Dirofilaria immitisandBrugia pahangi:Filarial Parasites Make Nitric Oxide

EXPERIMENTAL PARASITOLOGY ARTICLE NO.

90, 131–134 (1998)

PR984308

RESEARCH BRIEF Dirofilaria immitis and Brugia pahangi: Filarial Parasites Make Nitric Oxide

Lana Kaiser, Timothy G. Geary,* and Jeffrey F. Williams Michigan State University, East Lansing, Michigan 48824; U.S.A.; and *Pharmacia & Upjohn, Kalamazoo, Michigan, U.S.A.

Kaiser, L., Geary, T. G., and Williams, J. F. 1998. Dirofilaria immitis and Brugia pahangi: Filarial parasites make nitric oxide. Experimental Parasitology 90, 131–134. q 1998 Academic Press Index Descriptors and Abbreviations: nematode; lymphatic filariasis; heartworms; canine heartworm disease; neuroregulatory peptides; FMRFamide-related peptides; chemiluminescence; NO, nitric oxide; L-NMMA, NG-monomethyl-L-arginine; PF1, SDPNFLRF amide; PF4, KPNFLRF amide.

Once regarded only as a noxious atmospheric chemical, nitric oxide (NO) is now recognized as an important modulator and controller of a diverse array of crucial mammalian functions (Koshland 1992; Anggard 1994; Lowenstein et al. 1994). NO is involved in control of vascular tone, macrophage and immune function, neural transmission and memory, penile erection, gastrointestinal motility, cardiac and renal function, hormone release, and hemostasis. Thus, alterations in the production and/or release of NO could be important in the pathogenesis of many human and animal diseases, including parasitic infections. Although most attention has focused on mammals, NO is also produced by invertebrates (Dow et al. 1994; Bowman et al. 1995; Elphick et al. 1993; Elofsson et al. 1993; Geary et al. 1995; Gelperin 1994; Holmqvist et al 1994; Muller 1994), and in invertebrates, including non-tissuedwelling nematodes, NO is involved in neuromuscular control. However, if NO were to be produced and released by tissue-dwelling filarial parasites, the molecule could be intimately involved in dynamics of the host–parasite relationship. Therefore, experiments were designed to detect and quantify NO production by Dirofilaria immitis, the canine heartworm, and Brugia pahangi. The influence on filarial NO production of nematode neuroregulatory peptides (PF1, PF4, or FLRFamide) and the competitive NO synthase inhibitor, L-NMMA, was also examined Adult D. immitis and B. pahangi were obtained as previously described (Kaiser et al. 1990, 1992, 1995). Parasites were harvested

0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

immediately after death of the host (barbiturate overdose) and placed in medium (RPMI 1640) (Kaiser et al. 1990, 1992, 1995). Heartworms were sexed and divided into groups that included one male and one female and B. pahangi were divided into approximately equal numbers based on visual inspection. Parasites were placed in beakers containing fresh, warm (378C) RPMI 1640. To assess number of B. pahangi used in each experiment, parasites were air dried, and numbers estimated from the formula # 5 weight 2 0.001/0.000094 (r 5 0.9999; Kaiser et al. 1995). The experimental protocols did not cause death of the parasites, nor did they obviously affect mobility or activity. Parasites exposed to drugs during the experimental protocols were not reused. NO was measured by chemiluminescence, using a 270B nitric oxide analyzer (NOA, Sievers Instruments, Boulder, CO) and HP 3396-Series II integrator (Hewlett-Packard, Palo Alto, CA) (Kaiser and Williams, 1997; Archer and Hampl 1992; Archer et al. 1995). Samples were injected into a purge vessel containing glacial acetic acid and sodium iodide, which measures NO as nitrite. In every experiment a baseline sample was taken to represent “baseline contamination” and its value was subtracted from all subsequent samples (Kaiser and Williams 1997). Finally, all samples were analyzed on the day they were obtained (Kaiser and Williams 1997). To determine if filarial parasites produce NO, warm RPMI 1640 was placed in a beaker, the baseline sample was obtained, and then parasites (one pair heartworms or B. pahangi) were added. Samples for analysis of NO were taken at 0.5, 1, 1.5, 2, and 5 min after addition of parasites. Both heartworms and B. pahangi make NO and the pattern of NO production was similar for the two filariae. Peak NO production by heartworms was measured at 30 s (52.4 6 19.8 pmol/ml/min; range 0 to 232 pmol/ml/min) (Fig. 1). Each parasite made 106 6 39 pmol/ min NO (range 4 to 464). Peak NO production by B. pahangi also occurred at 30 s (150.5 6 101 pmol/ml/min); however, there was a wide range of NO production by B. pahangi (0 to 850 pmol/ml/min; n 5 8) which could not be accounted for by differences in the number of parasites (136 6 15). To determine if filarial NO production could be enhanced by exposure to parasite neuropeptides, parasites were equilibrated for 5 min,

131

132

KAISER, GEARY, AND WILLIAMS

FIG. 2. Effect of nematode neuropeptides PF1 (SDPFLRFamide), PF4 (KPNFLRFamide), and FLRFamide (Peptido Genic, San Diego, CA) on NO released by heartworms at 1.5 min after addition of peptides. PF1 (n 5 10), but not PF4 (n 5 7) or FLRFamide (n 5 7), significantly increased NO release when compared to control (n 5 7; P , 0.05).

FIG. 1. NO production (a) and NO release (b) by heartworms (n 5 11). Data are expressed as means 6 standard error of the mean with n representing the number of pairs of D immitis. Experiments were analyzed using a one-way analysis of variance and least significant difference post hoc test, with P, 0.05 taken as the criterion of statistical significance. To assure that heartworms actually produced NO, and that it was not leached from filarial tissues, identical experiments were done with dead worms (killed by exposure to 2708C for 30 min). Dead heartworms (n 5 3) did not release NO over the same time course.

in warm medium, and then the baseline sample was obtained. The peptide (10 mg/ml) was added and samples taken at 0.5, 1, 1.5, 2, and 5 min after addition of the peptide. When compared to control, neither PF4 nor FLRFamide increased NO measured at any time point (Fig. 2). PF1 significantly increased NO release by heartworms at 0.5, 1.5, and 2 min (P , 0.05); this effect disappeared by 5 min. The greatest difference was seen at 1.5 min (control NO release 5 10.4 6 3 pmol/ ml; range 0 to 19.9 pmol/ml vs PF1-induced release 23.2 6 5 pmol/ ml; range 0 to 41.7 pmol/ml). Although it appeared that PF1 increased NO production by Brugia, there was such marked variability in the amount of NO produced and the time of maximum production that statistical significance was not detected. There was no significant difference in the number of parasites used (control, 225 6 36, range 156 to 285, vs PF-1, 209 6 24, range 140 to 270) or the amount of NO produced per parasite (control, 0.31 6 0.07 pmol/ml/worm, vs PF-1, 0.43 6 0.22 pmol/ml/worm). Since PF1 increased NO production by heartworms, experiments

were done to determine if the NOS inhibitor L-NMMA could inhibit peptide-induced NO production by heartworms. To inhibit NOS, one pair of heartworms was incubated for 30 min in medium containing 100 mM L-NMMA (Mupanomunda et al 1997). Parasites were then washed and added to media and the experiment done as described above. Pretreatment of the heartworms with the competitive NOS inhibitor L-NMMA significantly depressed PF1-induced NO release at 0.5, 1, and 1.5 min (Fig. 3), but not at 2 or 5 min. These data indicate that filarial parasites produce and release NO. NO production in heartworms can be enhanced by the nematode peptide PF1 and inhibited by the known inhibitor of mammalian NOS, L-NMMA. Filarial NO may be involved not only in neural regulation, but also in maintaining a hospitable minienvironment for these blood and lymphatic dwelling parasites. In Ascaris suum, NOS activity, measured by conversion of [3H]arginine to [3H]citrulline, was detected in the hypodermis of the parasite (Bowman et al. 1995). NO synthesized in the hypodermis could freely diffuse to its site of action, the somatic musculature, prompting the suggestion that “the nematode hypodermis would play a role analogous to that of the vascular endothelium in vertebrate systems” (Maule et al. 1996). While there is a likely role for parasite NO in nematode

FIG. 3. Effect of the inhibitor of NOS, L-NMMA (Sigma Chemical Co., St Louis, MO), on PF1-induced NO released by heartworms at 1.5 min after addition of peptides. L-NMMA significantly decreased PF1-induced NO release from 23.2 6 5 to 6.1 6 3 pmol/ml (n 5 9; P , 0.05; range 0 to 18.7 pmol/ml).

NO AND D. immitis AND B. pahangi

neuromuscular control, one could speculate additional roles for the radical in the pathogenesis of diseases caused by blood-borne filariae and microfilariae. NO inhibits platelet aggregation; thus, parasitederived NO could bring this about as do parasite-derived prostanoids (Liu et al. 1990; Liu and Weller 1992; Kaiser et al. 1992). This would enable the parasite to modulate local hemostatic environments, clearly a benefit to the parasite. Filarial-derived NO might also influence blood or lymphatic flow by interacting with mammalian smooth muscle, thereby enhancing opportunities for nutrient uptake. Finally, filarial NO might interact with other mammalian cells to change their behavior in ways beneficial to the parasite. The role of filarial NO in the pathogenesis of disease remains to be elucidated. The short half-life of NO suggests a local effect may be more likely than a systemic one. NO produced by heartworms could directly dilate pulmonary arteries and arterioles, resulting in increased blood flow and decreased resistance. Conversely, filarial-derived NO could inhibit endotheliumdependent relaxation, by feedback inhibition of endothelial NOS, resulting constriction of pulmonary vessels and increased resistance. The overall effect of filarial NO on vascular function is likely the result of a complex interplay of mammalian and filarial factors and thus most probably defies simple classification. On exposure to PF1, but not PF4 or FLRFamide, heartworms produced more NO. This peptide, originally isolated from both Panagrellus redivivus and Caenorhabditis elegans, has inhibitory effects on A. suum (Bowman et al. 1995; Maule et al. 1996), relaxing both stimulated and unstimulated muscle strips. The mediator of this response appears to be NO (Bowman et al. 1995). While we were unable to assess the effect of PF1 on muscle tone in heartworms, the production of NO by heartworms, and its inhibition by L-NMMA, suggests that filarial nematodes have synthetic pathways and physiological systems similar to other nematodes. Our inability to measure a statistically significant PF1-induced increase in NO production by B. pahangi may reflect the worms’ smaller biomass, increased cuticular surface area, marked variability in response of these parasites, inability to control either the number or the sex of the parasites studied, or a fundamental difference in the behavior of this parasite compared to heartworms. Heartworms released more NO in response to the nonmammalian, nematode peptide PF1. Although both mammals and nematodes appear to use similar second messenger systems, there is no compelling evidence for a mammalian counterpart to the FMRFamide-like group of nematode peptides. Thus, characterization of unique filarial peptide receptors may provide a novel target for drug discovery and add options for chemotherapeutic interventions for preventing and treating human filarial infections. (The authors thank Daria Maksimowich, Mike Metzger, and S. Ptu for technical assistance and Dr. John McCall for filarial material. This work was supported by grants from the National Institutes of Health (NIH AI-35757 and AI-01082) and NIH Supply Contract AI-02642, US–Japan Cooperative Medical Science Program. L.K. is a recipient of an NIH Research Career Development Award.)

133 Archer, S. L., Schultz, P. J., Warren, J. B., Hampl, V., and DeMaster, E. G. 1995. Preparation of standards and measurement of nitric oxide, nitroxyl, and related oxidation products. Methods: A Companion to Methods in Enzymology 7, 21–34. Archer, S. L., and Hampl, V. 1992. NG-Monomethyl-L-arginine causes nitric oxide synthesis in isolated arterial rings: Trouble in paradise. Biochemical and Biophysical Research Communications 188, 590– 596. Bowman, J. W., Winterrowd, C. A., Friedman, A. R., Thompson, D. P., Klein, R. D., Davis, J. P., Maule, A. G., Blair, K. L., and Geary, T. G. 1995. Nitric oxide mediates the inhibitory effects of SDPNFLRFamide, a nematode FMRFamide-like neuropeptide in Ascaris suum. Journal of Neurophysiology 74, 1880–1888. Dow, J. A. T., Maddrell, S. H. P., Davies, S-A., Skaer, N.J.V., and Kaiser, K. 1994. A novel role for the nitric oxide-cGMP signaling pathway the control of epithelial function in Drosophila. American Journal of Physiology 266, R1716–R1719. Elofsson, R., Carlberg, M., Moroz, L., Neslin, L., and Sakharov, D. 1993. Is nitric oxide (NO) produced by invertebrate neurones? Neuroreport 4, 279–282. Elphick, M. R., Green, I. C., and O’Shea, M. 1993. Nitric oxide synthesis and action in an invertebrate brain. Brain Research 619, 344–346. Geary, T. G., Bowman, J. W., Friedman, A. R., Maule A. G., Davis, J. P., Winterrowd, C. A., Friedman, A. R., Klein, R. D., and Thompson, D. P. 1995. The pharmacology of FMRFamide-related neuropeptides in nematodes: New opportunities for rational anthelmintic discovery? International Journal for Parasitology 25, 1273–1280. Gelperin, A. 1994. Nitric oxide mediates network oscillations of olfactory interneurons in a terrestrial mollusc. Nature 369, 61–63. Kaiser, L., Tithof, P. K., and Williams, J. F. 1990. Depression of endothelium-dependent relaxation by filarial parasite products. American Journal of Physiology 259, H648–652. Kaiser, L., Tithof, P. K., Lamb, V. L., and Williams, J. F. 1991. Depression of endothelium-dependent relaxation in aorta from rats with Brugia pahangi lymphatic filariasis. Circulation Research 68, 1703– 1712. Kaiser, L., Lamb, V. L., Tithof, P. K., Watson, J. T., Gage, D. A., Chamberlin, B. A., and Williams, J. F. 1992. Dirofilaria immitis: Do filarial cyclooxygenase products depress endothelium-dependent relaxation? Experimental Parasitology 75, 159–167. Kaiser, L., Mupanomunda, M., and Williams, J. F. 1995. Brugia pahangi induced contractility of bovine mesenteric lymphatics studied in vitro: a role for filarial factors in the development of lymphedema? American Journal of Tropical Medicine and Hygiene 54, 386–390. Kaiser, L., and Williams, J. F. 1997. Possible problems in measuring nitric oxide. Journal of the American Veterinary Medical Association 210, 1584–1586.

REFERENCES

Anggard, E. 1994. Nitric oxide: Mediator, murderer, and medicine. Lancet 343, 1199–1206.

Koshland, D. E., 1992. The molecule of the year. Science 258, 1861. Liu, L. X., Serhan, C. N., and Weller, P. F. 1990. Intravascular filarial parasites elaborate cyclooxygenase-derived eicosanoids. Journal of Experimental Medicine 172, 993–996. Liu, L.X., and Weller, P. F. 1992. Intravascular filarial parasites inhibit

134 platelet aggregation: Role of parasite-derived prostanoids. Journal of Clinical Investigation 89, 1113–1120. Lowenstein, C. J., Dinerman, J. L., and Snyder, S. H. 1994. Nitric oxide: A physiologic messenger. Annals of Internal Medicine 120, 227–237. Maule, A. G., Geary, T. G., Bowman, J. W., Shaw, C., Halton, D. W., and Thompson, D. P. 1996. The pharmacology of nematode FMRFamide-related peptides. Parasitology Today 12, 351–357. Maule, A. G., Geary, T. G., Bowman, J. W., Marks, N. J., Blair, K. L., Halton, D. W., Shaw, C., and Thompson, D. P. 1995. Inhibitory effects of nematode FMRFamide-related peptides (FaRPs) on muscle-strips from Ascaris suum. Invertebrate Neuroscience 1, 255–265.

KAISER, GEARY, AND WILLIAMS

Maule, A. G., Shaw, C., Bowman, J. W., Halton, D. W., Thompson, D. P., Thrim, L., Kubiak, T. M., Martin, R. A., and Geary, T. G. 1995. Isolation and preliminary biological characterization of KPNFIRFamide, a novel FMRFamide-related peptide from the freeliving nematode Panagrellus redivivus. Peptides 16, 87–93. Muller, U. 1997. The nitric oxide system in insects. Progress in Neurobiology 51, 363–381. Mupanomunda, M., Williams, J. F., Mackenzie, C. D., and Kaiser, L. 1997. Dirofilaria immitis: Heartworm infection alters pulmonary artery endothelial cell behavior. Journal of Applied Physiology 82, 389–398. Received 12 December 1997; accepted with revision 6 April 1998