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* Department of Animal Sciences and
and
Department of Dairy Science, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge 70803-4210
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Abstract |
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Key Words: Horses • Prolactin • Somatotropin • Thyrotropin Releasing Hormone
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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), is also an effective secretagogue for prolactin in many species (Jacobs et al., 1971; Convey et al., 1973), including the horse (Thompson et al., 1986; Johnson, 1987). In some species, TRH stimulates GH secretion as well (Smith et al., 1977; Scanes and Harvey, 1989; Cabell and Esbenshade, 1990). In contrast, TRH administration at the doses used to stimulate TSH and prolactin secretion does not stimulate GH secretion in horses (Thompson et al., 1992), whereas simultaneous administration of TRH with GH secretagogues abolishes the expected increase in GH secretion (Kennedy et al., 2002). The present series of experiments was designed 1) to extend our observations on the TRH inhibition of GH secretion in horses, specifically its effects on exercise- and aspartate-induced GH secretion, the duration of the inhibition, and the effects on endogenous GH secretion, and 2) to test the hypothesis that stimulation of endogenous TRH in the horse (by diminishing thyroid function) would reduce the normal rate of GH secretion. |
Materials and Methods |
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Mature, light horse mares (Exp. 1 through 3), geldings (Exp. 5 and 6), and stallions (Exp. 4 and 7) were used. Mares and geldings were maintained on pasture and were in good body condition (6 to 8; Henneke et al., 1983). Stallions were kept in individual outdoor lots with limited access to native grasses and were fed sufficient pelleted commercial feed and grass hay daily to maintain average to good body condition (between 5 and 7). All horses were on a long-term herd health program that included routine deworming and yearly vaccinations for infectious diseases.
For the short-term experiments (Exp. 1 through 6), mares and geldings were brought in from pasture at approximately 0700 and were tethered inside a barn and/or shed; stallions in Exp. 4 were tethered inside their individual shelters. After insertion of a 14-gauge catheter in the left jugular vein, the horses were allowed to stand quietly for a minimum of 1 h before initiation of blood sampling. Blood samples were collected from, and treatment injections were administered through, the jugular catheters. In all experiments, blood samples were placed into disposable glass tubes containing heparin (20 IU/mL) and were kept at 5°C until centrifugation at 1,200 x g for 15 min. Plasma was harvested and stored at -15°C.
Experiment 1.
Twelve mares were used in a split-plot design to characterize the effects of TRH on the GH response to exercise, a known stimulus for GH in horses (Thompson et al., 1992; 1994). Stage of the estrous cycle was not taken into consideration. Mares were randomly allotted to one of two treatment groups (six per group). The treated mares received an i.v. injection of TRH (Catalog no. P2161; Sigma Chem. Co., St. Louis, MO) at 10 µg/kg BW (Kennedy et al., 2002). Control mares received an equivalent amount of saline i.v. Treatments were immediately followed by 5 min of exercise in a round pen (moderate to fast trot; Thompson et al., 1992). Jugular blood samples (5 mL) were collected at -30, -15, 0, 10, 20, 30, 40, 50, 60, 75, 90, 105, and 120 min relative to the onset of exercise for the measurement of GH and prolactin.
Experiment 2.
Twelve mares were used in a split-plot design to characterize the effect of TRH on the GH response to aspartate infusion (Sticker et al., 2001). Stage of the estrous cycle was not taken into consideration. Mares were randomly allotted to one of two treatment groups (six per group). The treated mares received an i.v. injection of TRH as described in Exp. 1; control mares received saline. Immediately after treatment, all mares were infused with sodium aspartate (2.855 mmol aspartate/kg BW; Catalog no. A9256; Sigma Chem. Co.) in water (Sticker et al., 2001). The aspartate solutions were made to 1.427 M, pH of 7.3 to 7.4, and were infused at 2 mL/kg BW.
Samples of jugular blood (5 mL) were collected at -30, -15, and 0 min relative to saline or TRH treatment. Aspartate infusion was started immediately following the 0-min sample and required about 5 min to complete. Subsequent blood samples were drawn at 10, 20, 30, 40, 50, 60, 75, 90, 105, and 120 min after the onset of aspartate infusion. Prolactin and GH were measured in all plasma samples.
Experiment 3.
Six mares were used in a 6 x 6 Latin square to characterize the duration of the TRH inhibition of the GH response to exercise. Stage of the estrous cycle was not taken into consideration. The six treatments were 1) control (saline) injection immediately before exercise, 2) TRH immediately before exercise (as described for Exp. 1), 3) TRH 0.5 h before exercise, 4) TRH 1 h before exercise, 5) TRH 2 h before exercise, and 6) TRH 3 h before exercise. Dose of TRH in each case was 10 µg/kg BW. Jugular blood samples were drawn at 15-min intervals starting 60 min before exercise and continuing through 90 min after onset of exercise. Treatment days were separated by at least 2 d of inactivity. Concentrations of GH were measured in all plasma samples.
Experiment 4.
Six stallions were used in a single switchback design (three replicates of a 2 x 2 Latin square) to determine the effect of a single TRH injection on endogenous GH secretion. The experiment was performed on 2 d, 1 wk apart, in order to minimize any carryover effects of treatment. On the first treatment day, three stallions received TRH (10 µg/kg BW) in saline and the remaining three received an equivalent amount of saline. On the second treatment day, the treatments were reversed. Blood samples were collected at 15-min intervals for 4 h to characterize basal GH secretion, and then the treatments were administered. Subsequent samples were drawn at 15-min intervals for 6 h. Stallions were loosely tethered inside sheds within their individual paddocks during the experiment. They had ad libitum access to hay and water except when blood samples were being drawn. Prolactin and GH were measured in all plasma samples.
Experiment 5.
Ten geldings were used in a split-plot design to determine the effects of multiple hourly TRH injections on endogenous GH secretion. Jugular blood samples were drawn from all geldings at 15-min intervals for a total of 25 h beginning at 0800 on d 1. Immediately after the fifth sample (0900), five geldings received TRH (10 µg/kg BW) in saline i.v. through the jugular catheter; the other five received an equivalent amount of saline. Injections were repeated hourly through 1900 (total of 11 injections). During the 25-h period, the geldings were loosely tethered in stalls in a barn and were given access to grass hay and water on a regular basis. Prolactin and GH were measured in all plasma samples.
Experiment 6.
Because the stimulatory effects of TRH on prolactin and TSH secretion seemed to wane after the first injection in Exp. 5, 10 geldings were used in a split-plot design to test the hypothesis that the inhibitory effects of TRH on GH secretion after exercise would also wane after multiple injections. Five geldings were administered five hourly TRH injections (10 µg/kg BW in saline i.v.) and five geldings were administered saline. Immediately after receiving the fifth injection, each gelding was exercised for 5 min as described in Exp. 1. Jugular blood samples were drawn at 15-min intervals starting 120 min before the first injection and were continued through 120 min after the onset of exercise (8 h total). Prolactin and GH were measured in all plasma samples.
Experiment 7.
Eight stallions were used in a split-plot design to test the hypothesis that reduced thyroid function, resulting in increased endogenous TRH secretion, would reduce endogenous GH secretion. To reduce thyroid activity, four randomly selected stallions were fed 6-n-propyl-2-thiouracil (PTU; Catalog no. P3755; Sigma Chem. Co.) at 6 mg/kg BW (B. Breuhaus, personal communication) in 250 g of a molasses-containing balanced grain ration on top of their regular pelleted feed; four control stallions were given the top dressing with no added PTU. Stallions were fed their diets daily at 0800 and were allowed to eat throughout the day; hay and water were available ad libitum throughout the experiment. Blood samples were collected twice weekly for the measurement of thyroxine and TSH to monitor thyroid function.
Once PTU-fed stallions were determined to have reduced thyroid function (d 52 of treatment), all stallions were brought from their paddocks and placed in stalls for an extensive period of blood sampling and secretagogue challenges (PTU feeding was continued throughout this period). They were allowed 3 d to equilibrate to their surroundings in the barn.
At 1200 on d 3, each stallion was fitted with a 14-gauge indwelling jugular catheter, which was secured to the neck. The stallions were tethered lightly within their stalls and were allowed ad libitum access to hay and water. After an hour of inactivity, 15-min blood sampling was started. The first 22 h of sampling was used to assess endogenous GH secretion. Immediately following the 22-h blood sample (1100 on the second day), each stallion received an i.v. injection of the dopamine antagonist sulpiride (0.10 mg/kg BW; Catalog no. S8010; Sigma Chem. Co.; Johnson and Becker, 1987), followed immediately by an infusion of 2.855 mmol sodium aspartate/kg BW in water (Sticker et al., 2001). Blood samples were collected thereafter at 15-min intervals for an additional 3 h for measurement of prolactin and GH. Immediately after the last sample was drawn, each stallion was given an i.v. injection of TRH (10 µg/kg BW), and blood samples were collected for another 2 h for the measurement of prolactin and TSH.
Sample Analyses.
Concentrations of GH (Thompson et al., 1992), prolactin (Colborn et al., 1991), TSH (Sticker et al., 2001), and thyroxine (ICN Pharmaceuticals, Inc., Costa Mesa, CA) were analyzed with RIA previously validated for horse samples. Intra- and interassay coefficients of variation and assay sensitivities were 8%, 11%, and 0.5 ng/mL for GH; 7%, 12%, and 0.2 ng/mL for prolactin; 5%, 10%, and 0.02 ng/mL for TSH; and 5%, 9%, and 2 ng/mL for thyroxine.
Statistical Analyses.
For each experiment, data were analyzed by univariate ANOVA for repeated measures (split plot; Gill and Hafs, 1971) within a completely random design (Exp. 1, 2, 5, 6, and 7), 6 x 6 Latin square design (Exp. 3; Steel and Torrie, 1980), or replicated 2 x 2 Latin square design (Exp. 4; Steel and Torrie, 1980) using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). For Exp. 1, 2, 5, 6, and 7, factors in the analyses were treatment, horses within treatment (error term used to test treatment), time, and treatment x time (tested with residual error). For Exp. 3, the factors were mare, day, treatment, mare x day x treatment (error term for the first three factors), time, and treatment x time (tested with residual error). For Exp. 4, the factors were squares, stallions within squares, days within squares, treatment, square x stallion x day x treatment (error term for the first four factors), time, and treatment x time (tested with residual error). Differences between treatment groups were tested within each time period by the LSD-test (Steel and Torrie, 1980).
For Exp. 3, the maximum GH concentration achieved within the first 30 min after exercise was selected from the overall data set, and the natural log of these data were analyzed in a 6 x 6 Latin square ANOVA due to heterogeneity of variances (Steel and Torrie, 1980). Differences among treatment means were assessed by the LSD-test (Steel and Torrie, 1980).
Retrospective Analysis.
Based on the estimated duration of inhibition from Exp. 3 and the possible waning of the inhibition over multiple TRH injections (Exp. 6), it was decided to do a pooled, retrospective analysis on all horses for which endogenous (no external stimuli) GH concentrations were available after TRH or saline. The data for blood samples collected at 0 to 60 min relative to treatment from horses in Exp. 4, 5 (first TRH injection only), and 6 (first TRH injection only) were combined and analyzed by split-plot ANOVA. Factors in the ANOVA were experiment, treatment, experiment x treatment, horse within experiment x treatment (error term to test the first three factors), time, experiment x time, treatment x time, and the three-way interaction (the latter four terms being tested with residual error). Because Exp. 4 was performed as a replicated Latin square, the appropriate degrees of freedom were subtracted from the error degrees of freedom in the pooled analysis. The first 60 min was chosen for this analysis based on the results of Exp. 3, which indicated a duration of inhibition of at least 60 min but less than 120 min. Subsequent pooled analyses were performed using GH data for the first 75, 90, and 120 min to estimate at what time point the treatment effect was no longer significant (P > 0.10).
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Results |
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There were treatment effects (P < 0.001) as well as treatment x time interactions (P < 0.0001) for both GH and prolactin concentrations in mares in response to i.v. administration of TRH and/or saline followed by 5 min of acute exercise (Figure 1). Injection of TRH stimulated prolactin concentrations, as expected, and saline did not. Exercise stimulated GH concentrations, as expected, in mares receiving saline, whereas prior injection of TRH abolished the GH response.
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There were treatment effects (P < 0.001) as well as treatment x time interactions (P < 0.0001) for both GH and prolactin concentrations in mares in response to i.v. administration of TRH and/or saline followed by infusion of sodium aspartate (Figure 2). Injection of TRH stimulated prolactin concentrations, as expected, and saline did not. Aspartate infusion stimulated GH concentrations, as expected, in mares receiving saline, whereas previous injection of TRH greatly diminished the GH response. In addition to the hormonal responses, the infusion of aspartic acid resulted in sweating and panting in all mares beginning within 5 min of infusion and lasting from 10 to 15 min.
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There was a treatment effect (P = 0.0002) as well as a treatment x time interaction (P < 0.0001) for GH concentrations in response to exercise in mares treated with saline or TRH at increasing times before exercise (Figure 3a). There was the expected GH response in mares pretreated with saline before exercise, whereas TRH treatment immediately before exercise abolished the GH response. There was no GH response to exercise in mares treated with TRH at 30 min and 1 h before exercise; however, at 2 and 3 h after TRH treatment, the GH response to exercise was present. Based on maximum GH concentration achieved in the first 30 min (Figure 3b), the responses at 2 and 3 h were greater (P < 0.05) than the response after saline treatment, and the responses at 0, 0.5, and 1 h were less (P < 0.05) than after saline treatment.
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Endogenous GH concentrations in stallions were not affected (P > 0.10) by a single injection of TRH administered during the 10-h period of frequent sampling (Figure 4a). As expected, there was a treatment effect (P < 0.0001) and a treatment x time interaction (P < 0.0001) for prolactin concentrations, which increased rapidly when the stallions were administered TRH (Figure 4b).
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Endogenous GH concentrations in geldings were not affected (P > 0.10) by 11 hourly injections of TRH during the 25-h period of blood sampling (Figure 5a). As expected, there was an effect of TRH treatment (P = 0.035) and a treatment x time interaction (P < 0.0001) for prolactin concentrations (Figure 5b), which increased soon after first TRH injection and then gradually declined throughout the rest of the sampling period. There was also a treatment x time interaction (P < 0.0001) for TSH concentrations (Figure 5c), which increased soon after the first TRH injection and then declined gradually over the next 8 h. For both prolactin and TSH concentrations, injections of TRH after the first one or two produced little response in the already elevated concentrations.
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Plasma GH concentrations were affected by time (P < 0.0001) in the geldings in Exp. 6 (Figure 6a), but there was no effect of multiple TRH treatment nor any interaction between treatment and time (P > 0.10); the GH response to exercise at 360 min was virtually identical in the two groups. There was the expected prolactin response to TRH injection (P < 0.0001; Figure 6b), with concentrations in TRH-treated geldings being higher than in control geldings from 15 min after the first injection through the onset of exercise. Exercise resulted in a sharp rise in prolactin concentrations in control geldings, but little to no change in TRH-treated geldings.
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There was a treatment effect (P < 0.0001) as well as a treatment x day interaction (P < 0.0001) for thyroxine concentrations (Figure 7a) when stallions were fed PTU. Although variable and apparently affected by environmental conditions, thyroxine concentrations in stallions fed PTU decreased over time and were approximately half of those in control stallions by d 52. In addition, there was an effect of treatment (P < 0.0001) as well as a treatment x day interaction (P < 0.0001) for TSH concentrations (Figure 7b), which increased gradually in PTU-fed stallions beginning approximately d 20 and were 7 to 8 times higher than in control stallions by d 52.
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). Immediately before TRH was injected at 24.5 h, concentrations of both TSH and prolactin (Figure 9) were already higher (P < 0.05) in PTU-fed stallions relative to controls. Injection of TRH increased (P < 0.001) concentrations of TSH and prolactin in both groups of stallions, and the TSH response was greater (P < 0.05) in PTU-fed stallions than in controls.
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There was an effect of treatment (P = 0.002) and a treatment x time interaction (P = 0.007) in the retrospective analysis of endogenous GH concentrations from horses administered TRH or saline in Exp. 4, 5, and 6 (Figure 10). Horses administered TRH had lower GH concentrations at 45 and 60 min after injection. Similar analyses on data for the first 75, 90, and 120 min after treatment indicated that the difference between groups was present through 75 min (P < 0.02) but was absent thereafter (P > 0.10; data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. Although the mechanism by which exercise and aspartate infusion stimulate GH release is not known, reports for other species indicate that many of these indirect stimuli act via GHRH secretion from the hypothalamus, which in turn stimulates GH secretion from the pituitary (Wehrenberg et al., 1985; Magnan et al., 1994). Alternatively, acute GH secretion may also be due to an abrupt decrease in somatostatin secretion from the hypothalamus (Miki et al., 1988; Guyton and Hall, 1996). Because TRH inhibits GH secretion induced by GHRH itself, it is likely that TRH either affects the somatotropes directly to block GH release or acts via stimulation of somatostatin, which is known to block GH release via inhibition of adenyl cyclase formation in the somatotrope (Bass et al., 1996). Receptors for TRH have been reported on somatotropes of the rat (Konaka et al., 1997) in numbers approximately equal to those on lactotropes. Receptors for TRH have also been localized in various areas of the brain and in peripheral tissues (Zabavnik et al., 1993).
Time analysis of the TRH inhibition of exercise-induced GH secretion indicated that the inhibition lasted at least 1 h but was gone by 2 h. In fact, a rebound-like recovery and/or potentiation was evident at 2 and 3 h, such that the responses to exercise at these times were greater than the uninhibited response. Gentry et al. (2002) described a similar rebound-like response in mares administered the GH secretagogue EP51389 and TRH simultaneously; mares had no GH response in the first 90 min after injection but had surges in GH concentrations between 90 and 180 min. Clark et al. (1988) described a similar rebound secretion of GH after withdrawal of somatostatin infusion in rats; that rebound was subsequently determined to be mediated by hypothalamic release of GHRH.
At first glance, the results of Exp. 4 and 5 seemed to indicate that there is no inhibitory effect of TRH on endogenous GH secretion. However, focusing on only the first 60 min after injection, the time during which TRH inhibition is now known to be present, neither TRH-treated nor control horses had much endogenous GH activity in either experiment. Extending the TRH exposure to several hours in Exp. 5 with multiple injections seemed a logical way to average over the inconsistent (infrequent) endogenous surges among horses; however, the fallacy of that approach was subsequently borne out by Exp. 6. That is, just as the stimulatory effects of TRH on prolactin and TSH secretion waned with multiple injections, so did the inhibitory effect of TRH on exercise-induced GH release. Thus, given the episodic nature of GH release in horses (Thompson et al., 1992) and the infrequent occurrence of episodes, determination of the effect of a single injection of TRH on endogenous episodic GH release would require averaging over more replications than presented in any one experiment. For example, endogenous episodes result in elevated GH concentrations lasting 60 min or less (Thompson et al., 1992), and between-episode intervals range from 3 to 8 h (Thompson et al., 1992; individual horses in Exp. 4 and 5); thus, the chance that a randomly drawn blood sample will be drawn during an endogenous GH episode is 12 to 33%. The retrospective analysis performed with the data from Exp. 4, 5, and 6 was an attempt to average over these infrequent episodes, and although the approach may be less than ideal from a statistical standpoint, the results are consistent with a TRH inhibition of endogenous GH secretion in the first 60 to 75 min after injection.
The loss of inhibitory effect of TRH on exercise-induced GH secretion, as well as the wane in stimulatory effect on prolactin and TSH secretion, is characteristic of a down-regulation of receptors and refractoriness commonly associated with high doses of hypothalamic hormones. Although the TRH dose used in the present experiments (10 µg/kg of BW) was higher than the dose needed to produce maximal TSH response in mares (4 µg/kg of BW; Thompson and Nett, 1984), Gentry et al. (2002) used the lower dose in mares and observed the same inhibition of GH release in response to EP51389.
Experiment 7 was performed in an attempt to raise physiologic levels of TRH by reducing thyroid hormone secretion and feedback. Given the long-term increase in TSH concentrations in stallions fed PTU and their exaggerated response to exogenous TRH, it is likely that TRH secretion from the hypothalamus was increased to some degree. However, the relative importance of the hypothalamus vs pituitary gland as sites of negative feedback by thyroid hormones in the horse has yet to be determined; thus, we can only assume that TRH secretion was increased. Moreover, the direct effects of reduced thyroid activity must also be taken into consideration. There was no alteration in basal GH or prolactin secretion associated with PTU feeding and hypersecretion of TSH, indicating that 1) reduction of thyroid hormone feedback does not affect the secretion of these two pituitary hormones or 2) little or no change in endogenous TRH secretion was induced by the feeding of PTU. In contrast, the enhanced responses in GH and prolactin secretion to aspartate and sulpiride infusion in stallions fed PTU may indicate an increased pituitary content of these hormones or an increased sensitivity to stimulation (although such an increase was not evident for prolactin after TRH injection 3 h later). The increase in prolactin secretion is consistent with the fact that TRH stimulates prolactin secretion and maybe production (Laverriere et al., 1988; Guyton and Hall, 1996), but the increase in GH secretion after aspartate infusion is contrary to our working hypothesis that increased endogenous TRH would reduce GH secretion.
In summary, the TRH inhibition of GHRH- and EP51389-induced GH release reported by Kennedy et al. (2002) has been extended to two other GH-releasing stimuli, exercise and aspartate infusion. The duration of the inhibition is at least 1 h but less than 2 h, and, like the stimulatory effect on prolactin and TSH, wanes with multiple injections. There is likely a TRH inhibition of endogenous GH episodes as well; however, more research on this question is needed to confirm our limited analysis. Although PTU feeding reduced thyroid feedback on the hypothalamic-pituitary axis, as evidenced by increased TSH concentrations, basal GH and prolactin concentrations were unaffected. The increases in aspartate-induced GH secretion and sulpiride-induced prolactin secretion in stallions fed PTU may indicate an alteration in pituitary content of these hormones or sensitivity to stimuli, but again, further research is needed to clarify these possibilities.
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Footnotes |
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2 Correspondence— phone: 225-578-3445; fax: 225-578-3279; E-mail: dthompson{at}agctr.lsu.edu.
Received for publication April 1, 2003. Accepted for publication May 29, 2003.
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