Male Worm Female Worm Piece of pig intestine containing Ascaris suum, along with a male and female...
1
Male Worm Female Worm Piece of pig intestine containing Ascaris suum, along with a male and female specimen Morphine produced a small transient hyperpolarization followed by a prolonged weak depolarization in intracellularly recorded muscle cells Application of 20 µL, 10 mM morphine produced a small transient hyperpolarization followed by a weak depolarization that was slow in onset and prolonged in duration (average amplitude approximately +1.5 mV; Fig. 3A,B. Recovery occurred over a 2-8 min period post-application. Since muscle has been shown to release nitric oxide (NO) in response to morphine (Pryor et al., 2004; Zhu et al., 2004), one explanation for the morphine-induced depolarization is that it reflects the direct and/or indirect action of NO. The depolarization was often accompanied by an increase in graded muscle spike frequency. Morphine HC Na 2 SO 4 The most consistent effect of morphine application (20 µL, 10 mM) to intracellularly recorded muscle cells was prolonged albeit weak depolarization, plotted here (Fig. 3B). Applications of the control salines, Hi CaMg (abbreviated HC) and the equimolar Na 2 SO 4 in HC to control for the morphine sulfate salt pentahydrate used, were similar in being without significant effect. Morphine- induced depolarizations were reproducible in both 1st and 2nd day worms, but 2nd day worms were much less likely to show the initial transient hyperpolarization. Morphine produced a transient depolarization in intracellularly recorded hypodermis Morphine 20 µL, 10 mM Hypodermis is a multi-purpose tissue that underlies and secretes the cuticle of the worm, underlies and helps to anchor body wall muscle cells and also envelops and probably supports the neurons and nervous tissue throughout the worm in a glial-like fashion. Morphine (20 µL, 10 mM) applied to intracellularly recorded lateral line hypodermis produced depolarization. This hypodermal depolarization was much faster in onset and much less prolonged than the morphine-induced muscle depolarization (cf. Fig. 4A and 3A). Morphine HC Na 2 SO 4 * The average morphine-induced depolarization of the hypodermis was approximately +2.2 mV, plotted here. Applications of the control salines, Hi CaMg (abbreviated HC) and the equimolar Na 2 SO 4 in HC to control for the morphine sulfate salt pentahydrate used, were similar in being without significant effect. Second day worms were much less likely to show hypodermal responses to morphine, with over a third of applications eliciting no response, though the average depolarization of those that did respond was similar to responses in first day worms. Unlike muscle, hypodermis does not appear to be an excitable tissue, though there is evidence for a glutamate transporter in the hypodermis (Davis, 1998). The faster onset and less prolonged nature of the hypodermal depolarization compared to the muscle depolarization suggest that a different mechanism of action may be involved in the two tissues. ↑ Morphine 20 µL, 10 mM 5. Sample Traces of Morphine Effects on Dorsal Excitatory Type-1 (DE1), Dorsal Excitatory Type-2 (DE2) and Dorsal Inhibitory (DI) Motorneurons 5A. DE1 motorneuron 5B. DE2 motorneuron 5C. DI motorneuron 5D-E. Summary data for morphine-induced changes in membrane potential (Fig. 5D) and input resistance (Fig. 5E) for the 3 classes of motorneurons; morphine is abbreviated “Mor” in Fig. 5D,E. The HC and Na 2 SO 4 control salines were without significant effects. To detect morphine-induced input resistance changes indicative of changes in membrane conductance, two microelectrodes were inserted in single neurons at minimal separation and uniform hyperpolarizing current pulses were injected into one microelectrode while recording with the other microelectrode to obtain steady-state input resistance data (cf. Fig. 5A-C). DE1 Motorneurons In dorsal excitatory type-1 (DE1) motorneurons, morphine produced short initial transient depolarizations (approximately 3 mV) followed by long, slow hyperpolarizations (approximately -5 mV; cf. Fig. 5A; hyperpolarizations are plotted in 5D). These responses were accompanied by a decrease in input resistance of ≥ 50% at 30 sec post- morphine application (Fig. 5A,E); the input resistance decrease gradually reversed to the pre-morphine value, though most preps had not fully recovered even by 15 min post- application in the Hi CaMg control saline wash (data not shown). DE2 Motorneurons Similar long, slow hyperpolarizations with prolonged recovery were obtained for morphine applications to dorsal excitatory type-2 (DE2) neurons, though the transient initial depolarization seen in DE1 motorneurons was not uniformly seen in DE2 neurons (Fig. 5B,D). DE2 input resistance also decreased, though not as dramatically as in DE1 motorneurons (Fig. 5B,E). Like DE1 motorneurons, DE2 recoveries from morphine were prolonged (>15 min). DI Motorneurons In contrast to the prolonged morphine-induced hyperpolarizations of the DE1 and DE2 excitatory classes of motorneurons, the dorsal inhibitory motorneuron class (DI) exhibited depolarizations (approximately +2.4 mV) upon morphine application (Fig. 5C,D), which were smaller in amplitude and recovered much faster than excitatory motorneuron responses (after about 5 min). A smaller decrease in DI input resistance (≤ 10%) suggests a weaker morphine effect on the inhibitory motorneurons than on the excitatory motorneurons (Fig. 5C,E). 6A 6B The pharynx of nematodes is a muscular tube used for feeding that connects the mouth and intestine. A glass extracellular suction electrode was gently placed on the surface of the pharynx for recording spontaneous or serotonin (5-HT) evoked spikes. Morphine applications (20 µL, 10 mM) were made as in the previous experiments. Representative traces of both complete abolition of spiking (Fig. 6A) and attenuation of spiking (Fig. 6B) upon morphine application are shown. Morphine either abolished or diminished both spontaneous and serotonin- induced pharyngeal spiking. (In cases where spontaneous activity was not present, spiking was brought up by application of serotonin.) In over half the experiments, spikes were abolished completely, usually immediately after morphine application (Fig. 6A). In a few experiments, morphine caused a decrease in both spike frequency (on average by 29%) and amplitude (on average by 45%), but did not abolish spikes completely (Fig. 6B). The morphine effects were not reversible in most preparations. Summary 1. Morphine has an inhibitory effect on whole worm behavior. Consistent with this, the µ-opioid receptor antagonist, naloxone, caused hyperactivity in whole worm behavior, suggesting that morphine may be tonically released as an internal signal/modulator of worm locomotion. If endogenously produced morphine is released by the worm to decrease host GI tract motility, morphine’s internal function may be to produce a corresponding decrease in worm motility, since the worm does not then need as strong or sustained locomotion to maintain its position in the GI tract. 2. Morphine caused hyperpolarization of two classes of dorsal excitatory motorneurons, depolarization of dorsal inhibitory motorneurons, and decreases in input resistance in the motorneurons, thus suggesting a net inhibitory effect of the drug on the locomotory motornervous system. The net effects of these motorneuron changes are consistent with a possible contributing mechanism to the overall suppression/inhibition of locomotory activity seen in the whole worm morphine injections. 3. Other internal targets of morphine include the muscle (weak depolarization), hypodermis (depolarization) and pharynx (spike abolition/suppression). The possible involvement of morphine-induced release of the volatile neurotransmitter NO (Pryor, et al., 2004; Zhu et al., 2004), makes it difficult to assess the precise source(s) of these effects. Different mechanisms of action may be involved in the morphine- and M6G-elicited effects reported here; we hope that on- going work will help to sort them out. 4. These results provide further evidence for an internal signaling system involving morphine and/or its metabolite, M6G, in the parasitic nematode, Ascaris suum. 5. This morphinergic signaling system potentially involves multiple sources and target tissues in the parasite’s motornervous, feeding and reproductive systems; it represents a novel target for possible opioid-related anthelmintic development and intervention in parasitic helminthiases. Support provided by NIH grant AI070584 to R.D. (Gouman et al., 2000; Pryor et al., 2004; Zhu et al., 2004; see Fig. 1 for structures). HPLC coupled to electrochemical detection as well as gas chromatography and mass spectrometry revealed large amounts of morphine within the worm (1168±278 ng/g worm wet weight; Gouman et al., 2000). Morphine has been identified within several tissues including subcuticle layers, hypodermis/lateral cords, nerve cords, muscle, intestinal and reproductive tissues; M6G has also been identified in A. suum muscle tissues (167±28 ng/g in females; 92±11 ng/g in males) (Gouman et al., 2000; Pryor et al., 2004; Zhu et al., 2004). It has been hypothesized that the parasite releases these opiates in order to suppress host gastrointestinal motility thus countering worm expulsion (Gouman et al., 2000). The worm may also release morphine to suppress the host immune system, thus escaping the consequences of an immune response (Gouman et al., 2000; Pryor et al., 2004, 2005). Release into the external environment is supported by the detection of morphine in the extra-worm fluid during incubation periods (Gouman et al., 2000; Zhu et al., 2004). Both morphine and M6G have been shown to stimulate release of the volatile neurotransmitter nitric oxide (NO) from A. suum muscle, suggesting that morphine may also play a role as an internal signaling molecule in the worm (Gouman et al., 2000 [morphine]; Zhu et al., 2004 [both]). In muscle, the general opiate receptor antagonist naloxone blocks morphine but not M6G release of NO, while the specific µ antagonist, CTOP, blocks M6G but not morphine release of NO (Pryor, et al., 2004; Zhu et al., 2004). While RT-PCR was unsuccessful at yielding an A. suum mRNA corresponding to the human neuronal µ opiate receptor (Gouman et al., 2000), the work with opiate antagonists suggests that one or more novel µ opiate receptors might be present in the worm. The present study seeks to further elucidate the targets and effects of morphine and the M6G metabolite as possible internal signaling molecules within A. suum. The human parasitic gastrointestinal nematode Ascaris lumbricoides infects roughly one fourth of the world’s population and is a major contributor to the three leading causes of human mortality – diarrheal disease, respiratory disease, and malnutrition. Although a separate species, A. lumbricoides is very similar to the pig species A. suum (Fig. 2). If morphine and/or M6G can be found to play a role in Ascaris neurophysiology, motor control, feeding, and/or reproduction, and especially if either of the opiates acts on a receptor(s) shown to be distinct from human opiate receptors, such findings may have significance for the potential development of a novel anthelmintic (antiparasite drug) that targets the opioid receptor system in parasitic nematodes. Parasitic nematodes are relatively similar across species, especially with respect to anthelmintic susceptibility. Endogenous morphine has been identified in several nematodes, but also appears to be present in other parasitic helminths, e.g., Schistosoma mansoni (Pryor & Elizee, 2000; Zhu et al., 2002; Pryor et al., 2005). An opioid-related anthelmintic may provide a novel approach for developing new drugs with a broad spectrum of parasite control. Novel anti-parasitic agents are needed to supplement or replace currently used anthelmintics which are unfortunately showing increasing ineffectiveness due to their susceptibility to parasite resistance, especially in veterinary applications. Ascaris suum worms (Fig. 2) were collected from a local pig slaughterhouse and stored in Ascaris phosphate-buffered saline. For whole worm injection experiments, worms received injections according to our standard injection protocol: 50 µL injected at 2 sites; total 100 µL, 10 mM of drug or Ascaris Hi CaMg control saline (final intra-worm concentration approx. 1 mM drug). For electrophysiology experiments, preparations were superfused with Ascaris Hi CaMg saline (1.5-2.0 mL/min at 37 o ± 2 o C). Morphine (20 µL, 10 mM) was applied using 20 µL fixed volume pipettes to dorsal muscle cells, the hypodermis, 3 classes of motorneurons, and the pharynx of A. suum. Controls for electrophysiology experiments included Hi CaMg saline without drug as well as equimolar Na 2 SO 4 in Hi CaMg saline, since the morphine sulfate salt pentahydrate was used. Intracellular recordings (and intracellular stimulations in two microelectrode intracellular experiments) were made with 3 M KCl-filled glass microelectrodes. Extracellular pharyngeal recordings were made using a glass suction electrode. Drugs were obtained from Sigma-Aldrich. Minimum sample size, N ≥ 4 for all data points, unless otherwise noted. Error bars, mean ±S.E.M. Morphine and naloxone injections were performed to determine whether there were any observable effects on whole worm behavior of these opioid-related drugs. Both male and female worms injected with morphine (approx. 1 mM final internal concentration) displayed significantly attenuated body movement and head searching compared to controls. These effects took several minutes to become definitive, but persisted up to 7 hours post- injection with complete recovery by about 9½ hours post-injection. In addition to these effects, male worms injected with morphine displayed the distinctive male tail curl that is seen during male copulatory behavior (cf. Fig. 2); this effect became apparent after several minutes, persisted for at least 6 hours and was not seen in the control saline-injected worms which showed the male tail curl only occasionally and transiently. Thus it is possible that endogenous morphine plays a role in male worm reproductive behavior, a finding that may also have anthelmintic implications. In contrast to morphine, worms injected with the μ-opioid receptor antagonist, naloxone (approx. 1 mM final internal concentration) exhibited increased high frequency head searching accompanied by “jerky/fitful” anterior body movements, and increased general locomotory and posterior body movements compared to Hi CaMg control injected or un-injected worms. This hyperactivity persisted at least 1¾ hours; full recovery occurred by 2½ hours post-injection. Naloxone-induced hyperactivity suggests that morphine may be acting tonically as an inhibitory signaling system in the worm; these naloxone effects may have potential anthelmintic implications as well. Morphine Morphine-6-glucuronide (M6G) Summary data for depolarizing effects of morphine on hypodermis phine Effects on Intracellularly Recorded Dorsal Muscle Cells 1 Morphine Effects on Intracellularly Recorded Hypodermis Morphine Effects on 3 Classes of Intracellularly Recorded Dorsal Motorneurons Morphine 20 µL, 10 mM Methods Introduction Whole Worm Injections of Morphine and Naloxone 2 Morphine Effects on Extracellularly Recorded Pharyngeal Spiking Activity 6. Morphine Abolished or Attenuated Spiking in the Pharynx 5A-C. Representative traces of typical changes in membrane potential and input resistance to application of 20 µL, 10 mM morphine for a motorneuron from each of the 3 classes of motorneurons studied. In these two microelectrode experiments, the current monitor trace is the top trace and the voltage recording microelectrode trace is the lower trace (the stimulating microelectrode trace is not shown). The three motorneuron types studied here were: dorsal excitatory type-1 motorneurons (DE1), dorsal excitatory type-2 motorneurons (DE2) and dorsal inhibitory motorneurons (DI). All three classes of motorneurons have synaptic output to and control of dorsal muscle cells. Morphine-6-Glucuronide (M6G) Effects on Intracellularly Recorded Muscle Cells, Hypodermis and Dorsal Motorneurons References Davis RE. (1998) Action of excitatory amino acids on hypodermis and the motornervous system of Ascaris suum: pharmacological evidence for a glutamate transporter. Parasitology, 116 :487-500. Goumon Y, Casares F, Pryor S, Ferguson L, Brownawell B, Cadet P, Rialas CM, Welter ID, Sonetti D, Stefani GB. (2000) Ascaris suum, an intestinal parasite, produces morphine. Journal of Immunology, 165 : 339-43. Pryor SC & Elizee R. (2000) Evidence of opiates and opioid neuropeptides and their immune effects in parasitic invertebrates representing three different phyla: Schistosoma mansoni, Theromyzon tessulatum, Trichinella spiralis. Acta Biologica Hungarica, 51 : 331-41. Pryor SC, Henry S, Sarfo J. (2005) Endogenous morphine and parasitic helminthes. Medical Science Monitor 11 :RA 183-189. Pryor SC, Putnam J, Hoo, N. (2004) Opiate alkaloids in Ascaris suum. Acta Biologica Hungarica, 55 :353- 361. Zhu W, Baggerman G, Secor WE, Casares F, Pryor SC, Fricchione GL, Ruiz-Tiben E, Eberhard ML, Bimi L, Stefano GB. (2002) Dracunculus medinensis and Schistosoma mansoni contain alkaloids. Annals of Tropical Medicine and Parasitology, 96 : 309-16. Zhu W, Pryor SC, Putnam J, Cadet P, Stefano GB. (2004) Opiate alkaloids and nitric oxide production in the nematode Ascaris suum. Journal of Parasitology, 90 : 15-22. Morphine 20 µL, 10 mM Morphine 20 µL, 10 mM C hange in m em brane potential(m V) -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 HC M 6G M6G has No Obvious Effect on Intracellularly Recorded Muscle Cells 7A C hange in m em brane potential (m V) 0 1 2 3 4 5 6 7 HC M 6G M6G Depolarizes Intracellularly Recorded Hypodermis 7B Application of M6G (20 µL, 10 mM), a bioactive metabolite of morphine that has been isolated in Ascaris suum (Pryor et al., 2004; Zhu et al., 2004), produced no obvious electrophysiological response in muscle cells (Fig. 7A), unlike morphine (cf. Fig. 3). M6G (20 µL,10 mM) application to hypodermis produced a strong depolarization, one that was similar in shape, but at least 2x larger than the hypodermal depolarization produced by morphine (Fig. 7B; cf. Fig. 4). C hange in m em brane potential (m V) -4 -3 -2 -1 0 1 M 6G HC M 6G HC M 6G HC DE1 DE2 DI -40 -20 0 20 M 6G HC M 6G HC DE1 DE2 % C hange in inputresistance M6G Effects on Input Resistance of Intracellularly Recorded Motorneurons 7D M6G Effects on Intracellularly Recorded Motorneurons 7C * Application of M6G (20 µL, 10 mM) to dorsal motorneurons produced hyperpolarizations in DE1 and DE2 motorneurons, albeit notably weaker than those elicited by morphine (Fig. 7C; cf. Fig. 5E). Unlike morphine, M6G- induced hyperpolarizations did not exhibit significant input resistance changes (Fig. 7D), suggesting the possibility that a non-conductance mechanism may be involved. (DI membrane potential and DE2 input resistance data are preliminary, N=1 and N=2 respectively.) Abolition of Spiking Attenuation of Spiking M or HC Na 2 SO 4 M or HC Na 2 SO 4 M orHC Na 2 SO 4 DE1 DE2 DI C hange in m em brane potential (m V) 4.0 2.0 0.0 -2.0 -4.0 -6.0 Summary Data for Morphine Effects on Membrane Potential of Intracellularly Recorded Motorneurons 5D. C hange in inputresistance (% ) 50 0 -50 M or HC Na 2 SO 4 M or HC Na 2 SO 4 M or HC Na 2 SO 4 DE1 DE2 DI Summary Data for Morphine Effects on Input Resistance of Intracellularly Recorded Motorneurons 5E. 3A. 4A. 4B. Morphine 20 µL, 10 mM Morphine 20 µL, 10 mM 3B. Summary Data for Depolarizing Effects of Morphine on Intracellularly Recorded Muscle Cells
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Transcript of Male Worm Female Worm Piece of pig intestine containing Ascaris suum, along with a male and female...
Male Worm
Female Worm
Piece of pig intestine containing Ascaris suum, along with a male and female specimen
Morphine produced a small transient hyperpolarization followed by a prolonged weak depolarization in intracellularly recorded muscle cells
Application of 20 µL, 10 mM morphine produced a small transient hyperpolarization followed by a weak depolarization that was slow in onset and prolonged in duration (average amplitude approximately +1.5 mV; Fig. 3A,B. Recovery occurred over a 2-8 min period post-application. Since muscle has been shown to release nitric oxide (NO) in response to morphine (Pryor et al., 2004; Zhu et al., 2004), one explanation for the morphine-induced depolarization is that it reflects the direct and/or indirect action of NO. The depolarization was often accompanied by an increase in graded muscle spike frequency.
Morphine HC Na2SO4
The most consistent effect of morphine application (20 µL, 10 mM) to intracellularly recorded muscle cells was prolonged albeit weak depolarization, plotted here (Fig. 3B). Applications of the control salines, Hi CaMg (abbreviated HC) and the equimolar Na2SO4 in HC to control for the morphine sulfate salt pentahydrate used, were similar in being without significant effect. Morphine-induced depolarizations were reproducible in both 1st and 2nd day worms, but 2nd day worms were much less likely to show the initial transient hyperpolarization.
Morphine produced a transient depolarization in intracellularly recorded hypodermis
Morphine20 µL, 10 mM
Hypodermis is a multi-purpose tissue that underlies and secretes the cuticle of the worm, underlies and helps to anchor body wall muscle cells and also envelops and probably supports the neurons and nervous tissue throughout the worm in a glial-like fashion. Morphine (20 µL, 10 mM) applied to intracellularly recorded lateral line hypodermis produced depolarization. This hypodermal depolarization was much faster in onset and much less prolonged than the morphine-induced muscle depolarization (cf. Fig. 4A and 3A).
Morphine HC Na2SO4
*
The average morphine-induced depolarization of the hypodermis was approximately +2.2 mV, plotted here. Applications of the control salines, Hi CaMg (abbreviated HC) and the equimolar Na2SO4 in HC to control for the morphine sulfate salt pentahydrate used, were similar in being without significant effect. Second day worms were much less likely to show hypodermal responses to morphine, with over a third of applications eliciting no response, though the average depolarization of those that did respond was similar to responses in first day worms. Unlike muscle, hypodermis does not appear to be an excitable tissue, though there is evidence for a glutamate transporter in the hypodermis (Davis, 1998). The faster onset and less prolonged nature of the hypodermal depolarization compared to the muscle depolarization suggest that a different mechanism of action may be involved in the two tissues.
↑Morphine
20 µL, 10 mM
5. Sample Traces of Morphine Effects on Dorsal Excitatory Type-1 (DE1), Dorsal Excitatory Type-2 (DE2) and Dorsal Inhibitory (DI) Motorneurons
5A. DE1 motorneuron 5B. DE2 motorneuron
5C. DI motorneuron
5D-E. Summary data for morphine-induced changes in membrane potential (Fig. 5D) and input resistance (Fig. 5E) for the 3 classes of motorneurons; morphine is abbreviated “Mor” in Fig. 5D,E. The HC and Na2SO4 control salines were without significant effects. To detect morphine-induced input resistance changes indicative of changes in membrane conductance, two microelectrodes were inserted in single neurons at minimal separation and uniform hyperpolarizing current pulses were injected into one microelectrode while recording with the other microelectrode to obtain steady-state input resistance data (cf. Fig. 5A-C). DE1 MotorneuronsIn dorsal excitatory type-1 (DE1) motorneurons, morphine produced short initial transient depolarizations (approximately 3 mV) followed by long, slow hyperpolarizations (approximately -5 mV; cf. Fig. 5A; hyperpolarizations are plotted in 5D). These responses were accompanied by a decrease in input resistance of ≥ 50% at 30 sec post-morphine application (Fig. 5A,E); the input resistance decrease gradually reversed to the pre-morphine value, though most preps had not fully recovered even by 15 min post-application in the Hi CaMg control saline wash (data not shown). DE2 MotorneuronsSimilar long, slow hyperpolarizations with prolonged recovery were obtained for morphine applications to dorsal excitatory type-2 (DE2) neurons, though the transient initial depolarization seen in DE1 motorneurons was not uniformly seen in DE2 neurons (Fig. 5B,D). DE2 input resistance also decreased, though not as dramatically as in DE1 motorneurons (Fig. 5B,E). Like DE1 motorneurons, DE2 recoveries from morphine were prolonged (>15 min). DI Motorneurons In contrast to the prolonged morphine-induced hyperpolarizations of the DE1 and DE2 excitatory classes of motorneurons, the dorsal inhibitory motorneuron class (DI) exhibited depolarizations (approximately +2.4 mV) upon morphine application (Fig. 5C,D), which were smaller in amplitude and recovered much faster than excitatory motorneuron responses (after about 5 min). A smaller decrease in DI input resistance (≤ 10%) suggests a weaker morphine effect on the inhibitory motorneurons than on the excitatory motorneurons (Fig. 5C,E).
6A
6B
The pharynx of nematodes is a muscular tube used for feeding that connects the mouth and intestine. A glass extracellular suction electrode was gently placed on the surface of the pharynx for recording spontaneous or serotonin (5-HT) evoked spikes. Morphine applications (20 µL, 10 mM) were made as in the previous experiments. Representative traces of both complete abolition of spiking (Fig. 6A) and attenuation of spiking (Fig. 6B) upon morphine application are shown. Morphine either abolished or diminished both spontaneous and serotonin-induced pharyngeal spiking. (In cases where spontaneous activity was not present, spiking was brought up by application of serotonin.) In over half the experiments, spikes were abolished completely, usually immediately after morphine application (Fig. 6A). In a few experiments, morphine caused a decrease in both spike frequency (on average by 29%) and amplitude (on average by 45%), but did not abolish spikes completely (Fig. 6B). The morphine effects were not reversible in most preparations.
Summary
1. Morphine has an inhibitory effect on whole worm behavior. Consistent with this, the µ-opioid receptor antagonist, naloxone, caused hyperactivity in whole worm behavior, suggesting that morphine may be tonically released as an internal signal/modulator of worm locomotion. If endogenously produced morphine is released by the worm to decrease host GI tract motility, morphine’s internal function may be to produce a corresponding decrease in worm motility, since the worm does not then need as strong or sustained locomotion to maintain its position in the GI tract.2. Morphine caused hyperpolarization of two classes of dorsal excitatory motorneurons, depolarization of dorsal inhibitory motorneurons, and decreases in input resistance in the motorneurons, thus suggesting a net inhibitory effect of the drug on the locomotory motornervous system. The net effects of these motorneuron changes are consistent with a possible contributing mechanism to the overall suppression/inhibition of locomotory activity seen in the whole worm morphine injections. 3. Other internal targets of morphine include the muscle (weak depolarization), hypodermis (depolarization) and pharynx (spike abolition/suppression). The possible involvement of morphine-induced release of the volatile neurotransmitter NO (Pryor, et al., 2004; Zhu et al., 2004), makes it difficult to assess the precise source(s) of these effects. Different mechanisms of action may be involved in the morphine- and M6G-elicited effects reported here; we hope that on-going work will help to sort them out. 4. These results provide further evidence for an internal signaling system involving morphine and/or its metabolite, M6G, in the parasitic nematode, Ascaris suum. 5. This morphinergic signaling system potentially involves multiple sources and target tissues in the parasite’s motornervous, feeding and reproductive systems; it represents a novel target for possible opioid-related anthelmintic development and intervention in parasitic helminthiases.
Support provided by NIH grant AI070584 to R.D.
The opiate alkaloid morphine and its metabolite, morphine-6-glucuronide (M6G), have both been identified in the parasitic gastrointestinal nematode, Ascaris suum (Gouman et al., 2000; Pryor et al., 2004; Zhu et al., 2004; see Fig. 1 for structures). HPLC coupled to electrochemical detection as well as gas chromatography and mass spectrometry revealed large amounts of morphine within the worm (1168±278 ng/g worm wet weight; Gouman et al., 2000). Morphine has been identified within several tissues including subcuticle layers, hypodermis/lateral cords, nerve cords, muscle, intestinal and reproductive tissues; M6G has also been identified in A. suum muscle tissues (167±28 ng/g in females; 92±11 ng/g in males) (Gouman et al., 2000; Pryor et al., 2004; Zhu et al., 2004). It has been hypothesized that the parasite releases these opiates in order to suppress host gastrointestinal motility thus countering worm expulsion (Gouman et al., 2000). The worm may also release morphine to suppress the host immune system, thus escaping the consequences of an immune response (Gouman et al., 2000; Pryor et al., 2004, 2005). Release into the external environment is supported by the detection of morphine in the extra-worm fluid during incubation periods (Gouman et al., 2000; Zhu et al., 2004). Both morphine and M6G have been shown to stimulate release of the volatile neurotransmitter nitric oxide (NO) from A. suum muscle, suggesting that morphine may also play a role as an internal signaling molecule in the worm (Gouman et al., 2000 [morphine]; Zhu et al., 2004 [both]). In muscle, the general opiate receptor antagonist naloxone blocks morphine but not M6G release of NO, while the specific µ antagonist, CTOP, blocks M6G but not morphine release of NO (Pryor, et al., 2004; Zhu et al., 2004). While RT-PCR was unsuccessful at yielding an A. suum mRNA corresponding to the human neuronal µ opiate receptor (Gouman et al., 2000), the work with opiate antagonists suggests that one or more novel µ opiate receptors might be present in the worm. The present study seeks to further elucidate the targets and effects of morphine and the M6G metabolite as possible internal signaling molecules within A. suum. The human parasitic gastrointestinal nematode Ascaris lumbricoides infects roughly one fourth of the world’s population and is a major contributor to the three leading causes of human mortality – diarrheal disease, respiratory disease, and malnutrition. Although a separate species, A. lumbricoides is very similar to the pig species A. suum (Fig. 2). If morphine and/or M6G can be found to play a role in Ascaris neurophysiology, motor control, feeding, and/or reproduction, and especially if either of the opiates acts on a receptor(s) shown to be distinct from human opiate receptors, such findings may have significance for the potential development of a novel anthelmintic (antiparasite drug) that targets the opioid receptor system in parasitic nematodes. Parasitic nematodes are relatively similar across species, especially with respect to anthelmintic susceptibility. Endogenous morphine has been identified in several nematodes, but also appears to be present in other parasitic helminths, e.g., Schistosoma mansoni (Pryor & Elizee, 2000; Zhu et al., 2002; Pryor et al., 2005). An opioid-related anthelmintic may provide a novel approach for developing new drugs with a broad spectrum of parasite control. Novel anti-parasitic agents are needed to supplement or replace currently used anthelmintics which are unfortunately showing increasing ineffectiveness due to their susceptibility to parasite resistance, especially in veterinary applications.
Ascaris suum worms (Fig. 2) were collected from a local pig slaughterhouse and stored in Ascaris phosphate-buffered saline. For whole worm injection experiments, worms received injections according to our standard injection protocol: 50 µL injected at 2 sites; total 100 µL, 10 mM of drug or Ascaris Hi CaMg control saline (final intra-worm concentration approx. 1 mM drug). For electrophysiology experiments, preparations were superfused with Ascaris Hi CaMg saline (1.5-2.0 mL/min at 37o ± 2oC). Morphine (20 µL, 10 mM) was applied using 20 µL fixed volume pipettes to dorsal muscle cells, the hypodermis, 3 classes of motorneurons, and the pharynx of A. suum. Controls for electrophysiology experiments included Hi CaMg saline without drug as well as equimolar Na2SO4 in Hi CaMg saline, since the morphine sulfate salt pentahydrate was used. Intracellular recordings (and intracellular stimulations in two microelectrode intracellular experiments) were made with 3 M KCl-filled glass microelectrodes. Extracellular pharyngeal recordings were made using a glass suction electrode. Drugs were obtained from Sigma-Aldrich. Minimum sample size, N ≥ 4 for all data points, unless otherwise noted. Error bars, mean ±S.E.M.
Morphine and naloxone injections were performed to determine whether there were any observable effects on whole worm behavior of these opioid-related drugs. Both male and female worms injected with morphine (approx. 1 mM final internal concentration) displayed significantly attenuated body movement and head searching compared to controls. These effects took several minutes to become definitive, but persisted up to 7 hours post-injection with complete recovery by about 9½ hours post-injection. In addition to these effects, male worms injected with morphine displayed the distinctive male tail curl that is seen during male copulatory behavior (cf. Fig. 2); this effect became apparent after several minutes, persisted for at least 6 hours and was not seen in the control saline-injected worms which showed the male tail curl only occasionally and transiently. Thus it is possible that endogenous morphine plays a role in male worm reproductive behavior, a finding that may also have anthelmintic implications. In contrast to morphine, worms injected with the μ-opioid receptor antagonist, naloxone (approx. 1 mM final internal concentration) exhibited increased high frequency head searching accompanied by “jerky/fitful” anterior body movements, and increased general locomotory and posterior body movements compared to Hi CaMg control injected or un-injected worms. This hyperactivity persisted at least 1¾ hours; full recovery occurred by 2½ hours post-injection. Naloxone-induced hyperactivity suggests that morphine may be acting tonically as an inhibitory signaling system in the worm; these naloxone effects may have potential anthelmintic implications as well.
Morphine
Morphine-6-glucuronide (M6G)
Summary data for depolarizing effects of morphine on hypodermis
Morphine Effects on Intracellularly Recorded Dorsal Muscle Cells
1
Morphine Effects on Intracellularly Recorded Hypodermis
Morphine Effects on 3 Classes of Intracellularly Recorded Dorsal Motorneurons
Morphine20 µL, 10 mM
Methods
Introduction
Whole Worm Injections of Morphine and Naloxone
2
Morphine Effects on Extracellularly Recorded Pharyngeal Spiking Activity
6. Morphine Abolished or Attenuated Spiking in the Pharynx
5A-C. Representative traces of typical changes in membrane potential and input resistance to application of 20 µL, 10 mM morphine for a motorneuron from each of the 3 classes of motorneurons studied. In these two microelectrode experiments, the current monitor trace is the top trace and the voltage recording microelectrode trace is the lower trace (the stimulating microelectrode trace is not shown). The three motorneuron types studied here were: dorsal excitatory type-1 motorneurons (DE1), dorsal excitatory type-2 motorneurons (DE2) and dorsal inhibitory motorneurons (DI). All three classes of motorneurons have synaptic output to and control of dorsal muscle cells.
Morphine-6-Glucuronide (M6G) Effects on Intracellularly Recorded Muscle Cells, Hypodermis and Dorsal Motorneurons
References
Davis RE. (1998) Action of excitatory amino acids on hypodermis and the motornervous system of Ascaris suum: pharmacological evidence for a glutamate transporter. Parasitology, 116:487-500.Goumon Y, Casares F, Pryor S, Ferguson L, Brownawell B, Cadet P, Rialas CM, Welter ID, Sonetti D, Stefani GB. (2000)
Ascaris suum, an intestinal parasite, produces morphine. Journal of Immunology, 165: 339-43.Pryor SC & Elizee R. (2000) Evidence of opiates and opioid neuropeptides and their immune effects in parasitic invertebrates representing three different phyla: Schistosoma mansoni, Theromyzon tessulatum, Trichinella spiralis. Acta Biologica Hungarica, 51: 331-41.Pryor SC, Henry S, Sarfo J. (2005) Endogenous morphine and parasitic helminthes. Medical Science Monitor 11:RA 183-189.Pryor SC, Putnam J, Hoo, N. (2004) Opiate alkaloids in Ascaris suum. Acta Biologica Hungarica, 55:353-361.Zhu W, Baggerman G, Secor WE, Casares F, Pryor SC, Fricchione GL, Ruiz-Tiben E, Eberhard ML, Bimi L, Stefano GB. (2002)
Dracunculus medinensis and Schistosoma mansoni contain alkaloids. Annals of Tropical Medicine and Parasitology, 96: 309-16.Zhu W, Pryor SC, Putnam J, Cadet P, Stefano GB. (2004) Opiate alkaloids and nitric oxide production in the nematode
Ascaris suum. Journal of Parasitology, 90: 15-22.
Morphine20 µL, 10 mM
Morphine20 µL, 10 mM
HC M6G (10 mM)
Ch
an
ge
in m
em
bra
ne
po
ten
tial (
mV
)
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
HC M6G
M6G has No Obvious Effect on Intracellularly Recorded Muscle Cells
7A
HC M6G (10 mM)
Ch
an
ge
in m
em
bra
ne
po
ten
tial (
mV
)
0
1
2
3
4
5
6
7
HC M6G
M6G Depolarizes Intracellularly Recorded Hypodermis
7B
Application of M6G (20 µL, 10 mM), a bioactive metabolite of morphine that has been isolated in Ascaris suum (Pryor et al., 2004; Zhu et al., 2004), produced no obvious electrophysiological response in muscle cells (Fig. 7A), unlike morphine (cf. Fig. 3). M6G (20 µL,10 mM) application to hypodermis produced a strong depolarization, one that was similar in shape, but at least 2x larger than the hypodermal depolarization produced by morphine (Fig. 7B; cf. Fig. 4).
M6G CS M6G CS M6G CS
Cha
nge
in m
embr
ane
pote
ntia
l (m
V)
-4
-3
-2
-1
0
1
HCM6G HC M6G HC M6G HC
DE1 DE2 DI
DE1 M6G DE1 CS DE2 M6G DE2 CS
% C
ha
ng
e in
in
pu
t re
sis
tan
ce
-40
-20
0
20
M6G HC M6G HC DE1 DE2
% C
hang
e in
inpu
t res
ista
nce
M6G Effects on Input Resistance of Intracellularly Recorded Motorneurons
7DM6G Effects on Intracellularly Recorded Motorneurons
7C
*
Application of M6G (20 µL, 10 mM) to dorsal motorneurons produced hyperpolarizations in DE1 and DE2 motorneurons, albeit notably weaker than those elicited by morphine (Fig. 7C; cf. Fig. 5E). Unlike morphine, M6G-induced hyperpolarizations did not exhibit significant input resistance changes (Fig. 7D), suggesting the possibility that a non-conductance mechanism may be involved. (DI membrane potential and DE2 input resistance data are preliminary, N=1 and N=2 respectively.)
Abolition of Spiking
Attenuation of Spiking
Mor HC Na2SO4 Mor HC Na2SO4 Mor HC Na2SO4
DE1 DE2 DI
Cha
nge
in m
embr
ane
pote
ntia
l (m
V)
4.0
2.0
0.0
-2.0
-4.0
-6.0
Summary Data for Morphine Effects on Membrane Potential of Intracellularly Recorded Motorneurons
5D.
Cha
nge
in in
put r
esis
tanc
e (%
)
50
0
-50
Mor HC Na2SO4 Mor HC Na2SO4 Mor HC Na2SO4
DE1 DE2 DI
Summary Data for Morphine Effects on Input Resistance of Intracellularly Recorded Motorneurons
5E.
3A.
4A.
4B.
Morphine20 µL, 10 mM
Morphine20 µL, 10 mM
3B. Summary Data for Depolarizing Effects of Morphine on Intracellularly Recorded Muscle Cells
