptors. However, potential application of MSBLW in the treatment of human biliary disorders needs further evaluation.     

Nucleus raphe obscurus participates in regulation of gallbladder motility through vagus and sympathetic nerves in rabbits

The aim of the present study is to investigate if the nucleus raphe obscurus (NRO) participate in regulating the gallbladder motility in rabbits. Rabbits were fasted for about 20-24 hours. After anesthetization with urethane, an incision was made at the middle of the abdomen and the gallbladder was exposed. A frog bladder connected with force transducer was inserted into the gallbladder through a small incision at the funds to record gallbladder motility (tonic contraction and phasic contraction). Glutamate and other chemicals were microinjected into NRO through a vitreous tube attached to a microsyringe. We found both the tonic contraction and phasic contraction of the gallbladder were enhanced after the glutamate was injected into NRO. GABA inhibited gallbladder motility if administrated in the same way. Microinjection of ketamine, NMDA (N-methyl-D-aspartate) receptor antagonist, into NRO inhibited the phasic contraction of gallbladder. Administration of CNQX (6-cyano-7-nitroquinoxaline-2, 3-dione), a non-NMDA receptor antagonist, enhanced the gallbladder tonic contraction. Pretreatment of ketamine into NRO attenuated the effect of glutamate, while pretreatment of CNQX had no effect on it. Intravenous injection of atropine or vagotomy completely abolished the effect of glutamate on gallbladder phasic contraction, while intravenous injection of phentolamine or transecting the spinal cord at T3-4 inhibited that on tonic contraction. Intravenous injection of propranolol did not influence the glutamate effect. These results suggested that glutamate in NRO participates in regulating the motility of the gallbladder through NMDA receptor. When excited, the NMDA receptors in NRO enhance the phasic contraction of the gallbladder through vagus nerve and peripheral M-receptors, and enhance the tonic contraction of the gallbladder through sympathetic nerve and peripheral alpha-receptors. GABA in NRO is also involved in the regulation of gallbladder motility.     

Reversed-phase liquid chromatographic method with fluorescence detection for the simultaneous determination of albendazole sulphoxide, albendazole sulphone and albendazole 2-aminosulphone in sheep plasma

A rapid and sensitive HPLC method for the simultaneous quantification of albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in sheep blood plasma has been developed. Plasma samples were extracted with ethyl acetate under alkaline conditions. Separation was achieved on a C18 reversed-phase analytical column, in the presence of positively- (tetra-n-butylammonium hydrogen sulphate) and negatively-charged (octanesulphonate sodium) pairing ions, while detection was performed fluorometrically. Excitation and emission wavelengths were 290 and 320 nm, respectively. Limits of quantification were defined at 39 ng/ml for ABZ-SO, 4.95 ng/ml for ABZ-SO2 and 4 ng/ml for ABZ-SO2NH2. Accuracy data, in terms of recovery efficiency showed overall values (+/- S.E.M.) of 85.6 +/- 1.0% for ABZ-SO, 100.0 +/- 1.0% for ABZ-SO2 and 89.1 +/- 0.6% for ABZ-SO2NH2. The method was successfully applied to quantitatively determine the three albendazole metabolites in plasma samples collected from sheep that had been orally administered albendazole.     

Comparative study on gastric absorption of albendazole and mebendazole in rats

1. A study was carried out to determine the kinetics of the gastric absorption of two wide spectrum benzimidazole anthelmintics, albendazole and mebendazole.2. The method used was gastric recirculation of solutions containing the drugs in concentrations ranging from 0.5 to 100 mM.3. The results obtained showed that absorption corresponds to first order kinetics, with diffusion constants of 0.0087 min⁻¹ for albendazole and 0.0077 min⁻¹ for mebendazole.4. Blood levels of the drugs for the whole range of concentrations were always higher in the case of albendazole.     

Comparative study on gastric absorption of albendazole and mebendazole in rats.

1. A study was carried out to determine the kinetics of the gastric absorption of two wide spectrum benzimidazole anthelmintics, albendazole and mebendazole. 2. The method used was gastric recirculation of solutions containing the drugs in concentrations ranging from 0.5 to 100 mM. 3. The results obtained showed that absorption corresponds to first order kinetics, with diffusion constants of 0.0087 min-1 for albendazole and 0.0077 min-1 for mebendazole. 4. Blood levels of the drugs for the whole range of concentrations were always higher in the case of albendazole.     

Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle

Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (nu(max)) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of nu(max) versus [MgADP] was significantly greater for tonic (-0.51 +/- 0.04) than phasic muscle myosin (-0.15 +/- 0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092 +/- 0.022 pN for phasic muscle and 0.084 +/- 0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state.     

Kinetics of mineralization of phenols in lake water.

The kinetics of mineralization of phenol and p-nitrophenol in lake water was determined at concentrations from 200 pg/ml to 5 micrograms/ml. The mineralization data were fit by nonlinear regression to equations for 14 kinetic models that describe patterns of biodegradation by nongrowing cells or by microorganisms growing on either the test chemical or other organic substrates. The kinetics od mineralization of phenol in water samples collected in July was best described by first-order models for 0.5 ng of phenol per ml; by Monod-without-growth, logistic, and logarithmic models for 1.0 and 2.0 ng/ml and 5.0 ng/ml to 1.0 micrograms/ml, respectively, if it is assumed that the mineralizing population uses phenol as the sole carbon source for growth; by models (for phenol at concentrations of 2.0 ng/ml to 1.0 micrograms/ml) that assume that the phenol-mineralizing populations do not grow or grow logarithmically or logistically on uncharacterized carbon compounds but metabolize the phenol when present at levels below and above Km, respectively, for that compound; and by a logarithmic model at 5.0 micrograms/ml. Under the test conditions, usually less than 10% of the phenol C that was metabolized was incorporated into microbial cells or retained by other particulate material in the water at substrate concentrations of 10 ng/ml or less, and the percentage increased at higher substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of mineralization of phenols in lake water

The kinetics of mineralization of phenol and p-nitrophenol in lake water was determined at concentrations from 200 pg/ml to 5 micrograms/ml. The mineralization data were fit by nonlinear regression to equations for 14 kinetic models that describe patterns of biodegradation by nongrowing cells or by microorganisms growing on either the test chemical or other organic substrates. The kinetics od mineralization of phenol in water samples collected in July was best described by first-order models for 0.5 ng of phenol per ml; by Monod-without-growth, logistic, and logarithmic models for 1.0 and 2.0 ng/ml and 5.0 ng/ml to 1.0 micrograms/ml, respectively, if it is assumed that the mineralizing population uses phenol as the sole carbon source for growth; by models (for phenol at concentrations of 2.0 ng/ml to 1.0 micrograms/ml) that assume that the phenol-mineralizing populations do not grow or grow logarithmically or logistically on uncharacterized carbon compounds but metabolize the phenol when present at levels below and above Km, respectively, for that compound; and by a logarithmic model at 5.0 micrograms/ml. Under the test conditions, usually less than 10% of the phenol C that was metabolized was incorporated into microbial cells or retained by other particulate material in the water at substrate concentrations of 10 ng/ml or less, and the percentage increased at higher substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dimethyl sulphoxide and some antibiotics on cultured human T-lymphoma cells as measured by microcalorimetry

The influence of dimethyl sulphoxide (I), penicillin/streptomycin (II), gentamicin (III), and amphotericin B (IV) on growing human T-lymphoma cells was measured by microcalorimetry. There was a dose-dependent decrease in the heat production rate of the cells after 24 h of incubation with I in concentrations ranging from 0-2% (v/v). At 3.6%, about half of the cells died. II and III had no effect on the cells after incubation for 6 days, at concentrations from 1 to 10 times that of the normal (50-500 IU/ml; 50-500 micrograms/ml). IV was used in combination with II (50 IU/ml; 50 micrograms/ml) and III (50 micrograms/ml), respectively, at concentrations between 0.25 and 7.5 micrograms/ml. After 6 days of incubation, the results were similar to those obtained with II and III separately.     

Influences of zinc on fertilisation and development of bovine oocytes in vitro.

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 micrograms added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 micrograms zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 micrograms added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 micrograms per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 microgram zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 microgram/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 microgram ml of zinc in vitro than in vivo because a concentration of 3.0 micrograms/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 micrograms/ml of zinc; however, higher concentrations were inhibitory.     

Nociceptive responses to altered GABAergic activity at the spinal cord

GABA agonists and antagonists were injected intrathecally at the spinal cord, to determine their effect on nociceptive thresholds. Tactile stimulation, applied against the flank by a medium diameter von Frey fiber (5.5 g force), elicited distress vocalizations after, but not before injection of the GABA antagonists, bicuculline MI or picrotoxin (0.25 and 1 microgram dosages). Vocalization threshold to tail shock was significantly reduced by bicuculline MI or picrotoxin. Tail flick withdrawal latency from radiant heat was not altered by GABA antagonists. The GABA agonist, muscimol, significantly elevated vocalization threshold to tail shock at a 5 micrograms dose. At a lower dose level (1 microgram), muscimol significantly reduced vocalization threshold to tail shock. Tail flick latency was significantly prolonged by the 5 micrograms dose of muscimol; however, flaccid paralysis of the hind limbs was also evident. Nociceptive thresholds were not altered by GABA or saline injection. These findings indicate that GABAergic activity contributes to the tonic modulation of nociception at the spinal cord.     

Effects of ivermectin and albendazole against Anisakis simplex in vitro and in guinea pigs

The activity of ivermectin and albendazole against larval Anisakis simplex was tested in vitro and in experimentally infected guinea pigs. Before drug exposure the medium for half of the larvae was adjusted to pH 2.0 with 1 N HCl, whereas the other half was held at pH 7.0. To these solutions, ivermectin was added to full concentrations of 1, 2, 5, 10, 50, 100, or 200 microg/ml, and for albendazole, 300, 400, and 500 microg/ml. Animals from group I were given 0.1 ml of 1% (3.3 mg/kg) ivermectin, whereas guinea pigs from group II were each given 5-7 mg (16.6-23.3 mg/kg) of albendazole orally. The efficacy of both drugs against L, A. simplex was high in vitro and in vivo against the larvae in different organs of guinea pigs.     

An evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs

Sodium ampicillin was administered subcutaneously to 350-550 g male Dunkin Hartley guinea pigs at doses of 6, 8 and 10 mg/kg tid for 5 days. Over a period of 12 days, the lowest ampicillin dose appeared to be tolerated well. However, significant body weight reduction and mortality occurred with the two higher dosage regimens. Cecal cultures of dead animals confirmed the presence of Clostridium difficile, an organism associated with antibiotic-induced enterotoxemia. Assay of serum collected from ampicillin-treated animals revealed ampicillin concentrations of approximately 10 micrograms/ml at 5 minutes post-dosing which fell precipitously to less than 0.2 micrograms/ml at 60 minutes. Determination of biliary ampicillin levels during the 60 minutes after administration of a single 10 mg/kg SQ dose revealed concentrations ranging from 18 micrograms/ml to 90 micrograms/ml. Estimates of total urinary ampicillin content after a single 10 mg/kg SQ dose were less than 500 micrograms/animal at 7.5 minutes, but increased to greater than 2000 micrograms/animal at 60 minutes after dosing. Results of this study indicated that due to its short serum half-life, sodium ampicillin probably has little systemic therapeutic efficacy in guinea pigs. Because high concentrations of ampicillin accumulated in the urine and bile, the antibiotic probably would have therapeutic efficacy for urinary and intestinal infections. However, its associated toxicity at large doses probably precludes its use. In view of the rapid clearance of ampicillin in guinea pigs in comparison to other species, the pharmacokinetics of other antibiotics, especially those reported to be less toxic for guinea pigs, should be considered.     

Differential effects of depolarization on the growth of crayfish tonic and phasic motor axons in culture

Previous studies have demonstrated neuron-specific differences in the inhibitory effects of depolarization upon neurite outgrowth. We examined whether there is a relationship between the normal impulse activity level of an axon and the effect of depolarization upon its growth. Inactive phasic motor axons and active tonic motor axons grow from crayfish abdominal nerve cord explants in culture. Depolarization of these axons with high K⁺ solutions produced greater inhibition of advancing growth cones from the phasic axons than from the tonic axons. During the period 20–40 min after the beginning of depolarization, tonic axon growth cones continued to advance, whereas phasic axon growth cones retracted. During chronic depolarization, all of the phasic axons retracted during the first day and approximately half of the phasic axons had degenerated after 4 days of depolarization. The majority of tonic axons continue to grow after 3 days of depolarization, and all of the tonic axon growth survived the 4 days of depolarization. The different responses of the growing phasic and tonic axons to depolarization appear to be Ca²⁺ dependent. The inhibitory effects of depolarization upon phasic axon growth were reduced by the Ca²⁺ channel blockers La³⁺ and Mg²⁺. Application of a Ca²⁺ ionophore, A23187, produces greater inhibition of phasic axon growth than tonic axon growth. This study demonstrates that depolarization produces greater inhibition of growth from inactive motor axons than from active motor axons. This is likely due to differences in Ca²⁺ regulation and/or sensitivity to intracellular Ca²⁺. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 85–97, 1997     

Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells

Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)