Leuprolide acetate affects intestinal motility in female rats before and after ovariectomy RAMAN KHANNA, RICHARD M. BROWNE, ANDREW D. HEINER, MARY H. CLENCH, AND JOHN R. MATHIAS Department of Internal Medicine, School of Medicine, University of Texas Medical Branch, Galveston, Texas 77550 Khanna, Raman, Richard M. Browne, Andrew D. Heiner, Mary H. Clench, and John R. Mathias. Leuprolide acetate affects intestinal motility in female rats before and after ovariectomy. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G185-Gl90, 1992.-Leuprolide acetate, a gonadotropin-releasing hormone (GnRH) analogue, is currently being proposed to control debilitating symptoms in women with functional bowel disease. Whether leuprolide alters gastrointestinal motility as part of its actions is unknown. This study was designed to assess, using myoelectric techniques in an animal model, the effects of leuprolide on potential mechanisms of neuromuscular function of small intestine. Female rats with (n = 6) or without (n = 8) bilateral ovariectomy were used to study jejunal motility before and after leuprolide therapy. Throughout the study, daily leuprolide dosages of 0.02, 0.2, or 0.4 pg/kg were injected into intact rats and 0.02, 0.2, 0.4, 1.0, or 2.5 pg/kg into ovariectomized rats. Recordings were made while the rats were fasted and postprandial and before and after leuprolide administration. Under control conditions, migrating myoelectric complexes (MMCs) were found in intact female rats, whether fasted or postprandial. After ovariectomy, postprandial controls and those treated with low-dose leuprolide (0.02, 0.2, and 0.4 pg) had typical fed-state patterns and no MMCs, but at 1.0 and 2.5 pg the fed state was inhibited and cycling MMCs occurred at a frequency similar to that of fasted controls. Reproductive hormones thus have a significant effect on gastrointestinal motility. leuprolide acetate; gonadotropin-releasing hormone; nervous system; migrating myoelectric complex
enteric
NATIVEGONADOTROPIN-RELEASING hormone(GnRH)is a decapeptide (pGlu’-His’-Trp3-Ser4-Tyr5-Gly”-Leu7Arg’-Prog-Gly’“-NH2) (1, 23) that is synthesized and stored in the neurosecretory cells of the medial basal hypothalamus and then secreted into the hypothalamicpituitary portal circulation (12). GnRH binds to specific receptors (3) on the gonadotrophs in the anterior pituitary to initiate secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (12, 23). FSH stimulates ovarian follicle growth and maturation in females and spermatogenesis in males, whereas LH stimulates ovulation and corpus luteum formation in females and testosterone secretion in males (12). In turn, these steroid hormones regulate GnRH secretion through closed-loop feedback mechanisms (32). In women, native GnRH is secreted in a pulsatile manner, its peak occurring in the middle of the menstrual cycle (6), but if GnRH is given in a continuous dose, as in a daily injection or depot form, there is an initial rise in LH and FSH levels, followed -7-10 days later by a decrease in both LH and FSH secretion which remains low thereafter (2, 9). This phenomenon, referred to as downregulation or “desensitization,” results in sex hormones also being significantly decreased or completely 0193-1857/92
$2.00 Copyright
inhibited (2, 9). The same phenomenon occurs in the female rat and most other female mammalian vertebrates (5, 17). Desensitization has been applied clinically in various conditions (e.g., hormone-dependent prostatic carcinoma in men, central precocious puberty, and endometriosis) and is currently being investigated for hypogonadism, contraception, and polycystic ovary syndrome (8) Various synthetic analogues of native GnRH have been made, with the predominant form possessing changes at the following three positions on the molecule: at position 6, substitution of a D-amino acid (7), which decreases the analogue’s susceptibility to enzymatic degradation by peptidases in the hypothalamus (14, 30) and pituitary (13, 18); at position 10, a des-Gly-NH, deletion; and at position 9, addition of an ethylamide to the proline, which increases the binding affinity to gonadotropin receptors (19). Because these analogues are more potent than native GnRH, they may be used in various clinical situations (8, 31). Leuprolide acetate (pGlu’-His2-Trp3Ser4-Tyr5-D-Leu’-Leu7-Arg’-Prog-NHCH2CH3) is one such analogue agonist, with a substitution of D-leucine at position 6 and of an ethylamide at position 9 (27). Leuprolide acetate is considerably more potent than native GnRH and causes downregulation or desensitization of pituitary gonadotropins in humans and laboratory animals, including male and female rats (5, 17, 27) “Functional” bowel diseases are emerging as neuromuscular motility disorders of the gastrointestinal tract; of common occurrence, they are estimated to affect 2% 31 million Americans (21). This laboratory recently reported preliminary results showing that leuprolide effectively treated the debilitating symptoms of these diseases (20). Because the symptoms are expressed -30 times more often in women than men, an interaction between the reproductive system and gastrointestinal motility is suggested (20). The present study was designed to investigate the myoelectric activity of the small intestine in the female rat in the fasted and postprandial states as follows: 1) under control conditions (in the intact reproductive state and without drug administration), 2) in the intact reproductive state after the administration of leuprolide acetate given subcutaneously daily, 3) after bilateral ovariectomy but without therapy with leuprolide acetate, and 4) after bilateral ovariectomy and with therapy with leuprolide acetate. MATERIALS AND METHODS The study model used was similar to one we and others have reported using in male rats (11). Female Wistar rats (Charles
0 1992 the American
Physiological
Society
G185
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
G186
LEUPROLIDE
AFFECTS
FEMALE
River, Wilmington, MA) were prepared for study with implanted electrodes. After the animals were anesthetized with ketamine hydrochloride (100 mg/kg ip), an incision was made in the skin at the back of the neck, and four miniature bipolar silver-silver chloride electrodes were tunneled subcutaneously to the abdomen, which was then opened by a vertical midline incision. After the proximal small intestine was identified, the electrodes were sewn onto the serosal surface of the jejunum, 5 cm apart, beginning 5 cm distal to the ligament of Treitz. The other end of the electrode assembly had previously been soldered to an electrical connector (ITT Cannon, Santa Ana, CA) and had a saddle of dental acrylic (Hygenic, Akron, OH) formed around it to fit the curvature of a rat’s thoracic spine and neck area. With the connector placed on the neck, the neck skin and abdomen were closed, and the rats were kept warm by a water-perfused heating blanket until fully recovered from the anesthesia. Each rat was then allowed to recover from surgery until completely healed; myoelectric recordings were not started until 8-10 days after surgery. In ovariectomized rats, the ovaries were removed bilaterally during the electrode implantation surgery. Eight intact and six ovariectomized rats were included in this study. The approximate age of the rats was 60 days at the time of surgery; weights ranged from 180 to 225 g. Throughout the study the animals were given water and food (Formulab 5008; Purina Mills, St. Louis, MO) ad libitum, except for occasional 16-h fasting periods when the food was withdrawn. The rats were weighed regularly to assure normal growth. Recordings were made on a SensorMedics R612 Dynograph with 9878 AC couplers (SensorMedics, Anaheim, CA) at a sensitivity of 0.5 mV/cm, a time constant of 0.3 s, a highfrequency filter of 30 Hz, and a paper speed of 1 mm/s. All recording was conducted during normal daylight hours, with the rats awake in their cages and unrestrained except for the connector lead. Studies were conducted with all rats in both postprandial and fasted conditions and before and after leuprolide administration; each animal acted as its own control. Four 2-h control recordings were begun 10 days after surgery and before leuprolide was administered, two while the animal was fasted and two under postprandial conditions: rats were fasted (overnight) for 16 h, recorded for 2 h, given two 5-g food pellets and allowed 1 h in which to eat them, and then recorded again for 2 h. In each instance, the rat was watched to confirm ingestion of both food pellets before the control recording of the postprandial condition began. When rats were recorded in the postprandial condition after leuprolide administration had begun, they had food continuously available in their cages; their body weight and defecation rate during the recording sessions suggested normal feeding behavior. Treatment with leuprolide (Lupron; TAP Pharmaceuticals, North Chicago, IL) was begun after the fasted and postprandial control recordings were completed. Once treatment had started (-10-12 days after the electrodes were implanted), leuprolide was administered subcutaneously each morning at the same time, regardless of whether the animal was to be recorded that day. On recording days, the session began after administration of the daily dose. Recordings were not made every day because the animals were allowed regular rest days, but multiple recordings of each rat under all its experimental conditions were obtained. Recordings of intact rats totaled 192 h, and those of ovariectomized rats totaled 230 h. Intact rats were administered leuprolide in amounts equivalent to therapeutic dosages established for humans as follows (in pg/kg body wt): 0.02 (n = 3), 0.2 (n = 3), or 0.4 (n = 2). In ovariectomized rats, the dosages were (in ,ug/kg body wt) 0.02 (n = 2), 0.2 (n = 2), 0.4 (n = 2), 1.0 (n = 3), or 2.5 (n = 2). The last recording in the series was with the rat in the postprandial
RAT
INTESTINAL
MOTILITY
condition. When that recording was complete, each animal was killed with a pentobarbital sodium overdose, and the viscera were examined to confirm the presence of food in the stomach and proximal small bowel. The recordings were analyzed visually by two independent observers (R. Khanna and M. H. Clench) for the number of migrating myoelectric complexes (MMCs) per hour, the duration of MMC phasesI, II, and 111, the propagation velocity of phase III, and the slow-wave frequency. The data for all myoelectric studies were first assessed for kurtosis and then by parametric analysis (25). Because the kurtosis values were close to 1.00, analysis was performed with the Student’s independent t test; P < 0.05 was considered statistically significant. RESULTS
Intact (Not Ovariectomixed) Rats Control. In previous studies with male rats as the experimental model, we and others have not seen cycling MMCs when the animals were studied while postprandial. (Note the terminology distinction between postprandial or “fed” animals, those recorded while food is present in the intestinal lumen, and “fed-state” myoelectric activity, with its characteristic pattern of random and frequent spike potentials.) Control recordings of the intact female rats in our study had from 4.4 to 4.7 MMC/ h while the animals were in the fasted condition (Table 1). A representative tracing from a control period, fastedintact female rat is shown in Fig. 1. When the animals were postprandial, cycling MMCs were also seen, although they were fewer than when the females were fasted (from 1.4 to 2.6 MMC/h for controls; Table 1). Leuprolide treatment. In all the intact rats treated with leuprolide, fed-state myoelectric activity was inhibited. Regardless of whether the treated animals were postprandial or fasted, the numbers of cycling MMCs were not significantly different from fasted-control values or from each other, ranging from 3.4 to 5.4 MMC/h (Table 1). Among the postprandial rats, all treatment group values were significantly higher than those of control periods (P values of ~0.01 to 0.0001; Table 1). Figure 2 shows a representative MMC in an intact rat treated with leuprolide and recorded while postprandial. Ovariectomized Rats Table 2 shows the number of MMCs recorded after ovariectomy. Groups of rats were studied at leuprolide doses of 0.02, 0.2, 0.4, 1.0, and 2.5 pg/kg body wt. The Table 1. Migrating myoelectric complexes in intact female rats Leuprolide Dosage
Controls
Acetate Group Fasted
0.02 pg/kg body wt 4.69t0.24 0.2 pg/kg body wt 4.43k0.25 0.4 pg/kg body wt 4.75k0.48
Postprandial
2.60&0.27* 1.40t0.48* 2.33&0.33*
Treatment Leuprolide Fasted
with Acetate Postprandial
4.60t0.12 4.83+0.65? 5.41k0.17 4.11&0.22f4.48kO.12 3.45&0.25-/-
Values are means I~I: SE and are in no. of migrating myoelectric complexes (MMC) per hour; n = 3 rats in 0.02- and 0.2-pg dosage groups, and n = 2 in 0.4-pg dosage group. Controls received no leuprolide acetate. * P < 0.05 for control postprandial vs. treated postnrandial. Inhibition of fed state.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
LEUPROLIDE
AFFECTS
FEMALE
RAT
INTESTINAL
G187
MOTILITY
5 cm JJ E3. lT 5 cm c
-u-
60
set
I)
Fig. 1. A representative tracing of a migrating myoelectric complex (MMC) in a fasted-intact, control female rat. EZectrodes l-4 are illustrated schematically on left; a sensitivity calibration of 0.5 mV/cm and a I-min time scale are also shown. An activity front of MMC is shown propagating over all 4 electrode sites. Slope of line indicates propagation velocity (mean = 2.90 cm/min).
numbers of MMCs in the ovariectomized fasted-control and the fasted-treated rats were similar to those in both groups of fasted-intact rats. After bilateral ovariectomy, the postprandial-control rats and the postprandialtreated rats given leuprolide at the lower dosageshad no cycling MMCs (the value of 0.88 t 0.58 MMCs in the postprandial-control column of Table 2 denotes activity seen in a single rat on a single occasion). Fed-state myoelectric activity (random and frequent spike potentials) consistently occurred after leuprolide administration at dosagesof 0.02, 0.2, and 0.4 pg/kg, but at dosages of 1.0 and 2.5 pg/kg the fed-state activity was inhibited as it had been with the lower dosagesof leuprolide before ovariectomy; under higher-dose leuprolide treatment, cycling MMCs once again occurred in postprandial rats. At the l.O-pg/kg dose, treated rats had 4.50 t 0.34 MMC/h in contrast to none during their postprandial-control period (P < 0.0001); at 2.5 pg/kg, the numbers were 5.00 L-
..-.
---
& 0.58 vs. 0.88 t 0.58 MMC/h (P < 0.001). This degree of activity at higher leuprolide dosagesin ovariectomized rats was not significantly different from that in treated intact females when postprandial during recording (Tables 1 and 2; P = 0.08). MMC Characteristics Intact rats. The characteristics of the MMCs recorded in intact female rats are shown in Table 3. When the animals were fasted, significant changes occurred in the group treated with leuprolide compared with controls; the duration of phase II was shorter (P < O.OOl), the duration of phase III was longer (P < 0.02), and the propagation velocity was slower (P c 0.001). When the rats were postprandial, phase I duration became longer in the treated group compared with controls (P < O.Ol), phase II became shorter (P < O.OOOl), and phase III ..-
-
-
-
. ..-_
h
h l-
-
-.-
....
.__
qi
-
-.-
-i
-
.-
-.i.--
- --
h
-.-
_.e---d\
h
5 cm
5 cm
w
60
set
I
-u-
5 cm
I
Fig. 2. A representative tracing of an MMC in a postprandial-intact, 1 for details. Mean propagation velocity of phase III of this complex was inhibited after leuprolide treatment.
leuprolide-treated was 2.46 cm/min.
female rat. See legend to Fig. Fed-state myoelectric activity
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
GM8 Table
LEUPROLIDE
AFFECTS
FEMALE
2. MMC in ovariectomized rats
Leuprolide Acetate Dosage Group
0.02 pg/kg 0.2 pg/kg 0.4 ,ug/kg 1.0 pg/kg 2.5 ,ug/kg
body body body body body
Treatment Leuprolide
Controls
wt wt wt wt wt
Fasted
Postprandial
3.86t0.14 5.17t0.31 4.50t0.22 4.OO_tO.17 4.5OkO.22
0 0 0.88&0.58*
0-t 0.88kO.58”“r
Fasted
RAT
INTESTINAL
MOTILITY
All rats, with or without ovariectomy, gained weight. No alteration in stool consistency was noted in any of the animals; the fecal pellets appeared normal in both the control and leuprolide-treated rats.
with Acetate Postprandial
4.4020.16 0 5.40to. 15 0 5.23t0.41 0 5.00t0.41 4.50+0.34$ 4.50t0.29 5.00&0.58$
Values are means * SE and are in MMC/h; n = 3 in 1.0~pg dosage seen in a single rat on a GOUP, n = 2 in all other groups. * Activity single occasion. ‘f P < 0.001 for control postprandial vs. treated postprandial. $ Inhibition of fed state.
duration and propagation velocity were unchanged. The slow-wave frequency increased in the postprandialtreated rats (P < 0.02). Ovariectomized rats. The characteristics of the MMC after ovariectomy are shown in Table 4. The fed-state activity was not inhibited when the rats were under postprandial-control conditions during recording, and no cycling MMCs were observed. When the rats were fasted, the durations of both phases II and 111 were shorter in the leuprolide-treated group (P < 0.0001 and 0.01, respectively, compared with controls), and the slow-wave frequency increased in both leuprolide groups (P < 0.02). Comparison of MMC characteristics before and after ovariectomy. The characteristics of the MMC before and after ovariectomy are shown in Tables 3 and 4. Data from the ovariectomized rats with those from the intact rats compare as follows. The duration of phase I increased in fasted-controls (P < 0.02) and in fastedtreated (P < 0.001) rats. The phase I duration did not change significantly (P = 0.11) in the postprandial leuprolide-treated rats. In contrast, all phase II durations decreased after ovariectomy (P < 0.01 in the fasted leuprolide-treated rats and P < 0.04 in the postprandialtreated). The control animals showed less decrease in phase 11 duration, but it was not significant. Phase III duration also decreased in all groups with ovariectomy (P < 0.0001 in both the fasted and postprandial leuprolide-treated rats). The fasted controls also decreased, but not significantly. The propagation velocity changed only in the fasted leuprolide-treated rats, where it increased (P < 0.0001). Changes in the propagation velocity were not significantly different in the treated rats when recorded postprandially, nor were they in the control groups. The slow-wave frequency increased in both the fasted and postprandial leuprolide-treated rats (P < 0.02 and 0.005, respectively).
DISCUSSION
Szurszewski (26) in 1969 first showed a myoelectric complex that migrated the entire length of the small bowel; this has come to be called the MMC. Since its description and characterization, considerable investigation has been performed to explain the origin and function of the MMC. That the complex appears to exist in all warm-blooded vertebrates (4) suggests the importance of this complex as a basic motility pattern, yet the exact function of this complex remains unknown. Even without an understanding of its function, however, the MMC is extensively used by investigators to study neuromuscular mechanisms (21). In humans, subprimates, dogs, and male rats, it occurs only during the fasting condition, but it is present in all ruminants whether fasted or postprandial and may be present during the postprandial condition in pigs and rats if the animals are accustomed to small frequent meals (4, 22). Although motility studies have almost exclusively made use of male animal models to avoid the complicating effects thought to be reflective of female reproductive cycles, the end results of GnRH analogue administration are well known to affect the two sexes in strongly different ways (15). Because functional bowel diseaseprimarily occurs in women (ZO), we elected to use a female rat model with and without an intact reproductive system and thus with and without the gonadal hormones present (postovariectomy status). Scott et al. (24) also showed changes in MMC periodicity in fasting ovariectomized and intact female rats during the various stages of the estrous cycle. Our study shows that MMCs occur in intact control female rats when they are in the postprandial condition, although at one-third to one-half the rate recorded when they are fasted (Table 1). These observations are in contrast to the pattern seen in the male rat. In our model, -4-5 MMC/h occur in fasted males and none are seen in postprandial males (11). Ruckebusch and Fioramonti (22) have found MMC-like activity in male rats that had been fed small and frequent meals, and this is true of other mammalian species as well. Although the presence of MMCs in postprandial female rats may result from their being accustomed to small frequent meals, all of our animals (male or female) have been fed ad libitum
Table 3. Characteristics of MMCs in intact female rats
Phase I, min Phase II, min Phase III, min Propagation velocity, Slow-wave frequency,
cm/min min-l
5.19kO.31 4.83t0.38 2.41t0.05 2.90&O. 17 39.30t0.41
Values are means t SE. Data were pooled from all dosages. postprandial compared with treated postprandial. (P < 0.02).
2.32t0.41 10.61t1.31 2.22t0.13 2.63kO.21 38.91t0.37 * Significant
values
5.36rt0.14 3.59&O. 16* 2.59t0.04* 2.57&0.05* 39.37kO.18 for control
fasted
compared
3.68t0.17* 8.19t0.66* 2.37t0.04 2.46kO.11 40.18t0.23* with
treated
fasted
or control
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
LEUPROLIDE
Table
AFFECTS
FEMALE
RAT
INTESTINAL
G189
MOTILITY
4. Characteristics of MMCs in ovariectomized female rats Controls Fasted
Phase I, min Phase II, min Phase III, min Propagation velocity, Slow-wave frequency,
cm/min min-l
6.25t0.31 4.61kO.31 2.33t0.07 2.65t0.13 39.3OkO.41
Values are means & SE. Data were pooled from all dosages. postprandial compared with treated postprandial (P < 0.02).
Leuprolide Postprandial
and thus the feeding regimen would not seem to have been an important variable. In contrast to our observations in intact females, ovariectomized control rats did not show MMCs when they had food in the intestinal lumen during recording (Tables 2 and 4). These observations indicate that the absence of ovaries and subsequent hormonal changes (inhibition of sex hormones) alter the expression of the MMC under postprandial conditions. When we treated postprandial intact rats with leuprolide acetate, the number of MMCs per hour increased to values similar to those during the fasting condition (Table 1). In postprandial leuprolide-treated ovariectomized rats, MMCs were also induced, but only at a high dose (~1.0 pg/kg; Table 2). In contrast, the MMC was not seen in ovariectomized rats in the postprandial-control condition (Table 2). These results indicate that leuprolide, in addition to its effect on the inhibition of gonadotropins and sex hormones, also stimulates cycling motor activity during either fasted and postprandial conditions through as-yet-unknown neural mechanisms. How leuprolide inhibits fed-state activity and restores cycling MMCs remains unknown. Progesterone, which is secreted by the corpus luteum after ovulation and during pregnancy and is also used as a contraceptive agent, has been shown to prolong small-bowel transit in humans (28, 29). The corpus luteum also secretes other hormones, such as relaxins A and B, whose effect on intestinal muscle and nerve remains unknown; but because relaxins have been shown to inhibit uterine smooth muscle by interfering with myosin light-chain kinase (20), we speculate that relaxins may have the same effect on gastrointestinal smooth muscle. The inhibition of the hypothalamic-pituitary-ovarian axis by leuprolide, and the resultant decrease in gonadotropin and sex hormones indicate that the various hormones alone or in combination significantly affect the motility of the small intestine. DuPont et al. (10) showed significant GnRH uptake in the small intestine of mice by distribution studies with tritiated GnRH. A GnRHlike peptide has also been demonstrated as a putative neurotransmitter in the prevertebral sympathetic ganglion of the bullfrog (Rana catesbeiana) (16). These two studies indicate that GnRH peptides may act in the nerves of the enteric nervous system either directly as a neurotransmitter or indirectly by modulating specific neurons. The present study shows that in the female rat, unlike the situation in the male rat, cycling MMC activity occurs (albeit less well organized) in either the fasted or
Postprandial
6.20t0.15 3.06t0.12* 2.17t0.03* 2.85kO.06 40.26t0.21*
0 0 0 0 38.4220.3 * Significant
Treatment
Fasted
values
for control
fasted
4.37t0.33 5.13t0.78 1.88t0.06 2.42kO.18 40.86t0.59"
compared
with
treated
fasted
or control
postprandial condition. This phenomenon in the female rat changes significantly when the ovaries are removed. Ovariectomized control female rats do not exhibit MMCs in the postprandial condition; but when treated with sufficiently high doses of leuprolide, they develop organized MMCs in either the postprandial or fasted conditions. Therefore ovariectomized female rats treated with leuprolide develop cycling MMC activity like intact females, but they require higher doses. The same requirement has been noted in humans. We suggest that gonadotropins and ovarian sex hormones significantly influence the motor activity of the gastrointestinal tract and determine the expression of motor activity; this influence may vary, depending on the phase of the reproductive cycle and which substances are secreted. Leuprolide acetate improves not only the quality but the quantity of cycling activity. We also suggest that this might be accomplished by at least two mechanisms of action: 1) inhibition of gonadotropins and ovarian sex hormones that act as endogenous antagonists on gastrointestinal nerve and muscle, and 2) stimulation of organized cycling motor activity through asyet-unexplained neural mechanisms. Understanding the mechanisms of action of GnRH analogues will facilitate a better understanding of the pathophysiology of neuromuscular dysfunction in gastrointestinal disorders and of how the reproductive system may interact with gastrointestinal tract function. We thank Alice W. Cullu for editorial assistance and Patricia A. Ciejka for checking the accuracy of our references. This research was supported by funds from The University of Texas Medical Branch at Galveston. Leuprolide acetate (Lupron) was kindly supplied by Takeda-Abbott Pharmaceuticals. Address reprint requests to J. R. Mathias. Received
2 February
1990; accepted
in final
form
5 July
1991.
REFERENCES Amoss, M., R. Burgus, R. Blackwell, W. Vale, R. Fellows, and R. Guillemin. Purification, amino acid composition and Nterminus of the hypothalamic luteinizing hormone releasing factor (LRF) of ovine origin. Biochem. Biophys. Res. Commun. 44: 205210,1971. Belchetz, P. E., T. M. Plant, Y. Nakai, E. J. Keogh, and E. Knobil. Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science Wash.DC202: 631-633,1978. Clayton, R. N., and K. J. Catt. Regulation of pituitary gonadotropin-releasing hormone receptors by gonadal hormones. Endocrinology 108: 887-895, 1981. Clench, M. H., V. M. Pifieiro-Carrero, and J. R. Mathias. Migrating myoelectric complex demonstrated in four avian species.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
G190
5. 6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16. 17.
18.
LEUPROLIDE
AFFECTS
FEMALE
Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G598G603, 1989. Conn, P. M., and W. F. Crowley, Jr. Gonadotropin-releasing hormone and its analogues. N. Engl. J. Med. 324: 93-103, 1990. Conn, P. M., A. J. W. Hsueh, and W. F. Crowley, Jr. Gonadotropin-releasing hormone: molecular and cell biology, physiology, and clinical applications. Federation Proc. 43: 2351-2361, 1984. Coy, D. H., J. A. Vilchez-Martinez, E. J. Coy, and A. V. Schally. Analogs of luteinizing hormone-releasing hormone with increased biological activity produced by D-amino acid substitutions in position 6. J. Med. Chem. 19: 423-425, 1976. Cutler, G. B., Jr., A. R. Hoffman, R. S. Swerdloff, R. J. Santen, D. R. Meldrum, and F. Comite. Therapeutic applications of luteinizing-hormone-releasing hormone and its analogs. Ann. Intern. Med. 102: 643-657,1985. Dowsett, M., B. Cantwell, L. Anshumala, S. L. Jeffcoate, and A. L. Harris. Suppression of postmenopausal ovarian steroidogenesis with the luteinizing hormone-releasing hormone agonist goserelin. J. C&n. EndocrinoZ. Metab. 66: 672-677, 1988. DuPont, A., F. Labrie, G. Pelletier, R. Puviani, D. H. Coy, E. J. Coy, and A. V. Schally. Organ distribution of radioactivity and disappearance of radioactivity from plasma after administration of [“Hlluteinizing hormone-releasing hormone in mice and rats. Neuroendocrinology 16: 65-73, 1974. Eaker, E. Y., G. B. Bixler, A. J. Dunn, W. V. Moreshead, and J. R. Mathias. Chronic alterations in jejunal myoelectric activity in rats due to MPTP. Am. J. Physiol. 253 (Gastrointest. Liver Physiol. 16): G809-G815, 1987. Hazum, E., and P. M. Conn. Molecular mechanism of gonadotropin releasing hormone (GnRH) action. I. The GnRH receptor. Endocr. Rev. 9: 379-386, 1988. Hazum, E., M. Fridkin, T. Baram, and Y. Koch. Degradation of gonadotropin-releasing hormone by anterior pituitary enzymes. FEBS Lett. 127: 273-276,198l. Hersh, L. B., and J. F. McKelvy. Enzymes involved in the degradation of thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH) in bovine brain. Bruin Res. 168:553-564,1979. Hsueh, A. J. W., and P. B. C. Jones. Extrapituitary actions of gonadotropin-releasing hormone. Endocr. Rev. 2: 437-461, 1981. Jan, L. Y., and Y. N. Jan. Peptidergic transmission in sympathetic ganglia of the frog. J. Physiol. Lond. 327: 219-246, 1982. Karten, M. J., and J. E. Rivier. Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocr. Rev. 7: 44-66, 1986. Kochman, K., B. Kerdelhue, U. Zor, and M. Jutisz. Studies of enzymatic degradation of luteinizing hormone-releasing hormone by different tissues. FEBS Lett. 50: 190-194, 1975.
RAT
INTESTINAL
MOTILITY
19. Loumaye, E., Z. Naor, and K. J. Catt. Binding affinity and biological activity of gonadotropin releasing hormone agonists in isolated pituitary cells. Endocrinology 111: 730-736, 1982. 20. Mathias, J. R., K. L. Ferguson, and M. H. Clench. Debilitating “functional” bowel disease controlled by leuprolide acetate, gonadotropin-releasing hormone (GnRH) analog. Dig. Dis. Sci. 34: 761766, 1989. 21. Mathias, J. R., and D. S. Finelli. Functional disorders of the small intestine. In: Contemporary Issues in Gastroenterology. Functional Disorders of the Gastrointestinal Tract, edited by S. Cohen and R. Soloway. New York: Churchill-Livingstone, 1987, vol. 6, p. 39-58. 22. Ruckebusch, Y., and J. Fioramonti. Electrical spiking activity and propulsion in small intestine in fed and fasted rats. Gastroenterology 68: 1500-1508, 1975. 23. Schally, A. V., A. Arimura, A. J. Kastin, H. Matsuo, Y. Baba, T. W. Redding, R. M. Nair, L. Debeljuk, and W. F. White. Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. Science Wash. DC 173: 1036-1038,197l. 24. Scott, L. D., R. Lester, D. H. Van Thiel, and A. Wald. Pregnancy-related changes in small intestinal,myoelectric activity in the rat. Gustroenterology 84: 301-305, 1983. 25. Snedecor, G. W., and W. G. Cochran. Statistical Methods (7th ed.). Ames: Iowa State Univ. Press, 1980, p. 507. 26. Szurszewski, J. H. A migrating electric complex of the canine small intestine. Am. J. Physiol. 217: 1757-1763, 1969. 27. Vickery, B. H. Comparison of the potential for therapeutic utilities with gonadotropin-releasing hormone agonists and antagonists. Endocr. Rev. 7: 115-124, 1986. 28. Wald, A., D. H. Van Thiel, L. Hoechstetter, J. S. Gavaler, K. M. Egler, R. Verm, L. Scott, and R. Lester. Gastrointestinal transit: the effect of the menstrual cycle. Gastroenterology 80: 1497-1500,198l. 29. Wald, A., D. H. Van Thiel, L. Hoechstetter, J. S. Gavaler, K. M. Egler, R. Verm, L. Scott, and R. Lester. Effect of pregnancy on gastrointestinal transit. Dig. Dis. Sci. 27: 1015-1018, 1982. 30. Wilk, S., M. Benuck, M. Orlowski, and N. Marks. Degradation of luteinizing hormone-releasing hormone (LH RH) by brain prolyl endopeptidase with release of des-glycinamide LH RH and glycinamide. Neurosci. Lett. 14: 275-279, 1979. 31. Yen, S. S. C. Clinical applications of gonadotropin-releasing hormone and gonadotropin-releasing hormone analogs. Fertil. Steril. 39: 257-266, 1983. 32. Yen, S. S. C., and A. Lein. Reproductive cycles of vertebrates. Mammals: man. In: Marshall’s Physiology of Reproduction (4th ed.), edited by G. E. Lamming. Edinburgh, UK: Churchill-Livingstone, 1984, vol. 11, p. 713-788.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
enteric
NATIVEGONADOTROPIN-RELEASING hormone(GnRH)is a decapeptide (pGlu’-His’-Trp3-Ser4-Tyr5-Gly”-Leu7Arg’-Prog-Gly’“-NH2) (1, 23) that is synthesized and stored in the neurosecretory cells of the medial basal hypothalamus and then secreted into the hypothalamicpituitary portal circulation (12). GnRH binds to specific receptors (3) on the gonadotrophs in the anterior pituitary to initiate secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (12, 23). FSH stimulates ovarian follicle growth and maturation in females and spermatogenesis in males, whereas LH stimulates ovulation and corpus luteum formation in females and testosterone secretion in males (12). In turn, these steroid hormones regulate GnRH secretion through closed-loop feedback mechanisms (32). In women, native GnRH is secreted in a pulsatile manner, its peak occurring in the middle of the menstrual cycle (6), but if GnRH is given in a continuous dose, as in a daily injection or depot form, there is an initial rise in LH and FSH levels, followed -7-10 days later by a decrease in both LH and FSH secretion which remains low thereafter (2, 9). This phenomenon, referred to as downregulation or “desensitization,” results in sex hormones also being significantly decreased or completely 0193-1857/92
$2.00 Copyright
inhibited (2, 9). The same phenomenon occurs in the female rat and most other female mammalian vertebrates (5, 17). Desensitization has been applied clinically in various conditions (e.g., hormone-dependent prostatic carcinoma in men, central precocious puberty, and endometriosis) and is currently being investigated for hypogonadism, contraception, and polycystic ovary syndrome (8) Various synthetic analogues of native GnRH have been made, with the predominant form possessing changes at the following three positions on the molecule: at position 6, substitution of a D-amino acid (7), which decreases the analogue’s susceptibility to enzymatic degradation by peptidases in the hypothalamus (14, 30) and pituitary (13, 18); at position 10, a des-Gly-NH, deletion; and at position 9, addition of an ethylamide to the proline, which increases the binding affinity to gonadotropin receptors (19). Because these analogues are more potent than native GnRH, they may be used in various clinical situations (8, 31). Leuprolide acetate (pGlu’-His2-Trp3Ser4-Tyr5-D-Leu’-Leu7-Arg’-Prog-NHCH2CH3) is one such analogue agonist, with a substitution of D-leucine at position 6 and of an ethylamide at position 9 (27). Leuprolide acetate is considerably more potent than native GnRH and causes downregulation or desensitization of pituitary gonadotropins in humans and laboratory animals, including male and female rats (5, 17, 27) “Functional” bowel diseases are emerging as neuromuscular motility disorders of the gastrointestinal tract; of common occurrence, they are estimated to affect 2% 31 million Americans (21). This laboratory recently reported preliminary results showing that leuprolide effectively treated the debilitating symptoms of these diseases (20). Because the symptoms are expressed -30 times more often in women than men, an interaction between the reproductive system and gastrointestinal motility is suggested (20). The present study was designed to investigate the myoelectric activity of the small intestine in the female rat in the fasted and postprandial states as follows: 1) under control conditions (in the intact reproductive state and without drug administration), 2) in the intact reproductive state after the administration of leuprolide acetate given subcutaneously daily, 3) after bilateral ovariectomy but without therapy with leuprolide acetate, and 4) after bilateral ovariectomy and with therapy with leuprolide acetate. MATERIALS AND METHODS The study model used was similar to one we and others have reported using in male rats (11). Female Wistar rats (Charles
0 1992 the American
Physiological
Society
G185
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
G186
LEUPROLIDE
AFFECTS
FEMALE
River, Wilmington, MA) were prepared for study with implanted electrodes. After the animals were anesthetized with ketamine hydrochloride (100 mg/kg ip), an incision was made in the skin at the back of the neck, and four miniature bipolar silver-silver chloride electrodes were tunneled subcutaneously to the abdomen, which was then opened by a vertical midline incision. After the proximal small intestine was identified, the electrodes were sewn onto the serosal surface of the jejunum, 5 cm apart, beginning 5 cm distal to the ligament of Treitz. The other end of the electrode assembly had previously been soldered to an electrical connector (ITT Cannon, Santa Ana, CA) and had a saddle of dental acrylic (Hygenic, Akron, OH) formed around it to fit the curvature of a rat’s thoracic spine and neck area. With the connector placed on the neck, the neck skin and abdomen were closed, and the rats were kept warm by a water-perfused heating blanket until fully recovered from the anesthesia. Each rat was then allowed to recover from surgery until completely healed; myoelectric recordings were not started until 8-10 days after surgery. In ovariectomized rats, the ovaries were removed bilaterally during the electrode implantation surgery. Eight intact and six ovariectomized rats were included in this study. The approximate age of the rats was 60 days at the time of surgery; weights ranged from 180 to 225 g. Throughout the study the animals were given water and food (Formulab 5008; Purina Mills, St. Louis, MO) ad libitum, except for occasional 16-h fasting periods when the food was withdrawn. The rats were weighed regularly to assure normal growth. Recordings were made on a SensorMedics R612 Dynograph with 9878 AC couplers (SensorMedics, Anaheim, CA) at a sensitivity of 0.5 mV/cm, a time constant of 0.3 s, a highfrequency filter of 30 Hz, and a paper speed of 1 mm/s. All recording was conducted during normal daylight hours, with the rats awake in their cages and unrestrained except for the connector lead. Studies were conducted with all rats in both postprandial and fasted conditions and before and after leuprolide administration; each animal acted as its own control. Four 2-h control recordings were begun 10 days after surgery and before leuprolide was administered, two while the animal was fasted and two under postprandial conditions: rats were fasted (overnight) for 16 h, recorded for 2 h, given two 5-g food pellets and allowed 1 h in which to eat them, and then recorded again for 2 h. In each instance, the rat was watched to confirm ingestion of both food pellets before the control recording of the postprandial condition began. When rats were recorded in the postprandial condition after leuprolide administration had begun, they had food continuously available in their cages; their body weight and defecation rate during the recording sessions suggested normal feeding behavior. Treatment with leuprolide (Lupron; TAP Pharmaceuticals, North Chicago, IL) was begun after the fasted and postprandial control recordings were completed. Once treatment had started (-10-12 days after the electrodes were implanted), leuprolide was administered subcutaneously each morning at the same time, regardless of whether the animal was to be recorded that day. On recording days, the session began after administration of the daily dose. Recordings were not made every day because the animals were allowed regular rest days, but multiple recordings of each rat under all its experimental conditions were obtained. Recordings of intact rats totaled 192 h, and those of ovariectomized rats totaled 230 h. Intact rats were administered leuprolide in amounts equivalent to therapeutic dosages established for humans as follows (in pg/kg body wt): 0.02 (n = 3), 0.2 (n = 3), or 0.4 (n = 2). In ovariectomized rats, the dosages were (in ,ug/kg body wt) 0.02 (n = 2), 0.2 (n = 2), 0.4 (n = 2), 1.0 (n = 3), or 2.5 (n = 2). The last recording in the series was with the rat in the postprandial
RAT
INTESTINAL
MOTILITY
condition. When that recording was complete, each animal was killed with a pentobarbital sodium overdose, and the viscera were examined to confirm the presence of food in the stomach and proximal small bowel. The recordings were analyzed visually by two independent observers (R. Khanna and M. H. Clench) for the number of migrating myoelectric complexes (MMCs) per hour, the duration of MMC phasesI, II, and 111, the propagation velocity of phase III, and the slow-wave frequency. The data for all myoelectric studies were first assessed for kurtosis and then by parametric analysis (25). Because the kurtosis values were close to 1.00, analysis was performed with the Student’s independent t test; P < 0.05 was considered statistically significant. RESULTS
Intact (Not Ovariectomixed) Rats Control. In previous studies with male rats as the experimental model, we and others have not seen cycling MMCs when the animals were studied while postprandial. (Note the terminology distinction between postprandial or “fed” animals, those recorded while food is present in the intestinal lumen, and “fed-state” myoelectric activity, with its characteristic pattern of random and frequent spike potentials.) Control recordings of the intact female rats in our study had from 4.4 to 4.7 MMC/ h while the animals were in the fasted condition (Table 1). A representative tracing from a control period, fastedintact female rat is shown in Fig. 1. When the animals were postprandial, cycling MMCs were also seen, although they were fewer than when the females were fasted (from 1.4 to 2.6 MMC/h for controls; Table 1). Leuprolide treatment. In all the intact rats treated with leuprolide, fed-state myoelectric activity was inhibited. Regardless of whether the treated animals were postprandial or fasted, the numbers of cycling MMCs were not significantly different from fasted-control values or from each other, ranging from 3.4 to 5.4 MMC/h (Table 1). Among the postprandial rats, all treatment group values were significantly higher than those of control periods (P values of ~0.01 to 0.0001; Table 1). Figure 2 shows a representative MMC in an intact rat treated with leuprolide and recorded while postprandial. Ovariectomized Rats Table 2 shows the number of MMCs recorded after ovariectomy. Groups of rats were studied at leuprolide doses of 0.02, 0.2, 0.4, 1.0, and 2.5 pg/kg body wt. The Table 1. Migrating myoelectric complexes in intact female rats Leuprolide Dosage
Controls
Acetate Group Fasted
0.02 pg/kg body wt 4.69t0.24 0.2 pg/kg body wt 4.43k0.25 0.4 pg/kg body wt 4.75k0.48
Postprandial
2.60&0.27* 1.40t0.48* 2.33&0.33*
Treatment Leuprolide Fasted
with Acetate Postprandial
4.60t0.12 4.83+0.65? 5.41k0.17 4.11&0.22f4.48kO.12 3.45&0.25-/-
Values are means I~I: SE and are in no. of migrating myoelectric complexes (MMC) per hour; n = 3 rats in 0.02- and 0.2-pg dosage groups, and n = 2 in 0.4-pg dosage group. Controls received no leuprolide acetate. * P < 0.05 for control postprandial vs. treated postnrandial. Inhibition of fed state.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
LEUPROLIDE
AFFECTS
FEMALE
RAT
INTESTINAL
G187
MOTILITY
5 cm JJ E3. lT 5 cm c
-u-
60
set
I)
Fig. 1. A representative tracing of a migrating myoelectric complex (MMC) in a fasted-intact, control female rat. EZectrodes l-4 are illustrated schematically on left; a sensitivity calibration of 0.5 mV/cm and a I-min time scale are also shown. An activity front of MMC is shown propagating over all 4 electrode sites. Slope of line indicates propagation velocity (mean = 2.90 cm/min).
numbers of MMCs in the ovariectomized fasted-control and the fasted-treated rats were similar to those in both groups of fasted-intact rats. After bilateral ovariectomy, the postprandial-control rats and the postprandialtreated rats given leuprolide at the lower dosageshad no cycling MMCs (the value of 0.88 t 0.58 MMCs in the postprandial-control column of Table 2 denotes activity seen in a single rat on a single occasion). Fed-state myoelectric activity (random and frequent spike potentials) consistently occurred after leuprolide administration at dosagesof 0.02, 0.2, and 0.4 pg/kg, but at dosages of 1.0 and 2.5 pg/kg the fed-state activity was inhibited as it had been with the lower dosagesof leuprolide before ovariectomy; under higher-dose leuprolide treatment, cycling MMCs once again occurred in postprandial rats. At the l.O-pg/kg dose, treated rats had 4.50 t 0.34 MMC/h in contrast to none during their postprandial-control period (P < 0.0001); at 2.5 pg/kg, the numbers were 5.00 L-
..-.
---
& 0.58 vs. 0.88 t 0.58 MMC/h (P < 0.001). This degree of activity at higher leuprolide dosagesin ovariectomized rats was not significantly different from that in treated intact females when postprandial during recording (Tables 1 and 2; P = 0.08). MMC Characteristics Intact rats. The characteristics of the MMCs recorded in intact female rats are shown in Table 3. When the animals were fasted, significant changes occurred in the group treated with leuprolide compared with controls; the duration of phase II was shorter (P < O.OOl), the duration of phase III was longer (P < 0.02), and the propagation velocity was slower (P c 0.001). When the rats were postprandial, phase I duration became longer in the treated group compared with controls (P < O.Ol), phase II became shorter (P < O.OOOl), and phase III ..-
-
-
-
. ..-_
h
h l-
-
-.-
....
.__
qi
-
-.-
-i
-
.-
-.i.--
- --
h
-.-
_.e---d\
h
5 cm
5 cm
w
60
set
I
-u-
5 cm
I
Fig. 2. A representative tracing of an MMC in a postprandial-intact, 1 for details. Mean propagation velocity of phase III of this complex was inhibited after leuprolide treatment.
leuprolide-treated was 2.46 cm/min.
female rat. See legend to Fig. Fed-state myoelectric activity
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
GM8 Table
LEUPROLIDE
AFFECTS
FEMALE
2. MMC in ovariectomized rats
Leuprolide Acetate Dosage Group
0.02 pg/kg 0.2 pg/kg 0.4 ,ug/kg 1.0 pg/kg 2.5 ,ug/kg
body body body body body
Treatment Leuprolide
Controls
wt wt wt wt wt
Fasted
Postprandial
3.86t0.14 5.17t0.31 4.50t0.22 4.OO_tO.17 4.5OkO.22
0 0 0.88&0.58*
0-t 0.88kO.58”“r
Fasted
RAT
INTESTINAL
MOTILITY
All rats, with or without ovariectomy, gained weight. No alteration in stool consistency was noted in any of the animals; the fecal pellets appeared normal in both the control and leuprolide-treated rats.
with Acetate Postprandial
4.4020.16 0 5.40to. 15 0 5.23t0.41 0 5.00t0.41 4.50+0.34$ 4.50t0.29 5.00&0.58$
Values are means * SE and are in MMC/h; n = 3 in 1.0~pg dosage seen in a single rat on a GOUP, n = 2 in all other groups. * Activity single occasion. ‘f P < 0.001 for control postprandial vs. treated postprandial. $ Inhibition of fed state.
duration and propagation velocity were unchanged. The slow-wave frequency increased in the postprandialtreated rats (P < 0.02). Ovariectomized rats. The characteristics of the MMC after ovariectomy are shown in Table 4. The fed-state activity was not inhibited when the rats were under postprandial-control conditions during recording, and no cycling MMCs were observed. When the rats were fasted, the durations of both phases II and 111 were shorter in the leuprolide-treated group (P < 0.0001 and 0.01, respectively, compared with controls), and the slow-wave frequency increased in both leuprolide groups (P < 0.02). Comparison of MMC characteristics before and after ovariectomy. The characteristics of the MMC before and after ovariectomy are shown in Tables 3 and 4. Data from the ovariectomized rats with those from the intact rats compare as follows. The duration of phase I increased in fasted-controls (P < 0.02) and in fastedtreated (P < 0.001) rats. The phase I duration did not change significantly (P = 0.11) in the postprandial leuprolide-treated rats. In contrast, all phase II durations decreased after ovariectomy (P < 0.01 in the fasted leuprolide-treated rats and P < 0.04 in the postprandialtreated). The control animals showed less decrease in phase 11 duration, but it was not significant. Phase III duration also decreased in all groups with ovariectomy (P < 0.0001 in both the fasted and postprandial leuprolide-treated rats). The fasted controls also decreased, but not significantly. The propagation velocity changed only in the fasted leuprolide-treated rats, where it increased (P < 0.0001). Changes in the propagation velocity were not significantly different in the treated rats when recorded postprandially, nor were they in the control groups. The slow-wave frequency increased in both the fasted and postprandial leuprolide-treated rats (P < 0.02 and 0.005, respectively).
DISCUSSION
Szurszewski (26) in 1969 first showed a myoelectric complex that migrated the entire length of the small bowel; this has come to be called the MMC. Since its description and characterization, considerable investigation has been performed to explain the origin and function of the MMC. That the complex appears to exist in all warm-blooded vertebrates (4) suggests the importance of this complex as a basic motility pattern, yet the exact function of this complex remains unknown. Even without an understanding of its function, however, the MMC is extensively used by investigators to study neuromuscular mechanisms (21). In humans, subprimates, dogs, and male rats, it occurs only during the fasting condition, but it is present in all ruminants whether fasted or postprandial and may be present during the postprandial condition in pigs and rats if the animals are accustomed to small frequent meals (4, 22). Although motility studies have almost exclusively made use of male animal models to avoid the complicating effects thought to be reflective of female reproductive cycles, the end results of GnRH analogue administration are well known to affect the two sexes in strongly different ways (15). Because functional bowel diseaseprimarily occurs in women (ZO), we elected to use a female rat model with and without an intact reproductive system and thus with and without the gonadal hormones present (postovariectomy status). Scott et al. (24) also showed changes in MMC periodicity in fasting ovariectomized and intact female rats during the various stages of the estrous cycle. Our study shows that MMCs occur in intact control female rats when they are in the postprandial condition, although at one-third to one-half the rate recorded when they are fasted (Table 1). These observations are in contrast to the pattern seen in the male rat. In our model, -4-5 MMC/h occur in fasted males and none are seen in postprandial males (11). Ruckebusch and Fioramonti (22) have found MMC-like activity in male rats that had been fed small and frequent meals, and this is true of other mammalian species as well. Although the presence of MMCs in postprandial female rats may result from their being accustomed to small frequent meals, all of our animals (male or female) have been fed ad libitum
Table 3. Characteristics of MMCs in intact female rats
Phase I, min Phase II, min Phase III, min Propagation velocity, Slow-wave frequency,
cm/min min-l
5.19kO.31 4.83t0.38 2.41t0.05 2.90&O. 17 39.30t0.41
Values are means t SE. Data were pooled from all dosages. postprandial compared with treated postprandial. (P < 0.02).
2.32t0.41 10.61t1.31 2.22t0.13 2.63kO.21 38.91t0.37 * Significant
values
5.36rt0.14 3.59&O. 16* 2.59t0.04* 2.57&0.05* 39.37kO.18 for control
fasted
compared
3.68t0.17* 8.19t0.66* 2.37t0.04 2.46kO.11 40.18t0.23* with
treated
fasted
or control
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
LEUPROLIDE
Table
AFFECTS
FEMALE
RAT
INTESTINAL
G189
MOTILITY
4. Characteristics of MMCs in ovariectomized female rats Controls Fasted
Phase I, min Phase II, min Phase III, min Propagation velocity, Slow-wave frequency,
cm/min min-l
6.25t0.31 4.61kO.31 2.33t0.07 2.65t0.13 39.3OkO.41
Values are means & SE. Data were pooled from all dosages. postprandial compared with treated postprandial (P < 0.02).
Leuprolide Postprandial
and thus the feeding regimen would not seem to have been an important variable. In contrast to our observations in intact females, ovariectomized control rats did not show MMCs when they had food in the intestinal lumen during recording (Tables 2 and 4). These observations indicate that the absence of ovaries and subsequent hormonal changes (inhibition of sex hormones) alter the expression of the MMC under postprandial conditions. When we treated postprandial intact rats with leuprolide acetate, the number of MMCs per hour increased to values similar to those during the fasting condition (Table 1). In postprandial leuprolide-treated ovariectomized rats, MMCs were also induced, but only at a high dose (~1.0 pg/kg; Table 2). In contrast, the MMC was not seen in ovariectomized rats in the postprandial-control condition (Table 2). These results indicate that leuprolide, in addition to its effect on the inhibition of gonadotropins and sex hormones, also stimulates cycling motor activity during either fasted and postprandial conditions through as-yet-unknown neural mechanisms. How leuprolide inhibits fed-state activity and restores cycling MMCs remains unknown. Progesterone, which is secreted by the corpus luteum after ovulation and during pregnancy and is also used as a contraceptive agent, has been shown to prolong small-bowel transit in humans (28, 29). The corpus luteum also secretes other hormones, such as relaxins A and B, whose effect on intestinal muscle and nerve remains unknown; but because relaxins have been shown to inhibit uterine smooth muscle by interfering with myosin light-chain kinase (20), we speculate that relaxins may have the same effect on gastrointestinal smooth muscle. The inhibition of the hypothalamic-pituitary-ovarian axis by leuprolide, and the resultant decrease in gonadotropin and sex hormones indicate that the various hormones alone or in combination significantly affect the motility of the small intestine. DuPont et al. (10) showed significant GnRH uptake in the small intestine of mice by distribution studies with tritiated GnRH. A GnRHlike peptide has also been demonstrated as a putative neurotransmitter in the prevertebral sympathetic ganglion of the bullfrog (Rana catesbeiana) (16). These two studies indicate that GnRH peptides may act in the nerves of the enteric nervous system either directly as a neurotransmitter or indirectly by modulating specific neurons. The present study shows that in the female rat, unlike the situation in the male rat, cycling MMC activity occurs (albeit less well organized) in either the fasted or
Postprandial
6.20t0.15 3.06t0.12* 2.17t0.03* 2.85kO.06 40.26t0.21*
0 0 0 0 38.4220.3 * Significant
Treatment
Fasted
values
for control
fasted
4.37t0.33 5.13t0.78 1.88t0.06 2.42kO.18 40.86t0.59"
compared
with
treated
fasted
or control
postprandial condition. This phenomenon in the female rat changes significantly when the ovaries are removed. Ovariectomized control female rats do not exhibit MMCs in the postprandial condition; but when treated with sufficiently high doses of leuprolide, they develop organized MMCs in either the postprandial or fasted conditions. Therefore ovariectomized female rats treated with leuprolide develop cycling MMC activity like intact females, but they require higher doses. The same requirement has been noted in humans. We suggest that gonadotropins and ovarian sex hormones significantly influence the motor activity of the gastrointestinal tract and determine the expression of motor activity; this influence may vary, depending on the phase of the reproductive cycle and which substances are secreted. Leuprolide acetate improves not only the quality but the quantity of cycling activity. We also suggest that this might be accomplished by at least two mechanisms of action: 1) inhibition of gonadotropins and ovarian sex hormones that act as endogenous antagonists on gastrointestinal nerve and muscle, and 2) stimulation of organized cycling motor activity through asyet-unexplained neural mechanisms. Understanding the mechanisms of action of GnRH analogues will facilitate a better understanding of the pathophysiology of neuromuscular dysfunction in gastrointestinal disorders and of how the reproductive system may interact with gastrointestinal tract function. We thank Alice W. Cullu for editorial assistance and Patricia A. Ciejka for checking the accuracy of our references. This research was supported by funds from The University of Texas Medical Branch at Galveston. Leuprolide acetate (Lupron) was kindly supplied by Takeda-Abbott Pharmaceuticals. Address reprint requests to J. R. Mathias. Received
2 February
1990; accepted
in final
form
5 July
1991.
REFERENCES Amoss, M., R. Burgus, R. Blackwell, W. Vale, R. Fellows, and R. Guillemin. Purification, amino acid composition and Nterminus of the hypothalamic luteinizing hormone releasing factor (LRF) of ovine origin. Biochem. Biophys. Res. Commun. 44: 205210,1971. Belchetz, P. E., T. M. Plant, Y. Nakai, E. J. Keogh, and E. Knobil. Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science Wash.DC202: 631-633,1978. Clayton, R. N., and K. J. Catt. Regulation of pituitary gonadotropin-releasing hormone receptors by gonadal hormones. Endocrinology 108: 887-895, 1981. Clench, M. H., V. M. Pifieiro-Carrero, and J. R. Mathias. Migrating myoelectric complex demonstrated in four avian species.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
G190
5. 6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16. 17.
18.
LEUPROLIDE
AFFECTS
FEMALE
Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G598G603, 1989. Conn, P. M., and W. F. Crowley, Jr. Gonadotropin-releasing hormone and its analogues. N. Engl. J. Med. 324: 93-103, 1990. Conn, P. M., A. J. W. Hsueh, and W. F. Crowley, Jr. Gonadotropin-releasing hormone: molecular and cell biology, physiology, and clinical applications. Federation Proc. 43: 2351-2361, 1984. Coy, D. H., J. A. Vilchez-Martinez, E. J. Coy, and A. V. Schally. Analogs of luteinizing hormone-releasing hormone with increased biological activity produced by D-amino acid substitutions in position 6. J. Med. Chem. 19: 423-425, 1976. Cutler, G. B., Jr., A. R. Hoffman, R. S. Swerdloff, R. J. Santen, D. R. Meldrum, and F. Comite. Therapeutic applications of luteinizing-hormone-releasing hormone and its analogs. Ann. Intern. Med. 102: 643-657,1985. Dowsett, M., B. Cantwell, L. Anshumala, S. L. Jeffcoate, and A. L. Harris. Suppression of postmenopausal ovarian steroidogenesis with the luteinizing hormone-releasing hormone agonist goserelin. J. C&n. EndocrinoZ. Metab. 66: 672-677, 1988. DuPont, A., F. Labrie, G. Pelletier, R. Puviani, D. H. Coy, E. J. Coy, and A. V. Schally. Organ distribution of radioactivity and disappearance of radioactivity from plasma after administration of [“Hlluteinizing hormone-releasing hormone in mice and rats. Neuroendocrinology 16: 65-73, 1974. Eaker, E. Y., G. B. Bixler, A. J. Dunn, W. V. Moreshead, and J. R. Mathias. Chronic alterations in jejunal myoelectric activity in rats due to MPTP. Am. J. Physiol. 253 (Gastrointest. Liver Physiol. 16): G809-G815, 1987. Hazum, E., and P. M. Conn. Molecular mechanism of gonadotropin releasing hormone (GnRH) action. I. The GnRH receptor. Endocr. Rev. 9: 379-386, 1988. Hazum, E., M. Fridkin, T. Baram, and Y. Koch. Degradation of gonadotropin-releasing hormone by anterior pituitary enzymes. FEBS Lett. 127: 273-276,198l. Hersh, L. B., and J. F. McKelvy. Enzymes involved in the degradation of thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH) in bovine brain. Bruin Res. 168:553-564,1979. Hsueh, A. J. W., and P. B. C. Jones. Extrapituitary actions of gonadotropin-releasing hormone. Endocr. Rev. 2: 437-461, 1981. Jan, L. Y., and Y. N. Jan. Peptidergic transmission in sympathetic ganglia of the frog. J. Physiol. Lond. 327: 219-246, 1982. Karten, M. J., and J. E. Rivier. Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocr. Rev. 7: 44-66, 1986. Kochman, K., B. Kerdelhue, U. Zor, and M. Jutisz. Studies of enzymatic degradation of luteinizing hormone-releasing hormone by different tissues. FEBS Lett. 50: 190-194, 1975.
RAT
INTESTINAL
MOTILITY
19. Loumaye, E., Z. Naor, and K. J. Catt. Binding affinity and biological activity of gonadotropin releasing hormone agonists in isolated pituitary cells. Endocrinology 111: 730-736, 1982. 20. Mathias, J. R., K. L. Ferguson, and M. H. Clench. Debilitating “functional” bowel disease controlled by leuprolide acetate, gonadotropin-releasing hormone (GnRH) analog. Dig. Dis. Sci. 34: 761766, 1989. 21. Mathias, J. R., and D. S. Finelli. Functional disorders of the small intestine. In: Contemporary Issues in Gastroenterology. Functional Disorders of the Gastrointestinal Tract, edited by S. Cohen and R. Soloway. New York: Churchill-Livingstone, 1987, vol. 6, p. 39-58. 22. Ruckebusch, Y., and J. Fioramonti. Electrical spiking activity and propulsion in small intestine in fed and fasted rats. Gastroenterology 68: 1500-1508, 1975. 23. Schally, A. V., A. Arimura, A. J. Kastin, H. Matsuo, Y. Baba, T. W. Redding, R. M. Nair, L. Debeljuk, and W. F. White. Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. Science Wash. DC 173: 1036-1038,197l. 24. Scott, L. D., R. Lester, D. H. Van Thiel, and A. Wald. Pregnancy-related changes in small intestinal,myoelectric activity in the rat. Gustroenterology 84: 301-305, 1983. 25. Snedecor, G. W., and W. G. Cochran. Statistical Methods (7th ed.). Ames: Iowa State Univ. Press, 1980, p. 507. 26. Szurszewski, J. H. A migrating electric complex of the canine small intestine. Am. J. Physiol. 217: 1757-1763, 1969. 27. Vickery, B. H. Comparison of the potential for therapeutic utilities with gonadotropin-releasing hormone agonists and antagonists. Endocr. Rev. 7: 115-124, 1986. 28. Wald, A., D. H. Van Thiel, L. Hoechstetter, J. S. Gavaler, K. M. Egler, R. Verm, L. Scott, and R. Lester. Gastrointestinal transit: the effect of the menstrual cycle. Gastroenterology 80: 1497-1500,198l. 29. Wald, A., D. H. Van Thiel, L. Hoechstetter, J. S. Gavaler, K. M. Egler, R. Verm, L. Scott, and R. Lester. Effect of pregnancy on gastrointestinal transit. Dig. Dis. Sci. 27: 1015-1018, 1982. 30. Wilk, S., M. Benuck, M. Orlowski, and N. Marks. Degradation of luteinizing hormone-releasing hormone (LH RH) by brain prolyl endopeptidase with release of des-glycinamide LH RH and glycinamide. Neurosci. Lett. 14: 275-279, 1979. 31. Yen, S. S. C. Clinical applications of gonadotropin-releasing hormone and gonadotropin-releasing hormone analogs. Fertil. Steril. 39: 257-266, 1983. 32. Yen, S. S. C., and A. Lein. Reproductive cycles of vertebrates. Mammals: man. In: Marshall’s Physiology of Reproduction (4th ed.), edited by G. E. Lamming. Edinburgh, UK: Churchill-Livingstone, 1984, vol. 11, p. 713-788.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
