Comparative Biochemistry and Physiology, Part A 151 (2008) 682–686

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Comparative Biochemistry and Physiology, Part A

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Neurotensin and cholecystokinin depress motility in isolated Lumbricus terrestriscrop-gizzard preparations

Sara Gibbs, Teresa F. DeGolier ⁎Department of Biological Sciences, Bethel University, 3900 Bethel Drive, St. Paul, Minnesota 55112, USA

⁎ Corresponding author. Tel.: +1 651 638 6798; fax: +E-mail address: t-degolier@bethel.edu (T.F. DeGolier

1095-6433/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.cbpa.2008.08.022

a b s t r a c t

a r t i c l e i n f o

Article history:

The effects of neurotensin (N Received 29 April 2008Received in revised form 16 August 2008Accepted 17 August 2008Available online 27 August 2008

Keywords:NeurotensinCholecystokininLumbricus terrestrisCrop-gizzardMotility

T) and cholecystokinin (CCK) were studied on isolated crop-gizzard preparationsof Lumbricus terrestris suspended in a smooth muscle organ bath. Changes in the amplitude and frequency ofcontractions associated with spontaneous motility were observed in response to neurotransmitters known tohave an excitatory effect (acetylcholine) and an inhibitory effect (serotonin); and to the hormones NT andCCK, which in vertebrate models have both been shown to inhibit gastric motility. The overall contractileamplitude and frequency of crop-gizzard contractions were decreased in response to increasingconcentrations of NT and CCK. In general, both hormone-induced responses were similar when comparedat equal molar concentrations. Cholecystokinin, however, did exhibit a greater reduction in contractilefrequency than NT. It is speculated that possible desensitization of earthworm NT-receptors to higherhormone concentrations resulted in a depressed maximal response in the concentration–response curve.Despite that possibility, the overall hormonal inhibition was statistically significant. These results infer thatNT- and CCK-induced inhibition of crop-gizzard motility may have a modulatory role in the transport ofnutrients and overall efficiency of worm metabolism.

© 2008 Elsevier Inc. All rights reserved.

1. Introduction

Neurotensin (NT) is a 13 amino acid brain-gut peptide first isolatedfrom bovine hypothalamus (Carraway and Leeman, 1973); its highestperipheral concentration is found in the gastrointestinal (GI) tract(Carraway and Leeman, 1976). Intestinal NT is produced in openenteroendocrine “N” cells lining the gut (Helmstaeder et al., 1977;Sundler et al., 1977) and luminal contents, principally long chain fattyacids (Rosell and Rokaeus,1979; Kihl et al., 1981) signal the “N” cells torelease NT into the bloodstream where it travels to and acts on anumber of target tissues including the stomach. In vertebrate models,NT has been shown to inhibit gastric motility in rats (Hellstrom, 1986;Parolaro et al., 1987), dogs (Andersson et al., 1976; Keinke et al., 1986;Siegle and Ehrlein, 1989), humans (Thor et al., 1980), and chickens(DeGolier et al., 1997).

Cholecystokinin (CCK) is a 33 amino acid brain-gut peptide (Jorpesand Mutt, 1973; Dockray, 1976; Rehfeld et al., 1979). Intestinal CCK isproduced by open enteroendocrine “I” cells of the proximal intestine(Buffa et al., 1976); its primary stimulus for release is also the presenceof long chain fatty acids as well as amino acids (Meyer, 1975).Cholecystokinin activity in vertebrate digestion is well documentedand historically is well understood to be a potent stimulus for both thecontraction of the gall bladder (Ivy and Oldberg, 1928) and pancreatic

1 651 638 6001.).

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activity (Harper and Raper, 1943). Cholecystokinin has also beenshown to mediate relaxation of the stomach, resulting in a delay ingastric emptying in humans (Liddle et al., 1989; Fried et al., 1991;Schwizer et al., 1997) and in rodents (Raybold and Tache, 1988; Younget al., 1996).

The physiological roles that both NT and CCK play in regulating fatdigestion have been acquired principally through vertebrate models.However, NT- and CCK-like immunoreactivities have also beendetected in the phylum Annelida (Carraway et al., 1982; Engelhardtet al., 1982, respectively), and specifically in the class Oligochaeta forthe common earthworm Lumbricus terrestris (Curry et al., 1989;Reglodi et al., 1999, respectively).

The isolated crop-gizzard preparation of the L. terrestris is a readilyavailable invertebrate model for investigating neurotransmitter andneuromodulator activity (Wu, 1939; Krajniak and Klor, 1999, 2001).The purpose of this project was to determine if the commonphysiologic role for NT and CCK to inhibit gastric motility invertebrates could similarly be observed as depressed crop-gizzardmotility in an invertebrate model. A resulting inhibition would serveto delay the passage rate of ingesta, potentially optimizing digestionefficiency and absorption of nutrients in the earthworm gut. Thespecific objectives of this investigation were 1) to determine whateffect NT and CCK had on the contractility of the crop-gizzardapparatus; 2) to investigate and quantify whether any contractileresponses to NT and CCK were concentration-dependent, and 3) atequal molar concentrations, to compare the effectiveness of NT-induced contractile responses to those induced by CCK.

Fig.1.Mean+S.E.M. contractile changes in amplitude and frequency of earthworm crop-gizzard tissue exposed to increasing concentrations of ACh 10−10 M to 10−4 M. Data arepresented as percent change from the control (n=8). Increasing concentrations of AChescalated the baseline tension of the crop-gizzard preparation by nearly 500%(Pb0.0001, not plotted). Changes in amplitude were masked by the rapid increase inbaseline preventing a repeatable and reliable measurement and appear to bestatistically unchanged. Increasing concentrations of ACh increased the frequency ofcontractions; at 10−6 M the rate was 65% greater than the baseline response (Pb0.0001).

683S. Gibbs, T.F. DeGolier / Comparative Biochemistry and Physiology, Part A 151 (2008) 682–686

2. Materials and methods

2.1. Tissue preparation

Forty-four earthworms (L. terrestris) were used in this investiga-tion. Each was anesthetized by placing it in a 5% solution of ethanol forapproximately 5 min. Next the earthworm was placed dorsal side upin a dissecting pan and an incision was made exposing the crop-gizzard apparatus which was then removed by cutting connectionsdistal to the esophagus and proximal to the intestine (Krajniak andKlohr, 2001). Ligatures were tied around both the proximal and distalends of the excised tissue. The gizzard end was attached to the distalend of a stationary rod and the anterior crop end was connected to anisometric force transducer coupled to an amplifier and fed intoPowerLab computer data acquisition software (AD Instruments, Inc.,Colorado Springs, CO, USA).

2.2. Smooth muscle bath

Each isolated crop-gizzard preparation was suspended along itslongitudinal axis in a 20 mL organ bath filled with worm Ringer'ssolution (g/5 L): 35.65 NaCl,1.47 KCl, 7.55 NaHCO3, 2.76 NaH2PO4,1.015MgCl2, and 1.11 CaCl2. A mixture of 95% O2/5% CO2 gas was bubbledinto the bath throughout the duration of the experiment. Themaintenance of the bath temperature at 20 °C helped reduce theamount of spontaneous motility and allowed more accurate measure-ment of the contractile changes due to drug application (Kitchen,1984; Percy, 1996).

2.3. Tissue testing

The crop-gizzard tensionwas adjusted to 0.5 g to induce consistentspontaneous motility. Once a regular wave pattern was observed,20 µL of 10−2 M carbachol (CCH) was added to the crop-gizzardsuspension for a total bath concentration of 10−5 M. An increase inwaveform amplitude and frequency indicated tissue viability. Anytissues not demonstrating these responses were not included in thedata set. Carbachol, a cholinergic receptor agonist, was given at boththe beginning and the end of the experiment to verify that musclefatigue was not a major factor in the interpretation of the results.

Acetylcholine (ACh), known to have an excitatory response in theearthwormmodel (Wu,1939;Millott, 1943b), was used as a control fora positive contractile response. Serotonin, known to have an inhibitoryresponse in the earthworm model (Gardner and Cashin, 1975;Vassileva et al., 1982), was used as a control for a negative contractileresponse. To test for contractile responses, the drugs were applieddirectly to the top of the suspended crop-gizzard apparatus inincreasing concentrations (10−10 M to 10−4 M) and left on the tissuefor 10 min before the next dose-cycle (i.e. adding of the next drugconcentration). Maximal responses were observed to occur within5 min of application.

The hormones NT and CCK were also applied directly to the top ofthe suspended crop-gizzard apparatus in increasing concentrations(10−10M to 10−7M), and initially left on the tissue for 10min before thenext dose-cycle. However, tissue responses to hormone applicationsappeared to become insensitive to greater concentrations once asignificant response occurred. In an attempt to minimize possibledesensitization of receptors, the tissues received awashout after 5min.

2.4. Drugs and peptides

All drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA).Neurotensin was solubilized in 0.1% bovine serum albumin in 0.01 Macetic acid. Cholecystokinin fragment 26–33 sulfated was solubilizedin oxygen free water. Acetylcholine, serotonin, and CCH were allsolubilized in distilled water.

2.5. Statistical comparisons

Changes in contractile amplitude, frequency, and overall baselinetension were observed for each concentration of CCH, ACh, serotonin,NT, and CCK. Contractile amplitude was determined from the wave-form baseline to the top of the drug-induced response. Meanamplitudes+S.E.M. were calculated for each drug concentration.

To measure frequency, crop-gizzard contractions were counted for5 min after either ACh or serotoninwere added. For the NT and CCK 5-minute dose-cycles, contractions were counted during the initial3 min of peptide exposure during which time maximal responseswere observed. Mean frequencies+S.E.M. were calculated for eachdrug concentration.

Differences in responses among varying concentrations werestatistically analyzed using ANOVA for multiple comparisons amongmeans. Following the rejection of the null hypotheses that ACh,serotonin, NT, and CCK would have no effect on crop-gizzardcontractions, the Tukey–Kramer Honestly Significant Difference test(JMP, 2001) was used as a post hoc test to determine which of theconcentrations responses were significantly different from each other.Results were considered to be significant at Pb0.05.

3. Results

Spontaneous motility was observed and recorded in all crop-gizzard preparations. A series of control experiments (n=4) was run toobserve whether fatigue was influenced by the length of anexperiment (∼1.5 h). Worm Ringer's solution (20 µL) was added attime intervals consistent with the experimental protocol. Sponta-neous motility patterns of the control trials were very regular and didnot statistically decrease over the time course of the experiments. Theaverage control frequency was 4.3+0.07 contractions per min, and theaverage control amplitude was 0.82+0.03 g of tension produced.

The amplitude and frequency of pre- and post-experiment CCH-induced contractions also did not statistically change during the NTand CCK trials (amplitude: P=0.2563; frequency: P=0.2563). Thisoverall consistency of crop-gizzard contractions attest to the robust-ness of the experimental tissue, and further substantiate thattreatment inhibitions were principally drug-induced as opposed totissue fatigue.

Fig. 2. Typical crop-gizzard response to an application of ACh 10−5 M. This illustration represents 5min of recorded time. The default y-axis is inmV, andwhen calibrated, 158mV=1 gof tension. The dotted line represents when ACh was applied to the tissue and in this sample, resulted in a maximal increase in the baseline of 0.128 g of tension.

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3.1. Bioactive mediators: acetylcholine and serotonin

Increasing concentrations of ACh escalated the baseline tension ofthe crop-gizzard preparation by nearly 500% (Pb0.0001). Increasingconcentrations of ACh also increased the frequency of contractions(Fig. 1); at 10−6 M the rate was 65% greater than the baseline response(Pb0.0001). However, changes in amplitude appear to be statisticallyunchanged (Fig. 1). When observing an actual recording (Fig. 2) itbecomes obvious that consistent measurable increases in amplitudeare somewhat masked by the rapid increase in baseline tension. Atequal molar concentrations (10−5 M), the average increase in baselineproduced by ACh (0.129 g of tension) was 61.1% of the force producedby CCH (0.211 g of tension).

Increasing concentrations of serotonin inhibited both contractileamplitude and frequency (Fig. 3). The responses were statically lowerbeginning at 10−5 M (Pb0.0001), demonstrating a 50% reduction inboth contractile amplitude and frequency.

Fig. 3. Mean+S.E.M. contractile changes in amplitude and frequency of earthwormcrop-gizzard tissue exposed to increasing concentrations of serotonin 10−10 M to 10−4

M. Data are presented as percent change from the control (n=8). Increasingconcentrations of serotonin inhibited both contractile amplitude and frequency. Theresponses were statically lower beginning at 10−5 M (Pb0.0001), demonstrating a 50%reduction in both contractile amplitude and frequency.

3.2. Gut hormones: neurotensin and cholecystokinin

Results from trials that included a washout between dose-cyclesgenerally showed a greater response when compared to trials with nowashouts. Neurotensin produced an average 36% decrease in tensionat concentrations 10−9 M–10−7 M (P=0.0008, Fig. 4). Higherconcentrations of CCK (10−8 M and 10−7 M) also inhibited meancontractile amplitudes by 32% and 39%, respectively; the changes weresignificant at 10−8 M–10−7 M (P=0.0004, Fig. 4).

There was an average 46% decrease in contractile frequencyfollowing applications of NT 10−9 M–10−7 M (P=0.0016, Fig. 5).Cholecystokinin 10−9 M–10−7 M had profound inhibitory effects (up to72%) on contractile frequency (Pb0.0001, Fig. 5).

At equal molar concentrations, NT demonstrated a greater overallinhibition of contractile amplitude as compared to CCK (Fig. 4), but thedifferences were not statistically greater. Cholecystokinin-induced

Fig. 4.Mean+S.E.M. contractile changes in amplitude of earthworm crop-gizzard tissueexposed to increasing concentrations of NT and CCK 10−10 M to 10−7 M. Data arepresented as percent change from the control (n=12). Neurotensin produced an average36% decrease in tension at concentrations 10−9 M–10−7 M (P=0.0008). Higherconcentrations of CCK (10−8 M and 10−7 M) also inhibited mean contractile amplitudesby 32% and 39%, respectively; the changes were significant at 10−8 M–10−7 M(P=0.0004). At equal molar concentrations, NT demonstrated a greater overallinhibition of contractile amplitude as compared to CCK but the differences were notstatistically greater. As hormone concentrations increased from 10−10 M to 10−7 M, thecontractile force in the NT trials decreased from 0.1 to 0.055 g of tension, and in the CCKtrials from 0.063 to 0.035 g of tension.

Fig. 5. Mean+S.E.M. contractile changes in frequency of earthworm crop-gizzard tissueexposed to increasing concentrations of NT and CCK 10−10 M to 10−7 M. Data arepresented as percent change from the control (n=12). There was an average 46%decrease in contractile frequency following applications of NT 10−9M–10−7M (=0.0016).Cholecystokinin 10−9 M–10−7 M had profound inhibitory effects (up to 72%) oncontractile frequency (Pb0.0001). At equal molar concentrations, CCK-induced inhibi-tion of frequency was more pronounced than NT, but only statistically greater at10−7

M (P=0.0374). As hormone concentrations increased from 10−10 M to 10−7 M, thecontractile frequency in the NT trials decreased from 0.79 to 0.65 contractions per min,and in the CCK trials from 1.68 to 0.51 contractions per min.

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inhibition of frequencywasmore pronounced than NT (Fig. 5), but wasonly statistically greater at 10−7 M (P=0.0374).

4. Discussion

The earthworm digestive tract has dual, antagonistic innervation(Millott, 1943a) and can be affected by neurotransmitters that eitherenhance or inhibit contractions. Acetylcholine evokes an excitatory, orparasympathetic response, causing food to be propelled through theearthworm's gut. In contrast, serotonin has an inhibitory responsethat presumably occurs when the organism needs to reduce itsperistaltic activity, or expend its energy in places with more lifethreatening consequences than digestion. The contractile responses ofthe isolated crop-gizzard experiments herein substantiate both theexcitatory responses of ACh and the inhibitory responses of serotonin,and are consistent with results described from previous investigations(Wu, 1939; Millott, 1943b; Anderson and Fange, 1967; Krajniak andKlohr, 1999, 2001).

In general, equal molar concentrations of NT and CCK-inducedsimilar responses from the earthworm crop-gizzard preparations:both hormones inhibited contractile amplitude and frequency. Once asignificant depression of motility occurred in the tissues, the plottedwashout concentration-curves appeared to plateau following theapplication of hormones with increasing concentrations (Figs. 4and 5). These patterns could partially be characterized as a depressionof themaximal response due to receptor desensitization (Tanaka et al.,1990; Hermans and Maloteaux, 1998).

Desensitization responses have been documented in a number ofvertebrate studies in which GI tissues exhibited a decrease inresponsiveness, especially when a second or successive applicationwas subjected to NT (rat fundus: Huidobro-Toro and Kullak, 1985; ratduodenum: Mule et al., 1992; chicken rectal muscle: Komori et al.,1986; porcine distal jejunum: Brown and Treder, 1989). While theunderlying mechanisms of NT-receptor desensitization are not wellunderstood, it is speculated that the pathways regulating intracellularsignaling, or the receptor itself are involved (Hermans and Maloteaux,1998).

The effectiveness of tissue washouts to reverse receptor desensi-tization is quite variable. In both guinea pig ileum (Huidobro-Toro andZhu, 1984) and rat proximal colon (Mule et al., 1995) tissues werereported to rapidly recover following a washout. However, chicken

rectal muscle took up to 90 min before tissues would demonstrate amaximal response (Komori et al., 1986).

Our results demonstrated that even in the isolated crop-gizzardapparatus, washouts were particularly effective in increasing theoverall magnitude of response to NT and CCK. This was most evidentwhen comparing responses induced from the maximal hormoneconcentration achieved (10−7 M) between the washout resultspresented herein and non-washout pilot studies. For example(presented as washouts vs. non-washouts): inhibition of contractileamplitude: NT, −41% vs. −24%; CCK, −39% vs. −26%; inhibition ofcontractile frequency: NT, −47% vs. 6%; CCK, −72% vs. −25%.

Regardless, it is noteworthy that both the NT and CCK peptidesused in this investigation were of vertebrate sequence; they boundand affected annelid receptors, implying that there is some conserva-tion of amino acid sequences across invertebrate to vertebrate species.

Since fat, particularly long chain fatty acids, is amajor trigger for therelease of NT and CCK in vertebrate models, the earthworm's diet maybe reviewed to discern the contribution that NT and CCK play in aregulatory role. A worm's diet consists primarily of dead organicmaterial mostly found from seeds, decaying plants, eggs, the larvae ofanimals, bacteria and fungi (Buchsbaum, 1974). These food sourcescontain adipocytes as well as cell membranes composed of fatty acids.By inhibiting crop-gizzard motility, NT and CCK may function to delaythe emptying of fatty contents into the anteriormidgut of the intestine,thus increasing the amount of time permitted for efficient digestion atthis site. Since the posteriormidgut is responsible for the absorption ofnutrients, the actions of NTand CCKwould act to optimize the amountof food exposed to the absorptive surface at any given time.

Furthermore, L. terrestris is an environmentally important inverte-brate particularly in the role it performs in bioremediation via casting,grazing, and dispersal (Brown, 1995). A greater knowledge of thephysiological factors affecting crop-gizzard motility may also beimportant from the perspective of terrestrial ecosystems.

Digestion is an important and complex function and it would seemthat multiple hormones are needed to monitor digestion in inverte-brates as well as in vertebrates. The results of this investigationcontribute to a growing body of knowledge about intestinal motility inearthworms. In an experimental set-up similar to the projectdescribed herein, Krajniak and Klohr (1999) found that FMRFamideactivity on the Lumbricus crop-gizzard contractions was primarilyinhibitory. Inhibitory pentapeptides, as isolated from the earthwormEisenia foetida, were demonstrated to inhibit crop-gizzard andintestinal contractions (Ukena et al., 1996). In contrast, annetocin, anoxytocin-related peptide, potentiated spontaneous contractions of thecrop-gizzard (Ukena et al., 1995) and substance P increased both theamplitude and frequency of intestinal spontaneous contractions(Kaloustian and Edmands, 1986).

In conclusion, the major findings of this study demonstratedthat: 1) both NT and CCK affected the contractility of the crop-gizzardapparatus by reducing both the tension developed during contractionsand the rate of spontaneous motility; 2) the inhibitory effects of bothNT and CCK were partially characterized by a depression of themaximal response in the concentration–response curve followingrepeated applications of the hormones, possibly indicating receptordesensitization; 3) at equal molar concentrations, the inhibitory effectof both hormone-induced responses were similar; CCK, however, didexhibit a greater reduction in contractile frequency than NT; and 4)analogous to NT- and CCK-induced gastric inhibition in vertebratemodels, NT and CCK may have a common physiologic role to depresscrop-gizzard motility in L. terrestris and thus modulate GI motility ininvertebrates.

Acknowledgements

This research was partially funded by a grant awarded from the C.Weldon Jones Memorial Research Fund, and contributions from the

686 S. Gibbs, T.F. DeGolier / Comparative Biochemistry and Physiology, Part A 151 (2008) 682–686

Division of Natural Sciences and the Department of BiologicalSciences, all at Bethel University.

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